Microbial Enzymes in Bioconversions of Biomass

Biofuel and Biorefinery Technologies 3
Vijai Kumar Gupta Editor
Microbial
Enzymes in
Bioconversions
of Biomass
Biofuel and Biorefinery Technologies
Volume 3
Series editors
Vijai Kumar Gupta, Molecular Glycobiotechnology Group, Department of
Biochemistry, School of Natural Sciences, National University of Ireland
Galway, Galway, Ireland
Maria G. Tuohy, Molecular Glycobiotechnology Group, Department of
Biochemistry, School of Natural Sciences, National University of Ireland
Galway, Galway, Ireland
More information about this series at http://www.springer.com/series/11833
Vijai Kumar Gupta
Editor
Microbial Enzymes
in Bioconversions
of Biomass
123
Editor
Vijai Kumar Gupta
Department of Biochemistry, School of
Natural Sciences
National University of Ireland Galway
Galway
Ireland
ISSN 2363-7609 ISSN 2363-7617 (electronic)

Biofuel and Biorefinery Technologies
ISBN 978-3-319-43677-7 ISBN 978-3-319-43679-1 (eBook)
DOI 10.1007/978-3-319-43679-1
Library of Congress Control Number: 2016947366
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Preface
This book summarizes the information on different biomass converting enzymes

and their potential use in the bioconversion of biomass into simple sugar to generate
bioenergy and other value added co-/by products.
Biofuel produced from agricultural crops, e.g. cereals, maize, sugarcane, sugar
beet, and sweet sorghum, is referred to as first generation biofuels while that
produced from lignocellulosic materials is referred to as second generation biofuels.
The global production of first and second generation biofuels is dependent upon the
lignocellulosic materials available for bioconversion.
Microbes have been established as important mediators in the production of
biofuels and other valuable biorefinery products. It is prevalent that microbes have
been playing a key role by producing key enzymes for bioconversion of various
biomasses for biorefinery applications. This selection of microbes is based upon
their ability to selectively or simultaneously degrade lignocellulosic materials, the
high redox potential of their enzymes, their engineering capabilities and/or their
thermostability. The key to their degradative capabilities is the extracellular
enzymes they secrete. These microorganisms are capable of producing an array of
enzymes such as 1) Cellulase systems, comprised of endo-1,4-β-glucanase, cel-
lobiohydrolase and β-glucosidases; 2) Hemicellulases include endo-1,4-β-
xylanases, β-xylosidases, endo-1,4-β-mannanase and β-mannosidase; 3) Lignin
degrading enzymes comprising laccase, lignin peroxidase, manganese peroxidase
and versatile peroxidase. There are also accessory enzymes which aid the hemi-
cellulases in their degradation of hemicellulose polymers through the cleavage of side
chain residues; these enzymes include α-glucuronidase, α-L-arabinofuranosidase,
acetylxylan esterase, ferulic acid esterase and β-galactosidase.
This book summarizes the information on different biomass converting enzymes
and their potential use in the bioconversion of biomass into simple sugar to generate
bioenergy and other value added co-/by products. The chapters have been con-
tributed by the experts of the area across the globe and these chapters offer clear and
concise information on both standard and new technologies and will serve as an
v
vi Preface
invaluable reference for undergraduates, post-graduates, researchers and practi-

tioners studying and working in the field of microbial enzymes for biofuel and
biorefinery applications.
Galway, Ireland Vijai Kumar Gupta

Contents
1 Microbial Enzymes for Conversion of Biomass to Bioenergy . . . . . . 1

M.P. Raghavendra, S. Chandra Nayaka and Vijai Kumar Gupta
Part I Cellulases
2 Cellobiohydrolases: Role, Mechanism, and Recent
Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Neelamegam Annamalai, Mayavan Veeramuthu Rajeswari
and Nallusamy Sivakumar
3 Endo-1,4-β-glucanases: Role, Applications and Recent
Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
and Thangavel Balasubramanian
4 The Role and Applications of β-Glucosidases in Biomass
Degradation and Bioconversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Hanlin Ouyang and Feng Xu
Part II Hemicellulases
5 Role and Applications of Feruloyl Esterases in Biomass
Bioconversion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Constantinos Katsimpouras, Io Antonopoulou, Paul Christakopoulos
and Evangelos Topakas
6 Endo-β-1,4-xylanase: An Overview of Recent Developments . . . . . . 125
Alexandre Gomes Rodrigues
7 Microbial Xylanases: Sources, Types, and Their Applications . . . . . 151
Hesham Ali El Enshasy, Subeesh Kunhi Kandiyil, Roslinda Malek
and Nor Zalina Othman
vii
viii Contents
8 Mannanase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Suttipun Keawsompong
9 The Role and Applications of Xyloglucan Hydrolase in Biomass
Degradation/Bioconversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
M. Saritha, Anju Arora, Jairam Choudhary, Vijaya Rani,
Surender Singh, Anamika Sharma, Shalley Sharma and Lata Nain
Part III Lignocellulose Oxidureductases

10 Role of Mushroom Mn-Oxidizing Peroxidases in Biomass
Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Mirjana Stajić, Jelena Vukojević, Ivan Milovanović,
Jasmina Ćilerdžić and Aleksandar Knežević
11 Role and Application of Versatile Peroxidase (VP) for Utilizing
Lignocellulose in Biorefineries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Nadine Busse and Peter Czermak
12 Fungal Aryl-Alcohol Oxidase in Lignocellulose Degradation
and Bioconversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Juan Carro, Ana Serrano, Patricia Ferreira and Angel T. Martínez
13 Monosaccharide Oxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Avnish Kumar, Monika Asthana, Hirawati Deval and Sarika Amdekar
Chapter 1
Microbial Enzymes for Conversion
of Biomass to Bioenergy
M.P. Raghavendra, S. Chandra Nayaka and Vijai Kumar Gupta
Abstract Microbial enzymes are capable of degrading a wide range of complex

substrates including carbohydrates into more useful energy source. The simple
sugars then can be converted into ethanol or other liquid biofuels by a large group
of fermentative microbes. Even though cellulose serves as an abundant source of
carbon and energy in the ecosystem, its exploitation as a source of biofuel is
hindered due to lack of effective microbial systems to break it down, including other
carbohydrates to simple sugars leading to more production of biofuels. If these
materials could be exploited, they would represent a massive new energy resource
for biofuel production. In continuous search for alternative energy sources, it is now
proven that electricity can be produced directly from the degradation of organic
matter in a microbial fuel cell and fermentation of lignocellulosic biomass to
ethanol, which is an attractive route to fuels that supplements the fossil
fuels. Studies have revealed that special group enzymes known as feruloyl esterases
produced by microorganisms are capable of breaking apart key links between the
polymers and helps in effective degradation of plant materials. This review covers
various known microbial approaches to convert different carbon sources to simple
soluble sugars en route to production of biofuels. The importance of the biofuel in
future is highlighted by the Renewable Fuel Standard of the United States Energy
Independence and Security Act (EISA) of 2007, which mandates that 36 billion
gallons of biofuels are to be produced annually by 2022, of which 16 billion gallons
are expected to come from cellulosic feed stocks. It is obvious fact that microor-
M.P. Raghavendra
Postgraduate Department of Microbiology, Maharani’s Science College for Women,
JLB Road, Mysore, Karnataka 570 005, India
S.C. Nayaka (&)
Department of Studies in Biotechnology, University of Mysore, Mysore,
Karnataka 570 005, India
e-mail: [email protected]
V.K. Gupta
Department of Biochemistry, School of Natural Sciences,
National University of Ireland, Galway, Ireland
© Springer International Publishing Switzerland 2016 1

V.K. Gupta (ed.), Microbial Enzymes in Bioconversions of Biomass,
Biofuel and Biorefinery Technologies 3, DOI 10.1007/978-3-319-43679-1_1
2 M.P. Raghavendra et al.
ganisms and its array of enzymes need to be effectively screened, identified and
employed in developing effective strategies for converting biomass to biofuel.
1.1 Introduction
Due to over reliance on petrochemicals, rising oil prices, pollution associated with
its usage, world is looking forward for alternative renewable resources of energy
from different sources. Biofuel obtained from carbon sources are gaining
momentum as a source of biofuel but the concern over production of ethanol from
food crops is creating a imbalance and there is a search for a resources, which are
not being used as food and should be available plenty in the ecosystem so that the
problems associated with the starchy material can be addressed. In this connection
cellulosic feed stock is considered to be excellent source for ethanol production, but
there are problems associated with degradation of cellulose also due to its recal-
citrance to biodegradation at least at the early stages of degradation. Liquid fuel
derived from biomass feedstock has been considered as a low-carbon substitute for
fossil hydrocarbons in vehicles, but many of the existing approaches are encoun-
tering significant challenges.
1.2 Importance of Biofuel Research
Search for new energy supplies is an important agenda for several countries in the
coming years. They are developing new energy infrastructure capable of meeting
growing demands for electric power and transportation fuels. Even unrest in oil
producing countries will have great impact on national security and economic
strength of several oil dependent countries. India in particular currently imports
80 % of its oils and it is expected to increase to 91 % by 2020. This overreliance on
oil import is the greatest burden on Indian economy and its security. On the other
hand, India is an agrarian country and known for its biodiversity that can be
exploited to convert biomass to biofuel. Biofuels employ recycling of agricultural
byproducts and dedicated energy crops, which offer opportunities for mitigation of
greenhouse gas emission as growing these leads to C sequestration through
photosynthesis.
Various plants and plant-derived materials are used for biofuels manufacturing
including grains (1st generation) and lignocellulosic biomass (2nd generation). The
second generation of biofuels is more important as they are based on the cheap and
abundant lignocellulosic biomass and do not compete with food crops. Wheat straw
and bagasse are considered that best example, which are inexpensive and widely
available ligonocellulosic resources containing 75–80 % polysaccharides (cellulose
and hemicelluloses). The monosaccharides glucose and xylose obtained from
hydrolysis of these can be used as substrate for production of industrially important
1 Microbial Enzymes for Conversion of Biomass to Bioenergy 3
Proteins and Secondary

Amino acids metabolites
Pretreatment, hydrolysis Microbial degradation
Lignocellulose and other carbohydrates
Lignin, Hydrolysates of
Cellulose hemicellulose
Enzymatic/acid hydrolysis
Xylose, mannose, glucose,

galactose, rhamnose, arabinose
Lignin Hexose
Fragmentation
Depolymerizatio Chemical conversion
Green gasoline or
diesel Fermentation
Gaseous fuels: H2, methane

Liquid fuels: methane, ethanol, dimethyl ether, butanol, alkanes
Fig. 1.1 Conversion of carbohydrate biomass into biofuel
products (Fig. 1.1). These products will have more potential economical, envi-
ronmental, and strategic advantages over traditional fossil-based products.
The era of advanced biofuels—cellulosic ethanol, biomass-based diesel,
biobutanol, bio-oil, green gasoline, and bio-based jet fuel—is also drawing nearer
and nearer. According to the International Energy Agency, biofuels have the
potential to meet more than a quarter of world demand for transportation fuels by
2050. Bioethanol is by far the most widely used biofuel for transportation world-
wide (Mohanram et al. 2013). It has been estimated that widespread use of biofuels,
biopower, and other bio-based products has the potential to conserve 1.26 billion
barrels of oil, 58 million tons of coal, and 682 million tons of carbon dioxide from
2020 to 2030.
1.3 Lignocelluloses and Its Structural Complexity
The cell walls of plants represent an enormous nutrient source, yet highly variable
in terms of amount, diversity, and botanical source. Plant carbohydrates are
chemically and structurally highly complex, and are arranged in a
three-dimensional network that has evolved to be intrinsically resistant to enzymatic
breakdown (Fontes and Gilbert 2010). Thus high molecular weight crystalline
cellulose microfibrils are intertwinned with hemicelluloses and pectins, which are a
whole range of homo- and heteropolysaccharides composed of dozens of different
monosaccharide units linked in a multitude of ways to resist degradation. Ester
substituents or non-carbohydrate, polymers, such as lignin, proteins, cutin, and
suberin, add a further layer of complexity. As a result, a single vegetable contains
hundreds of different bonds that need to be cleaved in order to unlock the assim-
ilable carbon of the cell wall constituents.
In general cell walls are intricate assemblages of celluloses, hemicelluloses (i.e.,
xyloglucans, arabinoxylans, and glucomannans), pectins (i.e., homogalacturonan,
rhamnogalacturonan I and II, and xylogalacturonans), lignins, and proteoglycans
(e.g., arabinogalactan-proteins, extensins, and proline-rich proteins). Most mass in
the plant cell wall is in the form of polysaccharides (cellulose and hemicelluloses).
The next most abundant polymer is lignin, which is composed predominantly of
phenylpropane building blocks.
Among these different carbon sources lignocellulosic biomass is the most
abundant, low-cost and unexploited biomass that is considered as a source for
industrial production of fuel ethanol. Considering its availability over the last
decades, researchers have been devoted to converting lignocellulosic materials to
bioethanol. Lignocellulosics are composed of heterogeneous complex of carbohy-
drate polymers (cellulose, hemicelluloses, and lignin). Majority of plant biomass is
locked up in 5- and 6-carbon sugars, comprised of mainly cellulose (a glucose
homopolymer, 40–60 % by wt.); less so, hemicelluloses (a sugar heteropolymer,
20–40 % by wt.); and least of all lignin (a complex aromatic polymer, 10–30 % by
wt). The major component cellulose, which is organized into microfibrils, each
containing up to 36 glucan chains having thousands of glucose residues linked by
β-(1,4) glycosidic bonds, is largely responsible for the plant cell wall’s mechanical
strength. Hemicelluloses are built up by pentoses (D-xylose, D-arabinose), hexoses
(D-mannose, D-glucose, D-galactose) and sugar acids. These include β-glucan,
xylan, xyloglucan, arabinoxylan, mannan, galactomannan, arabinan, etc. Hardwood
contains mainly xylans, while in softwood glucomannans are most common. Both
the cellulose and hemicelluloses can be broken down enzymatically into the
component sugars which may be then fermented to ethanol.
Cellulose can be classified into two types based on the arrangement of chains. In
type I cellulose chains are arranged in parallel direction of the long axis of the
microfibril, whereas in type II chains are in antiparrellel position. Among these,
type I is considered to be dominant that can be converted to type II by alkali
treatment. Studies show that cellulose I actually contain two distinct crystal lattices:
cellulose Iα, with triclinic symmetry, and cellulose Iβ, with monoclinic symmetry.
These two forms differ in the organization of their intermolecular hydrogen bonds
and lead to further complexity of cellulose structure. The percentage crystallinity of
cellulose varies from very low to almost 100 % and it depends on cellulose source.
However, it is clear that cellulose microfibrils are not uniformly crystalline;
imperfections in packing or mechanical damage result in a proportion of substrate
in which the lattice is disordered or paracrystalline (Ranjan 2014). Hence, microbes

have to produce cellulases with range of structures, activities, and stabilities.
1.4 Enzymatic Hydrolysis of Lignocelluloses
Even though lignocellulose is considered as the most abundant and renewable

natural resource available to man throughout the world, massive technological
difficulties must be overcome to exploit this for commercial purpose. In order that
cellulose hydrolysis becomes economically feasible, it is important to identify
methods that increase enzyme effectiveness and overcome barriers of enzymatic
hydrolysis (Kristensen et al. 2007). Major factors that influence enzymatic con-
version of lignocelluloses to fermentable sugars include accessible surface area of
lignocelluloses (Mansfield et al. 1998), enzyme loading, and presence of inhibitors
(Eriksson et al. 2002).
Pretreatment is a necessary step generally carried out to produce ethanol from
lignocellulosic materials by dilute sulfuric acid or enzymatically. The polysaccha-
ride chains, being tightly packed, require additional factors that would make the
substrate more accessible, as has been suggested since the 1950s (Horn et al. 2012).
The enzymatic hydrolysis is generally carried out by cellulolytic enzymes. Without
pretreatment, conversion is tedious and very slow. Enzymatic hydrolysis is con-
sidered to be effective because it happens in mild hydrolysis conditions and high
yields of hydrolysis, but it results in accumulation of inhibitory byproducts, costly,
and hydrolysis require long time and also results in product inhibition during
hydrolysis. The biological treatments can be achieved by enzymes of fungi and
actinomycetes, which results in delignification and reduction in degree of poly-
merization of hemicellulose and cellulose.
By the use of enzymatic hydrolysis, pure cellulose can be degraded to soluble
sugars, which can be fermented to form ethanol, butanol, acetone, single cell
protein, methane, and many other products. Enzymatic saccharification remains one
of the most costly steps in conversion of cellulosic biomass to ethanol and cellulase
preparations dedicated for bioethanol industry are hardly available. It has been
estimated that the greatest returns in cost savings will be realized by improving
conversions of biomass to sugars, increasing hydrolysis yields, reducing enzyme
loadings, and eliminating or reducing pretreatment (Lynd et al. 2008). A broader
suite of enzymes is required for hydrolysis of cellulose and hemicelluloses to
fermentable sugars (McMillan et al. 2011). Thus enzymatic hydrolysis can be
effectively carried out if a mixture of different cellulolytic and accessory enzymes is
used.
Enzyme hydrolysis is generally considered the most sustainable technology for
saccharification. Enzymatic hydrolysis of cellulose consists of three steps viz.,
1. Adsorption of cellulase enzymes onto the surface of the cellulose,

2. Biodegradation of cellulose to fermentable sugars, and
3. Desorption of cellulase (Sun and Cheng 2002).
Different strategies are also available for enzymatic hydrolysis and fermentation
of lignocelluloses such as separate enzymatic hydrolysis and fermentation (SHF),
simultaneous saccharification and fermentation (SSF), nonisothermal saccharifica-
tion and fermentation (NSSF), simultaneous saccarification, and co-fermentation
(SSCF) and consolidated bioprocessing (CBP) (Taherzadeh and Karimi 2007).
Among different strategies CBP is considered to posses outstanding potential,
ethanol together with all of the required enzymes is produced by single microbial
communities is also referred to as direct microbial conversion (DMC). It may be
involved single or multiple cultures of microorganism to directly convert cellulose
to ethanol in a single bioreactor (Wyman 2007).
The classical model for degradation of cellulose to glucose involves the coop-
erative action of endocellulases (EC 3.2.1.4), exocellulases (cellobiohydrolases,
CBH, EC 3.2.1.91; glucanohydrolases, EC 3.2.1.74), and betaglucosidases (EC
3.2.1.21). Endocellulases hydrolyze internal glycosidic linkages in a random
fashion, which results in a rapid decrease in polymer length and a gradual increase
in the reducing sugar concentration. Exocellulases hydrolyze cellulose chains by
removing mainly cellobiose either from the reducing or the nonreducing ends,
which lead to a rapid release of reducing sugars but little change in polymer length.
Endocellulases and exocellulases act synergistically on cellulose to produce
cello-oligosaccharides and cellobiose, which are then cleaved by beta-glucosidase
to glucose (Kumar et al. 2008).
Hydrolysis of hemicelluloses involve enzymes like glycoside hydrolases, car-
bohydrate esterases, polysaccharide lyases, endohemicellulases, and others, the
concerted action of which hydrolyze glycosidic bonds, ester bonds, and remove the
chain’s substituents or side chains (Sweeney and Xu 2012). These include endo-1,
4-β-xylanase, β,-xylosidase, β-mannanase, β mannosidase α-glucuronidase, α- L-
arabino furanosidase, acetylxylan esterase and other enzymes.
Glycohydrolases viz., cellulase and hemicellulase enzymes are involved in
hydrolysis of cellulose and hemicelluloses respectively. These are known to posses
15 protein families with other subfamilies. Hydrolysis of cellulose begins with
adsorption of it on to the surface of the enzyme then its biodegradation to glucose
with the synergistic action of three major classes of enzymes: endoglucanases,
exoglucanases and β-glucosidases which are generally represented as cellulase or
cellulolytic enzymes.
All these enzymes are available in the cellulosome complexes, which are intri-
cate multienzyme machines produced by many cellulolytic microorganisms and are
designed for efficient degradation of plant cell wall polysaccharides. In the past
several years, selected studies have addressed the conformational flexibility of the
cellulosome and its possible significance to biomass degradation. Early on, it was
evident that isolated cellulosomes could attain an overall tight or loose conforma-
tion (Mayer et al. 1987; Lamed et al. 1983a). The cellulolytic enzymes are either
secreted into the substrate or attached to the cell wall of the microorganism. The
former system is called noncomplexed and the latter is complexed cellulase system.
Noncomplexed cellulase systems are mostly found in filamentous fungi and acti-
nomycete bacteria, because they can penetrate the lignocellulosic material with their
hyphal extensions. The enzymes of noncomplexed cellulase systems are then just
released into the substrate and the free enzymes start hydrolyzing the cellulose. The
glucose and cellodextrins of a length of maximal four glucose molecules are taken
up by the microorganism and either used directly or cleaved further via intracellular
hydrolases. Organisms that produce noncomplexed cellulase systems are most often
used in the industrial production of cellulolytic enzymes, because the secreted
enzymes can easily be harvested (Lynd et al. 2002).
Cellulosome contains cellulose-binding domains, also called carbohydrate
binding modules (CBM), which bind to the cellulosic material (Fontes and Gilbert
2010; Sweeney and Xu 2012; Shoseyov et al. 2006). CBMs can also be found in
noncomplexed cellulase systems, where they are especially important for binding
and processing of exoglucanases. The main function of the cellulosome is to hold
the microorganism tight to the cellulose, and therefore reduce diffusion distances
and losses and to arrange the different cellulolytic enzymes in a favorable way.
More recently (Garcia-Alvarez et al. 2013), ultrastructural studies of a homoge-
neous mini-cellulosome containing three cohesin modules attached to three
matching cellulases suggested that the flexibility of the linkers connecting con-
secutive cohesin modules could control structural transitions and thus regulate
substrate recognition and degradation (Vazana et al. 2013).
One of the most potent cellulose degrading microorganisms is the well-studied
bacterium Clostridium thermocellum. This anaerobic thermophilic cellulolytic
bacterium secretes a multienzymatic complex called the cellulosome, first discov-
ered in 1983 (Lamed et al. 1983a, b). Later there are several reports on cellulosomal
oraganisms which possess an array of cellulosomal architectures (Belaich et al.
1997; Gal et al. 1997; Ding et al. 1999, 2000; Ohara et al. 2000; Rincon et al. 2003,
2004, 2010; Xu et al. 2003, 2004; Bayer et al. 2004, 2013; Dassa et al. 2012).
1.5 Microbial Enzymes for Degradation of Cellulose
The isolation and characterization of novel glycoside hydrolases from eubacteria

are now becoming widely exploited. There are several reasons for these shifts, for
one, bacteria often have a higher growth rate than fungi allowing for higher
recombinant production of enzymes. Secondly, bacterial glycoside hydrolases are
often more complex and are often expressed in multienzyme complexes providing
increased function and synergy. Most importantly, bacteria inhabit a wide variety of
environmental and industrial niches, which produce cellulolytic strains that are
extremely resistant to environmental stresses. These include strains that are ther-
mophilic or psychrophilic, alkaliphilic, or acidophilic, and, strains that are halo-
philic. Not only can these strains survive the harsh conditions found in the
bioconversion process, but also they can produce enzymes that are stable under
extreme conditions which may be present in the bioconversion process and this may
increase rates of enzymatic hydrolysis, fermentation, and, product recovery (Maki
et al. 2009). Along with these due to availability of novel techniques for gene
transfer, microbes can be genetically modified easily to suit the need of the
industrial processes and also the different substrates employed in the fermentation.
Considering these, decomposition of lignocelluloses by enzymes produced by
complex microbial communities represents a promising alternative for biomass
conversion. In particular, thermophilic consortia are a potential source of enzymes
adapted to adverse reaction conditions (Wongwilaiwalina et al. 2010; Gladden
et al. 2011). Members of the genus Caldicellulosiruptor can grow on and degrade
biomass containing high lignin content as well as highly crystalline cellulose
without conventional pretreatment (Blumer-Schuette et al. 2008; Yang et al. 2009)
and are also known to ferment all primary C5 and C6 sugars from plant biomass
and are the most thermophilic cellulolytic bacteria known, with growth temperature
optima between 78 and 80 °C raising the possibility of further economic
improvement of biofuel production from plant biomass by reducing or eliminating
the pretreatment step (Hamilton-Brehm et al. 2010).
In comparison with the anaerobic thermophilic bacteria few aerobic have been
described to produce cellulases and xylanses. The aerobic thermophiles R. marinus
produces a highly thermostable cellulose (Cel 2A), and three glycoside hydrolases
belonging to family GH10 of the carbohydrate active enzyme (CAZy) database
(http://www.cazy.org) (Cantarel et al. 2009). Aquifex aeolicus, isolated in the
Aeolic Islands in Sicily (Italy), represents one of the most thermophilic bacteria
since its growth temperature can reach 95 °C (Deckert et al. 1998).
Bacteria belonging to Clostridium, Ruminococcus, Bacteroides, Erwinia,
Acetovibrio, Microbispora, and Streptomyces can produce cellulases (Bisaria
1991). Cellulomonas fimi and Thermomonospora fusca have been extensively
studied for cellulase production. Similarly, other bacterial strains have the ability to
produce cellulase complexes aerobically as well as anaerobically. Cellulase pro-
ducing bacterial strains of Rhodospirillum rubrum, Cellulomonas fimi, Clostridium
stercorarium, Bacillus polymyxa, Pyrococcus furiosus, Acidothermus cellulolyticus,
and Saccharophagus degradans have been extensively reviewed (Kumar et al.
2008; Weber et al. 2001; Kato et al. 2005; Taylor et al. 2006). An extracellular
alkaline carboxy methyl cellulase (CMCase) from Bacillus subtilis strain AS3 has
been purified and characterized by Deka et al. (2013) for utilization of cellulosic
biomass. Although many cellulolytic bacteria, particularly the cellulolytic anaer-
obes such as Clostridium thermocellum and Bacteroides cellulosolvens produce
cellulases with high specific activity, they do not produce high enzyme titres (Lynd
et al. 2002).
Apart from the cellulolytic fungus T. reesei, many other fungi produce cellulases
and degrade treated cellulosic material or soluble cellulose derivatives such as
carboxymethylcellulose. However, they are not very effective on crystalline cel-
lulosic substrates. Mesophilic strains producing cellulases like Fusarium oxyspo-
rum, Piptoporus betulinus, Penicillium echinulatum, P. purpurogenum, Aspergillus
niger and A. fumigatus have also been reported (Martins et al. 2008; Valaskova and
Baldrian 2006). The cellulases from Aspergillus usually have high β-glucosidase
activity but lower endoglucanase levels, whereas, Trichoderma has high endo and
exoglucanase components with lower β-glucosidase levels, and hence has limited
efficiency in cellulose hydrolysis. Thermophillic microorganisms such as
Sporotrichum thermophile, Scytalidium thermophilum, Clostridium straminisolvens
and Thermomonospora curvata also produce the cellulase complex and can degrade
native cellulose (Kato et al. 2005; Kaur et al. 2004).
Even human gut microbiota encodes a huge diversity of enzymes for the
digestion of all components of plant cell wall polysaccharides including cellulose.
Turnbaugh et al. (2010) have shown that the distal gut microbiota of humans also
encodes dockerin-containing cellulolytic enzymes that indicate the presence of
cellulosomes (Fontes and Gilbert 2010; Bayer et al. 2004). The presence of
dockerin-containing proteins is also observed in the oral, nasal and vaginal samples
(Cantarel et al. 2012). In nondigestive sites, however, these dockerin domains may
have another role than attaching cellulases to form a cellulosome.
The interaction between loads of bacteria in gut with other bacteria has lead to
development of novel CAZymes. The recent outcome of the research by Hehemann
et al. (2010) proved the novel CAZymes added due to flexible microbial interac-
tions. They characterized the first porphyranases from a member of the marine
Bacteroidetes, Zobellia galactanivorans, active on the sulphated polysaccharide
porphyran from marine red algae of the genus Porphyra. Furthermore, they showed
that genes coding for these porphyranases, agarases and associated proteins have
been transferred to the gut bacterium Bacteroides plebeius isolated from Japanese
individuals. Their comparative gut metagenome analyses showed that por-
phyranases and agarases are frequent in the Japanese population and that they are
absent in metagenome data from North American individuals.
White Rot Fungi (WRF) are microorganisms of great interest that secrete
complex suites of nonspecific extracellular ligninolytic enzymes, i.e., lignin per-
oxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13), and laccase (EC
1.10.3.2) to biodegrade lignin or digest substrates required for their proliferation
and growth. β-glucosidase is among the suite of enzymes produced by WRF such as
Pleurotus ostreatus, Auricularia auricula, Polyporus squamosus, Trametes versi-
color, Lentinula edodes, and Grifola frondosa to biodegrade plant biomass. The
β-glucosidase family (EC 3.2.1.21) is a wide-spread group of enzymes that catalyze
the hydrolysis of a broad variety of glycosides (Berrin et al. 2003). While some
organisms secrete either endoglucanase or β-glucosidase, in other organisms;
β-glucosidase is either lacking or produced in insufficient quantities (Kumar et al.
2008). When β-glucosidase secretion is low, cellobiose accumulates instead of
glucose. Cellobiose accumulation acts as a feedback-inhibitor of cellulose
depolymerization by endo- and exoglucanases (Howell and Stuck 1975; Morais
et al. 2010), which is a critical factor in the industrial scale conversion of cellulose
to glucose (Cai et al. 1997). This situation can be alleviated during industrial scale
conversion of cellulosic biomass by exogenous incorporation of β-glucosidase
enzyme.
Among known cellulolytic microorganisms, cellulases of Trichoderma reesei or
T. viride are well studied and characterized. Cellulase of T. reesei comprises of two
major cellobiohydrolases, (CBHI and CBHII), two major endoglucanases, (EGI and
EGII) and at least two low molecular weight endoglucanases, EGIII, and EGV. The
mixture of these enzymes is capable of solubilizing native cellulose. Many other
aerobic filamentous fungi, including Agaricus bisporus, Humicola spp., Irpex
lacteus, and Sclerotium roysii, also use similar cellulase systems.
1.6 Novel Enzymes for Cellulose Degradation
In addition to enzymes that act directly on polysaccharides, lignocelluloses

degrading microorganisms are also found to secrete several proteins, which modify
cellulose and enhance its hydrolysis by cellulase. These include the no-enzymatic
proteins such as ‘expansins’ and their fungal and bacterial homologs, the ‘swol-
lenins’ expressed by T. reesei with sequence homology to plant expansins and the
similar ‘loosenins’ produced by Bjerkandera adusta. These interact with cellulosic
substrates resulting in expansion, slippage, or lengthening of the components,
thereby facilitating the access of glycosyl hydrolases (Sweeney and Xu 2012;
Banerjee et al. 2010). Recently, cellulose-induced proteins (CIP1 and CIP2) have
been found in a transcriptional analysis of T. reesei, with some synergistic activity
with both GH61 and swollenin and are thought to play a role in the cleavage of
hemicellulose–lignin crosslinks (Sweeney and Xu 2012; Scott et al. 2009). There
are various hydrolytic enzymes and proteins produced by fungi and bacteria, which
help to facilitate the lignocellulose bioconversion process.
Along with the catalytic core, many of the enzymes also possess noncatalytic
domains including CBM and dockerins which, respectively, anchor the enzyme to
targeted substrate or onto scaffolding to assemble a cellulosome and disrupt the
crystalline cellulose microfibrils (Kumar et al. 2008; Sweeney and Xu 2012).
Recently, a new type of bacterial proteins currently classified as CBM33 (family 33
CBM) in the CAZy database and fungal proteins classified as GH61 (family 61
glycoside hydrolases) that catalyze oxidative cleavage of polysaccharides have been

discovered (Horn et al. 2012; Sweeney and Xu 2012). These copper dependent
monooxygenases act on the surfaces of insoluble substrates where they introduce
chain breaks in the polysaccharide chains thereby increasing substrate accessibility
and potentiating hydrolytic enzymes (Horn et al. 2012). Recent data obtained from
CBM33 and GH61 families revealed that these enzymes disrupt the more recalci-
trant portions of the cellulose and make the substrate more accessible to digestion
by cellulases. The emerging research on disruptive proteins from the CBM33 and
GH61 is exciting because these proteins appear to have novel mechanism to attack
cellulose and synergize with cellulase system (Kostylev and Wilson 2012). Studies
related to synergistic activity with high throughput advanced screening will yield
efficient method to degrade cellulose with these enzymes.
Inspired by the findings for CBP21 and earlier indications that certain CBM33s
may act synergistically with cellulases (Moser et al. 2008) studies were conducted
to prove if certain CBM33s act on cellulose like CBP21 acts on chitin. In 2011 it
was then shown that CelS2, a CBM33 protein from Streptomyces coelicolor, indeed
cleaves cellulose, producing aldonic acids (Forsberg et al. 2011). Like CBP21, the
activity of CelS2 depended on the presence of divalent metal ions as shown by the
inhibitory effect of EDTA and the ability to restore activity by adding divalent metal
ions. Again like CBP21, purified CelS2 was active without the addition of metals,
probably due to high affinity binding. Both the initial study on CBP21 and the study
on CelS2 concluded that the enzymes could use several divalent metal ions, but the
most recent work clearly shows that these enzymes in fact are copper dependent
monooxygenases.
At the same time, a series of elegant studies showed that GH61s are functionally
very similar to CBM33s (Quinlan et al. 2011; Beeson et al. 2012; Phillips et al.
2011; Westereng et al. 2011; Li et al. 2012). Quinlan et al. (2011) described the
crystal structure of a GH61 from Thermoascus aurantiacus (TaGH61A) and
showed that this protein catalyzes oxidative cleavage of cellulose in the presence of
an external electron donor such as gallic acid. These authors were the first to
convincingly show that enzyme activity is copper dependent. These findings were
confirmed by work on a GH61 from Phanerochaete chrysosporium (PcGH61D) by
Westereng et al. (2011) and work on several GH61 proteins from Neurospora
crassa (Beeson et al. 2012; Phillips et al. 2011; Li et al. 2012). Thus, GH61s are
copper dependent lytic polysaccharide monooxygenases. Recent work on a
chitin-active CBM33, using experimental conditions that ruled out possible effects
of metal ions trapped in the substrate, showed that this CBM33 was copper
dependent too (Vaaje-Kolstad et al. 2005).
The list of enzymes and noncatalytic proteins associated with degradation of
lignocellulose is increasing every year, the recent list of novel microbial enzymes
and its functions are given in Table 1.1 (Mohanram et al. 2013).
Table 1.1 Novel enzymes and other proteins involved in degradation of biomass
Enzymes Function Microorganisms
Endo-1,4 β-D- Hydrolysis of the internal Acidothermus cellulolyticus,
glucanglucanohydrolase glycosidic linkages in a Pectobacterium
(EC. 3. 2. 1.4) random fashion, generating chrysanthami, Thermobifida
oligosaccharides of varying fusca, Thermotoga maritime,
lengths Bacillus, Pseudomonas,
Ruminococcus, Fibrobacter,
Clostrdium, Halomonas,
Streptomyces, Cellulomonas,
Mycobacterium, Aspergillus,
Trichoderma, Anaeromyces,
Pestalotiopsis,
Phanerochaete, Fusarium,
Orpinomyces, Piromyce
Exoglucanase or 1,4-β-D- Hydrolysis of beta-D- Bacillus, Pseudomonas,
glucan cellobiohydrolase glucosidic linkages by Clostridium, Paenibacillus,
(EC3.2.1.91) releasing mainly cellobiose Thermobifida, Cellulomonas,
either from the reducing or Mycobacterium, Ralstonia,
nonreducing ends of the Trichoderma, Penicillium,
chains Aspergillus, Chaetomium,
Fusarium, Pestalotiopsis,
Orpinomyces, Piromyces,
Rhizopus
β-glucosidases or β-D- Hydrolysis of terminal, Clostridium, Cellulomonas,
glucoside glucohydrolase nonreducing β-D-glucosyl Aerobacter, Leuconostoc,
(EC 3.2.1.21) residues with release of β-D- Aspergillus, Monilia,
glucose Phanerochaete, Sclerotium,
Saccharomyces,
Kluyveromyces
Cellodextrin phosphorylase Catalysis of the reversible Clostridium, Cellvibrio
or (1 → 4)-β-D-glucan: phosphorolytic cleavage of
phosphate α-D- cellodextrins ranging from
glucosyltransferase (EC cellotriose to cellohexoses
2.4.1.49)
Cellobiose phosphorylase Catalysis of the reversible Cellulomonas, Clostridium,
or cellobiose: phosphate phosphorolytic cleavage of Ruminococcus, Thermotoga,
α-D-glucosyltransferase cellobiose Cellvibrio, Fomes annosus
(EC 2.4.1.20)
Endo-1,4-β-D-xylanase or Endohydrolysis of (1 → 4)- Bacillus circulans,
1,4-beta-xylan beta-D-xylosidic linkages in Thermoanaerobacterium,
xylanohydrolase xylans to release xylose Ruminococcus, Geobacillus,
(EC 3.2.1.8) Thermopolyspora,
Cellulomonas, Streptomyces,
Aspergillus, Trichoderma,
Thermomyces, Fusarium,
Anaeromyces,
Neocallimastix,
Dictyoglomus
Thermophilum, Streptomyces
halstedii
(continued)
Table 1.1 (continued)

Xylan β-1,4-xylosidase Hydrolysis of (1 → 4)-beta- Bacillus,
(EC 3.2.1.37) D-xylans, to remove Thermoanaerobacterium,
successive D-xylose residues Geobacillus, Aspergillus,
from the non-reducing Fusarium, Talaromyces,
termini Trichoderma
Mannan Random hydrolysis of Cellulomonas, Bacillus,
endo-1,4-β-mannosidase (1 → 4)-beta-D-mannosidic Clostridium, Rhodothermus,
(EC 3.2.1.78) linkages in mannans, Aspergillus, Trichoderma
galactomannans and
glucomannans
α-L-arabinofuranosidase Hydrolysis of terminal Bifidobacterium,
(EC 3.2.1.55) nonreducing alpha-L- Thermobacillus, Bacillus,
arabinofuranoside residues in Clostridium, Streptomyces,
alpha-L-arabinosides Aspergillus, Penicillium,
Fusarium, Trichoderma
Acetyl (xylan) esterase Hydrolysis of ester linkages Bacillus, Clostridium,
(EC 3.1.1.72) of the acetyl groups in Streptomyces, Fibrobacter,
position 2 and/or 3 of the Pseudomonas,
xylose moieties of natural Thermoanaerobacterium,
acetylated xylan Penicillium, Aspergillus,
Trichoderma,
Phanerochaete,
Chrysosporium
α-L-Fucoside fucohydrolase Hydrolysis of O-glycosyl Thermotoga,
or α-L-fucosidase bond in xyloglucan to release Bifidobacterium,
(EC 3.2.1.51) l-fucose residues Streptococcus, Bacillus,
Cellulomonas, Clostridium,
Fusarium, Beauveria,
Penicillium, Trichoderma,
Neurospora, Aspergillus
α-D-Glucosiduronate Hydrolysis of O-glycosyl Thermotoga, Cellvibrio,
glucuronohydrolase or bond to release 4-O- Bacteroides, Bacillus,
α-glucuronidase methylglucuronic acid from Aspergillus, Trichoderma,
(EC 3.2.1.139) xylan Thermoascus
Novel proteins
Swollenins Homologous to plant Bacillus, Cellulomonas,
expansins which rapidly Clostridium,
induce extension of plant cell Myceliophthora,
walls by weakening the Thermomonospora,
noncovalent interactions; Streptomyces, Fibrobacter,
contain an N-terminal Trichoderma, Aspergillus,
carbohydrate binding module Neosartorya, Humicola,
family 1domain (CBD) with Fusarium, Penicillium,
cellulose-binding function Neurospora, Gliocladium,
and a C-terminal Candida, Pichia,
expansin-like domain Rhodotorula,
Sporobolomyces,
Bjerkandera adusta
(continued)

Cellulose induced proteins Contain a carbohydrate Streptomyces coelicolor,
(CIP1 and CIP2) binding module (CBM); Trichoderma, Schizophyllum,
Hydrolysis of the ester Pyrenophora
linkage between 4-O-methyl-
D-glucuronic acid of
glucuronoxylan and lignin
alcohols
Lossenins Facilitate acess of glycosyl Bjerkandera adusta
hydrolases
Dockerin and Cohesin Assembly of cellulase and Anaerobic microorganisms
hemicellulase proteins to
structural scaffoldings on
microbial surface
1.7 Swollenins: A Novel Protein for Cellulose Degradation
Expansins in plants have been proposed to disrupt hydrogen bonding between

cellulose microfibrils or between cellulose and other cell wall polysaccharides
without having hydrolytic activity (McQueen-Mason and Cosgrove 1994; Whitney
et al. 2000). In this way they are thought to allow the sliding of cellulose fibers and
enlargement of the cell wall. Saloheimo et al. (2002) reported the discovery of a
novel fungal protein having significant sequence identity to plant expansins. Unlike
plant expansins, this protein has a modular structure with an N-terminal CBD. The
protein was named swollenin due to its ability to swell cotton fibers and loosening
effect of swollenin makes lignocellulosic biomass more accessible and readily
hydrolyzable by cellulase, thereby promoting the degradation of lignocellulose
during fermentation (Arantes and Saddler 2010; Chen et al. 2010) without pro-
ducing detectable amounts of reducing sugars. Swollenin was later shown to be
capable of weakening and disrupting hydrogen-bond networks in lignocellulose
(Wang et al. 2011). Swollenin has an N-terminal fungal type cellulose-binding
domain connected by a linker region to the expansin-like domain. The protein also
contains regions similar to mammalian fibronectin type III repeats, found for the
first time in a fungal protein. The swollenin gene is regulated in a largely similar
manner as the T. reesei cellulase genes.
Efforts were made by different groups to express swollenin gene heterologously
in Saccharomyces cerevisiae and Aspergillus niger (Saloheimo et al. 2002) and
Kluyveromyces lactis (Jäger et al. 2011). In continuation to improve the direct
conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying
cellulase and expansin-like protein on the cell surface were constructed and
examined for direct ethanol fermentation performance. The cellulase and
expansin-like protein co-expressing strain showed 246 mU/g-wet cell of
phosphoric acid swollen cellulose degradation activity, which corresponded to

2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly
demonstrated that yeast cell surface expressed cellulase and expansin-like protein
act synergistically to breakdown cellulose. In fermentation experiments examining
direct ethanol production from PASC, the cellulase and expansin-like protein
co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a con-
centration that was 1.4-fold higher than that achieved by the cellulase-expressing
strain (2.5 g/L) (Nakatani et al. 2013).
Its exploitation for removal of detectable amounts of reducing sugars has been
recently reported by Kang et al. (2013). They have isolated a novel gene encoding a
swollenin-like protein, POSWOI, from the filamentous fungus Penicillium oxali-
cum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family
1 carbohydrate binding module (CBM1) followed by a linker connected to a family
45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombi-
nant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and
showed significant disruptive activity on crystalline cellulose. Simultaneous reac-
tion with both POSWOI and cellulases enhanced the hydrolysis of crystalline
cellulose Avicel by approximately 50 %. Using a POSWOI-pretreatment proce-
dure, cellulases can produce nearly twice as many reducing sugars as without
pretreatment. The mechanism by which POSWOI facilitates the saccharification of
cellulose was also studied using a cellulase binding assay.
1.8 CIP1 and CIP2
Cellulose-induced protein 1 (Cip1) protein consists of a glycoside hydrolase family

1 carbohydrate binding module connected via a linker region to a domain.
A calcium ion binding site was identified in a sequence conserved region of Cip1
and is also seen in other proteins with the same general fold as Cip1, such as many
carbohydrate binding modules. The Cip1 structure was analyzed and a structural
homology search was performed to identify structurally related proteins. The two
published structures with highest overall structural similarity to Cip1 found were
two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate
lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However,
initial trials did not detect significant lyase activity for Cip1 (Jacobson et al. 2013).
The function of CIP1 is not known yet. BLAST searches of CIP1 in public data-
bases show that it is similar to glycosyl hydrolases found in bacteria but that are not
yet characterized in detail, indicating that CIP1 could have a novel undescribed
enzymatic activity or mechanism. CIP2 was described to encode a CE15 glu-
curonoyl esterase, probably cleaving the ester linkage between 4-O-methyl-D-glu-
curonic acid of glucuronoxylan and lignin alcohols (Li et al. 2007).
1.9 Consolidated Bioprocessing (CBP) for Enhanced

Biofuel Production
CBP can be achieved by two ways by modifying cellulase producer to ferment

glucose or to modify glucose producer to produce celulase. CBP may result in poor
yield of alcohol and requires more incubation period and hence thermophilic strains
are considered to be best alternative and above said combination is appears to be
practically viable. It represents an alternative method with outstanding potential for
low-cost processing of lignocellulosic biomass (Lynd et al. 2008). Fujita et al.
(2004) constructed a whole-cell biocatalyst with the ability to induce synergistic
and sequential cellulose hydrolysis reaction through co display of three types of the
cellulololytic enzymes on the cell surface of S. cerevisiae. This strain achieved
successful conversion of cellulose to ethanol with good yield. In another attempt
Den et al. (2007) developed recombinant strain of S. cerevisiae which can be used
for CBP. Two cellulase-encoding genes, an endoglucanse of T. reesei and
β-glucosidase of Saccharomyces fibuligera in combination were expressed in
S. cerevisiae.
No ideal CBP microbe able to degrade efficiently lignocelluloses and at the same
time, to utilize the released sugars to produce ethanol is currently available.
A newly discovered thermophilic microorganism, Geobacillus sp. R7 (T 60 °C), is
a facultative anaerobic bacterium isolated from soil samples of the Homestake gold
mine, South Dakota. It produces a thermostable cellulose when grown on
extrusion-pretreated agricultural residues such as corn stover and prairie cord grass,
and ferments lignocellulosic substrates to ethanol in a single step (Zambare et al.
2011).
Cha et al. (2013) demonstrated recently the utility of gene deletion in the
pyruvate metabolic pathway for rational strain engineering of Caldicellulosiruptor
bescii while simultaneously creating a platform for further strain modification for
advanced production of fuels and chemicals from renewable plant feed stocks. The
method for creating a deletion of the ldh gene in the C. bescii chromosome was
efficient enough to allow targeted marker replacement using nonreplicating plas-
mids. The resulting mutant grew to a higher cell density and produced more
hydrogen than the wild-type strain. Using the tools developed by them, C. bescii
JWCB017 will serve as a platform for additional rational strain engineering for
production of fuels and chemicals from lignocellulosic feedstocks.
Efforts were also continued to transfer genes responsible for degradation of
cellulose to yeast. Cellulase genes from various kinds of microorganisms have been
expressed in the ethanol-producing yeast Saccharomyces cerevisiae with the aim of
directly producing ethanol from cellulose (Van and Van 1998; Fujita et al. 2002; Ito
et al. 2009; Wen et al. 2010; Yamada et al. 2013). In addition, yeast strains dis-
playing cellulase on the cell surface have also been developed for improving the
efficiency of direct ethanol production from cellulose (Yamada et al. 2013).
1.10 Recycling of Enzymes
A key factor that prevents the commercialization of enzymatic cellulose hydrolysis

is the high cost of cellulase enzymes. Enzyme cost is expected to account for more
than 20 % of ethanol production (Wooley et al. 1999). As much of it remains active
after hydrolysis, recycling of cellulases makes the overall conversion process more
economically feasible. Various methods have been used for recycling enzymes,
which include sedimentation followed by ultra filtration or micro centrifugation,
cation exchange chromatography, readsorption, and immobilization.
1.11 Potential Strategies for Stabilizing Cellulases
Enzyme stabilization and lifetime improvement has been achieved through point
substitution and chemical modification, of single catalytic domains, either through
rational, structure-based modeling, or through directed evolution. Another approach
to increase enzyme stability and enhance activity is insertion of protein domains
into stable scaffolds.
An effort was made to stabilize the cellulases by embedding cellulases of
extremophiles into hyperstable α-helical consensus. It was observed that catalytic
domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12;
Thermotoga maritima) to be stable in the context of ankyrin scaffold, and to be
active against both soluble and insoluble substrates. The ankyrin repeats in each
fusion are folded, although it appears that for C12A catalytic domain (CD) (where
the N- and C-termini are distant in the crystal structure), the two flanking ankyrin
domains are independent, whereas for CA (where termini are close), the flanking
ankyrin domains stabilize each other. Although the activity of CA is unchanged in
the context of the ankyrin scaffold, the activity of C12A is increased between two
and sixfold (for RAC and CMC substrates) at high temperatures. For C12A, activity
increases by with the number of flanking ankyrin repeats. These results show
ankyrin arrays to be a promising scaffold for constructing designer cellulosomes,
preserving or enhancing enzymatic activity and retaining thermostability.
In this type of scaffolding strategy, the distance between the N and C-termini of
the inserted protein (cellulase catalytic domains) is an important parameter.
Cellulases catalytic domains belong to various Glycoside Hydrolase (GH) families,
as reported in the CAZY database, with different folds, and thus have variable
N-to-C distances (Cantarel et al. 2009).
Insertion into a scaffold protein requires appropriate selection of insertion sites to
avoid disruption of structure of the inserted protein or scaffold (Betton et al. 1997;
Cutler et al. 2009). Proteins with linear, repeated architectures, such as ankyrin
repeats, have structurally modular architectures that should be able to accommodate
insertions locally, without affecting more distant regions of the scaffold. Ankyrin
repeat proteins are roughly linear arrays of 33 residues repeating units (Sedgwick
and Smerdon 1999; Zweifel and Barrick 2001). Each unit consists of two short
helices connected by a short, conserved turn, and connects to neighboring repeats
through an extended beta-hairpin containing loop (Zweifel et al. 2003). Adjacent
repeats strongly stabilize each other through highly stabilizing interfaces. The
repeating unit spans about 11 Å, approximately the size of each cellobiose unit
(2-units of glucose 10.4 Å). Studies have found that ankyrin repeats are greatly
stabilized by consensus sequence substitutions (Wetzel et al. 2008; Aksel et al.
2011). For a five repeat consensus ankyrin protein, the thermal denaturation mid-
point (Tm) was around 90 °C (Aksel et al. 2011; Aksel and Barrick 2009).
Therefore, consensus ankyrin seems to be an ideal scaffold to study the effects of
inserting cellulase CDs with various N–C distances.
1.12 Enzyme Cocktails
Catalytic activity of an enzyme is defined and mediated by its 3D structure and its
mobility. It is impossible to obtain complete comprehension of the enzymatic action
without detailed molecular studies which include structural, biophysical, bio-
chemical and genetic investigations, enzymatic assays, bioinformatics, and
molecular dynamics computations. It is not possible to innovate and to consistently
produce competitive enzymatic cocktails/mixtures for biomass transformation
without profound understanding of function and activity of glycoside hydrolases.
Therefore, we need to integrate our knowledge of biodiversity, genomics and
genetics studies, structural and molecular biology, biochemistry, enzymology, and
bioinformatics, focusing on bioindustrial applications.
Enzyme cocktails that hydrolyze plant cell wall polysaccharides are a critical
component of bioprocessing configurations designed to transform lignocellulosic
biomass into biofuels (Lynd et al. 2002; Gao et al. 2011). Specific characteristics of
an enzyme (activity, thermostability, etc.) is often based on homology to known
enzymes (Schnoes et al. 2009). Although free cellulases and cellulosomes employ
different physical mechanisms to break down recalcitrant polysaccharides, when
these system combined display dramatic synergistic enzyme activity on cellulose.
Two natural enzyme systems—one produced by fungi and the other by bacteria—
break down cellulose faster if used in combination. The resulting process shows
promise for less expensive biofuels (Resch et al. 2013).
Current commercial cocktails consist of preparations of fungus derived glycoside
hydrolases, primarily cellulases and hemicellulases (Bouws et al. 2008; King et al.
2009). However, fungal enzymes are often deactivated by elevated temperatures or
by residual chemicals from pretreatment (Bouws et al. 2008). For example, ionic
liquids (ILs), such as 1-ethyl- 3-methylimidazolium acetate ([C2mim][OAc]), can
dissolve lignocellulosic biomass and dramatically improve cellulose hydrolysis
kinetics, yet multiple studies have shown that fungal endoglucanases are deacti-
vated at low levels of ILs that may persist in the biomass after pretreatment (Dadi
et al. 2006; Turner et al. 2003). In contrast, thermophilic bacterial and archaeal
endoglucanases have been shown to be active in 20 % [C2mim][OAc], suggesting

that thermophilic prokaryotes may be an important source of enzymes for the
development of more robust enzyme cocktails (Datta et al. 2010).
Although free cellulases and cellulosomes employ very different physical
mechanisms to break down recalcitrant polysaccharides, when combined these
systems display dramatic synergistic enzyme activity on cellulose. Two natural
enzyme systems—one produced by fungi and the other by bacteria—break down
cellulose faster if used in combination. The resulting process shows promise for less
expensive biofuels (Resch et al. 2013).
1.13 Enzyme Engineering
The natural diversity of enzymes could provide a large reservoir that can be further
improved by engineering enzymes and strains for increased performance (King
et al. 2009). A number of designer enzymes called glycosynthases, including cel-
lulases and hemicellulases, have been engineered by replacing nucleophilic residues
resulting in higher yields of different oligosaccharides (Kumar et al. 2008).
1.14 Feruloyl Esterases (Faes)
Feruloyl esterases (Faes) represent a subclass of carboxyl esterases that can release
phenolic acids, such as ferulic acids or other cinnamic acids, from esterified
polysaccharides, especially xylan and pectin. Feruloyl esterases (Faes) constitute a
subclass of carboxyl esterases that specifically hydrolyze the ester linkages between
ferulate and polysaccharides in plant cell walls. Recently, a comprehensive set of
enzymes essential for decomposing plant cell walls, including feruloyl esterase
activity, in a newly described in yak ruminal bacterium, Cellulosilyticum
ruminicola H1. Strain H1 grew robustly on natural plant fibers, such as corn cob,
alfalfa, and ryegrass, as the sole carbon and energy sources, as well as on a variety
of polysaccharides, including cellulose, xylan, mannan, and pectin (Li et al. 2011).
Therefore, Faes are regarded as the key enzymes to loosen the internal crosslink of
plant cell walls by acting as the important accessory enzymes in synergy with
(hemi)cellulases in plant cell wall hydrolysis.
1.15 Future Scope
Urgent need of the alternate energy resources increased the novel ideas to achieve
success to convert lignocelluloses and other carbohydrates to ethanol. In this
connection development of systems biology approaches and integrated, predictive
modeling capabilities for metabolic and regulatory networks of biomass degrading

microorganisms is considered to be very important. The characterization of
microbes and microbial communities from environments with high rates of ligno-
cellulose degradation has to be continued, because microorganisms display varied
capacity for carbohydrate degradation.
The following are the thrust area of research already initiated by several
scientists
• Designing of optimized cellulosomes by synthesizing hybrid scaffolding
molecules that contain cohesins with different binding specificity from different
organisms is another recent approach to develop more active cellulose degrading
enzymes (Wilson 2009). Synergies between purified cellulases and xylanases
from the thermophilic bacterium, Thermobifida fusca displayed on designer
cellulosomes were found to possess higher activity on wheat straw than the
corresponding free enzymes (Morais et al. 2010).
• The recent trend is developing multifunctional chimeras by construction,
cloning and sequencing the chimeric gene and its expression followed by
purification of chimeric proteins (Nair et al. 2010).
• Brunecky et al. (2013) proposed a novel mechanism of cellulose digestion by
the dominant multimodular cellulase CelA of the thermophilic bacterium
Caldicellulosiruptor bescii and compared its hydrolytic performance with that
of a binary mixture containing Cel7A fungal exoglucanase (cellobiohydrolase I
from Trichoderma reesei) and bacterial Acidothermus cellulolyticus Cel5A (E1)
endoglucanase. The major finding of the report, that CelA is able to excavate
extensive cavities into the surface of the cellulosic substrate, is very interesting
and indeed reflects a novel cellulose digestion paradigm.
• There are efforts to develop, create, and investigate artificial cellulosomes with
high activity, and systematically evaluate the fundamental principles that
underlie their structure, dynamics, and catalytic functions. Ranjan (2014) have
developed a polycatalytic system consisting of cellulases covalently linked on
the surface of colloidal polymers with a magnetic nanoparticle (MNP) core.
MNP provides a convenient handle to separate the complex, while the colloidal
polymer would serve as a benign scaffold to attach the enzymes.
Although biomass may ultimately only supply a relatively small amount of the
world’s energy requirements, it will nevertheless be of immense overall value. In
some parts of the world, such as Brazil and other countries of similar climatic
conditions, biomass will surely attain wider exploitation and utilization. There may
still be some disadvantages when comparing it with coal or oil, but the very fact that
it is renewable and they are not must be the spur to further research. In time,
biomass will become much more easily and economically used as a source of
energy for mankind. Approaches like enzyme engineering, reconstitution of
enzyme mixtures and bioprospecting for superior enzymes are gaining importance.
The current scenario, however, also warrants the need for research and development
of integrated biomass production and conversion systems.
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Part I
Cellulases
Chapter 2
Cellobiohydrolases: Role, Mechanism,
and Recent Developments

and Nallusamy Sivakumar
Abstract Cellobiohydrolases or exoglucanases are produced by various bacteria

and fungi with catalytic modules belonging to families 5, 6, 7, 9, 48, and 74
glycoside hydrolases, which act at the chains end of cellulose resulting in release of
glucose as well as cellobiose. The CBH I and II works processively from reducing
and nonreducing ends of the cellulose chain, respectively. The catalytic module of
CBHs is the tunnel structure formed by two surface loops that may covers entirety
or part of active site evidenced that the mode of action proceeds in a processive
manner as cellobiohydrolase progresses along the cellulose chain. CBHs are able to
work actively in the crystalline region of cellulose, probably peeling them from the
microcrystalline structure of Avicel. Although several assays have been proposed,
no specific substrate as well as assay method to measure exoglucanases has been
described till date. In recent days, several new approaches such as δ-sequence
mediated integration, SCHEMA, and FoldX and a ‘consensus’ sequence have been
developed to improve activity and stability of CBHs.
2.1 Introduction
Considering the plant biomass, the main component from plant cell wall is the
cellulose, which is the more abundant carbohydrate constituted by glucopyranose
monomers linked by β-1,4 glycosidic bonds with two distinct regions such as
N. Annamalai (&)
Hawaii Natural Energy Institute, University of Hawaii at Manoa,
1680, East-West Road, Honolulu, HI 96822, USA
M.V. Rajeswari
Centre for Ocean Research, Sathyabama University, Jeppiar Nagar,
Chennai, TN 600119, India
N. Annamalai N. Sivakumar
Department of Biology, College of Science, Sultan Qaboos University,
PO Box 36, PC 123 Muscat, Oman

30 N. Annamalai et al.
crystalline and amorphous regions (Dashtban et al. 2009). Cellulosic biomass is the
largest amount of waste generated through human activities, which is the most
attractive substrate for ‘biorefinery strategies’ to produce high-value products such
as fuels, bioplastics and enzymes through fermentation processes (Mazzoli et al.
2012). The value of cellulose as a renewable source of energy has made cellulose
hydrolysis the subject of intense research and industrial interest (Bhat 2000). In
recent days, there is an increasing interest on use of biomass for biofuel production
is pointed-out as an important alternative for reduction of energetic and environ-
ment problems.
Cellulases are classified into three major categories based on their substrate
specificities and hydrolysis mechanism as (i) 1,4 β-D-glucanases or endoglucanases
(EC 3.2.1.4), (ii) exoglucanases or cellobiohydrolases (EC 3.2.1.91), and (iii) cel-
lobiase or β-glucosidases (EC 3.2.1.21). The most efficient hydrolysis of cellulose is
the result of combined synergistic actions of cellulases, whereby the enzymatic
activity of an enzyme mixture is significantly higher than individual enzyme
activity (Wood 1992; Irwin et al. 1993; Nidetzky et al. 1994).
2.2 Exoglucanases or Cellobiohydrolases (CBH)

(EC 3.2.1.91)
Exoglucanases, also known as cellobiohydrolases (CBHs) that act at the end of

cellulose chains and releasing glucose as well as cellobiose as product in a pro-
cessive fashion (Medve et al. 1998). CBHs are the most-studied exoglucanase,
which is about 70 % of the secreted cellulases by cellulolytic fungi belongs to
glycoside hydrolases (GH) 6 and 7, as well as 48 families (Teeri 1997). Different
CBHs are produced by various bacteria and fungi, with catalytic modules belonging
to families 5, 6, 7, 9, 48, and 74 glycoside hydrolases. Aerobic fungal CBHs are
belongs to families 6 and 7; aerobic bacterial CBHs belong to families 6 and 48;
anaerobic fungal CBHs are in family 48 and anaerobic bacterial CBHs are in family
9 as well as 48. In general, family 7 CBHs originate only from fungi, whereas
family 48 CBHs from bacteria (Zhang and Zhang 2013).
2.3 CBH I and II
CBHs mainly release cellobiose from cellulose derivatives, and the presence of
cellobiose competitively inhibits the rate of hydrolysis. The fungal CBHs belong to
glycosyl hydrolase families 6 and 7 (GH-6 and 7) and act most efficiently on highly
ordered crystalline cellulose, hydrolyzing from either reducing or non-reducing end
to liberate predominantly cellobiose (C2) with a minor amount of cellotriose (C3)
(Boer et al. 2000; Takahashi et al. 2010). There are two types of cellobiohydrolase:
2 Cellobiohydrolases: Role, Mechanism, and Recent Developments 31
CBH I and II that works processively from the reducing and non-reducing end of
the cellulose chain respectively. The cellobiohydrolases Cel6A (a CBH II) and
Cel7a (a CBH I) are much studied enzymes of fungal origin and are usually
expressed by the ascomycete Trichoderma reesei, accounts for 40 % of total pro-
tein and 70 % of cellulytic activity in the industrially relevant fungus Hypocrea
jecorina (T. reesei) which is used for industrial-scale production due to its ability to
produce more sugars from biomass through hydrolysis (Suominen et al. 1993).
2.4 Structure and Mode of Action of CBHs
The most significant topological feature of CBHs catalytic module is the tunnel
structure, formed by two surface loops that may cover the entirety (family 7 CBHs)
or part of the active site (family 48 CBHs) (Koivula et al. 2002; Vocadlo and
Davies 2008; Zhang and Zhang 2013). The CBH I (TrCBH I) is the major com-
ponent of the T. reesei cellulase system that belongs to the GH-7 family and
hydrolyzes the β-1,4-linkages of a cellulose chain from its reducing end (Claeyssens
et al. 1990). The catalytic core domain (CCD) structures of TrCBH I suggested that
a central structural motif constituted mainly by an antiparallel β-sandwich motif that
bears four surface loops, which decorate the concave face of the sandwich forming
the cellulose-binding tunnel (Divne et al. 1998; Stahlberg et al. 1996). During
enzymatic catalysis, the cellulose chain slides through the substrate-binding tunnel,
and every second glycosidic bond is correctly presented to the catalytic apparatus
located at the far end of the tunnel to progressively liberate disaccharide units
(cellobiose) from cellulose (Divne et al. 1994).
The three-dimensional (3D) structures of two GH-6 family members including
CBH of T. reesei and that of Humicola insolens in complex with glucose, cel-
looligosaccharide, and a non-hydrolyzable substrate analog (Rouvinen et al. 1990;
Varrot et al. 2002). It seems that the significant amino acids associated with cat-
alytic core domain, where the catalytic site is buried inside a tunnel-shaped cavity
and an enzyme–cello-oligosaccharide hydrogen bond network. This typical struc-
ture suggests that the mode of action proceeds in a processive manner as cel-
lobiohydrolase progresses along the cellulose chain (Boisset et al. 2000; Henrissat
1998; Reverbel-Leroy et al. 1997).
Cellobiohydrolases or exoglucanases is able to work effectively on microcrys-
talline cellulose, presumably peeling cellulose chains from the microcrystalline
structure (Teeri 1997). In general, CBHs are active on the crystalline regions of
cellulose; whereas, endoglucanases (1,4-β-glucanases) are typically active on the
more soluble amorphous region of the cellulose crystal (Rouvinen et al. 1990;
Divne et al. 1998). It seems that there is a high degree of synergy observed between
exo- and endoglucanases, which is required for efficient hydrolysis of cellulose
crystals (Abdel-Shakour and Roushdy 2009).
2.5 Substrates and Assay Methods
In general, enzymes in cellulase mixture which show relatively high activity on

Avicel and little activity on carboxymethyl cellulose (CMC) are identified as
exoglucanases (CBHs) (Maki et al. 2009). The processive activity of CBHs on
CMC is blocked or inhibited by the occurrence of derivatized glucose residues
which leads to severe substrate limitation. Avicel (microcrystalline cellulose or
hydrocellulose) is mainly used for measuring exoglucanase activity because of its
low degree of polymerization. However, Avicel contains some amorphous cellulose
and soluble cellodextrans which can act as substrates for both exo- and endoglu-
canases. Thus, there is no highly specific substrate to measure exoglucanase activity
(Wood and Bhat 1988).
There are several different assays have been proposed to measure exoglucanase
(CBH I and CBH II) activity; however, all of these assays have some sort of
limitations. CBHs acting from nonreducing end of the substrate can be assayed with
chromogenic arylcellobiosides, from which the chromogenic aryl residue, attached
to the reducing end of the substrate liberated. However, the processive enzymes
acting from the reducing end of the carbohydrate chain have been identified
recently, but chromogenic substrates to identify these enzymes are not yet com-
mercially available (Boisset et al. 1998). Measurement of the production of cel-
lobiose directly via high-pressure liquid chromatography (Gum and Brown 1976) or
via a cellobiose oxidoreductase assay (Kelleher et al. 1987) is complicated in that
the endoglucanases that are present in crude cellulase mixtures also produce cel-
lobiose. The substrate 4-methylumbelliferyl-β-D-lactoside was identified as an
effective substrate for assaying CBH I of T. reesei as lactose, phenol and
4-methylumbelliferone (a fluorescent signal molecule) are the hydrolysis products
(van Tilbeurgh et al. 1985). But, the activity of CBH II was not able to detect thus
the resulting activity is not an effective representation of true exoglucanase activity.
Alternatively, an assay for quantification of exoglucanase activity using
p-nitrophenyl-β-D-cellobioside as substrate to yield cellobiose and p-nitrophenol
has been developed; but (i) CBH II activity cannot be measured using p-
nitrophenyl-β-D-cellobioside, (ii) the specific activity of the available purified
endoglucanases may not be representative for all existing endoglucanases in the
mixture, and (iii) the product ratio from endoglucanase actions may be influenced
by the presence of exoglucanases (Deshpande et al. 1984).
A double-antibody sandwich enzyme-linked immunosorbent assay was devel-
oped for quantifying CBH I in cellulase complex of T. reesei. The antibody con-
figuration resulted in the highest specificity for the assay of CBH I employed a
monoclonal antibody to coat wells in polystyrene plates and peroxidase-labeled
polyclonal antibody to detect CBH I bound to the immobilized monoclonal anti-
body. Since there was no direct assay of CBH I activity in the presence of the other
enzymes in complex till date, it would be useful for quantifying this enzyme
between the ranges of 0.1–0.8 µg/ml (Riske et al. 1990).
2.6 Recent Developments
Recently, the cellulolytic yeast (Saccharomyces cerevisiae) with enhanced CBH

activity was developed by integrating three types of CBH-encoding genes (cbh1
from Aspergillus aculeatus, cbh1, and cbh2 from T. reesei) with a strong consti-
tutive promoter Ptpi were sequentially integrated into the S. cerevisiae W303-1A
chromosome via δ-sequence mediated integration. The three recombinant (W1, W2,
and W3) and control strains (W303-1A and AADY) produced ethanol (g/l) from
acid-and alkali-pretreated corncob was about 5.92 ± 0.51, 18.60 ± 0.81,
28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively (Hong et al. 2014).
The thermostable CBHs could offer potential benefits in the hydrolysis of pre-
treated lignocellulosic substrates because the harsh conditions often required by
several pretreatments can be harmful for conventional biocatalysts. Heinzelman
et al. (2010) investigated an efficient SCHEMA recombination-based approach for
screening homologous enzymes to identify stabilizing amino acid sequence blocks
which has been used to generate active, thermostable CBH I enzymes from the
390,625 possible chimeras that can be made by swapping eight blocks from five
fungal homologs. A total of 16 predicted thermostable chimeras, with an average of
37 mutations relative are more thermostable (>65 °C) than the most stable parent
CBH I of thermophilic fungus Talaromyces emersonii.
Likewise, Komor et al. (2012) attempted to produce thermostable chimeric
fungal CBH I by structure-guided recombination through FoldX and a ‘consensus’
sequence approach to identify individual mutations present in the five homologous
parent CBH I enzymes which further stabilize the chimeras. With an alignment of
41 CBH I sequences, effects on ΔG (Folding) was calculated using amino acid
frequencies at each candidate position and the mutations chosen using these
methods increased the T(50) of the most thermostable chimera by an additional
4.7 °C, to yield a CBH I with T(50) of 72.1 °C, which is 9.2 °C higher than that of
native CBH I, from Talaromyces emersonii.
2.7 Perspectives
The interest in enzymatic hydrolysis of cellulosic biomass for biofuel production is

increasing continuously due to availability and reutilization as cheaper source.
CBHs are one among the important enzymes which could produce more sugars
such as glucose and cellobiose from crystalline cellulose. (i) It is essential to
develop easy and suitable method with specific substrate in order to measure the
CBHs activity in the cellulose mixture. (ii) Engineering of CBHs is also needed in
order to enhance the specific activity to reduce the use of CBHs and also to increase
thermostability and reusability of exoglucanases. These approaches mentioned
above to enhance the activity and stability of CBHs would be useful in biorefinery
and biofuel industries.
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Chapter 3
Endo-1,4-β-glucanases: Role, Applications
and Recent Developments

and Thangavel Balasubramanian
Abstract The biomass conversion processes are highly dependent on the use of
efficient enzymes to degrade polymeric cellulose or hemicellulose into simple sac-
charides, sugars which can be fermented by microorganisms for the production of
valuable fuel and chemicals. The cellulose hydrolysis involves enzymes such as
endo-1,4-β-glucanases (EC 3.2.1.4), cellobiohydrolases (or exo-1,4-β-glucanases)
(EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). Endo-β-(1,4)-glucanases (EGs) or
β-(1,4)-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act randomly on soluble
and insoluble β-(1,4)-glucan substrates. EGs breakdown cellulose by attacking the
amorphous regions to produce more accessible new free chain ends for the action of
cellobiohydrolases. The EG-I, II, III, and V, respectively, GH7, 5, 12, and 45 are
most common in natural fungal cellulase mixes. EGs play an important role in
increasing yield of fruit juices, beer filtration, and oil extraction, as well as improving
the nutritive quality of bakery products and animal feed. Reducing sugar assay is the
most convenient and reliable method for EG estimation. However, it is very con-
ventional and time consuming. Recently, a specific and sensitive assay methods have
been developed using substrate mixture comprises of benzylidene end-blocked
2-chloro-4-nitrophenyl-β-cellotrioside (BzCNPG3) and 4,6-O-benzylidene-
4-methylumbelliferyl-β-cellotrioside (BzMUG3).
N. Annamalai (&)
Hawaii Natural Energy Institute, University of Hawaii at Manoa,
1680, East-West Road, Honolulu 96822, HI, USA
M.V. Rajeswari
Centre for Ocean Research, Sathyabama University, Jeppiar Nagar,
Chennai 600119, TN, India
T. Balasubramanian
CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University,
Parangipettai 608502, TN, India

3.1 Introduction
Lignocellulosic biomass contains morphologically different cellulose, complex

structural and compositional hemicellulose, recalcitrant lignin, diverse proteins and
lipids and other substances that interact with each other (Sweeney and Xu 2012).
Cellulose is a long homogenous linear polymer of β-D-glucosyl units linked by
1,4-β-D-glucosidic bonds. Over the last few years, the growing challenge has been
the setup of industrial-scale conversion of cellulosic biomass into fermentable
sugars, which can in turn be used as raw material for the production of biofuels and
other bio based high value-added products (Adsul et al. 2011; Du et al. 2011;
Somerville et al. 2010). Efficient conversion of cellulosic biomass into fuels and
other products is a fossil fuel-saving and environment-friendly biotechnology
(Lange 2007). The use of enzymes reduces the harsh chemicals and/or heat for the
production of fermentable sugars, raising the environmental feasibility of the
process.
The primary role of biomass-converting enzymes is to degrade polymeric cel-
lulose or hemicellulose into simple saccharides, sugars, which can be fermented by
microorganisms to, or serve as platform molecules for synthesis of valuable fuel or
chemicals. As potential industrial catalysts for biomass conversion, enzymes might
provide high specificity, low energy or chemical consumption, or low environment
pollution (Sweeney and Xu 2012). Cellulases and most hemicellulases belong to a
group of enzymes known as glycoside hydrolases (GH), which can degrade cellulose
and hemicellulose to constituent hexoses and pentoses. In general, biomass-
converting enzymes have to work in concert, to benefit from synergism among their
specificity (towards different components and regions of lignocellulose) as well as
mitigation of their inhibition (by different lignocellulose components or degradation
products) (Sweeney and Xu 2012). The classical scheme for cellulose hydrolysis
involves three main categories of enzymes: endo-1,4-β-glucanases (EC 3.2.1.4),
cellobiohydrolases (or exo-1,4-β-glucanases) (EC 3.2.1.91), and β-glucosidases (EC
3.2.1.21). The complete degradation of cellulose into glucose is a complex process
due to its amorphous/crystalline structure, which requires the cooperative action of
endo and exo-β-1,4-glucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21).
According to CAZy database (http://www.cazy.org), these enzymes are grouped into
15 of 132 glycoside hydrolase (GH) families, i.e., 1, 3, 5–9, 12, 27, 44, 45, 48, 51,
61 and 74 (Wang et al. 2014).
In this respect, the most important enzyme has long been realized to be an
endo-β-1-3,1-4-glucanase (EC 3.2.1.73), which hydrolyzes β-1-4 linkages on the
reducing side of β-1-3 linkages to produce primarily of oligosaccharides with
contiguous β-1-4 linkages that derive from cellulosic regions in the polymer as
substrates for an endo-β-1-4-glucanase (Kanauchi and Bamforth 2008).
3 Endo-1,4-β-glucanases: Role, Applications and Recent … 39
3.2 Endo-1,4-β-glucanases (EC 3.2.1.4, Endocellulase)
Endo-β-(1,4)-glucanases or β-(1,4)-D-glucan-4-glucanohydrolases (EC 3.2.1.4) act

randomly on soluble and insoluble β-(1,4)-glucan substrates. The endo-1,4-β-
glucanase (EGase, EC 3.2.1.4) often referred to as cellulase, hydrolyzes 1,4-β-D-
glucan chain molecules and plays a role in various physiological processes (Yu et al.
2013). Endoglucanases (EG) are also referred to as carboxymethylcellulases
(CMCase), named after the artificial substrate used to measure the enzyme activity.
The active sites of endoglucanases typically attain a cleft-like topology for random
fashion and bond cleavage can occur anywhere along the chain of the cellulose
(Himmel et al. 2010). Endoglucanase breakdown the cellulose by attacking the
amorphous regions of the cellulose to produce more accessible new free chain ends
for the action of cellobiohydrolases which has been shown by the effect of the
enzyme on carboxymethylcellulose and amorphous cellulose (Sandgren et al. 2005).
Degradation of amorphous cellulose can be carried out by endoglucanases
(EGs) (EC 3.2.1.4). Unlike cellobiohydrolases (CBH), EG hydrolyzes internal
glycosidic bonds in cellulose with a random, on-off fashion which make them
well-suited to less orderly or partially shielded cellulose parts, generating new
cellulose chain ends for CBH action. A few EGs can act “processively” on crys-
talline cellulose (Wilson 2008; Li et al. 2010). Also known as EG-I, II, III and V,
respectively, GH7, 5, 12 and 45 EG are most common in natural fungal cellulase
mixes. EGs genes are found in many prokaryotic and eukaryotic organisms,
including bacteria, fungi, slime molds, nematodes, animals and plants (Levy et al.
2002; Libertini et al. 2004). Most cellulolytic fungi and bacteria produce numerous
EGs. Although they all act on the same cellulose substrate, they do with different
mechanisms (“inverting” for GH6, 9, 45 and 48 EGs; “retaining” for GH5, 7, 12
EGs), which may relate to different EGs’ side-activities on hemicellulose in
degrading complex lignocellulose (Vlasenko et al. 2010), or synergism between
processive and conventional EGs (Wilson 2008). The active sites of most EGs are
cleft- or groove-shaped, inside which a cellodextrin or a cellulose segment may be
bound and acted on by EG. In addition to the catalytic core, EGs may possess
CBMs which is the direct host, but is not a pre-requisite, for EG’s action (Sweeney
and Xu 2012).
The fungal endoglucanases (EGs) are generally monomers with no or low
glycosylation and have an open binding cleft. They mostly have pH optima
between 4 and 5 and temperature optima from 50 to 70 °C. A typical cellulolytic
fungus secretes EGs at *20 % wt level in their secretomes (Sipos et al. 2010;
Chundawat et al. 2011). In addition, many of the fungi produce multiple EGs. For
example, T. reesei produces at least 5 EGs (EG I/Cel 7B, EG II/Cel5A, EG
III/Cel12A, EG IV/Cel61A and EG V/Cel45A) whereas three EGs were isolated
from white-rot fungus P. chrysosporium (EG28, EG34 and EG44) (Baldrian and
Valaskova 2008; Foreman et al. 2003). In addition, some EGs lack a CBM while
some other EGs with CBM have been described. For example, four of five EGs in
T. reesei including EG I, EG II, EG IV and EG V have CBM whereas EG III does

not have a CBM (Sandgren et al. 2005).
In plants, EGases are encoded by a large gene family, belonging to the glycosyl
hydrolase gene family 9 (GH9) (Henrissat 1991). According to their sequence
domain structure, this multigene family can be grouped into three subclasses: A
subclass (GH9A), membrane-anchored GH9; B subclass (GH9B), secreted GH9;
and C subclass (GH9C), secreted proteins with a carbohydrate binding module,
CBM49 (Urbanowicz et al. 2007). Alternatively, the plant EGase gene family can
be divided into α, β and γ subfamilies based on phylogenetic analysis (Libertini
et al. 2004). In Arabidopsis, the α-subfamily mainly consists of secreted GH9
proteins (including GH9B and GH9C subclasses) except for one GH9A subclass
protein. All γ-subfamily members are transmembrane proteins, belonging to GH9A
subclass (Yu et al. 2013).
3.3 Criteria for Optimized EG Production
In general, cellulase production accounts for 40 % of cost in bioethanol synthesis

(Sateesh et al. 2012). Carbon and nitrogen sources are the important factors for
endoglucanase production and synthesis of various cell wall lipids in fungi
(Sarria-Alfonso et al. 2013). The effects of different carbon sources (avicel, cel-
lobiose, CMC, xylan, rice straw, bagasse and palm kernel) on EG production by
various microorganisms were investigated till date. Among these, rice straw was
reported to be a cheaper and potential sole carbon source and yeast extract as the
nitrogen source for higher EG production. In addition, a significant increase in
enzyme production with an increased fermentation time, which was presumed to be
due to rapid hydrolysis of cellulose in the medium.
3.4 Industrial Applications of Endo-β-1,4-Glucanase
Cellulases are used in the textile industry, in detergents, pulp and paper industry,
improving digestibility of animal feeds and food industry. They account for a
significant fraction of industrial enzyme markets. Obviously, cellulase will be the
largest industrial enzyme as compared to the current market size for all industrial
enzymes ($ *2 billion) (Zhang and Zhang 2013). Among the cellulases,
endoglucanases play a key role in increasing the yield of fruit juices, beer filtration
and oil extraction, as well as improving the nutritive quality of bakery products and
animal feed (Bhat 2000). Endo-β-1,4-glucanase has been widely used in industries
to hydrolyze cellulosic materials to sugars in the biofuel production (Wilson 2009),
to increase the rate of wort filtration and reduce the possibility of β-glucan pre-
cipitation in beer and reduce the mash viscosity and turbidity in the brewing
industry (Celestino et al. 2006), to bioremediate pulp waste in the paper industry
(Ohmiya et al. 1997), and to increase β-glucan digestibility and improve feed
conversion efficiency in the feed industry (Mathlouthi et al. 2002). The
endo-1,4-β-glucanase is also used in the poultry and animal feed industry to
degrade β-glucan and thus increase the digestibility of feeds rich in barley and also
overcome the anti-nutritive effects of 1,3-1,4-β-D-glucan (Choct 2001). Further,
endo-1,4-β-glucanase is used for denim finishing and cotton softening in textile
industries, cleaning and colour care in the detergent industries and to modify fibre
and improves drainage in the paper industries (Cherry and Fidantsef 2003).
Thus, the enormous industrial applications demands endoglucanases with
varying pH and temperature optima, stabilities and substrate specificities. Although
cellulolytic enzymes of Trichoderma reesei have been investigated thoroughly
(Miettinen-Oinonen et al. 2004), the amount of cellulase secreted by this fungus is
insufficient for effective conversion of cellulose to glucose. Moreover, the industrial
demand is increasing day by day especially because of the emergence of second
generation-advanced biofuel industries, which require tremendous amounts of
various enzymes in their processes (Wilson 2009; Yeoman et al. 2010).
Thermostable endoglucanases/cellulases are more favourable for industrial
application due to their high activity and stability at high temperatures to decrease
process costs and increase the efficiencies (Yeoman et al. 2010). However, most
cellulases are not stable at high temperatures, and a number of thermostable cel-
lulases have been purified or cloned and characterized in recent years, some of them
display maximum activities even at 100 °C, such as GH 12 endoglucanase from
Pyrococcus furiosus (100 °C; Bauer et al. 1999), and endoglucanase CelB from
Thermotoga neapolitana (106 °C; Bok et al. 1998). As the main microbial source
of thermophilic endoglucanases, thermophilic fungi like Talaromyces emersonii
and Thermoascus aurantiacus have been reported to produce cellulases of GH 3, 5
and 7 families with temperature optima of 65–80 °C (Gomes et al. 2000; Grassick
et al. 2004).
3.5 Recent Developments on Endo-1,4-β-Glucanase Assay
The endo-1,4-β-glucanase in biological materials and microbial fermentation broths

can be specifically measured by the decrease in the viscosity of water soluble,
chemically modified cellulose derivatives such as CM-cellulose 7 M (McCleary et al.
2012). The reducing sugar assays method is most conventional and time consuming
(Somogyi 1952). The other methods such as measuring hydrolysis rate of substrates
such as cello-oligosaccharides, borohydride-reduced cello-oligosaccharides (McCleary
et al. 2012) or nitrophenyl-cello-oligosaccharides (Bhat et al. 1990; Claeyssens and
Henrissat 1992; Rahman et al. 2002) cannot be used to measure endo-1,4-β-glucanase
in the presence β-glucosidase and exo-1,4-β-glucanases. Hence, a specific and sen-
sitive substrate mixture comprises benzylidene end-blocked 2-chloro-4-nitrophenyl-β-
cellotrioside (BzCNPG3) was prepared as substrate for the assay of endo-1,4-β-
glucanase (cellulase). Hydrolysis by β-glucosidase and exo-1,4-β-glucanase is
prevented by the presence of the benzylidene group on the non-reducing end D-

glucosyl residue (McCleary et al. 2014).
In addition to this, novel fluorometric assay for endo-1,4-β-glucanase that is
based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside
(BzMUG3) in the presence of an ancillary β-glucosidase was developed for both
quantitative and qualitative measurements. The substrate BzMUG3 can be cleaved
in the presence of cellulase to generate a protected cello-oligosaccharide fragment
and a fluorogenic cello-oligosaccharide which will then be acted on by excess
β-glucosidase (EC 3.2.1.21) present in the reagent mixture to liberate
4-methylumbelliferone and 4,6-O-benzylidene protecting group prevents the action
of β-glucosidase on the parent substrate. The rate of formation of
4-methylumbelliferone is directly proportional to the cellulase activity in a given
sample (Mangan et al. 2014).
3.6 Future Prospective
Biomass saccharification is the largest technical and economic obstacle to biorefinery

and biofuels production. For the efficient conversion of cellulose from biomass and to
glucose for the subsequent production of fuel ethanol, protein engineering of
endo-1,4-β-glucanase should be focused to enhance specific activity towards pre-
treated biomass using enzyme cocktail and either rational design or directed evolu-
tion (Sathitsuksanoh et al. 2010; Zhang 2008), improve the stability for recycling and
reduce production costs ($ per kilogram of dry protein) (Zhu et al. 2009). In addition,
engineering of cellulolytic enzymes with improved catalytic efficiency and enhanced
thermostability is important to commercialize lignocelluloses biorefinery and further
improvement on cellulase performance needs the better understanding of hydrolysis
mechanisms as well as the relationship of cellulase molecular structure, function and
substrate characteristics. The consolidated bioprocessing (CBP) of microorganisms
or consortium would make the biomass hydrolysis easier and enhance the produc-
tivity. The improved simultaneous saccharification and fermentation (SSF) technol-
ogy would be useful to reduce end-product inhibition, investment costs and higher
yield efficient biofuel production.
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Chapter 4
The Role and Applications
of b-Glucosidases in Biomass Degradation
and Bioconversion
Hanlin Ouyang and Feng Xu
Abstract b-Glucosidases is known as the terminal enzyme in the synergy with

other cellulases for biomass degradation. It completes the final step of small
oligosaccharides (including cellobiose) conversion into glucose. However, this is
only a small part of the roles played by a subgroup of bacterial/fungal
b-glucosidases in cellulose biodegradation. To deepen our understanding about
the current challenges and limits in biomass conversion and to enlighten the future
for the industry, we take one step back and look into a broader range of
b-glucosidases that cross the life domains. b-Glucosidases from different subfam-
ilies are compared systematically in their distribution, phylogenetic relationship,
structure, in vivo function, mechanism of hydrolysis as well as substrate recogni-
tion, and kinetic profile. Protein engineering and application works on the enzymes
are discussed based on the knowledge herein.
4.1 Introduction
Biomass industry nowadays is developing and optimizing enzymatic solutions to

hydrolyze lignocellulosic plant-based substrates into monomeric sugars, which are
eventually fermented to bioethanol. Biomass substrate composition can be simplified
as cellulose, hemicellulose, and lignin in general while each component is targeted by
different enzymes. Glucan, or cellulose, consists of up to more than 40 % of biomass
substrates, and therefore cellulases are major players in the enzymatic hydrolysis of
glucan. Cellulases refer to a synergistic collection of three different groups of
enzyme: cellobiohydrolase (CBH, exo-b-1,4-glucanase), endo-b-1,4-glucanase
(EG), and b-glucosidase (BGL). While the exo- and endoglucanases cleave poly-
H. Ouyang (&)
Novozymes North America Inc., 77 Perrys Chapel Church Road, PO BOX 576, Franklinton,
NC 27525, USA
F. Xu
Novozymes Inc., 1445 Drew Ave, Davis, CA 95618, USA

48 H. Ouyang and F. Xu
meric cellulose into small oligosaccharides (cellodextrins), BGL is responsible to

release glucose (Glc) from these oligosaccharides, especially cellobiose. In addition,
different BGLs can have activity on many other glycosides, releasing the nonre-
ducing terminal glycosyl moiety from these substrates, which may be involved in
various physiological events (Ketudat Cairns and Esen 2010). The ‘Second
Generation’ (cellulosic) bioethanol industry locks their preference on BGLs from
microbes that feed on biomass (Rani et al. 2014; Sørensen et al. 2013; Teugjas and
Väljamäe 2013; Tiwari et al. 2013; Singhania et al. 2013; Sweeney and Xu 2012;
Krisch et al. 2010; Quinlan et al. 2010; Xu 2004, 2010a, b; Eyzaguirre et al. 2005;
Bhatia et al. 2002). The development of viable industrial BGLs has been challenging.
Native BGLs produced in many fungal strains occur in low abundance, which needs
to be greatly enhanced for the enzymes to become economically viable for industrial
application. The BGL’s product inhibition by Glc can be significant during the
industrial enzymatic cellulose hydrolysis. Besides, biomass-based ethanol producers
rely on thermochemical pretreatment of lignocellulosic substrates to improve enzyme
access to cellulose and cellulose hydrolysis often occurs at elevated temperature to
speed up the process. These two steps create harsh conditions for BGL’s ther-
mostability and inhibition resistance while the challenges have propelled active
studies on BGL.
4.2 Classification and Distribution
Historically, BGLs have been categorized into three groups based on the substrates:
aryl-BGLs, true cellobiases, and broad substrate specificity BGLs. Canonical BGLs
that catalyze the hydrolysis of b-glucopyranosides are assigned with the Enzyme
Commission (E.C.) number E.C.3.2.1.21. However, given the variety of b-linkage
terminal nonreducing glycosides in nature, many E.C. numbers have been assigned
to a larger group of enzymes that hydrolyze b-linkage glycosidic bond. For glucan
substrates, glucan 1,3-b-glucosidase (3.2.1.58) and glucan 1,4-b-glucosidase
(3.2.1.74) remove successively Glc from the nonreducing end with 1,3 and 1,4
b-linkage to the polysaccharide chain. Enzymes with hydrolytic activity on
b-glucosides with aglycon moieties also fall into E.C.3.2.1 family. These include
glucosylceramidase (3.2.1.45), steryl-b-glucosidase (3.2.1.104), 3a(S)-strictosidine
b-glucosidase (3.2.1.105), amygdalin-b-glucosidase (3.2.1.117), prunasin
b-glucosidase (3.2.1.118), vicianin b-glucosidase (3.2.1.119), raucaffricine
b-glucosidase (3.2.1.125), coniferin b-glucosidase (3.2.1.126), and b-D-glucopyr-
anosyl abscisate b-glucosidase (3.2.1.175). More complicated substrates like
thio-linkage, 7-[b-D-apiofuranosyl-(1, 6)-b-D-glucopyranosyloxy]isoflavonoid, b-D-
galactopyranose-(1, 4)-a-L-galactopyranose-6-sulfate in porphyran, or even 3-O-(N-
acetyl-b-D-glucosaminyl)-L-serine/threonine in proteins can be hydrolyzed by
thioglucosidase (myrosinases) (3.2.1.147), b-apiosyl-b-glucosidase (3.2.1.161),
b-porphyranase (3.2.1.178) and protein O-GlcNAcase (3.2.1.169) respectively.
E.C. numbering system is a classification based on the chemical reactions which
enzymes catalyze. It has little information on structure, mechanism, or phylogenetic
4 The Role and Applications of b-Glucosidases in Biomass … 49
relationship. A sequence-based classification has been developed since 1996. The

glycoside hydrolases (GH) are currently classified into 133 families in the
Carbohydrate Active enZyme, or CAZy database (www.cazy.org). Each GH family
contains proteins that are related by sequence, corollary, folding (based on the
hydrophobic cluster similarity). This allows the possibility of useful mechanis-
tic predictions as the motifs, key residues and geometry around the active sites
usually share high similarity for a conserved enzymatic mechanism, even when the
substrates (thus the E.C numbers) are different. Almost all the BGLs identified so
far belong to the GH1, GH3, GH5, GH9, GH30, and GH116 families. Based on the
CAZy as of June 2015, the GH1 family includes six BGLs from archaea, 194 from
bacteria, 210 from eukaryotes (especially fungi and plants), one from a virus and six
from unclassified organisms, along with 42 myrosinases and one glucosylcerami-
dase from eukaryotes. The GH3 family includes one BGL from an archaeon, 118
from bacteria, 141 from eukaryotes (especially fungi and plants), ten from
unclassified organisms, and several likely from animal (Gabrisko and Janecek
2015). The GH5 family includes one BGL from a virus, one from plant and four in
fungi, while the GH30 family includes one BGL from a fungus, two from animals
and one from human. The GH30 family also has two animal glucosylceramidases.
The GH9 family so far only covers two BGLs from the bacterium Vibrio cholera.
Most of these BGLs are intracellular or cytoplasmic enzymes. However, some of
them are membrane associated, such as bacterial Pyrococcus horikoshii bgl (GH1)
(Matsui et al. 2000) and Prevotella ruminicola cdxA (GH3) (Wulff-Strobel and
Wilson 1995). Some of them can reside in periplasmic space, such as bacterial
Azospirillum irakense salA, salB (GH3) (Faure et al. 1999), Bacillus subtilis bglA
(GH1) (Srivastava et al. 1999), Erwinia chrysanthemi bglx (GH3) (Vroemen et al.
1995), Erwinia herbicola bglA (GH1) (Marri et al. 1995) and fungal (mold)
Aspergillus kawachii bglA (GH3) (Iwashita et al. 1999). Some can be extracellular,
such as bacterial Ruminococcus albus bgl (GH3) (Takano et al. 1992), fungal
Aspergillus niger bgl1(GH1) (Dan et al. 2000), Trichoderma reesei (Hypocrea
jecorina) bgl1 (GH3) bgl2 (GH1) (Barnett et al. 1991; Takashima et al. 1999),
Saccharomycopsis fibuligera bgl1, bgl2 (both GH3) (Machida et al. 1988) and
barley (Hordeum vulgare) bgq60 (GH1) (Leah et al. 1995), Catharanthus roseus
strictosidine bgl (GH1) (Geerlings et al. 2000). The extracellular microbial BGLs
are of particular industrial interest because of their production costs (that can be
significantly lower than that of the intracellular counterparts).
4.3 In Vivo Function
4.3.1 Bacteria and Fungi
One of the functions for BGL is being a part of the synergistic cellulase system that
breaks down cellulose, deployed by a large population of organisms including
bacteria, fungi (molds, mushrooms, yeasts), and animals (e.g., termites). BGL is
used for energy/nutrition uptake in metabolism as polysaccharide is degraded into

Glc. During the same process, solid surfaces of substrate like plant cell walls are
penetrated to allow these organisms to establish pathogenic or symbiotic relation-
ships (Gilbert et al. 2008). There are generally two strategies for how the secreted,
cellulose-degrading enzymes work: to form a cell surface-anchored supramolecular
complex called cellulosome, or enzymes acting as cell-free entities. The first case
can been found with a collection of anaerobic cellulolytic bacteria like Bacteroides,
Clostridia, and Ruminococcus (Bayer et al. 2004) as well as some anaerobic cel-
lulolytic fungi like Neocallimastix frontalis (Wilson and Wood 1992) and
Piromyces (Ali et al. 1995). The second case can be found in most of the aerobic
cellulolytic fungi and bacteria. Along with multitudes of EGs, CBHs, and auxiliary
enzymes, extracellular BGLs are transported out of the cell and act in synergy with
the other cellulases to breakdown plant cell walls. Well-established examples are
BGL1 (GH3) and BGL2(GH1) from T. reesei and BGL1(GH1) from A. niger.
Some fungi, such as white rot Phanerochaete chrysoporium, may contain both
cytoplasmic and extracellular BGLs. Some of these may serve in the metabolism of
the organism’s own cell wall, while others may play a role in plant cell wall
metabolism (Igarashi et al. 2003; Tsukada et al. 2006).
4.3.2 Plants
Plant BGLs have been demonstrated with a wide functional diversity in defense,
symbiosis, cell wall catabolism and lignification (monolignol conversion), signaling
and secondary metabolism. The wide spectrum of biological activity is supported by a
large number of BGLs from different GH families. For instance, 48 GH1 genes are
found in Arabidopsis thaliana (Xu et al. 2004), while the number in rice is 40
(Opassiri et al. 2006). These GH1 enzymes cover a cluster of classical myrosinases
and another cluster of myrosinases and BGLs found in the endoplasmic reticulum
(ER) and peroxisome. Interestingly, GH1 enzymes from chloroplast of other plants
such as maize, sorghum and wheat are much more closely related to homologues in
thermophilic bacteria and archaea than rice and Arabidopsis enzymes (Thorlby et al.
2004). In addition, most plants also encode BGLs from GH families 3 and 5 (Opassiri
et al. 2007; Arthan et al. 2006). To defend themselves, plants have developed a wide
range of molecules. The first group is cyanogenic b-glycosides, including linamarin
from clover and cassava, dhurrin from sorghum and prunasin from cherry. These
cyanogenic glycosides can be hydrolyzed by BGL and release their a-hydroxynitrile
moiety, which then breakdown either enzymatically or spontaneously to yield cya-
nide (Poulton 1990; Morant et al. 2008a, b). The second group consists of non-
cyanogenic molecules, such as b- and c-hydroxynitriles (Morant et al. 2008a, b) and
isoflavones in legumes (Suzuki et al. 2006), flavonoids (Chuankhayan et al. 2005),
coumarins, hydroxaminic acids (e.g., 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-
3-one) in maize and wheat (Esen 1992; Sue et al. 2006; Nikus et al. 2001), and
saponins (Nisius 1988). These defensive molecules are also stored as b-D-glucosides
and unleashed by specific BGLs.
In the symbiosis between the endophytic fungus Piriformospora indica and
plant Arabidopsis, the fungus prevents Arabidopsis from overgrowing the roots and
ends up triggering a defense response (Sherameti et al. 2008; Nagano et al. 2005).
A special BGL in ER, AtBGLU23, is revealed to be critical in this symbiosis (Lipka
et al. 2005; Bednarek et al. 2009) by demonstrated activity on scopolin, the most
abundant coumarin glucoside in root (Ahn et al. 2010). Upon cell wall disruption,
plant defensive BGLs and myrosinases often bind to cytoplasmic aggregating
factors, which are believed to localize the otherwise soluble glucosidases at the site
and therefore generate the defense compounds on site (Nisius 1988; Esen and
Blanchard 2000; Kittur et al. 2007). In addition to the plant–microbe interaction,
BGL may also be involved in the plant–insect interaction.
BGLs also play an important role in cell wall metabolism. Several BGLs that
hydrolyze cell wall derived oligosaccharides have been identified. One BGL in
germinating barley seedlings showed activity toward b-1,3- and b-1,4-linked
oligosaccharides (Leah et al. 1995; Hrmova et al. 1998), preferentially man-
nooligosaccharides, which are found in barley endosperm cell walls (Hrmova et al.
2006). Rice seedling BGLs Os3BGlu7, Os3BGlu8, and Os3BGlu26 demonstrate an
increased activity as degree of polymerization (DP) increase from 2 to 6
(Kuntothom et al. 2009), while Os4BGlu12 (Opassiri et al. 2006) is not sensitive to
substrate DP, and Os3BGlu6 (Seshadri et al. 2009) is most efficient on disaccha-
rides. Os3BGlu7 and Os3BGlu8 are widely expressed in rice tissues and are thus
believed able to remodel cell wall by releasing Glc from oligosaccharides derived
from it. Some other BGLs play roles in lignification of secondary cell walls. Two
examples are coniferin BGL from lodgepole pine tree xylem found in differentiating
region of the xylem (Dhamawardhana et al. 1995) and BGL (AtBGLU45/46) from
Arabidopsis. The later has been found active in hydrolyzing coniferin, syringin and
coumaryl alcohol glucosides to release monolignols (Escamilla-Trevino et al.
2006).
Phytohormone activation in many cases also requires glucosides hydrolysis by
BGLs, such as the maize BGL Zm-p60.1 which hydrolyzes and activates cytokinin
b-glucosides (Brzobohaty et al. 1993), an extracellular BGL which hydrolyzes
abscisic acid glucosyl (Glc) ester transported from roots to leaves (Dietz et al.
2000), the BGL hydrolyzing auxin Glc ester 6-O-(4-O)-indole-3-ylacetyl-b-D-Glc
(Jakubowska and Kawalczyk 2005), and the Arabidopsis ER BGL AtBG1 that
hydrolyzes abscissic acid Glc ester in response to drought stress (Lee et al. 2006).
Mutation in this Arabidopsis AtBG1 leads to early germination and defective
stomata closing.
BGLs can also play metabolic roles to remove Glc protective group from
metabolic intermediates and consequently trigger the biosynthesis of various natural
products (phytochemicals) such as monoterpene alkaloids. For instance, one of the
downstream products of strictosidine is raucaffricine, a glucoside that could only be
further metabolized into ajmaline after deglucosylated by raucaffricine BGL
(Barleben et al. 2007).
Plant BGLs can also release volatile terpenols from glycosides as flower and
fruit scents (Reuveni et al. 1999) or attractants for parasitoid wasp to attack her-
bivores parasites (Mattiacci et al. 1995).
4.3.3 Animals
Some insects feeding on plants also developed GH1 enzymes in their larvae stage,
such as BGLs from Tenebrio molitor and Spodoptera frugiperda (Ferrieira et al.
2001; Marana et al. 2001) and myrosinases from cabbage aphid Brevicoryne
brassicae (Jones et al. 2001). These enzymes have hydrolysis capability on
gluco-oligosaccharides and plant cellobiose, gentiobiose and even amygdalin
(Marana et al. 2001) and share higher sequence similarity with vertebrate
lactase-phloridzin hydrolase (LPH) than with GH1 from plants, indicating their
further divergence from the same animal ancestor after their first divergence from
plants.
Mammalian cells contain BGLs for metabolism of glycolipids and dietary gluco-
sides. Human acid BGLs releases Glc ceramide; defects in this enzyme lead to gly-
coceramides accumulation and eventual Gaucher disease (Butters 2007). Human LPH
is involved in food digestion and displays activity on b-glucosides, b-galactosides and
phloridzin, a polyphenol carrying glycoside found in many fruits. Deficiency of this
enzyme ends up with the widespread lactose intolerance (Tribolo et al. 2007). Human
acid BGL GBA1 and bile acid BGL GBA2 belong to the GH30, and participate in the
metabolism of glycolipids and dietary glucosides (Berrin et al. 2002) (Fig. 4.1).
4.4 Structure
BGLs from various GH families can have amino acid sequences ranging from 420
to 840 units, some comprising signal peptides for secretion or other domains in
addition to the catalytic core. The structures of the catalytic core (Fig. 4.2) can be
divided into three groups. The first one is a (b/a)8-barrel found in GH1, GH5, and
GH30. The second one contains an (a/a)6-barrel found in GH9. The last one
consists of a (b/a)8-barrel and an a/b sandwich in which a 6-stranded b-sheet is
flanked by two sets of three a-helices, and is found in GH3. The (b/a)8-barrel group
features a catalytic domain ranging from 420 to 560 residues in length, depending
on the highly variable loop regions on the b-strands at the C terminus
(Sanz-Aparicio et al. 1998). Within the catalytic domain, two conserved Glu or Asp
from b-strands 4 and 7 respectively are identified as the catalytic acid/base and
nucleophilic residue (Ketudat Cairns and Esen 2010; Vallmitjana et al. 2001;
Henrissat et al. 1995). Monomers of GH1 enzymes can dimerize or form even
higher orders of aggregates. The GH5 BGLs have a (b/a)8-barrel core structure as
the GH1 BGLs (both belonging to the GH-A clan), and the rice GH5 BGL has a
OH OH
OH OH HO OH
HO
HO HO O
O O HO O O
HO HO HO
O OH
O HO O
O OH
OH OH CN
O
CN OH
NC CN
Linamarin Dhurrin D-Amygdalin

Prunasin
OH
OH
OH
O HO
HO HO O
HO O
O
O OH
OH OH
O N OH
HO
O
HO O O
R
O O HO Daidzin R = H
OH
OH
O Genistin R = OH
O
DIMBOA beta-glucoside Apigenin 7-O-beta-glucoside
OH OH
OH
OH
OH HO
OH HO O
HO O
O HO O
HO O OH
HO
O OH
OH O O
OH
OH
Coniferin Phloridzin
HO
O HO
OH
OH
HO
HO O HO
HO O
OH O NH
OH O
O
6
OH
OH
11
OH
HO O Salicin
O
Glucosyl ceramide
HO
O H
OH OH
OH OH
O O
Abscisic acid glucosyl ester
HO
O O OH
OH HO
H O O HO
OH N HO
O OH
HO H
HO N
HO O O Cellobiose
OH
HN
O
OH
OH
OH HO
HO
Indoxyl beta-glucoside Strictosidine HO O O
O
OH OH OH
Laminaribiose
Fig. 4.1 Structures of some BGLs substrates involved in various physiological processes. 1 Plant
defense: 1a cyanogenic glucosides: linamarin (clover/cassava), dhurrin (sorghum), prunasin (cherry)
and its precursor D-amygdalin. 1b noncyanogenic glucosides: 2,4-dihydroxy-7-methoxy-1,4-
benzoxazin-3-one 2-O-b-glucoside (DIMBOA-Glc, maize/wheat), apigenin 7-O-b-glucoside,
daidzin, genistin, phloridzin. 2 Plant cell wall metabolism: coniferin, p-coumaryl alcohol 4-O-
b-glucoside. 3 Plant phytohormone activation: abscisic acid glucosyl ester, indoxyl b-glucoside,
salicin. 4 Plant secondary metabolism: strictosidine, precursor to a wide range of monoterpene
alkaloids. 5 Fungal and bacterial degradation of plant primary cell walls: cellobiose, laminaribiose. 6
Animal (structural, signaling and metabolic functions): glucosylceramide
fascin-like domain (Opassiri et al. 2007). The (a/a)6-barrel group seen in GH9 is
dominated by a large number of endoglycosidases and leave only a few BGLs like
that from V. cholera. So far there is no conclusive identification of the catalytic
residues on the BGL from this family. However, the study on another GH9 exo-
cellulase has identified a Glu residue on the loop connecting a-helices 12 and 13 as
the catalytic acid/base, while a water molecule activated by two conserved,
hydrogen bond-forming Asp acts as the nucleophilic residue (Schubot et al. 2004).
A similar situation might be expected in BGLs from the same family. For the GH3
enzymes, the interface of the (b/a)8-barrel and a/b sandwich harbors the catalytic
nucleophilic residue (a conserved Asp) and acid/base, which may be variable in
terms of position and identity among different BGLs (Gabrisko and Janecek 2015;
Pozzo et al. 2010; Varghese et al. 1999). A fibronectin type III domain is found at
the C-termini of Thermotoga neapolitana and Aspergillus aculeatus BGLs, and the
domain’s function remains unclear.
Fig. 4.2 Structures of BGLs from different GH families. GH1: T. reesei BGL 2 (PDB: 3AHY),
GH3: T. reesei BGL 1 (PDB: 3ZYZ), GH5: Saccharomyces cerevisiae flavonoid BGL (PDB:
1H4P), GH9: Vibrio parahaemolyticus BGL-like protein (PDB: 3H7L), GH30: Homo sapiens acid
BGL (PDB: 1Y7 V)
4.5 Catalytic Mechanism
All BGLs mentioned so far are exoglycoside hydrolases working on the nonre-
ducing end of a cellodextrin or glucoside substrate. Glucosidases initiate
b-glucosidic bond cleavage with a nucleophilic attack on the anomeric carbon of
the Glc. This is carried out by either the side chain carboxyl of the conserved
catalytic Glu/Asp (Keresztessy et al. 1994), or a water molecule activated by a
conserved Asp. The oxygen in the b-glycosidic bond is activated by the conserved
proximal catalytic acid while the anomeric carbon and the ring oxygen form an
oxocarbenium-like ionic transition state (Fig. 4.3). When the water serves as the
nucleophile (as in the case of GH9), the transition moves forward as the aglycone
moiety leaves the anomeric carbon in concert with the hydroxyl (from the activated
water) displacement on the same carbon to release the Glc (Qi et al. 2008). This
leads to the inversion of chirality on the anomeric carbon and thus is referred as the
inverting mechanism. With the endogenous nucleophile, the first transition state
leads to the leaving of the aglycone moiety as well; however, the b-glycosidic bond
on the anomeric carbon is now displaced with the same carboxyl that initiates the
nucleophilic attack. This a-linked covalent enzyme-glycone intermediate then
undergoes another round of nucleophilic attack on the anomeric carbon. The cat-
alytic acid in the first step gets deprotonated after activating aglycone leaving group
and turns into a catalytic base. It then activates a water to attack the anomeric
carbon to release the Glc from the enzyme-glycone a-linkage. This retaining
mechanism allows the product keep the anomeric chirality. In myrosinases, the
conserved catalytic Glu in the second step is replaced by a Gln, and therefore an
ascorbic acid is required to fulfill the role of the catalytic base.
Unequivocal evidences of the covalent intermediates have been obtained through
incubation of Agrobacterium sp. GH1 BGL with 2,4-dinitrophenyl-2-deoxy-2-
fluoroglucoside (2F-DNPG) (Withers et al. 1987; Withers and Street 1988). The
electronegative property of fluoride destabilizes the transition state upon binding in
the active site, 2,4-dinitrophenyl aglycone moiety is a highly reactive leaving group;
both favor the formation of a lasting and stable enzyme-fluoroglycone intermediate.
The presence of fluoride was firstly supported by 19F-NMR (Withers and Street 1988)
and later observed in crystal structures (Hrmova et al. 2001; Chuenchor et al.
2008) and inactivated enzyme pepsin digest (Withers et al. 1990). In a similar
approach, [3H] conduritol B epoxide (CBE) forms a trapped, radioactively labeled
glycosyl-enzyme intermediate in Aspergillus wentii BGL A3. Radioactivity detected
in trypsin-cleaved fragments establishes the catalytic role of the nucleophilic Asp,
albeit CBE possesses less specificity than 2F-DNPG and labels nearby residues
(Bause and Legler 1980). Nevertheless, the majority of the catalytic residues have
been identified through homology, mutagenesis, chemical modification, and crystal
structure analyses. For instance, the catalytic acid/base residue and nucleophile
residue have been inactivated by mutagenesis, respectively, in the investigation of
retaining mechanism. Replacement of the nucleophile with a small non-nucleophilic
residue, e.g., Ala and Gly, inactivates the enzyme in attacking the anomeric carbon
(a) Inverting Mechanism
O
O O
H O
OH OH OH
OH O
RO
HO O O O
HO HO
HO OR HO HO
R OH
OH OH OH
O H O H OH
H H
O O HO
O O O
ES E+P
TS
(b) Retaining Mechanism
O O
OH
OH H O
OH
HO O
HO OR RO
HO O
OH HO
O OH O
R OH
O O
ES O
OH
O
TS
HO O H
HO O
OH H
O
O O O
OH
OH H O
OH
HO O Intermediate + P
HO OH O H
HO O
OH HO
O OH O
O O
E+P TS
Fig. 4.3 The inverting and retaining mechanisms of BGLs. The inverting mechanism in
Scheme A is found in the GH9 BGL where water-anomeric carbon bond is formed in a single step
of displacement reaction. However, in the retaining mechanism (Scheme B), water does not attack
the anomeric carbon in the first displacement step which is done in a similar way by the catalytic
nucleophile. This leads to a covalently bonded glycosyl intermediate with inverted anomeric
carbon conformation. The following hydrolysis step ends up with an invertion of anomeric carbon
conformation for the second time and a retaining glycoside product as a result. ES enzyme–
substrate complex, TS 6¼ transition state, E + P (resting) enzyme and product
(Trimbur et al. 1992). However, it can be rescued by exogenous small nucleophiles,

e.g., azide and fluoride, similar to the effect of the activated water in GH9, to form an
a-D-glucoside azide, a chirality-inverted product (Wang et al. 1994; Mackenzie et al.
1998). Alternatively, using a-fluoroglucoside as an intermediate analog could cir-
cumvent the nucleophilic attack and allow the enzyme to release a regular Glc product
after hydrolysis (Ly and Withers 1999). When the catalytic acid/base is replaced by
Gly, water cannot be activated for the hydrolysis while small nucleophiles like azide
again are able to fulfill this step and yield a b-D-glucoside azide (Wang et al. 1995).
4.6 Substrate–Enzyme Interaction
Compared with the thorough characterization of the catalytic residues and their
mechanism in b-glycosidic bond cleavage, less is known about how the interaction
between BGLs and their substrates gives rise to the tremendous substrate diversity
and how the interaction determines BGLs’ substrate specificity. Crystal structures
have revealed that the binding pockets are cavities composed of several subsites. The
subsite that binds the Glc (glycone) unit is assigned subsite −1, whereas the aglycone
moiety binds to the subsites +1, +2, +3 and so on. The substrate specificity of BGL is
a combination of both Glc and aglycone specificities (Marana 2006).
Besides glucosides and cellodextrins, BGLs may act on other glycosides or
oligosacchardies. Such non-BGL activities are related to the coexistence of other
glycosidases in the GH families that BGLs belong to, a likely divergent evolution
effect. The same BGL may display different activities toward substrates with dif-
ferent glycosyls or glycones. For instance, S. frugiperda BGL hydrolyzes fucosides
40 times more quickly than galactosides (Marana et al. 2001). Aspergillus oryzae
GH3 BGL binds Glc well, but mannose (2-epimer of Glc) much more weakly, and
no allose or galactose (3- and 4-epimer) (Langston et al. 2006). GH1 BGLs might
act on b-D-mannosides, b-D-galactosides, b-D-fucosides and b-D-glucuronides,
while GH3 BGLs might act on b-D-xylosides and a-L-arabinosides. Hydrogen
bonding plays a key role in the BGL-substrate interaction (Fig. 4.4). In a set of
studies, the hydrogen bonds involving the glycone hydroxyls and subsite −1 are
studied in three different GH1 BGLs: human LPH (Fernandez et al. 1995),
Agrobacterium sp. Agbglu (Namchuck and Withers 1995) and S. frugiperda Sfbglu
(Marana et al. 2002). For LPH and Agbglu, replacement of the glycone hydroxyls
by hydrogen and/or fluorine is evaluated using enzyme kinetics. In the case of
Sfbglu, a combination of site-directed mutagenesis and enzyme kinetics is deployed
to determine the energy of isolated interaction between subsite −1 and glycone OH
in the glycosylation transition state. The analysis leads to the conclusion that the
specificity of glycone is controlled by a hydrogen bond network involving at least
five amino acid residues and four substrate hydroxyls. A Glu bridging the glycone
hydroxyls 4 and 6 are key in differentiating fucosides, glucosides, and galactosides.
In addition, interactions with the hydroxyl 2 are essential to the BGL activity. In
another set of studies, one GH3 cellodextrinase and three b-xylosidases from rumen
bacterium Prevotella bryantii B14 are examined for their gylcone specificity by
comparing kinetic parameters on different substrates (Dodd et al. 2010). Mutations
of each conserved residues in subsite −1 severely decrease the catalytic efficiency
without changing the overall structure of the enzyme variants. Similar to GH1
enzymes, replacing the conserved Glu115 (Asp120 in barley glucan b-glucosidase)
leads to 1.1-fold increase in kcat/Km for para-nitrophenyl-b-D-glucopyranoside
(pNP-Glc) and 14-fold decrease for pNP-b-D-xylopyranoside. Glu115 is therefore
believed to contribute to the discrimination between b-xylosides and b-glucosides.
In BGL, the conserved Asp forms hydrogen bond bridging the hydroxyls 4 and 6 of
the Glc unit, while in b-xylosidase, the conserved Glu forms hydrogen bond with
the hydroxyl 4 (Fig. 4.4b).
Fig. 4.4 Elements which are important in glycone (Dodd et al. 2010) and aglycone binding
subsites (Czjzek et al. 2000; Verdoucq et al. 2004). a GH3 active site residues for the b-glucan
exohydrolase (ExoI) from barley bound to Glc are shown (PDB: 1EX1). b Models for GH3
enzyme’s xylosides and glucosides recognition (Dodd et al. 2010). Asp120 from barley ExoI
forms hydrogen bond with the hydroxyls 4 and 6 of Glc. Glu115 from P. bryantii B14 Xyl3B
forms hydrogen bond with the hydroxyl 4 of xylose and may discriminate between Glc and xylose
on the basis of steric interactions. c, d Active site configurations from aglycone side of maize
BGL1 (ZMGlu1, c) and sorghum dhurrinase 1 (Dhr1, d), which are shown for the structures of the
ZMGlu1 Glu189Asp mutant in complex with DIMBOA-Glc (PDB: 1E56) and Dhr1 Glu191Asp
mutant in complex with dhurrin (PDB: 1V03). Phe261 residue narrows the active site in Dhr1 and
is also shown in front of the catalytic nucleophile Glu404
The BGL’s interaction with the aglycone part of the substrate may be more
diverse or complex, as shown by various structure, mutagenesis and chimera
studies. In contrast to the highly conserved amino acid residues at the subsite −1, a
different though overlapping set of residues is involved in interacting with the
aglycone part of the substrate in BGL. An archetypical case is found in the maize
ZmGlu1, a GH1 enzyme, which is active on a wide range of aglycones including
DIMBOA. The structure of one of its inactive variant in complex with
DIMBOA-Glc, DIMBOA and the inhibitor dhurrin shows that the aglycone is
sandwiched by Phe198, Phe205 and Phe466 on one side and Trp378 on the other
(Czjzek et al. 2000). In addition, DIMBOA has a hydrophobic contact with Ala467.
All these residues, except Trp378, are variable among BGLs. For instance, sorghum
dhurrinase SbDhr1 conserves this Trp on the same side of the algycone subsite and
use Val196, Leu203 and Ser462 on the other side in replacement of the three Phe
residues for a tighter and more polar interaction (Verdoucq et al. 2004). The
mutagenesis of the aglycone binding residues in the Zm60.8 isoform of the ZmGlu1
further confirms the importance of these residues (Zouhar et al. 2001). It is thus
believed the conserved Trp is required to bind any aglycone and three or four
homologous positions on the other side would shape the pocket and therefore define
the aglycone specificity. Other factors may contribute to the aglycone binding as
well. It is discovered that the conserved Trp may also differentiate substrates like
dhurrin and strictosidine via its positional variation (Barleben et al. 2007; Verdoucq
et al. 2004; Cicek et al. 2000). Closely related wheat BGL (TaGlu) also hydrolyzes
DIMBOA-Glc, although a different set of residues are present in the binding pocket
(Sue et al. 2006). In the case of rice BGlu1, two of the three corresponding Phe
positions are replaced by Leu and Asn, which makes either no or indirect inter-
action with oligosaccharide substrates (Chuenchor et al. 2008). Instead, Asn245
plays a key role in binding to the third Glc moiety in the substrate, while the
corresponding residues in SbDhr1 and rice Os3BGlu6 appear to block the entrance
to their active sites, with no binding of a trimeric substrate (Opassiri et al. 2003;
Verdoucq et al. 2004). In general, the GH3 BGLs have fewer restraints on the
aglycone part of the substrate than the GH1 BGLs. For instance, A. oryzae
GH3 BGL has similar activities toward cellobiose, sophorose, laminaribiose, and
b-gentiobiose, in which the activated Glc unit (at the subsite −1) is linked to the
‘leaving’ Glc unit (corresponding to the aglycones of 4-glucosides) via 1,4-, 1,2-,
1,3-, and 1,6-glucosidic bond, respectively (Langston et al. 2006).
The GH1 BGLs have narrow and deep wells-like active sites, whose topology
(including space restricted subsites +1 or higher), electrostatic and hydrophobic
interaction properties impact the BGLs’ substrate specificity and product
(Glc) inhibition tolerance (de Giuseppe et al. 2014; Tribolo et al. 2007). The GH3
BGLs have crater-shaped active sites, whose depth, shape, and local amino acids
determine the BGLs’ substrate specificity. The topological difference is believed to
contribute to the difference in substrate reactivity and product inhibition between
the GH1 and GH3 BGLs (de Giuseppe et al. 2014).
4.7 Enzymatic Properties
4.7.1 Hydrolase Activity
When the substrate concentration is relatively low, BGLs’ main activity is hydro-
lytic, cleaving cellodextrins or oligosaccharides into smaller carbohydrate mole-
cules such as Glc, or glucosides into carbohydrate and aglycone subunits. Various
substrates may be used to assay BGLs’ properties. Chromogenic and fluorogenic
substrates, exemplified by pNP-Glc and 4-methylumbelliferyl-b-D-Glc, are quite
reactive and convenient for spectrophotometric detections, which are suitable for
microplate-based high-throughput or robotized studies (Perry et al. 2007). Because
the chromo- or fluorogenic aglycones are often unnatural, these substrates might not
yield data applicable for quantitative or comprehensive understanding of BGLs’
active sites, especially the subsites +1, +2, etc. Cellodextrins, other oligosaccha-
rides, and glucosides (such as that in Fig. 4.1), are able to reveal BGLs properties
under physiological or real applications conditions. BGLs’ action on these sub-
strates may be monitored with a range of detections including HPLC, mass spec-
trometry, coupled secondary chemical or enzyme reactions for the BGL reaction
products (such as a glucose oxidase or hexokinase assay on Glc), activity-overlay
electrophoresis (Saqib and Whitney 2006), or thermochemistry (Jeoh et al. 2005).
In general, BGLs’ hydrolase activity follows the Michaelis–Menten kinetics.
With cellobiose as the substrate, kcat, Km, and kcat/Km in the order of 101–102 s−1,
100–101 mM, and 101–102 mM−1 s−1 are often observed, while with pNP-Glc as
the substrate, kcat, Km, and kcat/Km in the order of 102 s−1, 100 mM, and
102 mM−1 s−1 are often observed (Ferrara et al. 2014; Uchiyama et al. 2013;
Frutuoso and Marana 2013; Sørensen et al. 2013; Teugjas and Väljamäe; 2013;
Singhania et al. 2013; Kuntothom et al. 2009; Mendonca and Marana 2008;
Eyzaguirre et al. 2005). Significant variations (tenfold or higher) may exist for these
parameters, depending on the BGL active sites (Tsukada et al. 2008) and assay
conditions (buffer, pH, temperature, enzyme and substrate concentrations), and no
significant statistical differences are observed between the GH1 and GH3 BGLs
(p = 0.19, 0.26, 0.59, 0.07 for the kcat (cellobiose), Km (cellobiose), kcat (pNP-Glc),
Km (pNP-Glc), from two-tail t Tests) (Fig. 4.5).
BGLs’ hydrolase activity has an optimal pH range from weakly acidic to neutral,
mainly determined by the actions of the catalytic Asp and/or Glu as the nucleophile
and general acid/base, but also with contributions from other components of the
active site (Tsukada et al. 2008). Most of the BGLs (from the mesophilic organisms)
have optimal temperatures at 50–70 °C, but the BGLs from extreme thermophiles
can have optimal temperatures close to or above 100 °C (Park et al. 2005).
Many molecules may inhibit or inactivate BGLs. Nonspecific, general
protein-denaturing inhibitors include ionic surfactants, heavy metal ions such as Hg+
and Ag+, and high concentration salts (including NaCl) which exert the typical
Hofmeister (salt-out) effect (on the enzymes’ apparent pKa, not the second-order rate
constant) (Bowers et al. 2007). Redox-active metal ions such as Cu2+ and Fe3+ may
1.E+04 1.E+04
kcat, s-1, on cellobiose Km, mM, on cellobiose
1.E+03 1.E+03
1.E+02 1.E+02
1.E+01 1.E+01
1.E+00 1.E+00
1.E-01 1.E-01
1.E-02 1.E-02
GH1 (29) GH3 (57) All (101) GH1 (29) GH3 (59) All (105)
1.E+04 1.E+02
Km, mM, on pNP-Glc
1.E+03
1.E+01
1.E+02
1.E+00
1.E+01
1.E-01
1.E+00
1.E-02
1.E-01
kcat, s-1, on pNP-Glc
1.E-02 1.E-03
GH1 (71) GH3 (61) All (163) GH1 (72) GH3 (70) All (175)
Fig. 4.5 Box-and-whisker plots of the kcat and Km of BGLs, including those from the GH1 and
GH3 family, for the hydrolysis of cellobiose or pNP-Glc. In the parentheses are numbers of BGLs
analyzed. Data are from Brenda enzyme database (www.brenda-enzymes.org) and other literature
sources
inhibit BGL by oxidation (Tejirian and Xu 2010; Jeng et al. 2011; Sharmila et al.
1998). Tris, a commonly used ‘biological’ buffer, may also inhibit BGL by binding
to the active site (Jeng et al. 2011). BGLs may be deactivated by various protein
adsorbents that include soil minerals (Lammirato et al. 2010), oligo/polyphenols
(Tejirian and Xu 2011) and lignin, whose effect can become significant in the
enzymatic biomass hydrolysis (Haven and Jørgensen 2013; Xu et al. 2007).
Specific BGL inhibitors include its substrate, products, and the transition-state
mimics. Because of BGL active sites’ topological features, the products (Glc,
aglycone, or shortened oligosaccharide), which may be regarded as equivalent to
the subunits of a substrate, can bind to certain subsites and impede the enzymatic
reaction. Structural analogs of the products or substrates, or even competing sub-
strates with low-reactivity (e.g., fluoroglucosides), can also inhibit BGLs. The
transition-state mimics can lock up the active site with their structural features
highly similar to that of the activated substrate, and alter the enthalpy–entropy for
the binding, hence exerting competitive inhibitions more potent than the other
1.E+04 1.E+02
Ki(Glc), mM
1.E+01
1.E+03
1.E+00
1.E+02 1.E-01
1.E-02
1.E+01 1.E-03
1.E-04
1.E+00
Ki(GlcL), mM
1.E-05
1.E-01 1.E-06
GH1 (15) GH3 (44) All (76) GH1 (12) GH3 (25) All (44)
Fig. 4.6 Box-and-whisker plots of the Ki of Glc and GlcL on the hydrolysis of pNP-Glc by BGLs,
including those from the GH1 and GH3 family. In the parentheses numbers of BGLs analyzed.
Data are from Brenda enzyme database (www.brenda-enzymes.org) and other literature sources
inhibitors. The highly inhibitory transition-state mimics include D-glucono-1,5-

lactone (GlcL, the first derivative from Glc oxidation, a side reaction often
accompanying cellulose degradation), deoxynojirimycin, and other
heteroatom-substituted carbohydrate molecules (Gloster and Davies 2010; Gloster
et al. 2007). As inhibitor, Glc and GlcL often show Ki in the order of 101–102 and
10−1–100 mM, respectively, on the pNP-Glc hydrolysis by BGL (Uchiyama et al.
2013; Sørensen et al. 2013; Teugjas and Väljamäe; 2013; Mendonca and Marana
2008; Seidle et al. 2005, 2006; Seidle and Huber 2005; Eyzaguirre et al. 2005;
Marana et al. 2001). Significant variations (10- to 100-fold or higher) may exist for
the parameter, depending on the BGLs, substrates and assay conditions (buffer, pH,
temperature, enzyme, and substrate concentrations) (Fig. 4.6). Statistically, the
GH1 BGLs appear to have higher Ki for Glc (more tolerance to the Glc inhibition)
than that of the GH3 BGLs (p = 0.023), but comparable Ki for GlcL to that of the
GH3 BGLs (p = 0.76, from two-tail t Tests).
4.7.2 Glycosyltransferase/Transglycosylase Activity
When the substrate concentration is high, BGLs (except the GH9 ones) may show
significant glycosyltransferase or transglycosylase activity to synthesize oligosac-
charides or glucosidases, opposing the hydrolase activity (Uchiyama et al. 2013).
The (trans)glycosylation activity is allowed by the reversibility of the retaining
mechanism, when the water is replaced by a carbohydrate or another acceptor with
higher activity for the deacylation of the enzymatic intermediate (donor)
(Fig. 4.3b), as in the cases of other retaining glycosidases. The (trans)glycosylation
may yield sophorosyl, laminaribiosyl, or even cellobiosyl, products via 1,2-, 1,3-, or
1,4-glucosidic bond, or various glucosides (de Giuseppe et al. 2014).
In general, a BGL’s hydrolase activity is controlled by thermodynamic factors

and water activity, while its glycosyltransferase activity is controlled by kinetic
factors and the glycosyl acceptors, with rates independent on pH (Seidle and Huber
2005). The (trans)glycosylation may be detected directly by the related products
formation, or indirectly by the apparent substrate or product inhibition of the
hydrolysis, based on the decrease of the hydrolysis products formation (Frutuoso
and Marana 2013; Kawai et al. 2004). The (trans)glycosylation may contribute to a
BGL’s tolerance to or stimulation by Glc, or maintain a BGL’s turnover. Many
examples of Glc stimulation for the GH1 BGLs, fewer for the GH3 BGLs, have
been reported (de Giuseppe et al. 2014).
4.7.3 Non-BGL Activities
BGLs may have significant side activities on non-Glc substrates (Brenda enzyme
database www.brenda.org, Uchiyama et al. 2013; Sørensen et al. 2013; Singhania
et al. 2013; Kuntothom et al. 2009; Harnipcharnchai et al. 2009; Mendonca and
Marana 2008; Tribolo et al. 2007; Kim et al. 2006; Seidle et al. 2005; Park et al.
2005; Eyzaguirre et al. 2005; Opassiri et al. 2004; Berrin et al. 2002; Hrmova et al.
2002; Vallmitjana et al. 2001; Marana et al. 2001; Perry et al. 2007). With pNP-b-D-
fucopyranoside as substrate, kcat, Km, and kcat/Km of 101–102 s−1, 100–101 mM, and
102 mM−1 s−1 are often observed for BGL’s b-fucosidase activity. With pNP-b-D-
xylopyranoside as substrate, kcat, Km, and kcat/Km of 100–101 s−1, 100 mM, and 100–
103 mM−1 s−1 are often observed for BGL’s b-xylosidase activity. With pNP-a-L-
arabinofuranoside as substrate, kcat, Km, and kcat/Km of 100–101 s−1, 100–101 mM,
and 100–102 mM−1 s−1 are often observed for BGL’s a-arabinosidase activity. With
pNP-b-D-galactopyranoside as substrate, kcat, Km, and kcat/Km of 100–101 s−1, 100–
102 mM, and 10−1–101 mM−1 s−1 are often observed for BGL’s b-galactosidase
activity. Close range of BGL’s activities on these substrates with different gly-
cones indicate that the interaction between the active subsite −1 and the substrate’s
nonreducing glycone subunits 4- and 6-OH (which are changed in the galactosyl,
fucosyl, xylosyl, and arabinosyl subunits of the non-Glc substrates) does not dom-
inate BGL’s activity. In GH1, BGLs coexist with b-fucosidase, b-xylosidase, and
b-galactosidase, as well as b-mannosidase, b-glucuronidase. In GH3, BGLs coexist
with b-xylosidase and a-arabinofuranosidase. Therefore, the side activities might
arise from divergent evolution.
4.7.4 Engineering
Recombinant DNA technology has been applied to BGLs. In combination with 3D

structure and enzymatic reaction studies, site-directed mutagenesis enables eluci-
dation of the structural–function relationship in BGL, especially the aspects on how
the composition and topology of the active site affect the substrate specificity, pH or
temperature profile, or substrate/product inhibition (Jeng et al. 2012; Khan et al.
2011; Liu et al. 2011 Chuenchor et al. 2011; Tsukada et al. 2008; Mendonca and
Marana 2008; Chuenchor et al. 2008; Vallmitjana et al. 2001).
BGL variants with improved activity, stability, or specificity have been made by
either rational design or directed evolution (Lee et al. 2012; Khan et al. 2011; Hong
et al. 2006; Hancock et al. 2005). Engineering the active site (especially the subsites
that bind the aglycone of a substrate) has been pursued for the abatement of the
product (Glc) inhibition; however, the increase in the Glc tolerance is often
accompanied by a decrease in the hydrolase activity, due to the contribution made
by the BGL-Glc interaction to both the catalysis and inhibition. Domain swap
between mesophilic and thermophilic BGLs may lead to improved thermal or other
properties for BGLs originated from mesophiles, such as the chimera constructed
from Agrobacterium tumefaciens or Cellvibrio gilvus and Thermotoga maritime
(Deog and Kiyoshi 2011; Kim et al. 2006).
Most BGLs have no distinct functional domains other than the catalytic core
(although some GH3 BGLs have a C-terminal FnIII domain). The enzymes may be
engineered to possess domains with targeted functions, such as the carbohydrate-binding
module potentially useful for the enzymatic biomass hydrolysis (Gundllapalli et al.
2007).
The transglycosylase activity of BGLs may be improved by engineering
(Frutuoso and Marana 2013; Jeng et al. 2012; Lundemo et al. 2013; Shim et al.
2012; Choi et al. 2008; Feng et al. 2005). For instance, the Trp49 and Trp262 of
A. niger GH3 BGL are found regulating the preference to the transglycosylase and
hydrolase activity, and substitutions of the two Trp result in BGL variants with
abated or enhanced relative transglycosylase activity (Seidle et al. 2005, 2006). The
regioselectivity of a transglycosylating BGL may also be modulated by site-directed
mutagenesis (Choi et al. 2008).
4.8 Application
4.8.1 Synthesis of Oligosaccharides and Glycosides
BGLs have the potential in biosynthesis of functional, performance, or therapeutic

oligosaccharides, glycol-conjugates, and glycosides, based on their glycosyltrans-
ferase activity. To increase the efficiency or selectivity, it is desirable to engineer
BGLs so that their hydrolase activity can be minimized. Engineering on BGL may be
carried out by site-directed mutagenesis or directed evolution (Shim et al. 2012). The
kinetic and thermodynamic determinants for the transglycosylation and ‘reverse
hydrolysis’ (formation of the compounds that would serve as the substrates for the
hydrolase activity), respectively, can be explored by the engineering or changes on
the reaction conditions (Bhatia et al. 2002). For instance, alterations of an amino acid
at the subsite +2 of T. neapolitana GH1 BGL are able to enhance the enzyme’s
glycosyltransferase-to-hydrolase activity ratio, enabling the enzyme to catalyze

more efficiently the synthesis of alkyl glycosides as attractive surfactants (Lundemo
et al. 2013). The alkyl glycosides may also be synthesized by alcohol-tolerant or
stimulating BGL in solvents high in alcohol (Karnaouri et al. 2013).
4.8.2 Beverage, Brewing, Food, Feed, and Dietary
Combination of flavor precursors and b-glucosidases is widely used in beverage and

food production. BGLs may be added in different stage of production and brewing
to act on specific precursors to release the desired aglycones (e.g., terpenols, benzyl
alcohols, and phenylethyl alcohols) for flavor enhancement and stability.
A combination of a-rhamnosidase and BGL may be applied to debitter citrus fruits
by hydrolyzing bitter glucosides such as prunin and naringin, while BGL may be
applied to decolorize juices or wines by hydrolyzing various anthocyanins such as
malvidin, peonidin, and cyanidin (Eyzaguirre et al. 2005). The astringent diadzin,
genistin, or other isoflavonoid glucosides in soybean products may be hydrolyzed
by BGL to daidzein, genistein, or other isoflavones with more pleasant taste. BGLs
are also used in nutrition enrichment of feeds, foods and beverages to generate
vitamins, antioxidants and other dietary molecules from their glycoside precursors.
For instance, vitamin B6 is stored predominantly in its glycoside form and can be
released by rice BGLs (Opassiri et al. 2003). Feeds for the monogastric farm
animals like pigs and chickens are often treated with crude BGLs to improve
nutrition availability together with the native enzymes in the animal’s small
intestine. Aglycones from soybean isoflavone glycosides and phenylthiocarbamide
derived from cruciferous vegetable glycosides have antioxidant and anticarcino-
genic effect. They are also direct or derivative products from BGL and myrosinase
hydrolysis, respectively (Chuankhayan et al. 2007; Esen 2003).
4.8.3 Biomass Conversion
Biomass conversions lay the basis for the bioenergy, biorefinery, and other
biomass-sustained industries. The enzymatic hydrolysis of lignocellulose is a key
step of the processes, and its development has benefited greatly from the basic
biochemical studies on the natural lignocellulose degradation (Payne et al. 2015;
Xie et al. 2014; Sweeney and Xu 2012; van den Brink and de Vries 2011; Quinlan
et al. 2010; Xu 2004, 2010a, b; McFarland et al. 2007; Teter et al. 2006). The
industrial enzymatic biomass conversion can differ significantly from the natural
process in various aspects, especially the requirement for high efficiency: the much
shortened reaction time (days instead of years), enhanced turnover (to minimize the
cost of enzymes), and close to 100 % yield (to maximize the output), need for
enzymes to tolerate high levels of end products and insoluble substrates that are
often altered in composition or structure by pretreatments. Cellulolytic microbes

secrete consortia of enzymes that can work cooperatively and synergistically, but
they need adjustment in industrial applications, in terms of composition and
specificity. T. reesei is one of the best known cellulolytic microbes and industrial
hemicellulases and cellulases producers. Although the filamentous fungus can
secrete more than 20 lignocellulose-active enzymes, including at least one BGL
(Herpoël-Gimbert et al. 2008), its enzyme consortium cannot effectively hydrolyze
various industrial biomass feedstocks (such as dilute acid pretreated corn stover),
unless supplemented with exogenous BGLs (such as A. oryzae GH3 BGL), as
demonstrated by the development of the first generation of the industrial biomass
enzymes products (Rani et al. 2014; Sørensen et al. 2013; Teugjas and Väljamäe
2013; Tiwari et al. 2013; Singhania et al. 2013; Krisch et al. 2010; Eyzaguirre et al.
2005; Xu 2004; Bhatia et al. 2002). During an industrial biomass hydrolysis, the
CBHs and EGs of T. reesei hydrolyze cellulose and produce high levels of cel-
lobiose. The cellobiose cannot be degraded effectively by the native BGL present in
the fungus’ secretome in low abundance, and thus a severe product inhibition
builds on the CBHs and EGs, resulting in an underperformance of the overall
enzymatic hydrolysis. The inhibition can be mitigated by increasing the BGL
abundance.
Supplementing the BGL activity to an industrial hemicellulase and cellulase
consortium by co-expressing the enzyme in the same host (that produces the
hemicellulase and cellulase) is more economically viable than producing the enzyme
separately. Often, heterologous expression of a non-native BGL is selected to take
advantage of the enzyme’s superior properties (in comparison with the endogenous
BGL). The selection is initiated by multiple rounds of screening of individual BGLs,
based on activity or bioinformatics, for either wild-type or engineered variants. The
initial screening is followed by synergy study with other hemicellulase and cellulase
acting under industrial conditions and by heterologous expression trials. The pre-
ferred properties of superior BGLs include high specific activity, high thermal sta-
bility, suitable pH profile, low production inhibition, low lignin absorption, low
glycosyltransferase side activity, and high expression yield. A BGL’s stimulation by
Glc or xylose may further improve the enzyme’s action in hydrolyzing high con-
centrations of cellulosic materials (Souza et al. 2010). A BGL’s tolerance of, or
stimulation by, ethanol or other alcohol may further improve the enzyme’s action in
the biomass conversion processes where elevated levels of ethanol are present, such
as the simultaneous hydrolysis (saccharification) and fermentation (SHF, SSF) or
consolidated bioprocessing (CBP) (Karnaouri et al. 2013).
BGLs may be applied in individual, free forms as in the secretomes of T. reesei
and most of the aerobic cellulolytic fungi. The relative abundance (molar ratio) of
BGL in biomass-active enzyme consortia may be roughly adjusted by co-expression
or post-production mixing. BGLs engineered with altered properties, including that
gained through fused functional domains, such as the carbohydrate-binding-module
(Gundllapalli et al. 2007), may be used. The claimed benefits of applying BGL in
free forms include the freedom a BGL has to act close to other enzymes or away
from them, pursuing diffused cellobiose (or other oligosaccharides) or avoiding
common deactivators.
BGLs may also be applied in associated, part of supramolecular assembly forms
as in the cellulosomes of Clostridium thermocellum and most of the anaerobic
cellulolytic bacteria. The molar ratio of the BGL in biomass-active cellulosomes
may be finely adjusted by specific dockerin–cohesin pairing. Non-native or non-
cellulosomal BGLs may be incorporated into designer cellulosomes (with selected
enzyme composition and molar ratio) with fused dockerins (Kellermann and
Rentmeister 2014; Hyeon et al. 2013). Working in the same supramolecular
assembly allows BGL to cooperate with other enzymes in close vicinity.
BGLs may also be applied in cell surface display forms. A BGL can be dis-
played, alone or with other biomass-active enzymes, on the surface of a fermen-
tative but noncellulolytic yeast or bacterium. The displayed BGL and other
enzymes can hydrolyze biomass to Glc and other fermentable sugars, which are
then fermented by the yeast or bacterium in situ. In the so-called consolidated
bioprocessing process, enzyme production and biomass-to-ethanol conversion are
done in a single unit process step, by engineered microbes such as the thermophilic
Kluyveromyces marxianus displaying A. aculeatus BGL and T. reesei EG
(Kellermann and Rentmeister 2014; Yamada et al. 2013; van Zyl et al. 2007).
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Part II
Hemicellulases
Chapter 5
Role and Applications of Feruloyl
Esterases in Biomass Bioconversion
Constantinos Katsimpouras, Io Antonopoulou, Paul Christakopoulos

and Evangelos Topakas
Abstract Ferulic acid esterases (FAEs) act synergistically with xylanases to

hydrolyze the feruloylated decorations of the hemicellulosic fraction of cell wall
material and therefore play a major role in the degradation of plant biomass. In this
review, their role in plant biomass degradation, their production, classification, and
structural determination are discussed. In addition, the production, physicochemical
properties, and molecular biology of the different type of FAEs are presented,
giving emphasis in their potential applications utilizing their hydrolytic and syn-
thetic activity. A detailed map of the reaction systems used to date is demonstrated,
underpinning the potential of these enzymes as biosynthetic tools in the synthesis of
bioactive compounds for use in food and cosmeceutical industries.
5.1 Introduction
The combustion of fossil fuels constitutes a great percentage of our current energy
supply, and with depleting crude oil reserves and climate change due to the
greenhouse gas (GHG) emissions, it is imperative that we focus on more renewable
energy sources such as biofuels (Damásio et al. 2013). Biofuels from lignocellu-
losic biomass, also referred to as second-generation biofuels, are considered to be
the key due to their potential to mitigate GHG emissions compared with conven-
tional fuels, reduce oil dependency, and eradicate public concerns about a trade-off
between foods and fuels.
C. Katsimpouras E. Topakas (&)

Biotechnology Laboratory, School of Chemical Engineering,
National Technical University of Athens, 15780 Athens, Greece
e-mail: [email protected]; [email protected]
I. Antonopoulou P. Christakopoulos
Biochemical and Chemical Process Engineering, Division of Sustainable Process
Engineering, Department of Civil, Environmental and Natural Resources Engineering,
Luleå University of Technology, SE-97187 Lulea, Sweden

80 C. Katsimpouras et al.
Production of fuel ethanol from renewable lignocellulosic materials, such as agri-

cultural residues, forest residues, and energy crops has been extensively studied in the
last few decades. Lignocellulosic raw materials, being low-cost and abundant as
residual biomass from agricultural and agroindustrial waste, provide an attractive
feedstock for the production of ethanol and other value-added products under the
biorefinery concept without competing with food and feed industry (Larsen et al. 2012).
Through enzymatic hydrolysis processes, commonly known as saccharification, the
main components of lignocellulosic biomass can be converted to sugars and subse-
quently fermented to bioethanol. However, the plant cell wall, developed in the span of
million of years of co-evolution as the plants’ defense system against pathogens,
bottlenecks the aforementioned processes and several improvements should be made in
order to produce cellulosic ethanol at competitive costs (Malinovsky et al. 2014).
A possible strategy that is being suggested requires the usage of hemicellulose-
degrading enzymes in order to generate more efficient enzyme cocktails for the sac-
charification of lignocellulosic biomass and reduce catalysts costs. In addition, the
cooperative action of enzymes could also lead to less severe, and thus more sustainable
and environmentally friendly, pretreatment processes (Couturier et al. 2011).
A set of enzymes that is considered to be a biotechnological key in cell wall
hydrolysis and in the extraction of phenolic acids is ferulic acid esterases (FAEs) or
feruloyl esterases, as they are also known. FAEs (E.C. 3.1.1.73) represent a sub-
class of the carboxylic ester hydrolases, which liberate phenolic acids such as
ferulic acid (FA) or p-coumaric acid and their dimers from naturally occurring
hemicelluloses (Fig. 5.1) and pectins, where they mainly occur as esters with
L-arabinofuranose-containing polysaccharides, such as L-arabino-D-xylans and
Fig. 5.1 Action of FAEs for the hydrolysis of ester bonds in plant cell wall arabinoxylan, as
indicated by black arrows. However, most of the characterized FAEs are acting on soluble
feruloylated fractions created by the action of endo-β-1,4-xylanases and not directly on the sugar
polymer showing strong synergistic interaction
5 Role and Applications of Feruloyl Esterases … 81
L-arabinans (Topakas et al. 2007). Besides FAEs’ role in hydrolytic reactions, they
are also applicable for the synthesis of bioactive compounds with antioxidant and/or
antimicrobial activity, and other compounds of high industrial interest. The present
review is an attempt to elucidate the role and applications of FAEs in lignocellu-
losic biomass degradation and as biosynthetic tools for the modification of plant
derived antioxidants.
5.2 Plant Cell Wall Functionality
Plant cell walls, besides their role as structural support to the plant body, also
restrict enzyme access rendering this complex polysaccharide network recalcitrant
to biochemical degradation. Cellulose, hemicellulose, lignin, and pectin are the
main components of lignocellulosic biomass along with proteins and aromatic
compounds at proportions that differ among plant species (de Oliveira et al. 2014).
In plants, two major types of cell walls could be distinguished: the primary cell
walls, which are composed of cellulose, hemicelluloses, pectins, and proteins, and
the secondary cell walls, consisting of cellulose, hemicelluloses, and lignin that are
formed after the end of cell expansion (Endler and Persson 2011). Cellulose is a
linear natural biopolymer consisting of glucose units linked by β-1,4-glycosidic
bonds forming crystalline microfibrils via hydrogen bonding and van der Waals
interactions (Horn et al. 2012).
After cellulose, hemicelluloses are the second most abundant polysaccharides on
earth with xylans to be their main representative. Hemicelluloses are a group of
heterogeneous polysaccharides consisting of a β-1,4-linked backbone of pentoses,
hexoses, and sugar acids, which is usually decorated with side branches (Saha
2003). The exact structure and abundance of hemicelluloses diversify widely
among different plant species and cell types with xyloglucans occurring mostly in
the primary walls of dicots and conifers, while arabinoxylans dominate in com-
melinid monocots (Scheller and Ulvskov 2010). Through interactions with cellu-
lose, and sometimes lignin, hemicelluloses have the ability to strengthen the cell
walls (Endler and Persson 2011).
Pectins are a group of heterogeneous and branched polysaccharides that can be
found only in the primary cell wall and consist of a backbone of α-1,4-linked D-
galacturonic acid residues that can be methyl-esterified or decorated with acetyl
groups. Homogalacturonans, xylogalacturonan, and rhamnogalacturonans (I & II)
are the main groups in which pectins could be classified according to their basic
structure (Wong 2008). There is also increasing evidence that pectin interacts
covalently with hemicelluloses providing structural and functional complexity to
the plant cell wall (Caffal and Mohnen 2009).
Cell walls are also fortified by the deposition of the phenylpropanoid polymer
lignin, which is one of the main contributors in recalcitrance of cell walls to enzy-
matic saccharification (Chen and Dixon 2007). The term lignin is employed to
describe a group of aromatic and nonsoluble heteropolymers consisting mainly of
phenylpropanoid units derived from the oxidative polymerization of three hydrox-

ycinnamyl alcohols (coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol)
derivatives (Vanholme et al. 2010; Vogt 2010). The aforementioned hydroxycin-
namyl alcohols are incorporated into the lignin polymer in the form of guaiacyl (G),
syringyl (S), and p-hydroxyphenyl (H) units. In gymnosperms G-units are mainly
present with small amounts of H-units, while lignin in angiosperm dicots consists of
G- and S-units. In grasses and softwoods H-units amounts are higher (Vanholme
et al. 2010). In lignocellulosic biomass, lignin is cross-linked with cell wall polymers
by ether or ester bonds with FA being the predominant connector as the FA that is
etherified to lignin is also esterified to polysaccharides (Iiyama et al. 1990).
Hydroxycinnamic acids such as FA, sinapic acid, and p-coumaric acid are com-
ponents of plant cell wall with the ability to esterify hemicelluloses due to a carboxylic
group at the end of their propenyl group. FA plays an important role in the plant cell
wall by cross-linking with polysaccharides by ester bonds and with lignin by ether
bonds regulating this way the cell wall extensibility and inhibiting pathogen invasion
(de Oliveira et al. 2014). Implication of FA and diferulates to function as nucleation
sites for the lignification process has also been described (Ralph et al. 1995).
FA is synthesized in the phenylpropanoid pathway from phenylalanine and in
nature exists mainly in E (trans) form since isomerization in Z (cis) form occurs
during extraction due to the light sensitivity of hydroxycinnamates (Faulds and
Williamson 1999). In monocotyledons such as grasses and cereals, the arabinofu-
ranoses are esterified by hydroxycinnamates with trans-FA, (E)-4-hydroxy-3-
methoxycinnamic acid to be the most abundant whereas dicots contain ferulated
pectic polysaccharides (Mueller-Harvey et al. 1986). FA is esterified at position O-5
to α-L-arabinofuranosyl side chains in arabinoxylans (Mueller-Harvey et al. 1986),
at position O-6 to β-D-galactopyranosyl residues in pectic rhamno-galacturonanans
(Colquhoun et al. 1994) and at position O-4 to a-D-xylopyranosyl residues in
xyloglucans (Ishii et al. 1990). In some dicots such as spinach (Fry 1983) and sugar
beet pulp (Colquhoun et al. 1994), feruloylation occurs at position O-2 to α-L-
arabinofuranosyl residues in arabinans and at position O-6 to β-D-galactopyranosyl
residues in pectic rhamno-galacturonanans. FA amount varies in the cell walls of
different plant materials ranging from 0.1 to 3.8 % (w/w, dry weight). In wheat
bran, FA (w/w, dry weight) account for 0.66 %, 3.1 % in maize bran, 1.24 % in
wheat straw, 0.9 % in rice endosperm cells, 0.14 % in barley grains, 0.32 % in
barley spent grain, 1.4 % in barley hulls, 2.2–3.8 % in oat hulls, and 0.87 % in
sugar beet pulp (Smith and Hartley 1983; Saulnier et al. 1995; Benoit et al. 2006;
Shibuya 1984; Nordkvisk et al. 1984; Bartolome et al. 1997a, b; Tenkanen et al.
1991; Garleb et al. 1988; Yu et al. 2002a, b; Kroon and Williamson 1996).
Covalent cross-linking between feruloylated polysaccharide chains or between
hemicellulose and lignin can be achieved through the formation of ferulate dimers.
Dimers of FA can be formed by radical coupling reactions or by photochemical
coupling reactions (Ralph et al. 1994; Ishii 1997). Photochemical [2+2]-cyclodi-
merization (Ford and Hartley 1990) produces cyclodimers, while peroxidase-
catalyzed coupling produces several forms of dehydrodimers such as 8-5-, 8-O-4-,
5-5-, 8-8-, and 4-O-5-coupled dehydrodimers (Bunzel et al. 2005) commonly known
as diferulates. In pectins isolated from sugar beet pulp, 8.8 % of the ferulates were
mostly 8-8- and 8-O-4-coupled dehydrodimers (Oosterveld et al. 1997).
Fry et al. (2000) suggested that trimers or even larger products contribute highly
to cross-linking between polysaccharides in culture maize cells. The first FA
dehydrotrimer was isolated from maize bran insoluble fiber and found to be a 5-5/8-
O-4-coupled dehydrotrimer (Bunzel et al. 2003). Since then, more dehydrotrimers
and dehydrotetramers have been identified and characterized (Rouaou et al. 2003;
Bunzel et al. 2005, 2006; Funk et al. 2005; Barron et al. 2007; Hemery et al. 2009).
5.3 Role of FAEs in Plant Biomass Degradation and Their

Synergistic Action
The complex and extended polysaccharide networks in plant cell walls, due to the
covalent cross-linking mediated by FA, raise hurdles in utilizing lignocellulosic
biomass for biofuel production, as it contributes to biomass recalcitrance to enzy-
matic degradation (Wong et al. 2013). In order to surmount these hurdles, along
with cellulases and hemicellulases, accessory enzymes such as FAEs should be
employed, enhancing this way the fermentable sugars yield from lignocellulosic
biomass (Faulds 2010). FAEs action depends highly on the type of xylanases that is
being used with, having an effect not only on the amount but also in the form of FA
released, with family 11 xylanases being more efficient in the hydrolysis of FA
whereas family 10 xylanases present a more synergistic effect on the release of
diferulic acid (Faulds et al. 2006). The synergism of a recombinant FAE isolated
from a rumen microbial metagenome (RuFae2), in association with GH10 and
GH11 endoxylanases was investigated by Wong et al. (2013). The results obtained
from the release of FA from several natural substrates such as corn fiber, corn bran,
wheat bran, wheat-insoluble arabinoxylan, and switchgrass indicated that the GH10
xylanase showed a greater synergistic effect than that of the GH11 xylanase (Wong
et al. 2013). The synergistic action between xylanases and FAEs on the release of
FA from feruloylated polysaccharides seems to render the biomass more vulnerable
to glycoside hydrolase enzymes (Yu et al. 2003).
Tabka et al. (2006) reported a synergistic effect of a recombinant FAE from
Aspergillus niger when combined with cellulases and xylanases from Trichoderma
reesei in the hydrolysis of wheat straw. Significant increase in the release of reducing
sugars (34.8 % increase in total reducing sugars) from oat hulls was also a result of
synergistic interaction between A. niger FAE and T. reesei xylanase (Yu et al. 2003).
The enzymatic saccharification of pretreated corn stover by a cellobiohydrolase from
T. reesei was significantly enhanced by the addition of small quantities of an
endoxylanase, a FAE, and an acetyl xylan esterase. FAE addition to cellobiohydrolase
achieved a 37 % synergistic improvement in glucan conversion to cellobiose, while a
substantial increase of 85 % was shown when all enzymes were added (Selig et al.
2008). Supplementation of a commercial mixture of cellulase and β-glucosidase with
pectinase and FAE preparations resulted in higher arabinose and xylose yields from
pretreated dried distiller’s grains with solubles, a co-product of corn ethanol pro-
duction (Dien et al. 2008). Kim et al. (2008) reported a 15 % increase in glucose yields
and 2–4 times enhancement for xylose yields in the enzymatic hydrolysis of pretreated
distiller’s grain when cellulases were supplemented with xylanase and FAE activities.
Two recombinant FAEs from Penicillium funiculosum expressed in Aspergillus
awamori (FaeA and FaeB) were able to enhance the cellulolytic activity of purified T.
reesei on pretreated corn stover by releasing 19 and 7 % more cellobiose, respectively
(Knoshaug et al. 2008). The synergistic action among cellulases, xylanases,
β-glucosidases, and FAEs produced by T. reesei and A. awamori was investigated for
the hydrolysis of steam-pretreated sugarcane bagasse, as reported by Gottschalk et al.
(2010). Addition of FAE and xylanase activities, produced by Aspergillus oryzae, in a
commercial enzyme preparation resulted in increased bioconversion of sugarcane
bagasse by 36 % (Braga et al. 2014).
Enzymatic release of FA from agroindustrial waste materials with the use of
FAEs raises considerable interest due to its antioxidant and anti-inflammatory
properties. FA is considered to alleviate oxidative stress in neurodegenerative
disorders such as Alzheimer’s disease (Sultana et al. 2005). Furthermore, FA could
be employed as a source for the bioconversion into value-added products such as
vanillin, which is one of the most used flavoring agents in the food, pharmaceutical,
and cosmetic industries (Priefert et al. 2001).
Two FAEs from Streptomyces sp. were tested on their ability to release FA from
corn bran, wheat bran, and defatted rice bran in combination with xylanase and α-L-
arabinofuranosidase activities from Streptomyces with the results suggesting that the
amount of FA released from biomass increased due to a synergistic effect between
these enzymes (Uraji et al. 2014). A recombinant FAE from Aspergillus usamii in
combination with a recombinant GH 11 xylanase from the same microorganism
exhibited a 27 % increase in FA release from destarched wheat bran compared to that
of the FAE alone (Gong et al. 2013). The amount of FA released from steam exploded
corn stalk, when a FAE from Aspergillus flavus (AfFaeA) combined with a GH 10
xylanase from Geobacillus stearothermophilus, was 13-fold higher than that released
by AfFaeA alone (Zhang et al. 2013). Wu et al. (2012) reported the characterization of
two FAEs of myxobacterial origin (Sorangium cellulosum) capable of releasing FA
from grass biomass, such as triticale bran, and from wheat bran yielding up to 85 % of
total alkali-extractable FA in presence of a Trichoderma viride xylanase.
A recombinant FAE (MtFae1a) from the thermophilic fungus Myceliophthora ther-
mophila (synomym Sporotrichum thermophile) was able to release up to 41 % of the
total alkali-extractable FA from wheat bran only with the presence of a xylanase,
indicating this way the synergistic interaction between MtFae1a and M3 Trichoderma
longibrachiatum xylanase (Topakas et al. 2012). Synergistic interaction between a
FAE (Tx-Est1) and a GH11 xylanase (Tx-Xyl11) both from Thermobacillus
xylanilyticus was also reported by Rakotoarivonina et al. (2011), where esterase’s
efficiency in releasing FA and diferulic acid from destarched wheat bran was seven-
to eightfold higher than that of Tx-Est1 alone. A maximum of 67 % of total FA was
released from destarched wheat bran by the combined action of a type C FAE from
Fusarium oxysporum and a M3 T. longibrachiatum xylanase (Moukouli et al. 2008).
5.4 FAE Classification and Structural Determination
FAEs appear to be a very diverse set of enzymes, with little unifying sequence and
physical characteristics to link them. In order to categorize and classify these car-
bohydrate esterases into different types sharing the same substrate specificity and/or
the same structure, several attempts have been made starting in 2004. Under this
scope, the use of multiple alignments of sequences or domains demonstrating FAE
activity, as well as related sequences, helped to construct a neighborhood-joining
phylogenic tree (Crepin et al. 2004a). The outcome of this genetic comparison
supported substrate specificity data and allowed FAEs to be subclassified into 4
types: A, B, C, and D, a classification that is still used in literature by many
researchers. Thus, there does appear to be an evolutionary relationship between
FAEs, acetyl xylan esterases, tannases and certain lipases. The substrate specificity
was based on their specificity toward mono- and diferulates, on substitutions on the
phenolic ring, and on their amino acid sequence identity (Crepin et al. 2004a). An
unofficial nomenclature for describing FAEs was followed based on this classifi-
cation, following both the source of the enzyme and the type of the esterases (e.g.,
the type-A FAE produced by F. oxysporum is termed FoFaeA).
It is extremely common for esterases to act on a broad range of substrates.
Type A FAEs exhibit relatively high sequence identity with fungal lipases, however
lipase activity is not detected. For example, the Type A FAE from A. niger
(AnFaeA; Faulds et al. 1997) shows homology with lipases from Thermomyces
lanuginosus TLL (30 % sequence identity) and Rhizomucor miehei (37 % sequence
identity). This subclass show preference for the phenolic moiety of the substrate
containing methoxy substitutions, especially at meta- position(s), as occurs in fer-
ulic and sinapic acids, while type B FAEs shows complementary activity, showing
preference to substrates containing one or two hydroxyl substitutions as found in p-
coumaric or caffeic acid. Type B FAEs, such as M. thermophila MtFae1a (Topakas
et al. 2012), P. funiculosum PfFaeB (Kroon et al. 2000) and Neurospora crassa
Fae-1 (Crepin et al. 2003a), show high sequence identity with acetyl xylan esterases
and are the only type of FAEs that are members of carbohydrate esterase
(CE) family 1 of the Carbohydrate Active enZymes (CAZy) database (http://www.
cazy.org/CE1.html; Lombard et al. 2013). In contrast to type B esterases, type A
FAEs appear to prefer hydrophobic substrates with bulky substituents on the
benzene ring (Kroon et al. 1997; Topakas et al. 2005a). One of the famous Τype of
FAEs, due to their synthetic potential, is type C that exhibit high sequence identity
with fungal tannases. Together with type D FAEs, these esterases exhibit broad
specificity against synthetic hydroxycinnamic acids (ferulic, p-coumaric, caffeic and
sinapic acid) showing difference only in the ability to release 5-5′ diFA (Crepin
et al. 2004a, b; Vafiadi et al. 2006b). Lastly, Type D FAEs have been found only in
bacteria, such as XYLD from Pseudomonas fluorescens (reclassified as Cellvibrio
japonicus) (Ferreira et al. 1993) that show sequence similarity with xylanases
(Crepin et al. 2004a). These four different types of FAEs have different evolutionary
origin, as found by phylogenetic analysis resulting in five major clades I to V
(Olivares-Hernández et al. 2010). Clade I contains Type A FAEs, such as the

characterized FAEs from A. niger, A. awamori and A. tubingensis that belong to
Eurotiomycetes, except one FAE sequence from Laccaria bicolor belonging to
Agaricomycetes class of basidiomycetes. Clade II contains Type B FAE sequences
from both Sordariomycetes and Eurotiomycetes; however, the clade does not reflect
any phylogenetic structure. Clade IV contains characterized Type B and C FAEs
including also putative FAE sequences from four taxonomic classes, with its basis
consisted of esterases from Magnaporthe, Pyrenophora, Phaeosphaeria, and
Fusarium.
Recently, a modification to this classification has been proposed by Benoit et al.
(2008), while a descriptor-based computational analysis with pharmacophore
modeling provided a different approach for the classification of FAEs (Udatha et al.
2011). According to them, FAEs could be classified into seven subfamilies
depending on the phylogenetic analysis of fungal genomes, whereas the
descriptor-based classification and structural analysis of experimentally verified and
putative FAEs proposed 12 families. FAEs are also members to ESTHER database,
a comprehensive and continuously updated database devoted to α/β hydrolase fold
proteins that hierarchically classifies more than 30,000 proteins by blocks and Rank
families (Lenfant et al. 2013). In this database, FAEs are members of five Rank 1
families, four in Block X (Abhydrolase_6, Antigen85c, Esterase_phb, Tannase) and
one in Block L (Lipase_3).
Relatively few studies have been performed to elucidate the functional rela-
tionships between sequence and diverse FAEs. To date, the crystal structures of
FAEs include a Type A or Lipase_3 (ESTHER) AnFaeA from A. niger (Hermoso
et al. 2004; related PDB entries: 1UWC, 1USW, 1UZA, 2BJH, 2IX9, 2HL6, 2IX9,
2HL6), a FAE (Est1E) from Butyrivibrio proteoclasticus (PDB_ID: 2WTM, 2WTN;
Goldstone et al. 2010) that belongs to Abhydrolase_6 family (ESTHER) and FAE
domains, XynY and XynZ (Prates et al. 2001; Schubot et al. 2001 with related PDB
entries: 1JJF, 1GKK, 1GKL, 1JT2, 1WB4, 1WB5, 1WB6) of the cellulosomal
enzymes included in the cellulosome complex from Clostridium thermocellum that
belongs to Antigen85c family (ESTHER). Recently, a cinnamoyl esterase (LJ0536)
from Lactobacillus johnsonii was characterized and its 3D structure was determined
that resembles the structure of the Est1E from B. proteoclasticus (PDB_IDs: 3PF8,
3PF9, 3PFB, 3PFC, 3QM1, 3S2Z; Lai et al. 2011). Unfortunately, none of the
proposed FAE classifications include and categorize LJ0536 FAE or any of its
homologs. In 2014, the first crystal structure of a type B FAE was solved, belonging
to the tannase family of the ESTHER database from A. oryzae (AoFaeB) with the
serine and histidine residues of the catalytic triad, a typical motif of serine hydro-
lases, to be directly connected by a disulfide bond of the neighboring cysteine
residues (Suzuki et al. 2014; PDB_ID: 3WMT). As reported also in the first attempt
of FAE classification by Crepin et al. (2004a), Type C FAEs show homology with
members of tannase family showing broad substrate specificity against four methyl
esters of hydroxycinnamic acids. However, only one member of Type C FAE from
Talaromyces stipitatus (TsFaeC; Vafiadi et al. 2006b) has a substrate specificity
profile that corresponds to Type C FAEs, while the rest of them show a specificity
profile of Type B FAEs with weak or no activity against methyl sinapate. Such
discrepancy is well documented in literature with typical examples of Type C FAEs
with B type mode of action, such as AnFaeB (Kroon and Williamson 1996), AoFaeΒ
(Suzuki et al. 2014) and FoFaeC (Moukouli et al. 2008) from Aspergillus and
Fusarium species. Based on sequence homology, the structure determination of
AoFaeB esterase underpins the elucidation of the first structure of Type C FAEs.
These Type B and C FAEs are also members of the tannase family of ESTHER
database, where most of them do not exhibit tannase activity, however, Tan410
discovered from a soil metagenomic library exhibits both FAE and tannase activities
(Yao et al. 2013). The different Types of fungal and bacterial FAEs that have been
structural determined to date, is shown in Fig. 5.2.
The aforementioned FAEs have a common α/β-hydrolase fold that is
well-known in literature for the diverse activities of the enzymes that belong to this
structural superfamily, such as hydrolases, lipases, dehalogenases and peroxidases
(Holmquist 2000). In addition, these enzymes also share a conserved catalytic triad
(Ser–His–Asp) composed of a nucleophile (Ser, Cys or Asp), a conserved histidine
Fig. 5.2 The structures of fungal FAEs, a AnFaeA from A. niger (Hermoso et al. 2004), b AoFaeB
from A. oryzae (Suzuki et al. 2014) and bacterial FAEs c EstE1 from B. proteoclasticus (Goldstone
et al. 2010), d LJ0536 from L. johnsonii (Lai et al. 2011), domains, e XynY (Prates et al. 2001) and
f XynZ (Schubot et al. 2001) of C. thermocellum cellulosome complex. The catalytic α/β hydrolase
domain (green) and the lid domain (magenta) are shown. GlcNAc residues of N-glucans and the
catalytic triad are shown as cyan and yellow sticks, respectively. A calcium ion is represented as an
orange sphere. The 3D structures were prepared using PyMOL (http://pymol.org)
and an acidic residue. These residues are sequenced in the order of nucleophile,
acidic and histidine residues, with the nucleophile to be located between the fifth
β-strand and the following helix of the nucleophile elbow (Nardini and Dijkstra
1999). The tetrahedral oxyanion intermediate formed during catalysis is stabilized
by the oxyanion hole that is formed by the contribution of the nucleophilic elbow.
The catalytic triad of the Type A FAE AnFaeA and the active-site cavity is confined
by a lid and a loop that confers plasticity to the substrate binding site in analogy
with lipases (Fig. 5.2). What is surprising is the fact that FAE’s lid exhibits a high
ratio of polar residues keeping it in an open conformation that gives the typical
preference of catalyzing the hydrolysis of hydrophilic substrates compared
to lipases. In addition, the open conformation is further stabilized by a
N-glycosylation site (Hermoso et al. 2004). These subtle aminoacid and confor-
mational changes are a result of an evolutionary divergence within the FAE family,
as further discussed by Levasseur et al. (2006). A functional shift followed by a
duplication event was suggested, which was occurred within the ancestral lipase
genes. Such a “neofunctionalization” was a result of positive selection during the
functional shift, possibly due to the drastic environmental changes happened by the
colonization of land by terrestrial plants, giving the selective advantage to
Euascomycetes (Aspergilli), as hypothesized by the authors (Levasseur et al. 2006).
The Type A FAE from A. niger together with Est1E from B. proteoclasticus exhibit
a small lid domain (16 and 46 amino acid residues, respectively) compared with the
AoFaeB (159 amino acid residues), as shown by the first solved FAE structure of
tannase family (Suzuki et al. 2014). In addition, the lid domain of BpEst1E has a
unique fold forming a flexible β-sheet structure around a small hydrophobic core,
while on the other hand, FAE domains of C. thermocellum do not possess a lid
domain (Fig. 5.2). This newly discovered lid underpins the continuing diversity of
insertions that decorate the common α/β fold of hydrolases that point toward a
dynamic mechanism for binding and release of substrates (Goldstone et al. 2010).
5.5 Production and Physicochemical Characteristics
Since 1987, when FAE activity was detected in Streptomyces olivochromogenes

cultures grown on wheat bran for the first time (MacKenzie et al. 1987), and the first
purified FAE from the same microorganism was reported (Faulds and Williamson
1991), many FAEs have been produced and characterized from fungi and bacteria,
and are considered to be a common component of hemicellulose preparations. In
the last decade, over 50 FAEs have been produced through various fermentation
processes employing different lignocellulose derived carbon sources such as maize
bran, corn bran, and destarched wheat bran but with the most interest focused on
production through heterologous expression (Table 5.1). An extended table on the
microbial production of FAEs is provided by Topakas et al. (2007) that complement
the present investigation.
Table 5.1 Microbial production of FAEs both from WT and recombinant sources
Organism Carbon source/ Cultivation conditions Assay FAE activity References
inducer (mU/mL) (mU/mg)
A. mucronatus YE505 1 mM IPTG Standard protocol MFA/EFA – 9940/7750 Qi et al. (2011)
(E. coli BL21)
A. awamori G-2 1 % WB 30 °C, 72 h 1-Naphthyl acetate 394,000 – Kanauchi et al. (2008)
A. awamori (E. coli BL21) 1 mM IPTG Standard protocol EFA – 1850 Fazary et al. (2010)
A. awamori 2B.361 U2/1 3 % WB V = 300 mL, 30 °C, EFA 170 – Gottschalk et al. (2013)
200 rpm, 7 days
A. clavatus NRRL1 0.5 mM IPTG Standard protocol MFA/MpCA/MCA – – Damásio et al. (2013)
(E. coli BL21)
A. flavipes 1 % WB 28 °C, 6 days MFA 6820 10,570 Mathew et al. (2005)
1 % MB 28 °C, 5 days MFA 33,180 6980 Mathew et al. (2005)
A. flavus CBE332.1 Methanol V = 250 mL, 30 °C, CNPF 500 – Zhang et al. (2013)
(P. pastoris X-33) 2 days
5 Role and Applications of Feruloyl Esterases …
A. nidulans (S. cerevisiae) 2 % (w/v) V = 500 mL, 30 °C, 20 h, MFA – 14,900 Shin and Chen (2007)
Galactose 250 rpm
A. nidulans (P. pastoris) Methanol 30 °C, 3 days MFA – 21,700 Debeire et al. (2012),
Bauer et al. (2006)
A. niger NRRL3 1 % CB V = 100 mL, 30 °C, MFA/CB 13.9/0 – Shin and Chen 2006
5 days
A. niger CFR 1105 WB 30 °C, 96 h/V = 100 mL, 4NPF – 12,800/11700a Hegde and Muralikrishna
30 °C, 105 rpma (2009)
A. niger (S. cerevisae 2 % Glycerol V = 1 L, 30 °C 4NPF – 8200 Wong et al. (2011)
DY150)
A. oryzae RIB40 Koseki et al. (2009)
(P. pastoris GS115)
(continued)
89
90

AoFaeB Methanol Manufacturer’s protocol MCA/MpCA – 3200/30,600
AoFaeC Methanol Manufacturer’s protocol MCA/MpCA – 34,900/14,300
A. oryzae 0.5 % (v/v) Multicopy Pichia MFA – 58,400 Zeng et al. (2014)
(P. pastoris GS115) Methanol Expression kit manual
A. tubingensis 1 % DSWB, 35 °C, 3 days DSWB 2440 – Tai et al. (2014)
1 % Pectin
A. usamii E001 1 % (v/v) 30 °C, 3 days MFA – 4490 Gong et al. (2013)
(P. pastoris GS115) Methanol
B. proteoclasticus IPTG Standard protocol EFA – 37,000 Goldstone et al. (2010)
(E. coli BL21)
C. ruminicola H1 Li et al. (2011)
FaeI (E. coli BL21) 1 mM IPTG Standard protocol MFA – 3780
FaeII (E. coli 1 mM IPTG Standard protocol MFA – 38,760
Rosetta-gami2)
FaeIII (E. coli Rosetta) 1 mM IPTG Standard protocol MFA – 56,880
C. japonicus IPTG Standard protocol MFA – 28 McClendon et al. (2011)
(E. coli BL21)
Chaetomium sp. CQ31 4 % CC V = 250 mL, 40 °C, MFA 2100 – Yang et al. (2013)
200 rpm, 6 days
Cotton soil metagenomic 0.5 mM IPTG V = 50 mL, 37 °C, pNFA – – Yao et al. (2013)
library (E. coli BL21)
D. dadantii 3937 Hassan and
(E. coli BL21) Hugouvieux-Cotte-Pattat
(2011)
(continued)
C. Katsimpouras et al.
FaeD 2 mM IPTG Standard protocol pNPA –
FaeT 2 mM IPTG Standard protocol pNPA –
F. oxysporumA 0.5 % (v/v) V = 150 mL, 30 °C, DSWB – – Moukouli et al. (2008)
(P. pastoris X-33) Methanol 6 days
Fusarium proliferatum 1 % CB V = 100 mL, 30 °C, MFA 33.46 – Shin and Chen 2006
NRRL 26517 5 days
Fusarium verticillioides 1 % CB V = 100 mL, 30 °C, MFA/CB 19.6/0 – Shin and Chen 2006
NRRL 26518 5 days
Humicola grisea var. 2 % BSG/2 % V = 100 mL, 45 °C, MFA 470/330b – Mandalari et al. (2008)
thermoidea WB 10 days
JSC-3 1 % WB 28 °C, 9 days MFA 6320 10,090 Mathew et al. (2005)
1 % MB 28 °C, 8 days MFA 7170 36,710 Mathew et al. (2005)
L. johnsonii 1 mM IPTG Standard protocol Esters – – Kin et al. (2009)

(E. coli BL21)
L. plantarum WCFS1 4 mM IPTG Standard protocol Esteban-Torres et al.
(E. coli BL21) (2013)
M. thermophila 0.5 % (v/v) V = 150 mL, 30 °C, DSWB 930 – Topakas et al. (2012)
(P. pastoris X-33) Methanol 6 days
P. brasilianum IBT 20888 20 % BSG 30 °C, 196 h, 1542c – Panagiotou et al. (2006)
(2.5 g) Vextr = 10 mL/1 g dry
material
P. funiculosum Knoshaug et al. (2008)
(A.awamori)
(continued)
91
92

FaeA Glucoamylase V = 2.8 L, 32 °C, 4– MFA – –
6 days
FaeB Glucoamylase V = 2.8 L, 32 °C, 4– MFA – –
6 days
Piromyces equi 30 °C, 120 rpm, 2 days MpCA 660 20,000 Poidevin et al. (2009)
(T. reesei RutC30)
P. eryngii SNL medium, 24 °C, 13 days, 150 rpm MFA – 650 Nieter et al. (2014)
0.4 % Tween
80
P. sapidus 8266 DSMZ SNL medium, 5 days pNPB 243 – Linke et al. (2013)
0.4 % Tween
80
Rumen microbial IPTG Standard protocol WB – 10,300 Wong et al. (2013)
metagenome
(E. coli BL21)
Russula virescens MFA Wang et al. (2014)
S. cellulosum So ce56 Wu et al. (2012)
(E. coli Tuner cells)
ScFAE1 1 mM IPTG Standard protocol MFA – 12,200
ScFAE2 1 mM IPTG Standard protocol MFA – 19,300
Streptomyces ambofaciens 1 % (w/v) MpCA/MFA Kheder et al. (2009)
DSWB
S. cinnamoneus FAE activity was Uraji et al. (2014)
(E. coli BL21) expressed on a dry weight
basis
(continued)
R18 IPTG Standard protocol MFA – 23.07
R43 IPTG Standard protocol MFA – 19.8
Streptomyces C-248 1 % DSWB V = 1 L, 37 °C, 3 days DSWB 80 300 Faulds et al. (2006)
Streptomyces S10 1.5 % DSWB V = 20 mL, 30 °C, 96 h DSWB 2.0 15.45 Mukherjee et al. (2007)
T. stipitatus 2 % BSG/2 % V = 100 mL, 37 °C, MFA 140/90b – Mandalari et al. (2008)
WB 10 days
Thermoanaerobacter 0.1 mM IPTG Standard protocol MFA/MpCA – 456/426 Abokitse et al. (2010)
tengcongencis (E. coli)
T. xylanilyticus (E. coli) IPTG Standard protocol MFA – 25410 Rakotoarivonina et al.
(2011)
Literature is cited after 2005, for earlier data see Topakas et al. (2007) for a review
WB wheat bran, MB maize bran, DSWB destarched wheat bran, BSG brewer’s spent grain, CB corn bran, CC corn cobs, IPTG isopropyl β-D-
1-thiogalactopyranoside, MFA methyl ferulate, MpCA methyl p-coumarate, EFA ethyl ferulate, MCA methyl caffeate, 4NPF 4-nitrophenyl ferulate, pNFA
p-nitrophenyl ferulate, pNPA p-nitrophenyl acetate, pNPB p-nitrophenyl butyrate, CNPF 2-chloro-4-nitrophenyl ferulate, SNL standard nutrition liquid
a
ssf/smf
b
BSG/WB
c
FAE activity was expressed on a dry weight basis
93
FAE production through native secretory pathways of microorganisms is mainly

performed with two types of fermentation. Submerged fermentation (SmF), where
the microorganism is in contact with the liquid growth medium, and solid-state
fermentation (SSF), where the microorganism grows on solid substrates in the near
absence of water. SSF main advantages over SmF include higher yields, simpler
technology with rare operational problems, and resemblance to microorganism’s
natural habitat (Katsimpouras et al. 2014). Among Aspergillus species, A. flavipes
(Mathew et al. 2005), A. niger (Johnson et al. 1989) and A. awamori (Kanauchi
et al. 2008) were reported as very efficient FAE producers under SmF in the
presence of lignocellulosic materials such as wheat bran and destarched wheat bran
while P. brasilianum was the more active producer under SSF conditions on
brewer’s spent grain (Panagiotou et al. 2006).
The choice of the optimal substrate stand to be crucial for the homologous
esterase expression as its role is not only to serve as a carbon and energy source but
also to provide the necessary inducing compounds for the microorganism.
Lignocellulosic carbon sources that contain high amounts of esterified FA such as
wheat bran (Mathew et al. 2005; Kanauchi et al. 2008; Mandalari et al. 2008;
Hegde and Muralikrishna 2009; Gottschalk et al. 2013), corn bran (Shin and Chen
2006), corn cobs (Yang et al. 2013), destarched wheat bran (Faulds et al. 2006;
Mukherjee et al. 2007; Kheder et al. 2009; Tai et al. 2014), maize bran (Mathew
et al. 2005) and brewer’s spent grain (Panagiotou et al. 2006; Mandalari et al.
2008), have been successfully employed for FAEs production. Furthermore, in
many cases the effect of removal of FA from these materials was investigated with
the results revealing that deesterified carbon sources lead to a decreased production
of FAE compared to untreated materials even when the media were supplemented
with free FA (Topakas et al. 2003a, b, c; Topakas and Christakopoulos 2004; Shin
and Chen 2006). In contrast to these results, production of FAE was enhanced with
the addition of free FA but also FA’s breakdown products and other aromatic
compounds could induce FAE production as well (Faulds et al. 1997; Faulds and
Williamson 1999; de Vries and Visser 1999; de Vries et al. 2002).
FAEs’ wide range of potential applications necessitates the efficient production
of novel recombinant enzymes, and the availability of more and more genome
sequences for several microorganisms constitutes a powerful tool facilitating their
discovery. Native enzyme mixtures production poses some difficulties due to their
high production cost, as there is a need for special culturing and induction condi-
tions (Lambertz et al. 2014). In order to overcome this obstacle, gene cloning and
heterologous expression have been employed, producing enzymes with superior
properties.
To date, host microorganisms such as bacteria and yeasts have been used for
FAEs expression. The most widely used bacterial expression host is Escherichia
coli and more specifically the strain BL21, which has the advantage of being
deficient in the Ion and ompT proteases. As shown in Table 5.1, E. coli BL21 cells
were employed in recombinant FAEs expression from several microorganisms such
as Streptomyces cinnamoneus, Thermobacillus xylanilyticus, C. japonicus,
L. plantarum, Aspergillus clavatus, Cellulosilyticum ruminicola, Anaeromyces
mucronatus, Dickeya dadantii, Thermoanaerobacter tengcongencis, A. awamori,

B. proteoclasticus and L. johnsonii. Li et al. (2011) used E. coli Rosetta-gami 2 and
Rosetta for the expression of two FAEs (FaeII and FaeIII, respectively) from C.
ruminicola as these particular strains enable the enhanced formation of disulfide
bonds in the cytoplasm and facilitate the expression of genes that encode rare
E. coli codons. Furthermore, ScFAE1 and ScFAE2 from S. cellulosum were
overexpressed using E. coli Tuner cells, which enable precise control of protein
expression with IPTG (Wu et al. 2012).
Besides bacterial expression systems, yeasts such as Saccharomyces cerevisiae
and Pichia pastoris constitute also attractive host organisms for heterologous pro-
duction of eukaryotic proteins exhibiting several advantages such as high growth
rate and protein titers using low-cost growth media (Várnai et al. 2014). Yeasts are
also capable of protein folding and posttranslational modifications such as glyco-
sylation, with P. pastoris having the advantage over S. cerevisae of not hypergly-
cosylating secreted proteins (Grinna and Tschopp 1989). In addition, P. pastoris
secretes low levels of native proteins simplifying the purification process of the
recombinant protein (Barr et al. 1992). Moukouli et al. (2008) described first the
successful cloning and expression of a type C FAE from F. oxysporum in P. pastoris
X-33 employing a bioinformatic-assisted functional screening in order to discover
and analyze the enzyme. FAEs were also cloned and expressed in P. pastoris
from different Aspergillus species such as A. oryzae (Koseki et al. 2009; Zeng et al.
2014), A. flavus (Zhang et al. 2013), Aspergillus nidulans (Bauer et al. 2006; Debeire
et al. 2012) and A. usamii (Gong et al. 2013) and from M. thermophila (Topakas
et al. 2012).
A type B recombinant FAE was cloned from the genomic DNA of A. nidulans
and expressed in S. cerevisiae exhibiting about 86 % of total FAE activity in the
culture medium indicating an efficient protein secretion (Shin and Chen 2007).
Wong et al. (2011) cloned a FAE gene from A. niger into S. cerevisae achieving an
efficient secretion and a yield of about 2 mg/L.
Recently, there are reports describing the production, purification, and charac-
terization of FAEs from basidiomycetes. The limited knowledge on FAEs from
basidiomycetes could be explained due to their high laccase activity as it interferes
with determination of FA (Nieter et al. 2014). Schizophyllum commune and
Pleurotus sapidus were the only basidiomycetes, which were associated with FAE
activity (MacKenzie and Bilous 1988; Linke et al. 2013). However, Haase-Aschoff
et al. (2013) came up with a new efficient analytical method, using multiple sub-
strates, for FAEs detection circumventing substrate and FA polymerization. After
screening 41 basidiomycete strains, more than half of them exhibited FAE activity
with different substrate specificity. Pleurotus eryngii was cultivated in standard
nutrition liquid supplemented with Tween 80 as inducer and reached maximum
FAE activity on the 13th day (Nieter et al. 2014).
Physical characteristics such as molecular weight, isoelectric point, and optimum
hydrolytic reaction conditions differ significantly among purified FAEs (Table 5.2).
FAEs exhibit a wide range of molecular weights and isoelectric points from 11 to
210 kDa and from 3.0 to 9.9 respectively. The extracellular FAE from
Table 5.2 Physicochemical properties of purified FAEs known to date
96
Microorganism Enzyme Gene Data bank FAE MW pHopt Topt pI References

type (kDa) (°C)
A. awamori FE – 112 3.7 Wang et al. (2004)
CE – 75 4.2 Wang et al. (2004)
– – 35 5.0 45 3.8 Koseki et al. (1998)
AwFAEA AwfaeA AB032760 A 37 5.01 Koseki et al. (2005)
– 78 5.012 4012 Kanauchi et al. (2008)
AwFaeA AwfaeA AB032760 A 35 5.513 5513 4.2 Fazary et al. (2010)
A. clavatus AcFAE D 31 71 301 Damásio et al. (2013)
A. flavus AfFaeA A 40 6.02 582 Zhang et al. (2013)
A. mucronatus Fae1A fae1A – 37 7.2 37 Qi et al. (2011)
A. nidulans AnidFAE AN5267.2 – 28 6.12 372 Debeire et al. (2012), Bauer et al.
(2006)
AN1772.2 B 130 7.02 452 Shin and Chen (2007)
a
A. niger FAE-I B 63 3.0 Faulds and Williamson (1993)
FAE-II A 29 3.6 Faulds and Williamson (1993)
FAE-III or faeA Y09330 A 36 5.02 552 3.3 Faulds et al. (1997)
AnFaeA
CinnAE or faeB AJ309807 Bb 75.8a 6.03 503 4.8 Kroon and Williamson (1996)
AnFaeB
CE – 120 McCrae et al. (1994)
FAE-1 50 9.014 4014 Hegde and Muralikrishna (2009)
FAE-2 55 6.014 40–5014 Hegde and Muralikrishna (2009)
(continued)
type (kDa) (°C)
– faeA XP_001393337 31 6–714 5014 Wong et al. (2011)
A. oryzae FAE – 30 4.5– 3.6 Tenkanen et al. (1991)
6.04
AoFaeB AofaeB XP_001818628 B 61 6.01 Koseki et al. (2009)
AoFaeC AofaeC XP_001819091 C 75 6.01 Koseki et al. (2009)
AoFaeA AofaeA A 37 5.02 502 Zeng et al. (2014)
A. pullulans – B 210 6.75 605 6.5 Rumbold et al. (2003)
A. tubingensis FaeA faeA Y09331 – 36 Zwane et al. (2014)
A. usamii E001 AuFaeA AufaeA A 36 5.02 452 Gong et al. (2013)
Butyrivibrio – cinI or U44893 – Barbe and Dubourdieu (1998)
fibrisolvents cinA
– cinII or U64802 – De Vries et al. (1997)

cinB
B. proteoclasticus Est1E est1E 31.6 Goldstone et al. (2010)
C. ruminicola FaeI faeI – 58 6–72 402 Li et al. (2011)
FaeII faeII – 32 82 352 Li et al. (2011)
FaeIII faeIII – 46 92 402 Li et al. (2011)
C. japonicus XLYD or xynD X58956 D 59 Ferreira et al. (1993)
CjXYLD
C. japonicus Fee1B fee1B D 61 6.52 35–402 McClendon et al. (2011)
2
Chaetomium sp. – B 30.2 7.5 602 Yang et al. (2013)
(continued)
97
98

type (kDa) (°C)
Chrysosporium FaeA1 JF826027 A 29 6–72 452 5.5 Kühnel et al. (2012)
lucknowense
FaeA2 JF826028 A 36 7.52 402 5.2 Kühnel et al. (2012)
2
FaeB2 JF826029 B 33 7 452 6.0 Kühnel et al. (2012)
C. stercorarium – C or D 33 8.02 652 Donaghy et al. (2000)
C. thermocellum XynZ XynZ M22624 – 45 4–76 50–606 5.8 Blum et al. (2000)
– XynY X83269 – Blum et al. (2000)
D. dadantii FaeT faeT 35 Hassan and Hugouvieux-Cotte-Pattat
(2011)
FaeD faeD 31, 35 Hassan and Hugouvieux-Cotte-Pattat
(2011)
F. oxysporum FoFAE-I or B 31 7.07 557 >9.5 Topakas et al. (2003a, b, c)
FoFaeB
FAE-II or A 27 7.08 458 9.9 Topakas et al. (2003a, b, c)
FoFaeA
F. proliferatum FAE B 31 6.5– 502 Shin and Chen (2006)
7.52
Lactobacillus – – 36 5.69 379 Topakas et al. (2004)
acidophilusc
L. johnsonii Lj0536 WP_004898050.1 31 7.8 20 Kin et al. (2009)
(continued)
type (kDa) (°C)
Lj1228 WP_011162057.1 31 6.7 30 Kin et al. (2009)
L. plantarum Lp_0796 lp_0796 – 28 715 30–3715 Esteban-Torres et al. (2013)
M. thermophila FoFaeC-12213 Foxg- C 62 6.02 652 6.8 Moukouli et al. (2008)
12213.2
M. thermophila MtFae1a fae1a 96,478 B 39 7.02 502 Topakas et al. (2012)
a 10
Neocallimastix MC-2 pCAE – 11 7.2 4.7 Borneman et al. (1993)
FAE-I – 69 Borneman et al. (1992)
FAE-II – 24 Borneman et al. (1992)
N. crassa Fae-1 Fae-1 AJ293029 B 35 6.02 552 Crepin et al. (2003a)
NcFaeD-3.544 faeD- D 32 Crepin et al. (2004a, b)
3.544
P. equi EstA estA AF164516 D 55 6.711 50–6011 Fillingham et al. (1999)

7
estA AF16516 30 6.5 50–607 Poidevin et al. (2009)
P. eryngii PeFaeA A 67 5.02 502 5.2 Nieter et al. (2014)
Penicillium expansum – possible B 57.5 5.62 372 Donaghy and McKay (1997)
P. funiculosum FAEB or faeB AJ291496 B 53 6.0 Kroon et al. (2000)
PfFaeB
FaeA faeA AJ312296 Knoshaug et al. (2008)
Penicillium pCAE/FAE – 57 6.02 552 4.6 Castanares et al. (1992)
pinophilum
P. sapidus A 55 6.02 502 5.7 Linke et al. (2013)
Rumen microbial RuFae2 C 29 7 50 8.5 Wong et al. (2013)
metagenome
(continued)
99
100

type (kDa) (°C)
R. virescens – 62 5.02 502 Wang et al. (2014)
17
Soil metagenomic Tan410 tan410 – 55 7 3517 4.7 Yao et al. (2013)
library
S. cinnamoneus R18 AB921569 D 38 7.513 5013 Uraji et al. (2014)
13
R43 AB921570 D 52 7 4013 Uraji et al. (2014)
S. cellulosum ScFAE1 sce1896 D 35 72 5.07 Wu et al. (2012)
ScFAE2 sce8927 D 34 6–82 4.67 Wu et al. (2012)
a
S. thermophile StFAEA or B 33 6.02 55–602 3.5 Topakas et al. (2004)
StFaeB
StFaeC C 23a 6.02 552 <3.5 Topakas et al. (2005a, b)
2 2
S. olivochromogenes FAE – 29 5.5 30 7.9 Faulds and Williamson (1991)
T. stipitatus TsFaeA A 35 5.3 Garcia-Conesa et al. (2004)
TsFaeB B 35 3.5 Garcia-Conesa et al. (2004)
TsFaeC faeC AJ505939 C 66 6–73 603 4.6 Garcia-Conesa et al. (2004), Crepin
et al. (2003b)
T. tengcongensis TtFAE A 33 8.016 Abokitse et al. (2010)
T. xylanilyticus Tx-Est1 Tx-est1 – 37.4 8.52 652 Rakotoarivonina et al. (2011)
Numbers in superscripts indicate the substrates used for pH and temperature optimum: 1. α-naphthylbutyrate; 2. methyl ferulate (MFA); 3. methyl caffeate (MCA); 4.
wheat straw (WS); 5. 4-nitrophenyl 5-O-trans-feruloyl-α-L-arabinofuranoside (NPh-5-Fe-Araf); 6. O-{5-O-(trans-feruloyl)-[O-β-D-xylopyranosyl-(1 → 2)]-O-α-L-
arabinofuranosyl-[1 → 3]}-O-β-D-xylopyranosyl-(1 → 4)-D-xylopyranose (FAXXX); 7. methyl p-coumarate (MpCA); 8. methyl sinapate (MSA); 9. [2-O-(trans-
feruloyl)-α-L-arabinofuranosyl]-(1 → 5)-L-arabinofuranose (FAA); 10. O-[5-O-(trans-p-coumaroyl)-α-L-arabinofuranosyl]-(1 → 3)-O-β-D-xylopyranosyl-(1 → 4)-D-
xylopyranose (PAXX); 11. O-[5-O-(trans-feruloyl)-α-L-arabino-furanosyl]-(1 → 3)-O-β-D-xylopyranosyl-(1 → 4)-D-xylopyranose (FAXX); 12. 1-naphthyl acetate; 13.
Ethyl ferulate (EFA); 14. 4-nitrophenyl ferulate (4NPF); 15. p-nitrophenyl butyrate; 16. pNP caprylate; 17. p-nitrophenyl ferulate
a
Dimeric proteins (molecular weight estimated with SDS-PAGE electrophoresis)
b
Phylogenetic analysis of AnFaeB indicated that this enzyme belongs to the type C subclass (Crepin et al. 2004a)
c
Typical human intestinal bacterium
Aureobasidium pullulans with an apparent molecular weight of 210 kDa, is the

largest single-subunit esterase of its kind due to an extensive Asp-linked glyco-
sylation reaching a degree of 48 % (Rumbold et al. 2003). Optimum hydrolytic
conditions also vary with pH ranging from 4.0 to 9.0 and temperature from 20 to
65 °C depending on the source microorganism. Comparing the physicochemical
characteristics and conditions for optimal activity of purified FAEs shown in
Table 5.2, there is no pattern indicating a correlation among them.
5.6 Applications of FAEs
During the last decade, FAEs have gained increased interest acquiring considerable
role in biotechnological processes. Except for their role in biofuel industry as
accessory enzymes for the degradation of lignocellulose, FAEs’ potential applica-
tions cover a broad spectrum, including the pulp and paper industry as bleaching
agents, the cosmetic and pharmaceutical industry for the synthesis of bioactive
compounds with antioxidant and/or antimicrobial activity or for the production of
flavor and fragrance precursors, the food industry as food additives or as feed
additives in animal feeds (Fig. 5.3).
5.6.1 Exploiting the Hydrolytic Activity
The utilization of the FAEs’ hydrolytic activity lies on the release of FA from
hemicellulose present in plant cells walls. In ruminal digestion, FAEs are important
as they deesterify dietary fibers releasing hydroxycinnamates but are also take part
in colonic fermentation where their activities in gut ruminal microorganisms
Fig. 5.3 Potential applications of FAEs in Industrial Biotechnology

enhance the breakdown of ester bonds in hydroxycinnamates (Esteban-Torres et al.

2013). This is why FAEs are used for the pretreatment of food and animal feeds or
even as food/feed additives, as they allow the in situ improved digestibility of
hemicellulose. In animal feed industry the advantages of their use include improved
feed utilization, control of body weight gain and higher milk yield in cattle and
sheep (Howard et al. 2003). In food industry, FAE preparations are also widely
used in the bakery industry along with glucanases and oxidases in order to solu-
bilize arabinoxylan fractions of the dough resulting to increased bread volume and
improved quality (Butt et al. 2008). Other applications of FAEs include the syn-
thesis of oligosaccharides that can be utilized as functional food additives and their
use for juice clarification. On the other part, FA is one of the major antioxidant
additives in food and beverages such as beer (Benoit et al. 2008). The pretreatment
of agricultural by-products with FAEs could offer an economic alternative for the
production of FA for these purposes. Being also a flavor precursor, FA can be
further converted to valuable compounds such as vanillin and vanillic acid. The
biotransformation of FA has been investigated using different processes and fila-
mentous fungi (Benoit et al. 2008; Walton et al. 2000). Apart from its use for
flavoring, vanillin is also a fundamental constituent for the synthesis of pharma-
ceuticals and is used extensively in the perfume and metal plating industries, while
its herbicidal activity is useful as ripening agent for the achievement of higher
yields of sucrose in sugar canes (Fazary and Ju 2008).
In paper pulp processing, environment-friendly approaches include the
replacement of chemicals with enzymes, resulting to the reduction of water pol-
lution and associated clean-up costs (Koseki et al. 2009). Xylanases and laccases
are mainly used for enzymatic delignification and biobleaching, respectively,
leading to the reduction or elimination of chlorine-based chemicals (Valls et al.
2010; Thakur et al. 2012). The use of FAEs along with other accessory enzymes
might enhance this process by removing substitutions and linkages between
polymers, resulting to the detachment of hemicellulose walls and the release of
lignin fragments. A recombinant FAEA from A. niger has been used for the first
time in 2003 in combination with laccase and xylanase for the efficient delignifi-
cation of wheat straw pulp, which yielded 78 % (Record et al. 2003). In addition, it
has been shown to boost delignification in flax pulp resulting in very low kappa
number and pulp brightness (Sigoillot et al. 2005). The commercial lipase A
“Amano” with significant FAE and other accessory activities, when tested for its
bleachability in kraft pulps, offered the first evidence that accessory enzymes from a
commercial preparation such as FAE and arabinofuranosidase can result to direct
bleaching effect (Nguyen et al. 2008). Other minor applications of the FAEs’
hydrolytic activity in paper industry is the removal of fine particles from the pulp
facilitating water removal and the enhancement of chemical and mechanical
paper-pulping methods allowing the easier solubilization of lignin–carbohydrate
complexes (Fazary and Ju 2008). A prerequisite for their use is that enzyme
preparations are free of cellulases, since cellulose degradation would result in a
reduction in the quality of the pulp.
5.6.2 FAEs as Biosynthetic Tools of Plant Derived

Antioxidants
Ester-linked substituents (ferulic, p-coumaric, caffeic, sinapinic acid) on plant cell

wall polysaccharides have widespread potential due to their antimicrobial, photo-
protectant, antitumour and antioxidant activities (Faulds 2010), while some have
long been used as food preservatives in order to inhibit microbial growth (Fazary
and Ju 2008). However, their application in oil-based food and cosmetics industry
is limited due to their relatively low solubility in aprotic media. The direct esteri-
fication of phenolic, including hydroxycinnamic, acids or the transesterification of
their esters with fatty alcohols or sugars has gained increased interest as it targets to
the production of biologically active compounds with improved properties
(Fig. 5.4). The synthesized phenolic fatty esters are generally more lipophilic and
allow their application in oil-based processes; on the other hand, the phenolic acid
sugar esters are more hydrophilic nonionic biosurfactants and have clear antitumor
activity, so the potential to be used in order to formulate antimicrobial, antiviral
and/or anti-inflammatory agents is promising (Fazary and Ju 2008). Enzymatic
transesterification offers an alternative to the poor selectivity of chemical synthesis.
It is a one-step process resulting to the synthesis of one product instead of a mixture
of esters thus there is no unwanted side reactions causing darkening or the for-
mation of odors and no need of by-product and catalyst residues removal.
Complementary to the attractive environment-friendly configurations of the pro-
cess, the use of enzymes derived from thermophilic fungi allows the implementa-
tion of lower temperature (50–60 °C) than that of the chemical process (160 °C)
resulting to lower operating costs. Enzyme-catalyzed synthesis has been widely
studied in lipases including hydroxycinnamate (trans)esterification in nonconven-
tional media (organic solvents, ionic liquid mixtures). Lipases are known to be
more stable than FAEs in such reaction media, display high activity at low water
Fig. 5.4 Modification of hydroxycinnamic acids by FAEs in nonconventional reaction media. FA

ferulic acid, pCA p-coumaric acid, SA sinapic acid, CA caffeic acid
content and give high yields (Zeuner et al. 2011). On the contrary, FAEs exhibit
higher substrate specificity constituting them a potential biosynthetic tool with high
efficiency.
As with lipases, a reaction medium with low or non-water content is essential in
order to boost the FAEs’ synthetic activity to the detriment of the hydrolytic one.
The first synthetic reaction catalyzed by FAE was achieved in a water-in-oil
microemulsion system for the synthesis of 1-pentyl-ferulate (Giuliani et al. 2001).
Since then, detergentless microemulsions, which are ternary systems consisted of a
hydrocarbon, a short-chained alcohol and water, have mostly been employed for the
synthesis of various alkyl and sugar esters (Table 5.3). They are low water content
media representing thermodynamically stable dispersions of aqueous microdroplets
in the hydrocarbon solvent. The spherical droplets are stabilized by alcohol
molecules adsorbed at their surface, while the enclosed in water enzyme is pro-
tected from contacting the outer organic phase and from denaturation due to a
water-rich layer (Khmelnitsky et al. 1988). An important advantage of these mix-
tures as reaction systems is that they allow the separation of reaction products and
enzyme reuse, while the solubility of relatively polar phenolic acids is high owing
to the presence of large amount of polar alcohol. In these media, transesterification
reactions using activated esters of FA as acyl donors have been proven to be faster
and more efficient compared to the direct esterifications (Olsson et al. 2011).
Another approach for efficient transesterification is the use of ionic liquid mixtures
(ILs), which comprise organic salts that remain liquid under ambient temperature
(Table 5.4). The interest in ILs as reaction media is mainly urged by their lack of
vapor pressure as they have the potential to replace volatile organic solvents
resulting to the development of greener processes. A major advantage of using ILs
is their tailorability as they exhibit increased selectivity and ability to solubilize
both polar and nonpolar substrates and products, as well as they can adjust other
physicochemical properties such as density, viscosity and solvating power through
alternating the cation and anion type to meet the needs of each system (Zeuner et al.
2011). The lack of volatility together with their chemical and thermal stability allow
simple recycling and reuse therefore could reduce significantly the cost of the
process with respect to organic-based ones. When referring to transesterification
with saccharides, the advantage of using ILs, as compared to organic solvents is the
increased substrate solubility. Furthermore higher yields in shorter reaction times
including regioselectivity may also be achieved in enzymatic acylation of saccha-
rides in ILs compared to organic solvents. Most of the research has used
second-generation ILs, which may be too expensive for commercial applications.
The availability of advanced ionic liquids- greener, inexpensive and biodegradable-
increases the likelihood that they will find commercial use in biocatalysis (Gorke
et al. 2010). Although synthesis in organic solvents has been widely studied in
lipases, their use in FAEs has been rarely reported in literature due to stability
restraints (Table 5.5). An important advantage is that they allow the implementation
of one phase reaction and the separation of reaction products, although they do not
consist a green approach for transesterification.
Table 5.3 Enzymatic synthesis of hydroxycinnamic acid esters in detergentless microemulsions reported in the literature
Hydroxycinnamate Acyl donor Enzyme Solvent system Yield (h) T (°C) References
Synthesis of aliphatic
esters
Methyl ferulate 1-Butanol StFaeC n-Hexane:1-butanol: MES-NaOH pH Up to 20 % 35 Topakas
6.0 (53.4:43.4:3.2 v/v/v) (120) et al. (2005a, b)
Methyl sinapate Up to 10 %
(*144)
Methyl p-coumarate Up to 8 %
(*144)
Methyl caffeate Up to 5.5 %
(*144)
Methyl ferulate 1-Butanol StFae-A n-Hexane:1-butanol: Up to 8 % 35 Topakas et al.
buffer (47.2:50.8:2.0 v/v/v) (144) (2004)
Methyl p-coumarate Up to 50 %
(144)
Methyl caffeate Up to 25 %
(144)
Methyl p-coumarate 1-Butanol FoFae-I n-Hexane/1-butanol/MES-NaOH pH Up to 70 % 35 Topakas et al.
6.0 (47.2:50.8:2.0 v/v/v) (144) (2003a)
Methyl caffeate Up to 22 %
(144)
Methyl ferulate Up to 13 %
(144)
Methyl sinapinate Up to 1 %
(144)
(continued)
105
106
p-hydroxyphenylacetic 1-Propanol FoFae-II n-Hexane/1-propanol/MES-NaOH pH 75 (224) 30 Topakas et al.
acid 6.6 (47.2:50.8:2.0 v/v/v) (2003b)
p- 70 (224)
Hydroxyl-phenylpropionic
acid
Methyl sinapate 1-Butanol AnFaeA+1 n-Hexane:1- or 2-butanol: 56 % (120) 35 Vafiadi et al.
MES-NaOH pH 6.0 (47.2: 50.8: 2.0 (2008a)
v/v/v)
78 %a (120)
Methyl ferulate AnFaeA 42 % (120)
Methyl p-coumarate 2 % (120)
Methyl sinapate 2-Butanol 9 % (120)
Methyl ferulate 1-Butanol Ultraflo L+1 n-Hexane/1-butanol/water 97 % (144) 37 Vafiadi et al.
(47.2:50.8:2.0 v/v/v) (2008b)
Ultraflo 3.6 % (144)
Methyl ferulate 1-Butanol Depol n-Hexane/1-butanol/water 87 % (144) 37 Vafiadi et al.
740L+1 (47.2:50.8:2.0 v/v/v) (2008b)
Depol 740L 2.6 % (144)
Methyl ferulate 1-Butanol Depol 670L+ n-Hexane/1-butanol/water 5 % (144) 37 Vafiadi et al.
(47.2:50.8:2.0 v/v/v) (2008b)
Synthesis of sugar esters
Methyl ferulate L-Arabinose StFaeC n-Hexane:t-butanol:piperazine- HCl 40 % (160) 35 Topakas et al.
pH 6.0 (53.4:43.4:3.2 v/v/v) (2005b)
Methyl ferulate Up to 50 %a Vafiadi et al.
(120) (2005)
(continued)
Ethyl ferulate 6.3 % (–)
n-Propyl ferulate 3.8 % (–)
n-Butyl ferulate 3.4 % (–)
Isopropyl ferulate 3.3 % (–)
2-Butyl ferulate 2.7 % (–)
Isobutyl ferulate 4.2 % (–)
Methyl ferulate D-Arabinose 45 % (–)
D-Glucose n.q. (120) Vafiadi et al.
(2007a)
D-Xylose n.q. (120) Vafiadi et al.
(2005)
D-Mannose n.q. (120)
D-Fructose n.q. (120)

D-Galactose n.q. (120)
D-Ribose n.q. (120)
pNP-arabinofuranoside n.q. (120)
n.q. (120)
pNP-anabinopyranoside n.q. (–)
n.q. (–)
L-Arabinotriose n.q. (–) 37 Vafiadi et al.
(2007b)
(continued)
107
108
L-Arabinotetraose n-Hexane:2-methyl-2-propanol: n.q. (–)
piperazine HCl pH 6.0 (47.2:50.8:2.0
v/v/v)
Methyl ferulate L-Arabinopentaose 24 %a (96)
Ethyl ferulate L-Arabinohexaose 3 % (96)
Propyl ferulate L-Arabinose 4 % (96) 35 Vafiadi et al.
(2006a)
Methyl ferulate L-Arabinose TsFaeC n-Hexane: t-butanol: MOPS-NaOH 21.2 %a (96) 40 Vafiadi et al.
pH 6.0 (53.4:43.4:3.2 v/v/v) (2006b)
Ethyl ferulate 1.4 % (96)
Ferulic acid D-Arabinose Multifect n-Hexane/1-butanol or 11.3– 35 Couto et al.
P 3000 2-butanone/MES-NaOH pH 6.0 36.7 % (2010)
(51:46:3 v/v/v) (144)
D-Galactose 5.6–8.6 %
(144)
D-Xylose 12.1–
30.8 %
(144)
Ferulic acid D-Arabinose Ceremix n-Hexane/1-butanol or 15.3– 35 Couto et al.
2-butanone/MES-NaOH pH 6.0 32.5 % (2010)
(51:46:3 v/v/v) (144)
Ferulic acid D-Galactose RP-1 n-Hexane/1-butanol or 10.2– 35 Couto et al.
(51:46:3 v/v/v) (144)
D-Xylose 4.4–16.3 %
(144)
(continued)
Ferulic acid D-Arabinose Depol 670 n-Hexane/1-butanol or 7.2–17.7 % 35 Couto et al.
2-butanone/MES-NaOH pH 6.0 (144) (2010)
(51:46:3 v/v/v)
D-Galactose 3.4–15.8 %
(144)
D-Xylose 20.9–
26.5 %
(144)
Ferulic acid D-Arabinose Flavourzyme n-Hexane/1-butanol or 21.9– 35 Couto et al.
(51:46:3 v/v/v) (144)
D-Galactose 36.2–
41.9 %
(144)
D-Xylose 20.1–
21.7 %
(144)
Ferulic acid Raffinose Depol 740L n-Hexane/2-butanone/MES-NaOH pH 11.9 %a 35 Couto et al.
6.0 (51:46:3 v/v/v) (168) (2011)
Xylobiose 9.4 % (144)
Arabinose 7.9 % (144)
Galactobiose 5.4 % (144)
(continued)
109
110
Sucrose 13.2 % (–)
Lactose 4.4 % (–)
XOS 2.8 % (–)
FOS n-Hexane/1,4-dioxane/MES-NaOH 9.6 % (–)
pH 6.0 (51:46:3 v/v/v)
Raffinose 3.1 % (144)
Xylobiose 4.2 % (144)
Galactobiose 26.8 %
(144)
a
After optimization of reaction conditions; +1: Immobilized with CLEAs methodology; n.q.: detected but not quantified; StFaeC: FAE type C from S.
thermophile ATCC 34628; StFaeA: FAE type B from S. thermophile ATCC 34628; FoFae-I/FoFae-II: FAE from F. oxysporum F3; AnFaeA: FAE type A
from A. niger; Ultraflo L/ Depol 740L: multienzymatic preparation from H. insolens; Depol 670L: multienzymatic preparation from T. reesei; TsFaeC: FAE
type C from T. stipitatus; Multifect P 3000: multienzymatic preparation from Bacillus amyloliquefaciens; Ceremix: multienzymatic preparation from Bacillus
spp.; RP-1: multienzymatic preparation from Bacillus subtilis; Depol 670L: multienzymatic preparation from T. reesei; Flavourzyme: multienzymatic
preparation from A. oryzae; Depol 740L: multienzymatic preparation from H. insolens. Systems with conversion <1 % were not included
Table 5.4 Enzymatic synthesis of hydroxycinnamic acid esters in ILs reported in the literature
Hydroxycinnamate Acceptor Enzyme Ionic liquid Yield (h) T (°C) References
system
Sinapic acid Glycerol AnFaeA [C2OHmim] 72.5%a (24) 50 Vafiadi et al.
[PF6] (85%), (2009)
MOPS-NaOH
pH 6.0 (15%)
[C5OHmim] 76.5%a (24)
[PF6] (85%),
MOPS-NaOH
pH 6.0 (15%)
Methyl sinapate AnFaeA+1 [ΒΜΙm][PF6] 13% (0.5) 40 Zeuner et al.
AnFaeA (85%), MOPS (2011)
pH 6.0 (15%)
[C2OHMIm] 21% (0.5)
[BF4] (85%),
MOPS pH 6.0
(15%)
[C5OHmim] 72.5%a (24) 50 Vafiadi et al.
[PF6] (85%), (2009)
MOPS-NaOH
pH 6.0 (15%)
[C2OHmim] Up to 2.5%
[PF6] (94 %), (24)
MOPS-NaOH
pH 6.0 (6%)
[C5OHmim] Up to 7%
[PF6] (94%), (24)
MOPS-NaOH
pH 6.0 (6%)
Sinapic acid Glycerol AndFaeC [ΒΜΙm][PF6] 1.1% (0.5) 40 Zeuner et al.
(85%), MOPS (2011)
pH 6.0 (15%)
Sinapic acid Glycerol Ultraflo L [ΒΜΙm][PF6] 1% (0.5) 40 Zeuner et al.
(85%), MOPS (2011)
pH 6.0 (15%)
Ferulic acid Raffinose Depol [ΒΜΙm][PF6] 2% (144) 35 Couto et al.
740L (96%), (2011)
MES-NaOH
pH 6.0 (3%)
D-xylose 4.2% (144)
D- 2.8% (144)
Arabinose
D- 1% (144)
Galactose
a
After optimization of reaction conditions; +1: Immobilized with CLEAs methodology; AnFaeA: FAE type
A from A. niger; AnFaeC: FAE type C from Aspergillus nidulans; Ultraflo L/ Depol 740L: multienzymatic
preparation from H. insolens. Systems with conversion <1 % were not included
Table 5.5 Enzymatic synthesis of hydroxycinnamic acid esters in organic solvents reported in the literature
112
Hydroxycinnamate Enzyme Solvent system Yield (h) T (°C) References

Ferulic acid Glycerol FAE-PL Glycerol/ DMSO/acetate buffer pH 4.0 or 5.0 81%a (–) 50 Tsuchiyama et al.
(85:5:10 v/v/v) (2006)
Sinapinic acid n.q. (0.25)
3,4-Dimethoxy-cinnamic n.q. (0.25)
acid
p-Coumaric acid n.q. (0.25)
Methyl ferulate n.q. (0.25)
Ethyl ferulate n.q. (0.25)
Methyl sinapate n.q. (0.25)
Methyl n.q. (0.25)
3,4-dimethoxycinnamate
Arabinose n.q. (0.5)
Fructose n.q. (0.5)
Ferulic acid Galactose n.q. (0.5)
Glucose n.q. (0.5)
Xylose n.q. (0.5)
Arabinose n.q. (0.03)
Fructose n.q. (0.03)
Ethyl ferulate Galactose n.q. (0.03)
Glucose n.q. (0.03)
Xylose n.q. (0.03)
Methyl ferulate 1-Butanol Depol 1-Butanol/MOPS pH 6.0 (92.5:7.5 v/v) Up to 90 %a 37 Thorn et al. 2011
740L+2 (*192)
Depol Up to 40 %
740L (*192)
a
After optimization of reaction conditions; +2: Immobilized in mesoporous silica MPS-9D; n.q.: detected but not quantified; FAE-PL: FAE from A. niger (purified from
the commercial preparation Pectinase PL “Amano”); Depol 740L: multienzymatic preparation from H. insolens. Systems with conversion <1 % were not included
Novel FAEs purified from various filamentous fungi such as F. oxysporum,

S. thermophile, and A. niger have been used for the synthesis of alkyl ferulates
(1-propyl ferulate, 1-butyl ferulate, 1-pentyl-ferulate, 1-glyceryl ferulate) and
feruloylated-arabino-oligosaccharides (with up to six arabinose units) showing
regioselectivity for the primary hydroxyl group of the nonreducing arabinofuranose
ring (Table 5.3). In detergentless microemulsions, FoFaeI from F. oxysporum has
achieved the synthesis of various 1-butyl hydroxycinnamates exhibiting highest
yield on 1-butyl coumarate (up to 70 %), while FoFaeII has esterified p-hydro-
xyphenylacetic and p-hydroxyl-phenylpropionic acid with 1-propanol (70–75 %
yield) (Topakas et al. 2003a, b). The thermophilic StFaeC from S. thermophile has
synthesized 1-butyl hydroxycinnamates but also a variety of hydroxycinnamic acid
sugar esters, including the feruloylation of both L- and D-arabinose, showing no
chiral selectivity. The synthesized D-enantiomer successfully inhibited the growth of
Mycobacterium bovis BCG, indicating that FAEs are novel chemoenzymatic tools
for the synthesis of new drug (Vafiadi et al. 2007a). Multienzymatic preparations
containing FAE activity such as Ultraflo L and Depol 740L from Humicola insolens
have shown high yields up to 97 % in the transesterification of methyl ferulate to
1-butyl ferulate when immobilized with Crossed Linked Enzyme Aggregates
(CLEAs) methodology (Vafiadi et al. 2008b), while immobilized Depol 740L in
mesoporous silica MPS-90 supported significantly higher butyl ferulate yield up to
90 % comparing to the free enzyme (Thörn et al. 2011). Direct esterification of FA
with monomer sugars or oligosaccharides has been achieved by FAE multienzy-
matic preparations with relatively lower yields (up to 40 %; Couto et al. 2010).
The modification of sinapic acid, which is the second strongest antioxidant in
nature after FA, has also been achieved. AnFaeA transesterified methyl sinapate to
1- and 2-butyl sinapate, while StFaeC and FoFae-I have successfully synthesized
1-butyl sinapate in detergentless microemulsions (Topakas et al. 2003a, b; Vafiadi
et al. 2008a). Although, immobilized AnFaeA with CLEAs methodology resulted to
22 % lower conversion of methyl sinapate than that of the free enzyme. ILs have
been used for the synthesis of glyceryl-sinapate from sinapic acid and methyl
sinapate, where free and CLEAs AnFaeA also resulted to 76.5 and 72.5 % con-
version yield of sinapic acid, respectively (Vafiadi et al. 2009; Table 5.4). Butyl
esters of sinapic acid exhibit higher antioxidant activity against Low Density
Lipoprotein (LDL) oxidation comparing to their free acid or 1-butyl ferulate,
showing that sinapate esters are more promising antioxidants in vitro (Vafiadi et al.
2008a). On the contrary, sinapoylated glycerol has been shown to be a slower
antioxidant, when compared to free sinapic acid in LDL oxidation (Tsuchiyama
et al. 2006). It has not been established yet why hydroxycinnamate glycerols are
poorer antioxidants compared to their starting acids, although they are more
water-soluble than FA thus have a wider range of applications in water-based
industries, such as food, beverage and cosmetic industries. Sinapic acid and its
derivatives have the potential to be used in antiaging skin cosmetics, sun screen and
agents to improve DNA repair. They have also been implicated in cosmetic
preparations for the treatment of skin and dietary diseases and stimulation of hair
growth ailments (Faulds 2010).
5.7 Conclusions
A clean technology for the sustainable production of biofuels and fine chemicals is
anticipated, utilizing the agricultural and agroindustrial residual biomass. The use of
enzymes is essential for the production and bioconversion of bioactive compounds
from waste residues resulting in a competitive price of biofuels through the
biorefinery concept. FAEs will have a profound role in utilizing waste biomass for
the production of energy and high-added value compounds. Since their discovery in
1990s, novel FAEs have been isolated and characterized showing different varia-
tions in physical characteristics that result in a diverse specificity for the hydrolysis
of model and natural substrates. In addition, many attempts have been made to
elucidate the functional relationships between sequence-diverse enzymes in respect
to their corresponding structure; however, the limited number of crystal structures
available complicates this mission. In this review, FAEs are shown to be involved
not only in the degradation of residual plant biomass, but also as tools for the
synthesis of novel bioactive components for use in health and cosmeceutical
industries. The improvements in molecular biology and process engineering permit
the overproduction of FAEs in different expression hosts, opening the way for their
commercial production that is prerequisite for many industrial applications.
Acknowledgments Paul Christakopoulos and Io Antonopoulou would like to thank the EU

Framework 7 project OPTIBIOCAT for the financial support.
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Chapter 6
Endo-b-1,4-xylanase: An Overview
of Recent Developments
Alexandre Gomes Rodrigues
Abstract This chapter presents the main important aspects about the enzyme
endo-beta-1,4-xylanase. It starts with the biological grounds for the use of the
enzyme. Moving on, the advancements in the production process and optimisation
are substantially described, along with the microorganisms and a broad range of
molecular techniques used to obtain best performance enzyme. Finally, it delineates
the most common applications related to xylanase, such as in biobleaching, baking,
feedstock and the enzymatic role in juice and ink industries, among others.
Furthermore, the material brings updated research performed during the last few
years combined with essential established facts along the time.
6.1 Introduction
Since ancient times the mankind has made used of enzymes to produce goods as
wine, beer and bread. The gained knowledge in fields as chemistry, biology,
engineering and biotechnology have enabled more precise and efficient processes to
obtain and apply such biomolecules not only in the existing products but also to
create new ones, exploring fully the potential of a material and expanding its roles.
In this regard, plants serve us in many aspects, with their fundamental biological
and microbiological functions, and have been increasingly exploited to provide
valuable biomaterials, which are employed to boost advancements in daily life with
better and suitable products, while reducing the damage caused to the environment
thereby making our lives more comfortable. Hemicelluloses are the second most
present polysaccharide in plant cell wall and are made up of important polymers as
xylan (Schaedel et al. 2010). Xylanases are enzymes secreted by microorganisms,
mostly fungi and bacteria are used in a broad range of important manufacturing
pathways in the papermaking industry as well as in the food industry and in the
A.G. Rodrigues (&)

Institute of Pharmacy, Martin-Luther University Halle-Wittenberg,
Halle, Germany

126 A.G. Rodrigues
production of chemicals such as xylitol, in addition to their natural function. Due to

its key role the xylanases have reached an outstanding market position. The esti-
mative was that the market in which the enzyme is present represents about 200
million dollars (Sonia et al. 2005). In 2005, xylanases along with cellulases and
pectinases sliced 20 % of the global industrial enzyme market (Polizeli et al. 2005).
The innumerous applications of xylanases have raised interest and attracted many
researches and resources towards its whole production chain (Wong et al. 1988;
Coughlan and Hazlewood 1993; Beg et al. 2001; Harris and Ramalingam 2010; Juturu
and Wu 2012). Xylanase transforms water insoluble hemicellulose into soluble form,
which binds water in the dough, therefore decreasing the dough firmness, increasing
volume and creating finer and more uniform crumbs (Butt et al. 2008). The enzymes
break down xylan into oligosaccharides, lowering the viscosity, resulting in more
appropriate processed food and drink, as dough in baking, in animal feed to increase
digestibility, wood pulp, liquid fuels, solvents and other chemicals (Bhat 2000; Butt
et al. 2008). The current technological knowledge and available facilities provide
means by which the processes regarding the obtaining of xylanases and process
optimisation are improved. A wide range of techniques from molecular biology to
analytical chemistry and biochemical engineering have been reported as enabling
researchers to progress and provide better products continuously.
This chapter comprises the fundamental aspects related to endo-b-1,4-xylanase,
namely its early appearance and applications which have encouraged much effort
and investment, along with the most recent studies conducted worldwide and their
main outcomes leading relevant improvements and achievements in the field.
6.2 Plant Cell Wall Structure
Plant cell wall contains a variable amount of different chemical compounds making
up their structures. The cell wall itself is composed of three layers. The compounds
present in a plant cell wall are cellulose and hemicellulose, pectin and lignocellu-
lose. After cellulose, hemicellulose is the most abundant natural polysaccharide in
plants and accounts to one-third of plant biomass. However, it varies according to
plant species due to environmental conditions (Caffall and Mohnen 2009; Schaedel
et al. 2010; Yin et al. 2010). Hemicellulose contributes not only to biological
processes in nature but also in our daily life, through the employment of its com-
ponents in several industrial sectors from feedstock to paper making and bread
baking (Yin et al. 2010).
Schulze (1891) first introduced the term ‘hemicellulose’ for the fractions isolated
or extracted from plant materials with diluted alkali. It is an amorphous
heteropolymer formed by aldopentoses (xylose and arabinose) and aldohexoses
(glucose, galactose, mannose), forming a short polymer chain presenting lower
molar mass, when compared to cellulose (Jacobsen 2000). It is present in hardwood
(angiosperm) as well as in softwood (gymnosperm). Hardwood hemicelluloses are
xylans, whereas softwood consists of glucomannans. The portion present in
6 Endo-b-1,4-xylanase: An Overview of Recent Developments 127
hardwood is the most important due to its higher amount. Xylan backbone is highly
branched by side chains consisting of xylose, arabinose and galucuranic acids (Saha
2003). According to the type of sugar residue hemicelluloses are framed into four
categories: xylans, secondary cell walls of hardwood and herbaceous plants;
xyloglucans, primary cell walls of higher plants (bound to cellulose); mannans,
secondary cell walls of conifers and Leguminosae (Schaedel et al. 2010).
The degradation of hemicelluloses is mostly carried out by microorganisms that
can be found either free in nature or as part of the digestive tract in higher animals.
Such degradation is accomplished with the aid of enzymatic complexes and leads to
the production of several important industrial compounds.
6.3 Endo-b-1,4 Beta Xylanases
Xylan is the major component in hemicellulose in hardwood, but is less abundant in

softwood and accounts for one-third of all renewable organic carbon available on
earth (Prade 1996; Kulkarni et al. 1999). Yet, it is a polysaccharide formed by
monomers of 1,4-b-D-xylose with side chain components, such as arabinosyl,
glucuronosyl, methylglucuronosyl, acetyl, feruloyl and p-coumaroyl residues,
depending on the plant source (Biely 1985; Knob and Carmona 2008).
In the biodegradation of xylan the molecule is broken down into its units,
namely xylose, a pentose monosaccharide that serves as a primary source of car-
bons to bacteria and fungi, and was the first sugar isolated from wood in 1886
(Koch 1886; Sohpal et al. 2010). Xylose itself is composed by arabinofuranosyl,
glucuronyl and acetyl groups. Due to its heterogeneity and complex nature, the
complete breakdown of xylan requires the synergistic action of a large variety of
hydrolytic enzymes (Biely 1985; Coughlan and Hazlewood 1993). The sugar is
hydrolysed into its monomers by action of different enzymes, among them
endo-beta-1,4-xylanase.
Xylanases (EC 3.2.1.8) are classified into glycoside hydrolase (GH) families and
specific information can be found in the Carbohydrate-Active Enzyme (CAZy)
database (Coughlan and Hazlewood 1993; Biely et al. 1997; Pastor et al. 2007). The
protein presents more conserved folds in some families than their amino acid
sequences and these families are grouped into clans (Coughlan and Hazlewood
1993; Biely et al. 1997). Endo-xylanases are found in families 5, 7, 8, 10, 11, 26 and
43. The enzymes from families 10 (formerly known as F) and 11 (formerly known as
G) have been widely described in the literature. Family 10 endo-xylanases posses
higher molecular weight, compared to the ones of family 11, structurally composed
of a cellulose-binding domain and a catalytic domain connected by a linker peptide
(Biely et al. 1997). Family 11 endo-xylanases contain lower molecular weight
enzymes. Based on their isoelectric points, family 11 xylanases are further
sub-grouped into alkaline and acidic enzymes. It is considered that, due to their
relatively small sizes, these endo-xylanases can pass through the pores of hemi-
celluloses network performing efficient hydrolysis (Törrönen and Rouvinen 1997).
128 A.G. Rodrigues
These xylan degrading enzymes are expressed by a wide variety of fungi (Wase
et al. 1985; Bakir et al. 2001; Li et al. 2006) and bacteria (Mamo et al. 2006), but
also actinomycetes (Ball and McCarthy 1988), and yeast (Hrmova et al. 1984).
Even if there is a great variety of xylanases and their folds, mechanisms of action,
substrate specificities, hydrolytic activities and physicochemical characteristics
being diverse, mostly attentions have been driven to two of the xylanases con-
taining carbohydrolase families from families 10 and 11 and are based on their
amino acid sequence homologies (Henrissat 1991). These enzymes catalyse the
1,4-beta-D-xylosidic linkages in xylan (Collins et al. 2005). As described in the
literature (Biely et al. 1983), the enzymes not only hydrolyse the substrate but also
catalyse various biomolecular reactions, such as transglycosylation and degradation
of oligosaccharides. The authors explained the dependence of enzymatic activity on
the substrate concentration and the preference of xylanases from different sources,
as those examined from A. niger and C. albidus, for cleaving glycosidic bonds.
The thermophilic fungus Thermomyces lanuginosus, first isolated in 1899 by
Tsiklinskaya from a potato, is referred as the most suitable microorganism for the
cellulose-free xylanases production both in shake flask and bioreactor cultivations;
however, due to intraspecies variability the enzymatic expression may be affected.
According to extensive studies on the molecular, physiological and ecological
properties of T. lanuginosus strains have been carried out (Singh et al. 2003).
Furthermore, the cultivation conditions of each strain require optimisation since the
strains are influenced by nutrient and growth conditions used in their cultivation. In
addition, Aspergillus niger, Trichoderma reesei and Streptomyces have been fre-
quently explored for the production of xylanases (Pradeep et al. 2013). With the
advancements of recombinant DNA technology, the gene responsible for the
enzyme started being cloned and expressed in other microorganisms, enabling
researchers to end up with purer products, presenting higher enzymatic activity and
in increased amounts. The enzyme has been cloned in bacteria, mostly Escherichia
coli, and filamentous fungi such as Trichoderma reesei and Fusarium oxysporum
have also been employed for this purpose (He et al. 2009; Moukouli et al. 2011). In
most cases, when it comes to the heterologous expression of xylanase, the host
models used are widely applied yeast Pichia pastoris and in some cases E. coli (He
et al. 2009; Cai et al. 2011; Moukouli et al. 2011).
6.4 Molecular Features in Process and Process

Optimisation
As described by Harris and Ramalingam (2010) endo-xylanases cleave the glyco-

sidic bonds in the xylan backbone. The enzymes have been differentiated according
to the end products they release from the hydrolysis of xylan (for instance xylose,
xylobiose and xylotriose and arabinose). Therefore, xylanases may be classified as
non-debranching (arabinose non-liberating) or debranching (arabinose liberating)
enzymes.
Due to the absent of standardisation of the various existing sources of the

enzyme and process parameters Bailey et al. (1992) compared the measurement of
xylanase activity among twenty different laboratories. According to the authors, the
major source of variation between apparent xylanase activities was probably the
substrate chosen, although small differences in protocols were also significant. After
standardisation of substrate 260 and method, the interlaboratory standard variation
of the results decreased from 108 to 17 % from the mean.
Based on its technical and economic importance and vast range of applications,
endo-xylanases have been extensively investigated regarding its expression and
process optimisation, particularly over the last few years. Due to the current con-
ditions of operations it is desired that the employed enzyme presents thermostability
and an adequate pH range in the production process. The molecular features are of
upmost concern since detailed knowledge is required to obtain more efficient
processes and appropriate amounts of product with high quality. In order to
overcome enzyme limitations under diverse conditions and enhance stability,
among other important issues related to enzyme yield and industrial scale pro-
duction, many research groups have focused on molecular engineering techniques
in search for enzymes with superior performance, and substantial results have been
reported. Moreover, hybrid xylanases have been created and tend to be increasingly
applied in the coming years to enable such progress. Because the processes in
which endo-xylanases are applied are undertake in harsh conditions, such as the pH
in the case of pulp bleaching, there are specific requirements to improve the process
conditions (Biely et al. 1983; Nakamura et al. 1993; Stephens et al. 2007).
Therefore, it is crucial to understand the properties of the enzyme, its structure and
tune its features as possible in order to achieve optimal efficiency (van Dyk et al.
2010).
An endo-xylanase isolated from the soil showed high sequence homology with
the xylanases from Bacillus pumilus and Clostridium acetobutylicum in the
N-terminal region, with stability in alkaline pH (9.0) and optimum temperature for
the activity around 50 °C (Nakamura et al. 1993). The influence of medium
compounds in the induction and repression of xylanase has been described (Gomes
et al. 1994) demonstrating that arabinose, glucose, lactose and xylose were inef-
fective in the induction of xylanase from Thermoascus aurantiacus. The study
showed yet that while wood xylan, birch wood glucuronoxylan and methyl/3-D-
xylopyranoside (MXP) induced xylanase, glucose acted as a repressor in that case.
Purkarthofer and Steiner (1995) also studied the effect of carbohydrates on the
production of inducible b-xylanase of Thermomyces lanuginosus, concluding that
D-pentoses as well as xylan and xylobiose served as inducers, but D-lyxose, D-
arabinose, D-ribose and L-arabinose were significantly not as pronounced as xylose,
xylobiose and xylan. The crystallographic structure of endo-xylanase I from family
G, which typically exhibit acidic pH, resulting in a proposed model to serve as an
explanation of the mechanisms that govern the low pH optimum of the enzyme
(Krengel and Dijkstra 1996). By solving the structure of an endo-xylanase from
Aspergillus niger employing molecular replacement Krengel and Dijkstra
130 A.G. Rodrigues
(1996) revealed aspects of the enzyme conformation and purposed that two glu-
tamate residues would be involved in the catalysis.
De Groot et al. (1998) cloned the gene encoding xylanase from Agaricus bis-
porus and determined its regulation by heterologous screening techniques. Georis
et al. (1999) presented a recombinant xylanase from Streptomyces sp. The authors
studied the cleavage site between the signal peptide and the protein and compared
the similarities and strict identities of the enzyme with other family 11 xylanases.
By means of crystallography the group of Professor Mizuno in Japan (Fujimoto
et al. 2000) depicted the structure of the xylan-binding domain endo-beta-xylanase
family 10 from Streptomyces olivaceoviridis. Turunen et al. (2001) reported the
synergistic effect of a xylanase II from Trichoderma reesei brought about by sta-
bilising mutations to access the information about its thermal stability, catalytic
function and pH-dependent properties. Zhengqiang et al. (2001) isolated two
xylanases genes from the bacterium Thermotoga maritima. The genes were sub-
sequently cloned and expressed in E. coli and the authors found that the optimum
condition for the enzyme activity was at 90 °C and pH of 6.14. A disulphide bridge
into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase was
engineered by Fenel et al. (2004), increasing the enzyme thermostability by about
15 °C. Subsequently Fenel et al. (2006) achieved high stability using a mutant
xylanase from Trichoderma reesei, which increased brightness in sulphate pulp
bleaching. Brutus et al. (2004) showed the cloning, purification and expression of
endo-xylanase from Penicillium funiculosum in E. coli and Pichia pastoris as hosts.
Wakiyama et al. (2008), Jun et al. (2009) and Do et al. (2013) also published studies
related to the expression of xylanase in the same host. The group of Professor
Murray contributed to the elucidation of the structure of a endo-xylanase (family
10) from Thermoascus aurantiacus, showing that the glycone subsite of the enzyme
makes extensive direct and indirect interactions with the arabinose side chain and
concluding that xylan side chains are not just accommodated but can actually
constitute significant substrate specificity determinants in the enzyme under study
(Vardakou et al. 2005).
Another study explored the function of three enzymes, namely xylosidase–ara-
binosidase from Thermoanaerobacter ethanolicus and xylanase from Thermomyces
lanuginosus, with the aim to achieve more efficiency in the decomposition of ara-
binoxylan agricultural residue for the bioconversion into fuels or industrial chemi-
cals (Xue et al. 2009). The multifunctional enzyme was expressed in E. coli and
exhibited enhanced enzymatic activity in the degradation of arabinoxylan into its
monomer constituents when compared to the corresponding free enzymes. Vieira
et al. (2009) reported the effect of temperature and binding of substrate on the
dynamics of the family 11 xylanase from Bacillus circulans and its molecular
dynamics simulations in the presence of xylobiose. The study indicates that the
thermophilicity of the 11/G xylanases might be increased by rational design based on
two variables: mutations that lead to changes in the flexibility of the thumb loop and
mutations of residues in the substrate-binding cleft that aim to optimise the xyla-
nase–substrate intermolecular interaction. With that the authors purposed that the
residues may be considered as preferential targets for site-directed mutagenesis
experiments that modulate the enzyme substrate affinity, thus increasing the catalytic
efficiency of the enzyme. The work Michaux et al. (2010), based on crystallographic
techniques, with an acidophilic xylanase from Scytalidium acidophilum, a fungi
isolated from an uranium mine in acidic waters (pH 2.0), revealed features about the
stability and acidophilic adaptation. The conclusions were that various sequence and
structure modifications may be responsible for the acidophilic characteristic of the
enzyme. They would be the presence of aspartic acid hydrogen bonded to the
acid/base catalyst; the nature of specifically conserved residues in the active site; the
negative potential at the surface and the decreased number of salt bridges and
hydrogen bonds in comparison with highly alkaline enzymes.
A xylanase cloned and expressed in E. coli by Hwang et al. (2010) had its activity
not affected by most salts, such as NaCl, LiCl, KCl, NH(4)Cl, CaCl(2), MgCl(2),
MnCl(2) and CsCl(2) at 1 mM; however, the enzymatic activity was affected by
CuSO(4), ZnSO(4) and FeCl(3). As described, xylobiose was the major product
obtained. The presence of NaCL at 12.5 proved to be not only beneficial but
also the optimum condition to an endo-xylanase from thermophilic bacterium
Thermoanaerobacterium saccharolyticum isolated from a hydrothermal vent (Hung
et al. 2011). Furthermore, the expression of endo-xylanase has been described in
homologous (Anasontzis et al. 2011) and heterologous (Damásio et al. 2011) models
and applied in the production of ethanol and functional foods, respectively. In 2012
Trevizano et al., by means of direct evolution, improved the thermostability of
endo-xylanase. The mutant enzymes generated through error-prone PCR presented a
good performance in extreme pH conditions, retaining their ability to hydrolyse
birchwood and oat spelt xylans, according to the authors. Kim et al. (2012) studied
the intra-molecular hydrophobic interactions of xylanase, applying network analysis
to interpret such events, and noticed that the enzyme half-life was increased by
78-fold, highlighting the advantages of interpreting collective hydrophobic inter-
action patterns. Verma and Satyanarayana (2012) also reported a thermostable gene
from Geobacillus thermoleovorans expressed in E. coli with enzymatic production
of 27-fold higher than the wild strain. Thermostabilisation was once more investi-
gated by Li et al. (2013). Using Dictyoglomus thermophilum as an enzymatic source
of xylanase, the researchers stabilised the enzyme by an engineered N-terminal
disulphide bridge. The stabilisation was then tested against high temperatures, being
achieved at 100–110 °C and the ionic liquid used in the study appeared to affect the
functioning of the enzyme’s active site to a greater extent than the stability of an
already thermostable protein structure. Recently, Mander et al. (2014) produced a
cellulase-free xylanase from Streptomyces sp. using an agricultural residue (wheat
bran) as a growth substrate. The outcome was that the enzyme was not only able to
hydrolyse commercially available pure beechwood xylan to xylose, xylobiose and
xylotriose, but also abundantly available lignocellulosic agricultural residues in
nature such as wheat bran to xylooligosaccharides. Other studies have also
approached the thermal and alkaline stability, as well as enzyme–substrate interac-
tion of hybrid xylanases, as the ones reported by Song et al. (2014), Stephens et al.
(2014) and Chen et al. (2014).
132 A.G. Rodrigues
Lately, the enzymatic stability was investigate by Li et al. (2015a, b) employing

a disulphide mutant at pressure and temperature as high as 500 MPa and 80 °C,
respectively. Likewise, Qian et al. (2015) found that an engineered xylanase was
stable at 55 °C in a broad active pH range (5.5–10), being purposed as a candidate
for industrial applications given its high specific activity, stability and soluble
protein yield. Huy et al. (2015) observed the synergistic action of a endo glucanase
together with endo-xylanase, with increased reducing sugar release of birchwood
xylan (6.4 %), beechwood xylan (13 %) and arabinoxylan (15.8 %) and larger
increase of reducing sugar release from pretreated barley straw that addition at the
start or by treatment with endo-xylanase alone, showing a great potential applica-
tion for hemicellulose saccharification.
6.5 Techniques Used in Xylanases Process
A specific range of techniques has been used to enable researchers and producers
transforming what nature provides us, making important and always better goods in
daily life. From molecular biology to analytical chemistry and biochemistry engi-
neering, these methods and their improvements assure better results and more
efficient processes to be conducted.
Regarding the recombinant DNA techniques used in the process of obtaining,
characterising and optimising key factors of the enzyme, polyacrylamide gel
electrophoresis (SDS–PAGE) is a widely used technique to determine molecular
mass of proteins, thus aiding the characterisation of such biomolecules (Pribowo
et al. 2012). The technique has been applied through the decades as a key tool to
identify xylanases and is part of the essential technique repertoire (Biely et al. 1983;
Coughlan and Hazlewood 1993; Takahashi et al. 2013). Polymerase chain reaction
(PCR) and gene cloning comprises the paramount employed in the analysis and
improvement of the enzyme production and analysis of its properties (Stephens
et al. 2014).
Among the analytical chemistry techniques, UV/vis spectrophotometry has been
employed to analyse the enzymatic activity of xylanases (Sonia et al. 2005).
Chromatographic methods, namely high performance liquid chromatography
(HPLC), have been used to purify the enzyme as demonstrated in a recent work
(Pribowo et al. 2012). Column chromatography is another method to purify the
enzymes from cultivation (Heck et al. 2006), as well as thin layer chromatography
(TLC) (Chanwicha et al. 2015), used in the analysis of xylanases. Along with the
above-mentioned methods, mass spectrometry finds its use in the xylanases process
as described by Jänis et al. (2007) for the determination of steady-state kinetic
parameters for a xylanase-catalysed hydrolysis of xylooligosaccharides. Puchart
and Biely (2008) carried out the production of endo-beta-1,4-xylanase using
Thermomyces lanuginosus, and the authors studied the compounds formed
employing mass spectrometry, suggesting that the fungus might be of importance
regarding the conversion of xylan into xylooligosaccharides.
6.6 Biomass Production Methods
Xylanolytic enzymes from various sources, and most importantly from microor-
ganisms, have been studied along the last decades to understand their physical and
biochemical characteristics, due to their applications in several fields (Polizeli et al.
2005).
Xylanases from natural sources are produced only to relatively low yields;
therefore, the enzyme production cannot meet the demand of our societies. In view
of this, heterologous expression approaches have been implemented to design
xylanases with the desired characteristics for the production at the industrial scale.
The expression of fungal xylanase genes in Saccharomyces cerevisiae, Pichia
pastoris and other microorganisms has been reported in the literature (Karaoglana
et al. 2014). The enzyme properties and yield somehow vary depending on the gene
source, host system and secretion signal sequences, besides the process parameters.
Until the advent of recombinant DNA technology, enzymes were produced by
fermentation of the microorganisms that express the enzymes. Cultivation of fila-
mentous fungi for large-scale protein production is very complex, often ending up
in many interfering enzymes. Purification of target enzymes from a pool of proteins
requires several purification steps thereby increasing their costs (Kormelink et al.
1993). Recombinant DNA technology allows large-scale expression of these
enzymes in both homologous and heterologous protein expression hosts (Puchart
and Biely 2008; Juturu and Wu 2012).
Regarding the methods of obtaining endo-b-1,4-xylanases, solid-state fermen-
tation (SSF) is the most present in the literature (Filho et al. 1993; Pandey 2002;
Sonia et al. 2005; Chanwicha et al. 2015). SSF is the most suitable method for
xylanase production, since it deals with low water activity environment and allows
the use of agro waste substrates like corn and sugarcane bagasse. The method
received more attention after during 1950–1970 with the steroid transformation
using fungal cultures and the production of proteins for cattle feed enrichment. Later
on SSF would gain more importance with the design of bioreactors and the microbial
production of food, feed and enzymes such as lipase, phytase, cellulase, ligninase,
amylase, glutaminase, pectinase and xylanase, besides the production of organic
acids, secondary metabolites, fatty acids and biofuel (Pandey 2002; Bhargav et al.
2008). Compared to submerged fermentation (SmF), it facilitates the recovery of the
final product, requires less energy (no need for vigorous agitation) and has high
volumetric production, being thus eco-friendly and industrially favourable (Filho
et al. 1993; Pandey 2002; Bhargav et al. 2008). The method is applied under
microbial conditions in which cultures are closer to their natural habitat, considering
that fungi grow in nature on solid substrates such as pieces of wood, seeds, stems and
roots, therefore having their activity increased. The process can still be classified on
the grounds of substrate nature, which can be pure or mixed (Bhargav et al. 2008).
Key factors have to be satisfied if appropriate production is intended to be
achieved. Among these are moisture and water contend. This is due to the fact that
water activity of the substrate influences the microbial activity, spore production
134 A.G. Rodrigues
and metabolites secreted. Another issue to be addressed is the removal of heating

since in SSF a large amount of heat if generated based on the low thermal con-
ductivity of the substrates used. As important are the biomass determination and the
mass transfer during the process, where O2 and CO2 diffusion, nutrient absorption
and metabolites formation, airflow into and out of the SSF system, types of sub-
strate, mixing of substrate, bioreactor design, space between particles, variation in
particle size and microorganisms all play an important role (Bhargav et al. 2008).
Among the aspects to be considered when working with solid-state fermentation
are the selection of the suitable microorganism and substrate, optimisation of the
process parameters and isolation and purification of the product. Studies have been
conducted in this direction as reported by Sonia et al. (2005), who investigated the
endo-xylanase production by Thermomyces lanuginosus in SSF using sorghum
straw as substrate, obtaining high levels of xylanases. In another study the method
was employed by Irfan et al. (2014), in order to obtain endo-xylanase from
Trichoderma viride. The authors evaluated the enzymatic production using different
substrates such as wheat bran, rice polish, rice husk, soybean meal, sunflower meal,
sugarcane bagasse or corn cobs and found that sugar cane was the best substrate in
that occasion.
For practical reasons, immobilisation of microorganism or enzymes on solid
materials offer several advantages, including repeated usage of the catalyst, ease of
product separation and improvement of enzyme stability (Beg et al. 2001).
Endo-xylanase has also been successfully immobilised in other microorganisms as
Trichoderma reesei (Haapala et al. 1996) and Bacillus pumilus (Kapoor et al.
2008).
6.7 Technological Developments

of Endo-Beta-1,4-xylanase
Apart from its use in the pulp and paper industry, xylanases are also used as food
additive, in wheat flour for improving dough handling and quality of baked
products, for the extraction of coffee, plant oils and starch, and in the improvement
of nutritional properties of animal feed, and in combination with pectinase and
cellulase for clarification of fruit juices (Biely 1985; Wong et al. 1988; Maat et al.
1992; Beg et al. 2001).
6.8 Biobleaching
Xylanases and other side-cleaving enzymes have been used in pulp bleaching
primarily to reduce lignin and increase the brightness of the pulp. This process is
necessary due to the presence of residual lignin and its derivatives in the pulping
process, which causes the resultant pulp to gain a characteristic brown colour. The
use of xylanase in bleaching pulp requires the use of enzymes with special char-
acteristics (Beg et al. 2001).
A key requirement is to be cellulose-free, to avoid damaging the pulp fibres, as
cellulose is the primary product in the paper industry (Srinivasan and Rele 1999;
Subramaniyan and Prema 2000). In pulp bleaching, the enzyme must tolerate high
temperature and pH after alkaline cooking to be economical (Turunen et al. 2001).
The process of lignin removal from chemical pulps to produce bright or completely
white finished pulp is called ‘bleaching’. It is necessary for aesthetic reasons and for
improvement of paper properties, because the left-over residual lignin after sulphite
pulping imparts an undesirable brown colour to the paper. The bleaching of kraft
pulp uses large amounts of chlorine-based chemicals and sodium hydrosulfite.
These bleaching chemicals cause several effluent-based problems in the pulp and
paper industries. Byproducts from using these chemicals are chlorinated organic
substances, some of which are toxic, mutagenic, persistent and bioaccumulate, and
cause numerous harmful disturbances in biological systems (Onysko 1993). In
response to public interests, paper industries are currently changing practices to
minimise the use of chlorine-based chemicals. The available options are oxygen
delignification, extended cooking and substitution of chlorine dioxide for chlorine,
hydrogen peroxide and ozone. But most of these methods involve high capital
investment for process change. Thus, an alternative and cost-effective method, i.e.,
use of enzymes, has provided a very simple and economic way to reduce the use of
chlorine and other bleaching chemicals. Biobleaching involves using microorgan-
isms and enzymes for bleaching pulp. It relies on the ability of some microor-
ganisms to depolymerize lignin directly and on the use of microorganism or
enzymes that attack hemicellulose and hence favour subsequent depolymerization
(Jimenez et al. 1997).
Several criteria are essential for choosing a microorganism to produce xylanases.
To give the desired bleaching effect, the resulting enzyme preparation must be
completely free of any cellulase activity (Srinivasan and Rele 1999; Subramaniyan
and Prema 2000; Beg et al. 2001), since any cellulase activity will have serious
economic implications in terms of cellulose loss, degraded pulp quality and
increased effluent treatment cost.
Use of hemicellulolytic enzymes was the first large-scale application of enzymes
in the pulp and paper industry (Viikari et al. 1986). Limited hydrolysis of hemi-
cellulose in pulps by hemicellulases (mainly xylanases) increased the extractability
of lignin from the kraft pulps and reduced the chlorine required in subsequent
bleaching. The xylanase from T. reesei has been reported to act uniformly on all
accessible surfaces of kraft pulp and to be effective during biobleaching (Saake
et al. 1995; Suurnakki et al. 1996; Bhat 2000).
The enzyme is already on the market and has been successfully applied in the
bleaching process as demonstrated by Shatalov and Pereira (2007), nevertheless
many efforts have been done with the aim to improve its performance.
An xylanase with highly specific activity towards xylan was reported by Li et al.
(2005), with a narrow optimum pH range (7.0–7.5) and decrease in the chlorine
136 A.G. Rodrigues
consumption of just over 28 %. Battan et al. (2007) employed Bacillus pumilus to

produce xylanase, achieving an increase of 13-fold after process optimisation. The
enzyme was used in the biobleaching of eucalyptus kraft pulp. It was observed an
improvement in the brightness as well as in the whiteness, besides a decrease in the
yellowness of the material. Other parameters such as viscosity tensile strength,
breaking length, burst factor and burstness also presented better properties.
Moreover, the reduction in the consumption of chloride used was of 20 %. Another
study, from the group of professor Hazlewood, in California, screened 200 xyla-
nases and assessed their performance in biobleaching of softwood and hardwood
from different regions in the US, Canada and South America (Esteghlalian et al.
2008). The enzyme with the best properties was a xylanase from family 11,
although alkali-tolerant enzymes from the family 10 were also identified and tested.
The later enzyme, however, presented less effective bleachability, leading to a
minor reduction of chemicals at the same enzyme activity level when compared
with the selected candidate of family 11.
Maalej-Achouri et al. (2012) reported a reduction of 40 % of chloride con-
sumption using xylanase from Talaromyces thermophilus for the bleaching of kraft
pulp. The synergism of xylanase and a commercial laccase resulted in a higher
enzymatic activity when the sample was treated for 3 h at 50 °C. The enzyme
showed optimal activity under slight basic pH (7–8). Zheng et al. (2012) obtained
50 % reduction of chloride consumption and 3.65 % increase in the brightness on
cotton stalk pulp using a recombinant xylanase. The researchers also used the
enzyme in the saccharification of paper sludge in combination with mixed cellu-
lolytic enzymes and found that the efficiency of recycled material was increased by
10 %. Besides that, the recombinant enzyme presented thermal stability in the range
of 30–80 °C and in the pH from 4 to 9 being, as proposed by the group, suitable for
industrial applications in pulp biobleaching and bioethanol production. Other
studies have demonstrated the usefulness and importance of using endo-xylanases
in the biobleaching process with enhanced brightness, whiteness and significant
reduction of chemicals in the process, contributing thereby to better environmental
practices (Saleem et al. 2009; Kumar et al. 2009; Li et al. 2010; Ko et al. 2011; Lin
et al. 2013).
6.9 Baking
Hemicellulases, especially endo-xylanases have also been used to improve the

quality of dough, bread, biscuits, cakes and other bakery products (Poutanen 1997;
Butt et al. 2008). Although the endo-xylanases are known to exhibit many bene-
ficiary effects during dough handling and baking, improving the texture and shelf
life of bread, their actual mechanism of action is not well understood. It has been
hypothesised that the ability of endo-xylanases to hydrolyse arabinoxylan present in

dough facilitates the redistribution of water in both dough and bread, and is
responsible for the observed favourable effects on dough handling, bread volume,
texture and stability.
The enzyme was first introduced in the baking industry in the 1970s. They affect
rheological properties of dough, as well as the organoleptics properties of the bread
(Qi Si and Drost-Lustenberger 2002; Collins et al. 2006). Besides that, the addition
of endo-xylanases during dough processing is expected to increase the concentra-
tion of arabino-xylo-oligosaccharides in bread, which have beneficiary effects on
human health. Recently, arabinases, a-L-arabinofuranosidases, arabinoxylan a-L-
arabinofuranohydrolases and esterases have been reported to play important roles in
improving the texture, quality and sensory attributes of bakery products (Poutanen
1997). However, a suitable combination of these enzymes is vital for achieving
maximum benefit during dough processing and baking.
The hole of endo-xylanase purified from Trichoderma reesei in rye baking was
investigated by Autio et al. (1996), using two rye varieties, Danko (Polish culti-
vated in Sweden) and Muskate (Canadian). The authors found a lesser viscosity in
the bread mass after adding endo-xylanase. In relation to the fermentation assay, the
dough in both varieties tested increased with the use of xylanase with slight dif-
ference between the samples analysed; however, the bread volume was not
increased. This fact might be related to the degradation of arabinoxylans and beta
glucans, leading to the release of the gas during the process, which plays an
important role in the bread volume. The enzyme also influenced the softening of the
dough. Besides that, it was observed that the amount of xylan present in the rye also
influences properties as the molecular weight when xylanase was used, since it
affects the amount of xylooligosaccharides generated. Collins et al. (2006) studied
the properties of endo-xylanase family 8 in the baking of Argentinean long breads
and Belgian hard rolls. The increase in the bread loaf volume was higher when
using xylanase 8 than with the compared xylanase 10. Furthermore, the cut width
was also increased with xylanase 8.
6.10 Hydrolysis of Xylan into Xylooligosaccharides
The hydrolysis of xylan by endo-xylanases has already been reported during the last
decades in order to elucidate the structure of such xylooligosaccharides and the
enzymatic activity (Gorbacheva and Rodionova 1977; Meagher et al. 1988;
Kormelink et al. 1993; Anand and Vithayathil 1996; Vardakou et al. 2005; Rantanen
et al. 2007; Yang et al. 2011; Li et al. 2012, 2014).
Recently, the manufacture of xylooligosaccharides (XOS) from lignocellulosic
materials as novel sweeteners and functional foods attracts growing interest.
Oligosaccharides are generally defined as saccharides containing between 2 and 10
sugar moieties (Wang et al. 2009; Damen et al. 2012). As one of the non-digestible
oligosaccharides (NDOs), XOS exhibit many important and interesting functional
138 A.G. Rodrigues
properties, including non-toxicity, non-metabolism by human digestive system,

promotion of bowel function, calcium absorption, lipid metabolism, and growth of
beneficial intestinal bacteria, and reduction of colon cancer by forming short-chain
fatty acids. Because xylan is stable in alkaline media, it can be obtained by dis-
solving in caustic liquors and then precipitating with organic solvents. Then
enzymatic hydrolysis was extensively employed to degrade xylan to produce XOS,
due to less undesirable byproducts and specialised equipments. During this bio-
conversion process, the branch structure of natural xylan requires the coordination
of several hydrolases to breakdown. 1,4-b-D-xylanase xylanohydrolase hydrolyses
1,4-b-D-xylosidic bonds within the b-(1,4)-linked D-xylosyl backbone of xylan
randomly and liberate b-anomeric XOS (Belkacemi and Hamoudi 2003).
Due to the fact that chemical methods are less specific and may result in removal
of side groups and considering the importance of xylooligosaccharides in xylan,
arabinoxylans have been investigated and its production has been optimised, in this
way, resulting in better process conditions. Enzymes, specific glycosidic linkages
can be split, resulting in unmodified oligosaccharides (Kormelink et al. 1993;
Vardakou et al. 2005). The arabinoxylan backbone can be randomly hydrolysed
using endo-xylanases (EC 3.2.1.8), producing a mixture of various oligosaccha-
rides. Arabinose side groups are liberated by beta-L-arabinofuranosidases (EC
3.2.1.55) and exo-xylosidases (EC 3.2.1.37) release xylose units from the
non-reducing end of xylo-oligosaccharides (Biely 1985). The fragments released by
xylanase represent usually a complex mixture of arabinoxylooligosaccharides, as
reported by Puchart and Biely (2008).
Guerfali described the synergistic action of an immobilised endo-beta-
1,4-xylanase from Talaromyces thermophilus (Guerfali et al. 2011). The potential
of the combined use of endo-xylanase and arabinofuranosidase was highlighted as a
potential catalyst source and final application in food processing. Likewise, Várnai
et al. (2011) studied the combined action of endo-xylanase and endo-b-mannanase,
leading to a higher liberation of glucose than that theoretically expected in gluco-
mannan in softwood.
The obtainment of xylooligosaccharides using a plethora of microorganisms as
endo-xylanases source as well as its engineering in order to optimise the process
and improve efficiency has been continuously explored, especially in the last few
years in order to provide the needs the modern societies demand (Gonçalves et al.
2012; Verma and Satyanarayana 2012; Zhao et al. 2012; Chen et al. 2013; Kim
et al. 2014; Bibi et al. 2015; McCleary et al. 2015; Yang et al. 2015).
6.11 Feedstock
The animal feed industry is an important sector of agro-business with an annual

production of 600 million tons of feed, worth 50 billion US dollars. Of the total feed
produced, the major share is taken by poultry, pigs and ruminants (up to 90 %),
while the pet foods and fish farming account for 10 % (Bhat 2000).
Xylans are major constituents in the non-nutritional constituent of feed in

monogastric animals since the animals do not naturally produce the enzymes
required for the breakdown of these components. Another reason for the use of the
enzyme is that, with increasing concerns associated with antibiotic resistance, there
is pressure to reduce the use of antibiotics in feed. Alternatively probiotics can be
added to feed as a replacement for antibiotics. Since probiotics are effective in
enhancing the immune system, increasing body weight gain, reducing diarrhea and
improving feed conversion efficiency. Cereals (namely barley, wheat, rye and oats)
are major feed components for monogastric animals. The cell walls of cereals are
primarily composed of carbohydrate complexes referred to as nonstarch polysac-
charides, such as b-glucans in barley and wheat and arabinoxylans in rye and oats.
Due to low digestibility of b-glucans and arabinoxylans and their propensity to
form high-molecular-weight viscous aggregates in the gastrointestinal tract, these
polymers exhibit in antinutritive effect. Therefore, addition of specific enzymes
such as xylanase or b-glucanase into wheat- or barley-based diets for nonruminant
animals decreases viscosity and consequently reduces the antinutritional effect of
nonstarch polysaccharides, leading to better production performance (Liu et al.
2005). Xylanases have been extensively used to improve the performance in animal
feed (Beachemin et al. 1999; Deng et al. 2006; Cai et al. 2011). In feed application,
the use of the enzyme requires a set of parameters. It has to be stable in high
temperature during feed preparation, but the catalytic activity is needed at the body
temperature of domestic animals. The addition of xylanase in animal feed to
degrade xylans converts the more complex polysaccharides into fermentable energy
sources. This helps to maintain a lower pH, which helps exclude potential enter-
opathogens. The enzyme has a major economic importance in developing countries,
where the potential of improved dairy of ruminants is of significant importance
(Phakachoed et al. 2012). Improvements in animal performance due to the use of
enzyme additives can be attributed mainly to improvements in ruminal fibre
digestion resulting in increased digestible energy intake (Phakachoed et al. 2012).
Grounded on role of the enzyme in the animal feed, several researchers have
concentrated their efforts in the investigation properties and optimisation process of
xylanases in this regard. Liu et al. (2005) cloned and expressed three rumen microbial
fibrolytic enzyme genes in a strain of Lactobacillus reuteri and investigated the
probiotic characteristics of these genetically modified lactobacilli. The genes of
Neocallimastix patriciarum xylanase, Fibrobacter succinogenes b-glucanase and
the Piromyces rhizinflata cellulase were cloned in a strain of L. reuteri isolated from
the gastrointestinal tract of broilers. The enzymes were expressed and secreted under
the control of the Lactococcus lactis lacA promoter. According to the authors the L.
reuteri transformed strains not only acquired the capacity to break down soluble
carboxymethyl cellulose, b-glucan, or xylan but also showed high adhesion efficiency
to mucin and mucus and resistance to bile salt and acid. Other studies followed this
pattern and investigated in more detail the performance of xylanase in the field
(Panwar et al. 2013; Pedersena et al. 2015).
140 A.G. Rodrigues
6.12 Xylitol
Xylitol is the second most abundant polyol derived from xylose (Kirilin et al.
2012). Its application hits a broad spectrum in the actual scenario and more are
promising to come true (Aranda-Barradas et al. 2010; Kaialy et al. 2014). The sugar
alcohol is metabolised in the body by insulin-independent pathways and presents
good sweetness as sucrose, however, being less caloric. For these reasons the
molecule has been applied in the food industry (present in chewing gums, sweets,
soft drinks and ice creams, for instance) as an artificial sweetener in the treatment of
diabetes and erythrocytic glucose-6-phosphate dehydrogenase deficiency (Ping
et al. 2013).
It has also been used as a starting material in organic chemistry reactions, as a
potential anticancer compound, in the biomedical field, as a dental caries inhibitor
and oral biofilm formation (Huang et al. 2011; Li et al. 2015a, b; Cardoso et al.
2014; Ma et al. 2014). The molecule has a strong market position, in the range of
90–340 million dollars a year, with a production of 20,000–40,000 tons per year,
and therefore has been extensively studied regarding its production parameters
(Granström et al. 2007; Aranda-Barradas et al. 2010; Li et al. 2015a, b).
Kirilin et al. (2012) described the aqueous phase reforming (APR) process to
produce hydrogen. The work led to interesting results about the stability of
hydrogen formed and the conversion of xylitol. Apart from that, the researchers also
obtained higher yield and selectivities of xylitol than sorbitol towards hydrogen.
Methods to improve the performance in the food industry have also been attempted
as the microencapsulation of xylitol in gum arabic by complex coacervation method
(Santos et al. 2015). Saleh et al. (2014) explored the obtainment of xylitol through
olive stones. The researchers employed response surface methodology (RSM) to
optimise the hydrolysis conditions, analysing treatment temperature and process
time as key factors. Another study (Zhang et al. 2015) conducted at high temper-
ature, using engineered Kluyveromyces marxianus, resulted in high yield, demon-
strating the potential of thermo-tolerant organisms in the biomass conversion of
xylitol, using glycerol as the best substrate in this case. The simultaneous of ethanol
and xylitol production by pinch analysis was performed by Franceschin et al.
(2011).
The group of professor Miyatake in Japan recently reported synthesis of xylitol
microcapsules to encapsulate phase-change materials (PCM), a material capable of
storing and releasing large amount of energy, thereby improving its durability while
adding the advantages of smaller particles in relation to the bulk material (Makuta
et al. 2015).
6.13 Juice and De-inking Processes
Together with pectinases, cellulases and other hemicellulases, xylanases is part of

complex macerate enzymes, used in the extraction and clarification of juices. These
enzymes improve the texture and decrease the viscosity of the juices, contributing
to a more palatable end product. These enzymes participate in two steps of juice
production: after crushing, to macerate the fruit pulp either to partial or complete
liquefaction, which not only increases the juice yield and reduces the processing
time, but also improves the extraction of valuable fruit components, and after the
juice extraction, where mainly pectinases are used for its clarification, thereby
lowering the viscosity of fruit juice prior to concentration and increasing the fil-
tration rate and stability of the final product (Bhat 2000).
The application of enzymes in de-inking has been intensively studied in both
laboratory and pilot scales, but the technique has not yet been commercialised
(Buchert et al. 1998). The two principal approaches in using enzymes for de-inking
include the hydrolysis of soy-based ink carriers by lipase and the release of ink from
fibre surfaces by cellulases, xylanases and pectinases. The main advantage of
enzymatic de-inking is the avoidance of the use of alkali. De-inking, using enzymes
at acidic pH, also prevents the alkaline yellowing, simplifies the de-inking process,
changes the ink particle size distribution and reduces the environmental pollution. In
addition, the enzymatic de-inking improves the fibre brightness, strength properties,
pulp freeness and cleanliness as well as reduces fine particles in the pulp. Xylanase
treatment has been reported to increase the strength properties, while cellulase
treatment improved the brightness and freeness of the pulp (Prasad 1993).
6.14 Final Remarks
Endo-beta-1,4-xylanases comprise a series of enzymes obtained mostly in fungi and

applied in a wide range of processes. The roles of the enzyme in the chemical, food
and paper industries position this biomolecule as an important and interesting
biomaterial. The complexity of its obtaining and production optimisation has
gathered researchers from various fields during the last decades to collaborate and
contribute to advance towards better manufacturing processes.
Efforts have been applied in order to push through advancements that may result
in a finer understanding of the functionality and behaviour of the xylanases and its
relation with other molecules. Back in 1988, Meagher et al. determined the subsite
map of these hydrolases secreted by A. niger in order to get insights in the bond
cleavages in oligosaccharides and much progress have been noticed since then.
Molecular biology has been used as an important technique to perform such tasks
as enhancing the thermo stability of the enzyme, thereby enabling the makers of
goods to obtain the best possible products with maximum efficiency and expand the
range of applications to the ever growing needs and opportunities (Zhang et al. 2010;
142 A.G. Rodrigues
Deesukon et al. 2011; Karaoglana et al. 2014). The advancements in molecular

biology have provided tools to clone and express xylanases in heterologous hosts
and produce hybrid enzymes, thereby enhancing the quality and increasing the yield
of the product by tuning specific features, key factors in its production.
In short, the function of endo-xylanase in the production of paper, chemicals and
in the food industry has proved to be one of the central points in such fields and will
indeed continue to expand, fostering technological advancements and benefiting us
the best products through the most efficient processes.
Acknowledgments The author thanks Prof. V.K. Gupta for the invitation to write this chapter.
The previous work in the field was supported by the Coordination for the Improvement of Higher
Level Personnel (CAPES) and São Paulo Research Foundation (FAPESP), Brazil.
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Chapter 7
Microbial Xylanases: Sources, Types,
and Their Applications
Hesham Ali El Enshasy, Subeesh Kunhi Kandiyil, Roslinda Malek

and Nor Zalina Othman
Abstract Biomass conversion to an utilizable energy sources such as monomer

sugars using enzymatic hydrolysis has been emerged as the current technology
which promises the future energy. In nature, bioconversion process of biomass is
mediated by a group of biofunctional hydrolytic enzymes. These enzymes generally
work in cooperative synergetic action to facilitate enhanced effective degradation of
biomass. Xylanase is one of the crucial hydrolytic enzymes involved in hydrolysis
of xylan, the hemicellulose which constitutes 15–30 % of the plant biomass. This
chapter discusses in detail about the enzymatic hydrolysis of xylan by the xylolytic
enzyme endo-1,4-b-xylanase, its occurrence in nature and mode of action, structure
and classifications, current methods for its production, purification, and character-
ization. In addition, the major and recent industrial applications of this enzyme were
highlighted as well.
7.1 Introduction
Nowadays, the governments promote extensive researches for the development of

an alternative transportation fuel from a renewable energy sources, realizing the
upcoming major energy crisis due to the depletion of petroleum-derived fuels. As
an initiative, the department of energy in US has started producing biofuels with a
set target of 60 billion gallon per year by 2030. Europe also has the similar targets
by that time to replace 25 % of petroleum-based liquid transportation fuel by
biofuels (Himmel et al. 2007). However, it is a challenging target since the biofuel
production using substrates such as sugar cane and corn has limited capacity to
supply such a huge volumes. Apart from this, in many countries people protest
against the use of food crops for biofuel production and many debates that it leads
to a major food crisis. As a remedy, researchers found out that the lignocellulose
H.A. El Enshasy (&) S.K. Kandiyil R. Malek N.Z. Othman

Institute of Bioproduct Development (IBD), Faculty of Chemical Engineering (FKK),
University Teknology Malaysia (UTM), 81310 Johor Bahru, Malaysia

152 H.A. El Enshasy et al.
biomass which consists of 75 % polysaccharide sugars can be used as a significant

feedstock for biofuel production (Lynd et al. 1991). If the attraction is more, lig-
nocellulose biomasses are renewable energy source and exist abundant in nature
and it can be derived predominantly from agricultural wastes (Gomez et al. 2008).
Lignocelluloses are heteropolysaccharides that give structural rigidity to all kinds of
plant. They account for approximately more than 50 % of the total biomass in the
nature, synthesized (estimated 10–50 1012 tons year−1) by plants through pho-
tosynthesis (Claassen et al. 1999). Lignocelluloses are composed of three major
polymeric constituents such as cellulose (an unbranched linier polymers of long
b-1-4 glucan between 2000 and 27,000 units), hemicellulose (a heterologous
polysaccharides which are complex in nature with sugar monomeric subunits such
as D-xylose, D-mannose, D-galactose, and L-arabinose) and lignin (complex phenolic
structure act as linkage between cellulose and hemicellulose.
The name hemicellulose was first introduced by Schulze (1891). These
heteropolysaccharides are abundant in plant cell wall and have complex structures
when compared to cellulose due to the enormous number of sugar monomeric side
chains (500–3000). It creates a cross-linked network between the cellulose
microfibrils and lignin and plays a major role in structural integrity of plant cell
walls. It also regulates the rate of expansion of primary cell wall and acts as a shield
over cellulose to protect it from enzymatic hydrolysis. Hemicellulose is the second
most abundant renewable biomass that is available after cellulose within the lig-
nocellulose matrix representing about 20–35 % of total biomass (Saha 2003).
Table 7.1 summarizes the approximate composition of various biomass materials.
Classification of hemicellulose was made based on the types of sugar monomer
units present on them. As mentioned above, hemicelluloses are composed of xylan,
mannan, galactan, and arabinan polymers. Among these xylan is the most fre-
quently occurring polymer composed mainly of b-D-xylopyranosyl residues which
linked by b-1,4-glycosidic bonds (Beg et al. 2001).
In hardwoods plants, hemicellulose primarily consists of xylans and gluco-
mannans whereas in softwood plants, it is composed of arabinoglucuronoxylans
Table 7.1 Approximate composition (as a percentage) of various biomass materials

Biomass Cellulose Hemicellulose Lignin References
(%) (%) (%)
Bermuda grass 47.8 13.3 19.4 Li et al. (2010)
Reed canary straw 42.6 29.7 7.6 Bridgeman et al. (2008)
Corn cobs 35–39 38–42 4.5–6.6 Okeke and Obi (1994)
Rice straw 41 21.5 9.9 Lee (1997)
Bagasse 38.1 26.9 18.4 Lee (1997)
Coffee pulp 24.0 8.9 19.4 Dijkerman et al. (1997)
Coconut fiber 17.7 2.2 34 Dijkerman et al. (1997)
Rice hulls 24–29 12–14 11–13 Okeke and Obi (1994)
Corn stove 39 19.1 15.1 Lee (1997)
Wheat straw 36.6 24.8 14.5 Lee (1997)
Sawdust 45.0 15.1 25.3 Dijkerman et al. (1997)
7 Microbial Xylanases: Sources, Types, and Their Applications 153
Table 7.2 The hemicellulolytic enzymes and their substrates (Shallom and Shoham 2003)
Enzymes Substrates
Endo-b-1,4-xylanase b-1,4-Xylan
Exo-b-1,4-xylosidase b-1,4-Xylooligomers
a-L-Arabinofuranosidase a-Arabinofuranosyl, (1–3) xylooligomers, a-1,5-arabinan
Endo-a-1,5-arabinanase a-1,5-Arabinan
a-Glucuronidase 4-O-methyl-a-glucuronic acid, (1–2) xylooligomers
Endo-b-1,4-mannanase b-1,4-Mannan
Exo-b-1,4-mannosidase b-1,4-Mannooligomers, mannobiose
b-Galactosidase a-1,6-Galactopyranose, mannooligomers
b-Glucosidase b-1,4-Glucopyranose, mannopyranose
Endo-galactanase b-1,4-Galactan
(xylans), xyloglucans, glucomannans, and arabinogalactans. Xylans are the major

form of hemicellulose made of D-xylopyranosyl units which are linked by
b-1,4-glycosidic bonds. b-mannan-based polymers are the second major form of
cellulose which are made of b-1,4-linked mannose and/or glucose residues.
Galactoglucomannans which consist of a-1,6-linked galactose side chains are also
grouped under these polymers. They are found abundant in hardwood hemicellu-
lose, which comprised varying ratios of D-mannopyranose units a-(1–6)-substituted
galactopyranose side chains with O-acetyl side chains and b-1-4-linked-D-gluco-
pyranose units. Arabinan and arabinogalactans are also considered as hemicellu-
loses which are generally composed of a-1,5-linked L-arabinofuranosyl units.
Table 7.2 summarizes the major enzymes involved in the breakdown of
cross-linked hemicelluloses. Those groups of enzymes which are involved in
hydrolysis of xylan are generally known as xylanases. The current book focuses on
one of the crucial xylolytic enzymes known as endo-1-4-b-xylanase.
7.2 Enzymatic Hydrolysis of Xylan
In most terrestrial plant species, secondary cell wall thickening takes place by the
deposition of xylan and they are found in-between cellulosic fiber sheets and lignin
with complex binding relationship through hydrogen bonding. Xylan thus forms a
think wall over cellulose and enables the protection from degradation by different
cellulose degrading enzymes. Xylan constitutes 15–30 % of the plant biomass in
hardwood and 7–12 % of the plant biomass in softwood. Since they are the major
hemicellulose group in most of the plant species, they have been considered as one
among the renewable source of energy in the form of biomass. Structure of xylan is
complex in nature as they are composed of b-1,4 linked-D-xylopyranose units with
a-L-arabinofuranose and 4-O-methyl-a-D-glucuronopyranosyl acid side chains.
Figure 7.1 shows the major enzymes involved and their site of action during the
enzymatic hydrolysis is xylan (Polizeli et al. 2005).
Fig. 7.1 Enzymatic hydrolysis of xylan (Polizeli et al. 2005)
Hardwood xylan (e.g., birchwood xylan) consists of more than 70 b-1,4 linked-
D-xylopyranose units linked by b-1,4 glycosidic bonds and 4-O-methyl-a-D-glu-
curonopyranosyl acid side chains which are found attached at second carbon of
every tenth xylopyranose units. Acetylation rate is higher in these groups and it is
quite frequent at second and third carbon atom on xylopyranose ring. In softwood
xylan, acetylation rate is zero but instead of an acetyl group they possess a-L-
arabinofuranose linked by a-1-3 glycosidic bond at the third carbon atom on
xylopyranose ring. Based on the side chains of the xylan backbone, they are
classified into different groups such as linear homoxylan, arabinoxylan, glu-
curonoxylan, and glucuronoarabinoxylan. These four groups are heterogenic in
terms of their degree and nature of branching. In many plants, xylans are found
partially acetylated, which protect them from complete degradation by xylolytic
enzymes. This could be the reason behind the fact that the complete degradation of
xylan is accomplished only by synergetic interaction of acetyl xylan esterase and
endo-xylanase enzymes.
Due to the heterogeneity and complex chemical structure of polymer, the

complete breakdown of xylan requires the synergetic action of several hydrolytic
enzymes which are specific in their mode of actions. Generally, xylans are not
attacked randomly, but the bonds selected for hydrolysis depend on several bio-
chemical factors that determine the nature of the substrate molecules such as the
chain length, the degree of branching, and the presence of substituents.
The major xylolytic enzymes involved in xylan hydrolysis are as follows:
• Endo-1,4-b-xylanase—cleaves long xylan backbone into short
xylooligosaccharides.
• b-xylosidases—also attacks the b-1,4-glycosidic linkages to liberate xylose
from short oligosaccharides.
• a-L-arabinofuranosidases—remove L-arabinofuranose side chains.
• a-D-glucuronidases—hydrolyze the methyl glucuronate residues.
• Acetyl xylan esterases—hydrolyze acetate groups on xylan backbone.
• Ferulic acid esterases—hydrolyze the aromatic acids groups linked on arabi-
nofuranoside residues.
Endo-1,4-b-xylanase: (EC 3.2.1.8) cleaves the glycosidic bonds between the D-
xylopyranose units on the xylan backbone and as a result, short xylooligosaccharides
are formed. b-D-xylosidases: (EC 3.2.1.37) liberate xylose from short xylooligosac-
charides which are formed during xylan hydrolysis by endo-1,4-b-xylanase. These
enzymes are classified based on their relative affinity for the xylooligosaccharides into
distinct entities such as xylobiases and exo-1,4-b-xylanases. Even though they are two
different enzymes, they have been recognized as b-D-xylosidases in general, and
releases b-D-xylopyranosyl residues from small xylooligosaccharides and xylobiose.
Their affinity for the xylooligosaccharides is inversely proportional to its complexity.
Xylobiose was found to be the best substrate for these enzyme groups. These groups of
enzymes play important role in xylan hydrolysis when there is endo-1,4-b-xylanase
inhibition due to the accumulation of short oligomers of b-D-xylopyranosyl units, by
hydrolyzing these products and remove the cause of inhibition thereby enhancing the
xylan hydrolysis (Andrade et al. 2004). The molecular weights of these enzyme
groups are relatively high, between 60 and 260 kDa and their pH optima is between
4.0 and 5.0. Generally, these enzymes are thermophilic in nature and their optimum
temperature varies from 40° to 80°. However, in most cases they exhibit highest
activity around 60 °C (Rizzatti et al. 2001). a-L-arabinases: These enzymes remove L-
arabinose side chains which are substituted at second and third carbon on b-D-xylo-
pyranosyl ring of arabino xylan. They have been classified as exo-a-L-arabinofur-
anosidase (EC 3.2.1.55) and endo-1,5-a-L-arabinase (EC 3.2.1.99), based on their
distinct mode of action. The first group, which is most common, degrades branched
arabinans and p-nitrophenyl-a-L-arabinofuranosidase, whereas the second group
hydrolyzes only the linear arabinans (de Vries et al. 2000). The a-D-glucuronidases
(EC 3.2.1.131) can hydrolyze the a-1,2 linkages between glucuronic acid and xylose
residues found in glucuronoxylan. They are classified under glycoside hydrolase
family 67 and rarely found in nature. During biodegradation of glucuronoxylan, these
enzymes are able to release glucuronic acid. However, the enzyme activity is only
limited on short xylooligomers due to the partial hindrance by acetyl group present in
xylan backbone (Puls et al. 1991). Acetylxylan esterase: (EC 3.1.1.6) it acts by
removing the O-acetyl groups from second and third carbon on b-D-xylopyranosyl
ring of acetyl xylan. This enzyme plays important role in xylan hydrolysis by
removing O-acetyl side chains, since O-acetyl groups in acetyl xylan interfere the
activity of endo-1,4-b-xylanase and b-D-xylosidases on xylan backbone. Xylan
extraction from hardwoods is usually mediated by alkali treatment, resulting in acetyls
hydrolysis, and probably this could be the reason of the late discovery of this enzyme
(Shao and Wiegel 1995; Blum et al. 1999). Ferulic acid esterase: Ferulic acid esterases
(EC 3.1.1.-) act by hydrolyzing the bond between arabinose side chain and ferulic acid
group attached (Crepin et al. 2004). The cooperative functioning among the
above-mentioned xylolytic enzymes enhance the complete biodegradation of xylan. It
was also observed that acetylxylan esterase activity on xylan results in deacetylation
of xylan backbone and these deacetylated xylans are more easily hydrolyzed by
endo-xylanase. Similarly, there was a significant increase in hydrolysis efficiency
when arabinoxylan was pretreated by a-arabinofuranosidase. This was due to the
removal of arabinan side chains which act as hindrance to endo-xylanase activity (de
Vries et al. 1999).
7.3 Occurrence of Endo-1,4-b-xylanase
Endo-1,4-b-xylanase are also known as endo-(1!4)-b-xylan 4-xylanohydrolase,

endo-1,4-xylanase, xylanase, b-1,4-xylanase, endo-1,4-xylanase, endo-b-1,4-
xylanase, 1,4-b-xylan xylanohydrolase, b-xylanase, b-1,4-xylan xylanohydrolase,
endo-1,4-b-xylanase, b-D-xylanase, 4-b-D-xylan xylanohydrolase, endo-(1!4)-
beta-xylan 4-xylanohydrolase, beta-1,4-xylanase, endo-beta-1,4-xylanase, endo-1,
4-beta-D-xylanase, 1,4-beta-xylan xylanohydrolase, beta-xylanase, beta-1,4-xylan
xylanohydrolase, endo-1,4-beta-xylanase, beta-D-xylanase, and 4-beta-D-xylan
xylanohydrolase. In nature, the endo-1,4-b-xylanase enzymes are widely distributed
among eukaryotes and prokaryotes. The occurence of these enzymes was also
reported in higher eukaryotes such as plants, protozoa, small insects, and several
marine species. A Japanese pear fruit was reported producing endo-xylanase during
its over-ripening period. Endo-xylanase of molecular weight 55 kDa was also iso-
lated from wheat flour (Cleemput et al. 1997). Apart from these sources, members of
higher animals such as insects and fresh water mollusc were also reported as xylanase
producers (Yamura et al. 1997). However, bacteria, fungi, and actinomycetes were
found to be the largest source of xylanase enzyme.
There have been several reports on microbial endo-1,4-b-xylanase since 1960.
Nevertheless, most of these xylanase enzymes remained unnoticed. During 1980s
first studies on xylanase-mediated bio-bleaching in pulp industry was reported and
only since then the scientific world realized the great impact of xylanase enzymes in
industrial applications. As mentioned above, a number of microorganisms including
bacteria and fungi have the capacity for hydrolyzing xylans by synthesizing a
variety of xylolytic enzymes. Early reports reveal that many of these microbial
species are plant pathogens as they play an important role in degradation and
invasion of plant tissues. It also shows that xylanases can elicit defense mechanisms
in plants by a collective functioning with other cellulolytic enzyme groups.
7.3.1 Fungal Endo-1,4-b-xylanase
Many of the fungal species that are pathogenic to plants produce plant cell wall
polysaccharide degrading enzymes. Endo-1,4-b-xylanase is one among the major
group of such enzymes and they result in partial degradation of cell wall structures
to the region of penetration (Subramaniyan 2000). A hypothetical model of fungal
invasion on plant tissue is shown in Fig. 7.2.
A number of fungal species have been used to produce xylanase since they are
the major producer of xylanases in nature. Table 7.3 shows the major fungal species
used to produce xylanases. Phanerochaete chrysosporium, a potent plant pathogen,
reported to produce xylanase activity of 15–20 U mL−1 along with considerable
amount of cellulose activity (Copa-Patino et al. 1993). Other study also showed that
the thermophilic fungus Thermomyces lanuginosus has the capacity of high xyla-
nase production up to 3576 U mL−1 (Singh et al. 2000). Many fungal species have
been reported as xylanase producers; however, species with high xylanase activity
and negligible cellulase activity are found to be very rare in nature. It was also
observed that the pH optima of many of these fungal xylanases are between pH 5
and 6 even though they are stable at pH between 3 and 8. On the other hand,
bacterial xylanases showed slightly higher pH optima that makes them suitable for
many of the industrial applications. Thermal tolerance of the majority of fugal
xylanase reported so far has been found below 50 °C and in most cases this
Fig. 7.2 Fungal invasion on plant tissue by xylolytic degradation (Prade 1996)
Table 7.3 Major fungal xylanase and its characteristics

Source Molecular weight Optimum conditions References
(kDa) Temperature pH
(°C)
Aspergillus 39, 23, 26 45–55 4.0–5.5 Kormelink et al. (1993)
awamori
A. fumigatus 19, 8.5 55 5.5 Silva et al. (1999)
A. kawachii 35, 26, 29 60, 55, 50 5.5, 4.5 Ito et al. (1992)
A. nidulans 55 8 Taneja et al. (2002)
A. oryzae 35 60 5 Kitamoto et al. (1999)
A. nidulans 22, 34 62, 56 5.5, 6.0 Fernández-Espinar et al.
(1994)
A. sojae 32.7, 35.5 60, 50 5.0, 5.5 Kimura et al. (1998)
A. sydowii 33 50 4 Ghosh and Nanda (1994)
particular property of fungal xylanase makes them less favorable for application in
harsh industrial conditions. Reduced xylanase yield in fermenter studies is another
major problem associated with fungi xylanase. This was due to poor oxygen
transfer, shear force during fermentation, and the enzyme production process in
fungi is highly regulated by growth morphology (El Enshasy et al. 1999, 2006; El
Enshasy 2007). Generally, the fungal species has filamentous growth pattern, the
fungal growth in fermenters is restricted due to the shear stress, and eventually this
results in poor xylanase production (Dean et al. 1991).
7.3.2 Bacterial Endo-1,4-b-xylanase
Since 1980s, several bacterial strains which are capable of producing alkaline and
highly thermophilic xylanase enzymes have been reported. Among these wide
spectrum of bacteria, Bacilluss species are the most predominant producers of
endo-1,4-b-xylanase enzyme with negligible amount of cellulose activity under
optimized growth conditions. Other author showed a high yield of xylanase up to
506 IU mL−1 by Bacillus SSP-34 (Subramaniyan and Prema 2002). In general,
bacterial endo-1,4-b-xylanase is suitable for industrial application since they have a
wide pH optima and improved thermostability when compared to fungal xylanases.
Ratto et al. (1992) reported Bacillus circulans strain with xylanase activity of
400 IU mL−1. They observed the optimal enzyme activity was at pH 7 and retained
40 % of activity at high pH up to 9.2. Many other researchers reported
cellulose-free xylanase enzyme from Bacillus stearothermophilus strain T6 (Khasin
et al. 1993; Lundgren et al. 1994). A relatively high xylanase activity was reported
in Bacillus sp. strain NCL when it was grown in zeolite-induced medium
(Balakrishnan et al. 2000). Thermophile bacteria such as Rhodothermus marinus
produced approximately 1.8–4.03 IU mL−1 of thermostable xylanase along with
Table 7.4 Major bacterial xylanase and its characteristics

Source Molecular weight Optimum conditions
(kDa) Temperature pH
(°C)
Aeromonas caviae ME1 20 50 7
Bacillus amyloliquefaciens 18.5–19.6 80 9
B. circulans WL-12 85 – –
Bacillus sp. strain SPS-0 99 75 –
Bacillus sp. W1 (JCM2888) 21.5, 49.5 65, 70 4.5–10, 4.5–7
Bacillus sp. strain 41-1(36) 36 50 9
Bacillus sp. strain TAR-1 40 75 6
Bacillus stearothermophilus T-6 43 75 6.5
Streptomyces T-7 20.6 60 4.5–5.5
Streptomyces sp. No 3137 50, 25, 25 60–65 6-May
Thermotoga maritima – 85 6.5
T. thermarum – 80–100 7
Modified from Subramanya and Prema 2000
detectable amount of cellulose activity. Other strain such as Bacillus circulans

showed high yield of xylanase (400 IU mL−1) which has pH optima of 7 but with a
high stability at alkaline pH (Beg et al. 2000). The concept of TCF (total chlorine
free) bleaching of pulp was developed during this period and it was achieved with
xylanase from Bacillus stearothermophilus strain T6 which has optimum activity at
pH 6.5 (Kohli et al. 2001). A detailed description of major bacterial xylanase and
their characteristics are given in Table 7.4
7.4 Biosynthesis and Regulation of Microbial

Endo-1,4-b-xylanase
Biosynthesis of xylanase enzyme by microorganisms and the phenomenon of

xylanase induction are less investigated at molecular level due to the difficulty in
setting up a cell-free system under experimental conditions. However, Srivastava
and Srivastava (1993) introduced a hypothetical model of xylanase biosynthesis and
regulation to describe similar conditions. Xylans are comparatively complex in
structure with high molecular weight and thus it is usually hard to be utilized
directly by microbes. Xylans are converted into smaller molecules such as xylose,
xylobiose, xylotriose, or xylooligosaccharides by the constitutively produced small
amount of endo-1,4-b-xylanase enzymes. These simpler molecules can be easily
utilized by microbial cell as the carbon source (Zhao et al. 1997). These low
molecular weight molecules can act as inducer for further xylanase expression and
this has been one of the possible explanations for direct induction of xylanase gene.
Xylanases are usually secreted in the media which contains pure xylan or xylan
residues in cultures of different species such as Aspergillus awamori, Trametestrogii,
and Streptomyces sp. QG-11-3 (Beg et al. 2001). However, several exceptional cases
have also been reported such as in Cellulomonas flavigena; xylan acts as a poor
inducer of xylanase gene (Avalos et al. 1996). In some rare cases, for example, in
yeast strain Trichosporon cutaneum, xylanase biosynthesis is induced by certain
positional isomers. In many bacterial species, the xylanase induction is also possible
with various sugars such as D-xylose, D-maltose, D-glucose, and D-arabinose. On the
other hand, in many fungal species, the natural lignocelluloses such as corn cobs,
rice straw, sugarcane bagasse, and wheat bran were found to be capable of triggering
the xylanase induction (Gupta et al. 2000). Other study conducted by Kumar and
Satyanarayana (2011) reported on the wheat bran-mediated xylanase induction in
Bacillus halodurans TSEV1 strain. However, the hypothetical mechanism of direct
induction is questionable when the transportation across the cell wall can be blocked
by larger molecules. Based on the study of Gomes et al. (1994), there has been a
universally supported concept based on intracellular b-xylosidases. They explains
that the larger xylooligomers formed during xylan hydrolysis are directly transported
into the cell matrix and it was further degraded into xylose residues by b-xylosidase
which are present in the cytoplasm.
Hydrolysis of xylooligomers by hydrolytic transporter proteins during their
transportation through cell membrane is another possible explanation for above
phenomenon. There are also rare cases in which the high xylose concentration in
the media inhibits the xylanase gene expression (Strauss et al. 1995). Exclusion of
inducer transport across the cell membrane is another possible reason for poor gene
expression. In similar cases such as in E. coli it has been observed that the presence
of glucose in the media prevents the lactose transportation through the cell mem-
brane which is inducer for lac operon (Borralho et al. 2002). It was also observed
that the xylanase biosynthesis is mediated by complex metabolic pathway in which
the inducer level and the level of repressor molecule to that particular inducer vary
with the organism and their growth medium. For example, the xylanase production
by Streptomycetes sp. was increased when cellulose substrates are used in the
growth media, whereas in Cellulomonas favigena, sugarcane bagasse was found to
be the best inducer of xylanase enzyme (Alejandro et al. 2007). Addition of xylose
in fermentation media increased xylanase production to a significant amount in
Bacillus sp. (Gupta and Kar 2009). However, other studies showed that the pres-
ence of readily utilizable sugars such as glucose, xylose, and galactose can suppress
xylanase biosynthesis in other strains such as in Streptomyces sp. (Bajaj and Singh
2010) and in T. reesei (Mach-Aigner et al. 2010).
7.5 Classification and Structures of Xylanase
Xylanases are considered as one of the major hydrolyzing enzyme groups as they
mediate the xylan degradation to simpler utilizable units. Activity of these enzymes
depends on the substrate specificity as well as substrate complexity. Wong et al.
(1988) classified xylanases into two major groups based on the end product of
hydrolysis reaction. They were debranching enzymes which liberate arabinose and
non-debranching enzymes which do not liberate arabinose residues from a,1,3,L-
arabinofuranosyl. A few years later, another classification system was introduced
based on the physiological properties of xylanase enzymes such as isoelectric point
(PI) and molecular weight (MW). Therefore, xylanases were classified into two
major groups: end xylanase enzymes with molecular weight lower than 30 kDa and
basic pH and those with molecular weight higher than 30 kDa and acidic pH.
However, few years later, this classification system was found incorrect since it
matches for only a few xylanase enzymes.
Henrissat and Bairoch (1993) proposed a broad classification system based on
the similarities in amino acid sequence and catalytic module. The glycoside
hydrolases are further classified into different families. This classification system
has got wide acceptance since it gives information regarding the structural prop-
erties, the catalytic mechanisms of the enzyme, and their evolutionary relationship
within the group. Xylanases were grouped under glycoside hydrolases (GH) 5, 8,
10, and 11 families. However, the enzymes with multi-domain that exhibit
detectable amount of xylanase activity are grouped into GH7, 16, 43, and 62
families (Cantarel et al. 2009). Table 7.5 comprises major xylolytic enzymes, their
classification into CE, GH families, and their current crystallographic structure.
Some of these GH families are further classified into super families that represent a
more distant common evolutionary ancestor. Major GH family xylanase and their
general structures are shown in Fig. 7.3 (Table 7.6).
Table 7.5 Xylolytic enzymes, their classification into CE, GH families, and the their current
crystallographic structure status
Enzyme Enzyme family Structure status
EC GH
Endo-b-1,4-xylanase 3.2.1.8 5, 8, 10, 11 and GH5-Cryst. (1BQC)
43 GH8-Cryst. (1IS9)
GH10-1FH7,
1CLX, 1XYZ
GH11-1H4G, 1BCX,
1IGO
GH43-(1GYD)
Exo-b-1,4-xylosidase/b-xylosidases 3.2.1.37 3, 39, 43, 52 GH3-Cryst. (1EX1)
and 54 GH39-Cryst.
GH43-(1GYD)
a-L-Arabinofuranosidase 3.2.1.55 3, 43 and 51 GH3-(1EX1)
GH43-(1GYD)
GH51-Cryst
Endo-a-1,5-arabinanase 3.2.1.99 43 1GYD
a-Glucuronidase 3.2.1.139 67 1K9D, 1GQI
Acetyl xylan esterase 3.1.1.72 1 (1JJF)
5 1QOZ, 1G66
Ferulic acid esterases 3.1.1.73 1 1JJF, 1GKK
Source Shallom and Shoham 2003
Fig. 7.3 Structures of xylanase major GH family xylanase (Collins et al. 2005a, b)
Table 7.6 CBMs of known structure linked to xylanases (Berrin and Juge 2008)
CBMs family Source of xylanase GH family PDB code
CBM-2 Cellulomonas fimi 10A, 11A 1EXG, 2XBD, 1HEH
CBM-4 Rhodothermus marinus 10A 1K45
CBM-6 Clostridium thermocellum 11A 1UXX, 1NAE, 1UY4
CBM-9 Thermotoga maritima 10A 1I8A
CBM-10 Cellvibrio japonicus 10A 1QLD
CBM-13 Streptomyces olivaceovidris 10A 1XYF
CBM-15 Cellvibrio japonicus 10C 1GNY
CBM-22 Clostridium thermocellum 10B 1DYO
CBM-36 Paenibacillus polymyxa 43A 1UX7
7.5.1 GH10 Xylanases
Most of the glycoside hydrolases which are classified under GH10 are
endo-b-1,4-xylanases. Apart from these enzyme, G10 group also consists of a small
number of endo-b-1,3-xylanases (EC 3.2.1.32), which cleave b-1,3-glycosidic
linkages randomly in b-1,3-xylans backbone. A typical GH10 xylanases has a wide
spectrum of substrate specificity as they are able to hydrolyze various forms of xylans
in nature. These enzymes are capable to attack not only the linear forms of xylan but
also the highly branched heteroxylans and xylosomes. Kolenova et al. (2006)
analyzed the hydrolysis product from glucuronoxylan and confirmed that GH10
xylanases can attack xylan at its non-reducing end if there are two or more unbran-
ched xylose residues which are present on backbone. It was also found that GH10
xylanases can even hydrolyse the highly branched arabinoxylan into small fragments.
Plant xylanases which have been identified so far are grouped under GH10 family. In
recent years many studies were carried out to characterize these plant xylanases and it
was observed that these enzymes have limited substrate specificity (Chithra and
Muralikrishna 2008). The study of Van Campenhout et al. (2007) was focused on the
characterization of GH10 xylanase isolated from barley and they reported that the
enzyme can release small xylose units from various substrates. GH10 xylanases, in
addition to their xylolytic activity, are also active against glucose-derived substrates
like aryl-cello-oligosaccharides (Charnock et al. 1997; Andrews et al. 2000).
Three-dimensional structure of xylan was first described by Atkins (1992).
Subsequently, more studies were carried out using NMR spectrometry and X-ray
crystallography techniques to obtain more carbohydrate structure details. It was
found that under crystallized condition, the xylan backbone exhibits a threefold
left-handed confirmation and the geometry showed that there is no effect on b-1,4
glycosidic bonds by the side chains of those attached. Electron diffraction pattern
study confirmed the structure and the hexagonal morphology. The hydrogen at the
fifth carbon atom on xylose ring has binding properties either intra- or interchain.
The study also suggested that the structural confirmation of D-xylose ring indicated
the di-equatorial binding property of both 1–4 and 1–3 glycosidic linkages.
The X-ray crystallographic image of endo-b-1,4-xylanases enzyme isolated from
Thermoascus aurantiacus has been used to explain the general structure of GH
family 10 xylanase (Fig. 7.4). It was found that the 32.5 kDa long polypeptide
chain with a b/a-fold TIM barrel structure consists of eight major b strands which
are arranged side by side and parallel, forming a cylinder in the center followed by
eight major a helixes. In addition, around six short helixes are also found attached
with the polypeptide chain. The catalytic domains of family 10 xylanases are found
in cylindrical shape and the overall side view resembles a ‘Salad bowl’ (Fig. 7.5).
In this configuration, the catalytic sites are seen relatively closer to the carboxyl
terminus end of xylan backbone. Since the molecular weight of these xylanase is
higher, they always exhibit low degree of oligosaccharide polymerization. Both the
disulfide bond and salt bridges present on the xylan structure improve its ther-
mostability. In the overall structure, the top phase of the molecule which is b-barrel
side has larger than the bottom face, the a-b turns. This was due to the elaborate
architecture of b–a loops (Natesh et al. 1999).
7.5.2 GH11 Xylanases
GH11 xylanase unlike the other xylanase families, they only consist of
endo-b-1,4-xylanases that are exclusive enzymes cleaves b-1,4-xylosidic bonds
between xylose monomers. These enzymes are often sub-classified based on their
Fig. 7.4 a A front view

showing the (a/b) 8 TIM
barrel fold, b salad bowl view
exposing the substrate
binding cleft of
endo-1,4-b-xylanase from T.
aurantiacus (Natesh et al.
1999)
Fig. 7.5 Three-dimensional

structure of NpXyn11A. The
protein schematic is color
ramped from the N terminus
(blue) to the C terminus (red)
(Vardakou et al. 2005)
PI values into acidic and alkaline xylanases (Joshi et al. 1997). Many studies
demonstrated the correlation between the pH optima and the amino acid residues
which are present adjacent to the catalytic site of these enzymes. In many cases, it
was observed that the acidic xylanase (pH < 5) consists of asparagine, whereas in
alkaline xylanase has arginine (Krengel and Dijkstra 1996; Fushinobu et al. 1998).
This was also confirmed by other study which reported that arginine introduction to
bacterial xylanase shifts the pH optimum to acidic range (Pokhrel et al. 2013).
Heteroxylan, xylobiose, and xylotriose are the major subunits formed during GH11
xylanase-mediated xylan hydrolysis. Further hydrolytic activities on these subunits
were found negligible; however, hydrolysis products such as xylotetrose,
xylopentose, and xylohexose are further hydrolyzed by GH11 xylanases
(Cervera-Tison et al. 2009). These enzymes cleave the xylose backbone at
unsubstituted regions which are quite away from the branched xylose present at
non-reducing end. It was also found that GH11 xylanases require minimum of three
unsubstituted consecutive xylose residues for the primary binding and initiation of
hydrolysis (Biely et al. 1997; Katapodis and Christakopoulos 2008).
Ko et al. (1992) reported the first structural description of a GH11 xylanase that
was isolated from Bacillus pumilus. But his analysis was not very precise and he
failed to deposit the 3D structure in protein database. To date, more than 100 GH11
xylanase 3D structures from different fungal and microbial species have been
solved and made available in CAZy database. Xylanases isolated from a ther-
mophilic fungi Thermomyces lanuginosus are widely used to explain the general
structure of GH11 xylanases. The structure shows a globular protein composed of
two b-sheets (b-a, b-b) with a molecular weight of 25 kDa. The outer b-sheet (b-a)
is composed of five antiparallel b strands with polar and uncharged amino acids
such as threonine and serine. The inner b-b sheet is made up of nine antiparallel b
strands of which the outer sides are developed to catalytic sites and the inner sides
are found attached with b-a forming a hydrophilic core (Torronen et al. 1994). In
general, most of the GH11 xylanases are made of only a single catalytic domain and
found in b-jelly roll structure. It also resembles the shape of partially closed right
hand in which two b-sheets represent the ‘fingers,’ the loops between b-strands of
b-b such as B7 and B8 represent the ‘thumb,’ and the twisted inner sides of b-b and
a-helix represent the ‘palm’ of the hand (Gruber et al. 1998).
While comparing these two major GH families, the GH11 family consists only
of ‘true xylanases’ displaying substrate specificity toward D-xylose containing xylan
substrates. Their catalytic versatility is lower when compared to GH10 xylanases
and the products from its action such as xylobiose and xylotriose usually required
further hydrolysis by other xylanase enzymes. The GH10 xylanases can mediate
hydrolysis until the release of xylose as end product. Another interesting feature of
GH10 xylanases is their ability to tolerate glucose-derived to a certain extend in
addition to their xylanolytic activity that exhibits in addition to their xylanolytic
activity (Collins et al. 2005a). It is generally accepted that GH10 can cleave dec-
orated regions on AX backbone and its activity is less hampered by a-L-arabino-
furanosyl and acetyl or 4-O-methyl-D-glucuronate side chains present on xylan
backbone, whereas GH11 xylanases are very specific in their action as they cleave
only at the unsubstituted regions (Biely et al. 1997). This property is also reflected
in the shape of their active binding sites, where GH10 xylanase has a shallow
groove active site which has less affinity toward the unsubstituted regions and
GH11 xylanases possess a cleft-shaped active site which has higher affinity toward
unsubstituted consecutive xylose.
7.5.3 GH8 and GH5 Xylanases
In addition to the above-mentioned GH families, GH8 family xylanase is another

most studied xylanase which consists of both endo- and exo-xylanases enzymes.
Exo-xylanases are those enzymes which reduce xylooligosaccharides (XOS) and
release xylose subunits. A very few number of endo-xylanase have been identified
so far that belong to GH8 family such as xylanases isolated from
Pseudoalteromonas haloplanktis (Collins et al. 2005a); GH8 exo-oligoxylanases
was also isolated from Bifidobacterium adolescentis (Lagaert et al. 2007) and from
Bacillus halodurans (Honda and Kitaoka 2004). In general, the diversity in sub-
strate specificity of these enzymes is large in nature and rXyn8 isolated from
P. haloplanktis is the best example which shows highest activity on mixed linkage
(b-1,3- and b-1,4-bonds) homoxylan (Pollet et al. 2010). Studies on
three-dimensional structure of GH8 xylanase isolated from P. haloplanktis xylanase
show that it is a single-domain molecule with six (a/a)-fold barrel which is found
similar to GH48 enzymes (De Vos et al. 2006). Glycoside hydrolases 5 (GH5) is
another largest enzyme family which consists of more than 1800 entries in the
CAZy database. Under this family, several enzymes with apparent activity on xylan
have been reported. Endo-xylanases from Aeromonas punctuate (Suzuki et al.
1997) and Meloidogyne incognita (Mitreva-Dautova et al. 2006) are the major
reported GH5 xylanases so far. However, certain enzymes in this group such as
xylanase from H. jecorina also exhibit exo-activity. Larson et al. (2003) was the
first one who reported the structure of GH5 xylanase (XynA) isolated from E.
chrysanthemi. In recent years, many other X-ray analysis of GH5 xylanase has been
reported. St John et al. (2009) reported X-ray crystallographic analysis of XynC
from B. subtilis, which shows a multidomain protein (consist of a catalytic domain
along with a C-terminal CBM) with (a/b) 8-fold barrel.
7.6 Catalytic Mechanisms of Endo-1,4-b-xylanase
Hydrolysis of glycosidic bonds is carried out mainly by two different catalytic

residues that are present in the active site of the enzyme. Hydrolysis takes place at
the anomeric center of the substrate molecules either by inversion or retention of the
configuration and this has been depended on factors such as the structure and
Fig. 7.6 A hypothetical model of catalytic mechanism of GH xylanase. The double-displacement

reaction mechanism for retaining glycosidases (Koshland mechanism). Modified from Paës et al.
(2012)
position of catalytic residues on the enzymes (Collins et al. 2005b). Many of the
endo-1,4-b-xylanase exhibit a double-displacement mechanism in their operation
on specific substrate molecules (Fig. 7.6). In most cases, the first acid/base catalytic
residue initiate the hydrolysis by protonating the xylopyranosyl linkages between
the xylan monomers and this process is termed as glycosylation. The second cat-
alytic residue which acts as a nucleophile attacks the same linkage and results in the
formation of enzyme–glycosyl intermediate while passing through an
oxocarbenium-ion-like transition state. In consecutive steps which are also termed
as deglycosylation, the first catalytic residue exhibits basic properties by activating
the incoming water molecule and abstracting a proton from it. The activated water
molecule readily attacks the anomeric carbon of the enzyme–glycosyl complex
formed in the previous step and release the product with an a-configuration at the
anomeric carbon (Sidhu et al. 1999; Chiku et al. 2008).
7.7 Factors Affecting Endo-1,4-b-xylanase Functionality
7.7.1 GH Family Origin
Among the above-mentioned families, endo-xylanases GH10 and GH11 are the
most studied. Endo-xylanase enzymes with relatively higher molecular weight are
grouped into family F/10 and they are found with cellulose-binding domain and a
catalytic domain that are usually connected by linker peptide. Enzyme belonging to
this family also found to have a (b/a)-fold TIM barrel structure (Biely et al. 1997),
whereas the family G/11 xylanase is generally of low molecular weight and is further
classified into two major groups based on their PI value. Similarly, b-xylosidases,
another major xylolytic enzyme is grouped under GH classification system into
various families such as 3, 39, 43, 52, and 54. Studies showed that b-xylosidases
from the families 3, 39, 52, and 54 use a retaining mechanism to hydrolyze
xylooligomers, while those from family 43 mediate the hydrolysis by inverting the
anomeric configuration. Advanced bioinformatics tools such as Basic Local
Alignment Search (BLAST) and pair-wise alignments of the protein sequences have
been used to identify the xylanase enzymes and compared with closely related
enzymes within the families. The sequences of family 10 xylanase are generally used
to identify mutually exclusive enzymes using BLAST search. Further confirmations
are also made using X-ray crystallographic studies. A continuous update to all these
GH family classifications is provided by the carbohydrate-active enzyme database
(CAZy) since there is a direct relationship between the protein folding and the
sequence. This system is universally accepted as it can provide structural features of
enzymes that help to predict their sole substrate specificity; it helps to identify and
reveal the evolutionary relationships between the enzyme groups, and also act as a
convenient tool to derive mechanistic information.
7.7.2 Carbohydrate-Binding Modules (CBMs)
The X-ray crystallographic studies on the xylanase structure reveal that most of
them have a modular structure composed of a catalytic site and one or more
carbohydrate-binding modules (CBMs) that are interconnected by flexible linkages
(Kulkarni et al. 1999; Subramaniyan and Prema 2002; Collins et al. 2005b).
A CBM is defined as a specially arranged amino acid sequence with high affinity to
carbohydrate molecules within a carbohydrate-active hydrolytic enzyme. These
unique sites are designed to bind with specific polysaccharides on plant tissue and
mediate the structural damage by enzymatic hydrolysis (Bolam et al. 2001;
Boraston et al. 2002). Similar to the catalytic modules of glycoside hydrolases,
CBMs are classified into 64 families according to their amino acid sequence sim-
ilarity and this classification is available in CAZy database (Tomme et al. 1994).
A study of Boraston and his group reported a detailed overview about the structure
and functions of CBMs (Boraston et al. 2000). However, a very few members of
GH11 xylanases carry CMB. Among these only a few of them have been experi-
mentally demonstrated, but others are hypothetically explained based on their
sequential similarities with well-studied ones. It have been also reported that certain
CBMs from Xyl-11 are specific for cellulose and this characteristic feature helps the
xylanase to localize the substrates indirectly even when they are in close association
with cellulose. Major CBMs of known structure which are linked to xylanases are
presented in Table 7.7. Xylanases isolated from fungi such as Penicillium funicu-
losum XynB (Brutus et al. 2004) and Neocallimastix patriciarum XynS20 (Liu
et al. 2008) are found to have CMB1 modules. It was also reported that the
Table 7.7 Sensitivity of Xylanase source XIP-1 TAXI-1 TLXI

GH11 xylanases to
proteinaceous inhibitors Fungi
(XIP-1, TAXI-1 and TLXI) Penicillium purpurogenum − + −
(Gebruers et al. 2008) Fusarium graminearum + − −
P. funiculosum + + +
P. funiculosum + + +
Trichoderma + + −
longibrachiatum
T. viride + + +
Bacteria
Bacillus agaradhaerens − − −
B. subtilis − + −
Fibrobacter succinogenes − − −
Fig. 7.7 Spatial

conservation of residues
paving the catalytic cleft of
Xyl-11 (Paës et al. 2012)
occurrence of a C-terminal CBM1 in Xyl-11 isolated from Podospora anserina

found to have significantly improved ability to reduce the sugar during the
hydrolysis of wheat straw (Couturier et al. 2010). Figure 7.7 shows the
three-dimensional structure of NpXyn11A which belongs to GH11. Some GH11
xylanases, apart from the CBMs, also consist of dockerin domains that are key
elements which help in the formation of cellulose-binding multi-enzyme complex
known as cellulosomes. These enzyme complexes are originally observed in
Clostridium thermocellum (Lamed et al. 1983). In addition to cellulolytic enzymes,
cellulosomes are also found with hemicellulase such as xylosidases, xylanases, and
arabinofuranosidases. Other research showed also the similar results in XynC
isolated from Fibrobacter succinogenes S85 (Marrone et al. 2000).
7.7.3 Xylanase Enzyme Inhibitors
Almost all the plant species have developed a self-defense mechanism as response to
the pathogenic microbial attack by hydrolases enzymes. Xylanase inhibition by the
proteinaceous inhibitors is one among those defense response widely seen in both
soft- and hardwood plants. A numerous studies have been carried out to understand
this defense mechanism since 1990s and it was found that Triticum aestivum
xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP) are the two major
groups of proteins responsible for Xyl-11 in specific (Debyser et al. 1999; Rouau
et al. 2006). Apart from the above two groups, the third group of inhibitor known as
thaumatin-like xylanase inhibitor (TLXI) also been isolated from wheat. It was also
found that none of these xylanase inhibitors are effective against plant origin xyla-
nase enzymes (Dornez et al. 2010a, b). Occurrence of these inhibitors seems to be a
significant hindrance in xylanase-mediated industrial process such as bread making
and brewing as they inhibit effective hydrolysis of substrates (Sorensen and Sibbesen
2006). Therefore, it has been an important topic of research to understand the mode
of action of these inhibitors. Sensitivity of GH11 xylanases to proteinaceous inhi-
bitors (XIP-1, TAXI-1, and TLXI) are as given in Table 7.7 (Fig. 7.8).
Fig. 7.8 a Structure of the inhibition complex between P. funiculosum Xyl-11 (in green) and
XIP-I inhibitor (in blue). XIP-I inhibiting loop is in red, catalytic residues are in yellow.
b Structure of the inhibition complex between B. subtilis Xyl 11 (in green) and TAXI-IA inhibitor
(in blue). His374 residue is in red, catalytic residues are in yellow (Paës et al. 2012)
7.7.3.1 Triticum Aestivum Endo-xylanase Inhibitor (TAXI)
These are proteins with pI values of 8.0, which occur in two different molecular
forms known as A and B (Gebruers et al. 2008). Form A is seen as full-length
proteins with molecular mass near to 40 kDa, whereas the form B consists of two
polypeptide chains (a longer chain and a shorter chain of size 28–30 and 10–
12 kDa, respectively) that bound together by disulfide bonds (Croes et al. 2009).
These inhibitors are seen largely in wheat grains during the early stages of grain
development and specifically inhibit GH11 xylanase disrespect to their origin
(Tables 7.8, 7.9). Further analysis of originally identified TAXI inhibitor proteins
revealed that it is a mixture of two different proteins of different pI values. They are
named as TAXI-I and TAXI-II and have different inhibition activities on GH11
endo-xylanase isolated from A. niger (Gebruers et al. 2004). Other research carried
out by Raedschelders et al. (2004) identified and isolated the genes coding for these
two inhibitor proteins from ray and barley grains and based on this study Fierens
et al. (2004) developed a Pichia pastoris-based expression system for the
large-scale production. By 2005, two more new TAXI class named TAXI-III and
TAXI-IV were also identified (Igawa et al. 2005). TAXI-type inhibitors are found
to be very specific in their inhibitory characteristics. The available research data
shows that they inhibit only GH11 endo-xylanases of bacterial and fungal origin.
However, the subclass TAXI-II was turned to be an exception, as they are unable to
inhibit GH11 endo-xylanases such as XynBc1 and ExlA isolated from B. cinerea
and A. niger, respectively (Brutus et al. 2005).
Table 7.8 Occurrence of TAXI-type inhibitors in different cereals (Raedschelders et al. 2004)
Cereal Inhibitor proteins
TAXI-I TAXI-II Inhibitor level (ppm)
Wheat + + ca. 38
Durum wheat + + ca. 11
Rye + − ca. 21–37
Barley + − ca. 4–5
Maize, rice, oats, buckwheat − − −
Table 7.9 Occurrence of Cereal Inhibitor proteins

XIP-type inhibitors in
XIP-I Inhibitor level (ppm)
different cereals
(Raedschelders et al. 2004) Wheat + ca. 80
Durum wheat + ca. 14
Rye + ca. 95
Barley + ca. 3
Maize + ca. 2
Rice, oats, buckwheat − −
Sorghum − −
7.7.3.2 Endo-xylanase Inhibiting Proteins (XIP)
Basically, these are glycosylated single-chained proteins with molecular weight of

30 kDa and its pI value falls between 8.0 and 9.0. The crystallography study shows
that it has similar structural features of GH18 with typical (a/b) 8-fold TIM barrel
shape (Payan et al. 2003). However, they are found to be passive on GH18 enzymes
such as chitinases due to the high degree of mismatch at its enzyme biding site. In
general, these inhibitors consist of two independent enzyme-binding sites that
mediate binding with almost all GH10 and GH11 endo-xylanases that are fungal
origin. However, GH11 endo-xylanase from Neocallimastix patriciarum (Payan
et al. 2004) and GH10 endo-xylanase from Aspergillus aculeatus (Flatman et al.
2002) are found to be insensitive to XIP. Similar to TAXI-type inhibitors, XIP-type
inhibitors are also categorized into different polymorphic families based on the
recent proteomics studies. Followed by identification of Xip-I (the most common
inhibitor in the XIP) class, Durand et al. (2005) identified Xip-II class in T. tur-
gidum ssp. In addition to this, Xip-III type inhibitor encoding gene was discovered
in T. aestivum and was observed that these genes are transcribed very rarely under
stressed leaves. Later on, several other XIP-type genes were also identified known
as Xip-R genes, OsXip that are of plant origin (Takahashi-Ando et al. 2007). Biely
et al. (2008) confirmed the existence of XIP-type inhibitors in maize leaves and
roots during the first week after germination.
7.7.3.3 Thaumatin-like Xylanase Inhibitor (TLXI)
Thaumatin-like endo-xylanase inhibitors (TLXI) consist of a single amino acid

chain with molecular weight of 18 kDa. This type of inhibitor was first discovered
in wheat by Fierens et al. (2007). These proteins have high pI and usually exhibit
glycosylation at varying extents that found to have a very high stability (no
denaturation was observed at 100 °C at extreme pH ranges even after 120 min).
These inhibitors are grouped into class 5 pathogenesis-related proteins as they show
very high sequential similarity with certain antifungal proteins (Trudel et al. 1998).
Even though there are no crystallographic studies that have been reported on
structure of TLXI inhibitor, the structure has been predicted and shows the exis-
tence of five disulfide bridges and three b-sheets (Fierens et al. 2009). The inhi-
bitory mechanism of these proteins was found to be specific against Xyl-11. The
xylanase inhibitors were recently reviewed by Dornez et al. (2010a, b).
7.8 Recombinant Endo-1,4-b-xylanase
The advances in molecular biology and genetic engineering opened up several new
applications of recombinant DNA technology. The new opportunity for the con-
struction of GM microbial strains with selected enzyme machinery was one of the
greatest breakthroughs. On the basis of this, more efficient endo-1,4-b-

xylanase-producing microorganisms were constructed to improve the biodegrada-
tion of hemicellulosic residues and thus find many industrial applications. In this
section, we will highlight in detail the recent advance in recombinant xylanase
production. Moreover, studies on xylanase gene regulation and biosynthesis at
molecular level and various tools applied to enhance the novel characteristic of
xylanase enzymes will be discussed.
Several xylanases have been isolated and identified from various sources.
However, very limited numbers of these enzymes are found to be suitable for
industrial applications due to their poor performance in harsh process conditions.
Poor thermostability is the biggest issue associated with most of the xylanases.
Enzymes with higher thermostability can enhance the efficiency of industrial pro-
cess with an increased residual time. Therefore, there has been a huge demand for
thermostable xylanases in pulp and paper industry as they are applied during pulp
bleaching process which usually carried out at temperatures more than 90 °C. Most
of the xylanases we have today are not able to withstand this high temperature.
However, highly thermophilic xylanases have been reported from thermophilic
bacteria such as G. thermoleovorans and Thermotoga sp. (Sharma et al. 2007).
Alkali stability is also another major problem associated with industrial applica-
tions. It was observed that most of the highly thermophilic xylanases are less stable
under alkaline conditions which limit their potential use in industries. Most of the
xylanases reported so far have optimal activity at either acidic or neutral pH (Wang
et al. 2010). Exceptional cases are also reported, such as xylanases from Bacillus
halodurans which exhibit high alkali tolerance (Mamo et al. 2006), but these
enzymes are found to have poor thermostability. Cellulose-free xylanase is another
desirable characteristics required in pulp bleaching industries to mediate efficient
hydrolysis without disturbing the cellulosic content of the pulp. Studies also shows
that low molecular weight xylanases are more efficient in pulp bleaching as they can
easily diffuse into pulp fibers. In practical and economical point of view, growing
these extremophilic microorganisms in large scale is not feasible. Moreover, it is
difficult to have a single enzyme with all desired characteristics. Conventional
enzyme stabilization techniques such as helix capping and salt bridging (Puchkaev
et al. 2003) have been practiced to improve the enzyme characteristics. However,
these methods are found to be ineffective when the enzyme is required in large
quantities. To overcome these problems and enhance the large-scale production for
the industrial application, recombinant xylanases were introduced using gene
cloning and expression techniques. rDNA technology has been widely used
nowadays to make alteration or modifications in existing wild-type xylanase to
improve their specific characteristics such as thermostability and alkaline stability.
A number of xylanase coding genes from different microbial species have been
isolated and expressed successfully in suitable expression hosts. The expression
level of xylanase gene in host cell usually depends on several factors such as
cloning strategies used, host cell, biosynthesis mechanism, gene regulation, fer-
mentation media, and many bioprocessing parameters. Bacterial and fungal species
are generally used to express xylanases in large scale. The cloning vector selection
is usually carried out based on the expression host and a wide variety vectors have
been introduced in last few decades. Even though the expression rate is higher in
recombinant strain, the enzyme activity is usually less than in the native producer
strain. Several reasons have been identified for this activity loss and the intercellular
accumulation of the enzyme due to the lack of post-translational modification is
suggested to be the key reason (Schlacher et al. 1996).
7.8.1 Cloning and Expression of Fungal

Endo-1,4-b-xylanase Gene
It has been more than three decades since filamentous fungi are used in the pro-
duction of xylanase enzyme. Fungal cells are the widely used biofactories in
large-scale production of xylanases and have been considered as more potent
xylanase producers as they are capable of secreting much higher amounts of xyla-
nolytic enzymes in fermentation media than bacteria or yeast (Bergquist et al. 2002;
Fang et al. 2008). Large numbers of fungal strains are widely used for xylanases
production such as many species from Penicillium, Aspergillus, Fusarium,
Trichoderma, and Disporotrichum. However, in many of these fungi, the difficulty
in isolating pure form of xylanase was the major problem since they generally
exhibit a low amount of cellulase activity. Recombinant DNA technology has been
widely applied to overcome this drawback and production of xylanase expression
systems which are free of cellulase activity which improved its acceptability in
industrial applications. Apart from the bacterial expression hosts, filamentous fungi
are also attractive hosts for xylanase expression. It is because of their ability to
secrete the proteins into the fermentation media in large quantities. Aspergillus and
Trichoderma species are the widely used expression hosts in homolog expression
system. Kitamoto et al. (1999) successfully overexpressed XynF1 gene in A. oryzae
under a strong TEF1 promoter in glucose-based submerged fermentation. XynF1,
XynF3, and XynG2 are another important xylanase genes expressed efficiently in
A. oryzae under stronger promoter called P-No 8142 in similar fermentation con-
ditions. Aspergillus niger is another attractive host for recombinant xylanases
production due to its ability to produce and secrete protein in high capacity up to
30 g L−1 in the fermentation media (Hessing et al. 1994). Xylanase coding gene
from T. reesei was successfully expressed in A. niger cD15 stain under
G6P-dehydrogenase promoter and glaA terminator which is isolated from
Aspergillus awamori (Rose and van Zyl 2002). This recombinant xylanase retained
its 75 % activity even after 3 h of incubation at 50 °C. The optimal condition of the
enzyme activity was observed at temperature between 50 and 60 °C and pH 5–6.
Levasseur et al. (2005) also reported on the potential use of Aspergillus niger as host
to express xynB under promoter sequence isolated from Aspergillus nidulans. They
showed that, A. nidulans can be used as efficient host for xylanase expression and
production. Kimura et al. (1998) expressed xynG1 in A. nidulans under its own
promoter showed that the xylanase expression was induced in the presence of
Table 7.10 Major fungal xylanase expression system

Source of xylanase gene Expression host Vector MWt References
(kDa)
Heterologous expression host
Aspergillus niger P. pastoris GS115 pHIL-D2 30 Berrin et al.
(2000)
A. usamii E001 E. coli BL21 pET-28a 20 Zhou et al.
(+) (2008a)
Thermobifida fusca NTU22 P. pastoris KM71H pPICZa A 36 Cheng et al.
(2005)
Cochliobolus sativus E. coli SOLR Lambda 25 Emami and Hack
ZAP (2001)
A. niger BCC14405 P. pastoris KM71 pPICZaA 21 Ruanglek et al.
(2007)
Aureobasidium pullulans E. coli pYES2 – Ohta et al. (2001)
Penicillium sp. 40 E. coli DH5a pUC119 20.7 Kimura et al.
(2000)
Trichoderma reesei Rut E. coli BL21 (DE3) pET-28a 24 Jun et al. (2008)
C-30
T. reesei PC-3-7 E. coli JM109 pT7Blue-T 33.1 Ogasawara et al.
(2006)
Homologs expression host
A. oryzae KBN 616 Aspergillus oryzae pNAN8142 32 Kimura et al.
KBN616 (2000)
A. niger BRFM281 Aspergillus niger pAN52.3 23 Levasseur et al.
D15#26 (2005)
Acrophialophora nainiana Trichoderma reesei pHEN11 19 Salles et al. (2007)
Rut C-30 exp
Orpinomyces sp. PC-2 Hypocrea jecorina pT3C 28 Li et al. (2007)
glucose in the media. Filamentous fungi enable extracellular secretion of heterolo-

gous protein which facilitates the downstream processes and ease the purification
and characterization of the produced enzyme. Moreover, most genes from fungi have
introns and filamentous fungi are able to recognize them and express with minimal
miscoding. In addition, fungal proteins are glycosylated and the protein expression
in another filamentous fungi results in similar glycosylation pattern. For expressing
highly hemophilic xylanase, many researches have been selected filamentous fungi
because of above-mentioned advantages when compared to other expression hosts
like bacteria. Table 7.10 shows major fungal xylanase expression system.
Yeast-based heterologous gene expression is considered as an excellent alter-
native system when based on their ease scaling up compared to fungi. The presence
of eukaryotic post-translational modification mechanism is one of the main reasons
behind the wide application of yeast expression systems in industry (Cheng et al.
2005). S. cerevisiae is one of the important hosts for xylanase production by cloning
of xylanase cDNA obtained by RT-PCR through crossing over. The presence of
intron sequence (lake of proper splicing of introns) that hinders the xylanase gene
expression is the major disadvantage of these expression systems (Moreau et al.
1992). It has been also observed that a significant increase in xylanase expression in
yeast transformed with the xynA gene without intron (26.2 U mL−1) when compared
to the expression from the gene with intron (16.7 U mL−1) (Li and Ljungdahl 1996).
It is also worthy to note that Chavez et al. (2004) made the first successful attempt to
clone and express the xynA gene which is isolated from P. purpurogenum in S.
cerevisiae. Isolated xynA gene that consists of eight introns regions was spliced
correctly and was integrated on yeast chromosome. The Xln A gene was then
expressed under transcriptional control (XlnR and CreA) where xylose or xylan acts
as inducer and glucose acts as repressor (van Peij et al. 1997). The methanotropic
yeast Pichia pastoris is another widely used xylanase expression host which has
many advantages over S. cerevisiae such as improved secretion efficiency, ease to
attain high cell density culture in inexpensive media, and relative ease in scaled up
fermentation process (Cereghino and Cregg 2000). Moreover, the presence of strong
and regulatory alcohol oxidase (AOX I and AOX 11) promoters which are involved in
methanol metabolism promotes the overexpression of xylanase gene upon the
addition of methanol in the fermentation media (Tsai and Huang 2008). Table 7.11
summarizes some examples of successful cloning of fungal xylanase in yeast.
7.8.2 Cloning and Expression of Bacterial

Endo-1,4-b-xylanase Gene
La Grange et al. (2000) successfully co-expressed xylanase genes (xln B from

Bacillus pumilus along with xyn2 isolated from T. reesei) in yeast S. cerevisiae. E
coli-based expression systems are widely used to express the xylanase.
Endo-1,4-b-xylanase coding genes isolated from A. usamii were cloned in E. coli
BL-21 with the help of PET-28a plasmid vector and expressed successfully and a
maximum xylanase activity of 49.4 U mg−1 was reported. The gene expression was
induced by IPTG and the protein was expressed with 6-X histidine tags that
facilitate the purification of expressed protein (Zhou et al. 2008a). Similarly, for the
xylanase gene from B. circulans Teri-42 when cloned in E. coli DH5-alpha using
Table 7.11 Cloning of different fungal xylanase genes in yeast

Xylanase source Expression host Vector MWt (kDa) References
Aspergillus niger P. pastoris GS115 pPIC9K 20 Liu et al. (2006)
A. niger P. pastoris GS115 pHIL-D2 30 Berrin et al.
(2000)
A. niger BCC14405 P. pastoris KM71 pPICZaA 21 Ruanglek et al.
(2007)
A. sulphureus P. pastoris X33 pGAPZaA – Cao et al. (2007)
Aureobasidium pullulans P. pastoris GS115 pPIC3.5 39 Tanaka et al.
(2004)
A. pullulans S. cerevisiae INVSc1 pYES2 – Ohta et al. (2001)
Thermobifida fusca NTU22 P. pastoris KM71H pPICZa A 36 Cheng et al.
(2005)
T. fusca P. pastoris GS115 pPIC9K 31 Sun et al. (2007)
pUC19 plasmid vector, the xylanase expression was found to be reduced. However,
when the same gene is expressed in B. subtilis using plasmid pBA7, 14-fold
increase in expression was observed. Therefore, Bacillus became more attractive
hosts compared to E. coli and thus used widely in heterogeneous protein produc-
tion. Bacillus sp. used in industrial fermentation are non-pathogenic in nature and in
bacillus-based expression, the proteins are usually secreted out of the cell.
Moreover, they are gram-positive bacteria and therefore, they do not produce
endotoxins (lipopolysaccharides). These features could be of great advantage by
reducing some purification steps in terms of breaking the cells and extraction from
large number of intracellular proteins.
7.8.3 Overexpression in Escherichia coli
For the past several decades, E. coli has been recognized as the ideal platform for
expression of recombinant proteins. Factors such as rapid growth on inexpensive
media, non-pathogenicity, easy to make gene modification without effecting the cell
growth, easy methods required for isolation and purification of expressed proteins
made E. coli the most successful expression host for industrial use. In spite of these
advantages, due to the lake of specific post-translational modification such as
glycosylation and disulfide bond formation, many heterologous proteins were not
successfully expressed in E. coli. Xylanase was one of those which require
N-glycosylation during post-translation stage, whereas E. coli only can perform
O-glycosylation. However, many exceptional cases such as successful expression
of a glycosylated b-xylosidase gene isolated from thermophilic fungi
P. thermophile have been reported (Teng et al. 2011). It was also observed that the
endo-1,4-b-xylanase expression levels are significantly higher in gram-positive
hosts such as Lactobacillus species and B. subtilis compared to E. coli as they can
perform N-glycosylation.
In general, the recombinant xylanases expressed in E. coli are accumulated in the
cytoplasm or in periplasmic place in the absence of a proper secretory channel on cell
wall (Schlacher et al. 1996). However, in many cases, xylanase activity has also been
detected outside the cell (Karlsson et al. 1997; Ebanks et al. 2000). Xylanase
expression rate in E. coli mainly depends on the efficiency of transcription of
xylanase gene which is cloned under promoter sequence. Even though there are
several cloning vectors and host strains have been introduced, eukaryotic gene
expression in E. coli is unable to carryout due to the absence of a functional promoter
sequence. Basaran et al. (2001) isolated b-xylanase gene from Pichia stipites and
successfully cloned and expressed in E. coli. The recombinant enzyme activity was
low (4 U mg−1) when compared to the activity original enzyme activity (30 U mg−1)
in wild stain. Although a variety of cloning vectors have been used, pET expression
vector systems were reported to be the most efficient for protein expression in E. coli.
Xylanase genes isolated from different sources are cloned under the control of a
strong T7 promoter which is isolated from bacteriophage genome. This promoter
Table 7.12 Major cloning vectors used in E.coli-based expression system

Source of xylanase gene Cloning vector References
Bacteria
AeromoA75:C93nas sp. 212 pBR 322 Kudo et al. (1985)
Bacillus circulans pUC 19 Yang et al. (1988)
B. polymyxa pUC 13, pBR 322 Yang et al. (1988)
B. ruminicola pUC 18 Whitehead and Hespell (1989)
Butyrivibrio fibrosolvens pUC 19 Mannarelli et al. (1990)
Caldocellum saccharolyticum k1059, pBR 322 Luthi et al. (1990)
Cellulomonas sp. pUC 18 Bhalerao et al. (1990)
Chainia sp. pUC 8, kgt 10 Chauthaiwale and Deshpande (1992)
Clostridium stercorarium F9 pBR 322 Sakka et al. (1991)
C. thermocellum pUC 8 Hu et al. (1991)
Neocallimastix patriciarum kZAP II Lee et al. (1993)
Fungi
Aspergillus oryzae KBN 616 pNAN-d Kimura et al. (2000)
A. usamii E001 pET-28a(+) Zhou et al. (2008b)
Aureobasidium pullulans pCRII, k ZapII Li and Ljungdahl (1996)
Y-2311-1
Cochliobolus sativus k ZAP Emami and Hack (2001)
Helminthosporium turcicum H-2 pBluescript SK(+) Degefu et al. (2004)
Neocallimastix patriciarum pBTac2 Xue et al. (1992)
Penicillium sp. 40 pUC119 Kimura et al. (2000)
Trichoderma reesei Rut C-30 pET-28a Jun et al. (2008)
T. reesei PC-3-7 pT7Blue-T Ogasawara et al. (2006)
T. reesei C30 pGEM5Z(+) Torronen et al. (1994)
remains silent until T7 RNA polymerase is produced by the host cell genome.
Table 7.12 shows the major cloning vectors used in E. coli-based expression system.
Ogasawara et al. (2006) amplified xynIII gene from T. reesei genomic DNA
using a RT-PCR and was cloned and expressed in E. coli JM109 using pAG9-3
plasmid vector. When the xylanase gene expression was induced by IPTG, they
could produce 26 mU mL−1 of xylanase activity in cellular extract, which was very
low. However, the XynIII activity increased by about 500-fold (13.2 U mL−1)
compared to the activity in soluble supernatant, when the xylanase inclusion bodies
were refolded in 8 mM urea solution. Zhou et al. (2008a) reported the maximum
xylanase activity up to 49.4 U mg−1 when they cloned and expressed xynII cDNA
isolated from A. usamii in E. coli BL21-Codon plus (DE3) RIL strain using
pET-28a (+) expression vector. The xylanase protein expressed is attached to
6X-His-tag which ease protein purification by affinity chromatography. Jun et al.
(2008) also used the same pET vector to clone and express a b-xylanase gene, xyn2,
isolated from T. reesei in E. coli and reported the enhanced xylanase activity of 650
U mg−1. In some cases, the heterologous cloning and expression results in the
change in original characteristics of xylanase gene. For example, a

non-glycosylated XynA gene when expressed in E. coli exhibited significant loss in
its substrate specificity when compared to the glycosylated xylanase from the wild
strain P. stipites. The stability of the enzyme also decreased due to the change in
optimum temperature rage. However, improved thermostability and pH stability
have been reported when Xyn2 from T. reesei was expressed in E. coli. Table 7.13
shows the properties of major endo-b-1,4-xylanase enzyme expressed in E. coli.
Table 7.13 Properties of major endo-b-1,4-xylanase enzyme expressed in E. coli

Source of xylanase gene MWt Optimum Optimum temperature References
(kDa) pH (°C)
Geobacillus 45 8.5 80 Verma and
thermoleovorans Satyanarayana
(2012)
Paenibacillus macerans 205 4.5 60 Dheeran et al.
IIPSP3 (2012)
Thermoanaerobacterium 50 6.5 63 Hung et al.
saccharolyticum NTOU1 (2011)
Clostridium sp. TCW1 53, 60 6 75 Lo et al. (2010)
Actinomadura sp. S14 21, 30 6 80 Sriyapai et al.
and 40 (2011)
Alicyclobacillus sp. A4 42.5 7 55 Bai et al. (2010)
Anoxybacillus sp. E2 38.8 7.8 65 Wang et al.
(2010)
Nesterenkonia 22 7 55 Kui et al. (2010)
xinjiangensis CCTCC
AA001025
Bacillus sp. JB 99 20 8 70 Shrinivas et al.
(2010)
Paenibacillus sp. DG-22 20 6 60 Lee et al. (2007)
Bacillus licheniformis 77-2 40 8 75 Damiano et al.
(2003)
Enterobacter 43 9 100 Khandeparkar
sp. MTCC-5112 and Bhosle
(2006)
Geobacillus sp. MT-1 36 7 70 Wu et al. (2006)
Bacillus sp. 44 6.5, 8.5 50 Sapre et al.
and 10.5 (2005)
Bacillus thermantarcticus 45 5, 6 80 Lama et al.
(2004)
Bacillus thermoleovorans
K-3d 40, 69 7 70, 80 Sunna et al.
(1997)
Bacillus flavothermus 80– 7 70 Sunna et al.
LB3A 130 (1997)
Thermotoga sp. FjSS3-B. 31 5.5 80 Simpson et al.
1 (1991)
7.9 Improvement of Endo-1,4-b-xylanase Characteristics
In general, enzymes are classified based on their conserved regions on them.

Xylanases are specific in their identity due to the random variations in amino acid
sequence and it was found that some very specific stretches of amino acids defines
the structural and functional behavior of enzyme. Existing xylanases can be
improved in terms of their activities and other novel characteristics such as substrate
specificity, thermostability, and pH stability using protein engineering approaches.
Critical amino acid exchanges are usually practiced in order to enhance the char-
acteristics of existing xylanase enzymes. Therefore, before amino acid sequence
modification a detailed studies on the native xylanase amino acid sequences are
required to fully understand the structural and functional behaviors such as the
presence of catalytic domains, specificity of substrate-binding sites, occurrence of
thermo-stabilizing domains, and the presence of covalent/non-covalent interactions.
The significance of a particular acid or a set of amino acids for novel enzyme
characteristics can be identified easily using specific amino acid inhibitors such as
N-bromosuccinimide (tryptophan), phenylmethylsulfonyl fluoride (serine), and iodo
acetate (cysteine). It was also found that cysteine plays critical role in the formation
of covalent glycosyl enzyme intermediate during most bacterial xylanase activity.
Many of the family 10 xylanases possess three different amino acids such as Phe4,
Trp6, and Tyr 343. They were identified as crucial in protein folding and enzyme
stability even under extreme conditions. Torronen and Rouvinen (1997) studied the
significance of asparagines and aspartic acid on the pH optima of family 11 xyla-
nases. The studies conducted on xylanase isolated from B. circulans revealed that
the presence of asparagine at 52nd position of amino acid sequence increased the
thermal fluctuations at unstable regions. However, when it is substituted with tyr-
osine, the overall thermostability of the enzyme exhibited significant increased (Joo
et al. 2011). It was also reported that the catalytic activity of B. circulans xylanase
increased when the Asn at position 35 is mutated to Asp (Li and Wang 2011). Other
study showed also a significant increase in thermostability (10–15 °C) as a result of
point mutation (aspartic acid to asparagine) at position 56 (Yin et al. 2010).
However, in many other cases, the point mutation was reported to have negative
effects such as loss in substrate-binding affinity, alkaline stability, and thermosta-
bility. Simpson et al. (1999) reported the complete loss of carbohydrate-binding
module affinity toward the xylan when the arginine was mutated to glycine. In case
of B. circulans xylanase, change in pH optima was reported when R73V and
Q167M were mutated (Yang et al. 2008). Table 7.14 comprises significant
achievements made in xylanase protein engineering to increase enzyme stability.
Table 7.14 Significant achievements made in xylanase protein engineering (Verma and
Satyanarayana 2012)
Xylanase source Approaches/alterations Significant inferences References
Bacillus sp. NG-27 Deletion of Phe4 and Complete loss in activity, Bhardwaj
Trp6 while Trp6 and Tyr343 et al.
affected folding (2010)
B. circulans Amino acid Slight increase in half-life Joo et al.
modification (N52Y) and Tm as compared to (2011)
wild type
B. circulans Asn35Asp Improvement in catalytic Li and
activity Wang
(2011)
N. patriciarum D57N Enhancement in You et al.
thermostability by 10– (2010)
15 °C
G. stearothermophilus Substitution with Significant improvement Sun et al.
N-terminal of T. fusca in thermostability (2007)
T. reesei Improvement of Half-life of the mutant Turunen
C-terminal packaging xylanase was increased et al.
by 63 min (2001)
T. lanuginosus Introduction of Improvement in Gruber
DSM5726 disulfide bond thermostability et al.
between Cys100 and (1998)
Cys154
B. stearothermophilus Introduction of Enhancement in Jeong
No. 236 between Ser and Cys) thermostability by 5 °C et al.
100 and (Asn to Cys) (2007)
150
A. niger BCC144505 Substitution of Enhancement in the Sriprang
arginine in place of thermostability et al.
Ser/Thr (2006)
Thermobifida fusca Error prone PCR to Improved stability at Wang and
obtain a mutant having alkaline pH, 4.5-fold Xia (2008)
T21A, G25P, V87P, increase in Km and
I91T, and G217L 12-fold enhancement in
Kcat/Km
Thermomyces Error prone PCR Alkali-stable mutant Stephens
lanuginosus A54T et al.
(2009)
7.10 Industrial Production of Endo-1,4-b-xylanase
Although many microbial species such as bacteria, fungi, and actinomycetes have
been reported as xylanase producers, a few number of strains are acceptable by the
industrial society. To meet the current and forecasting market demands, xylanase
production need to be increased by many folds through efficient production
Table 7.15 Major commercial xylanases produced by SmF techniques (Polizeli et al. 2005)
Commercial Distributors Microorganism Application
name
Allzym PT Alltech Aspergillus Animal feed
niger improvement
Bio-Feed Plus Novo Nordisk Humicola Animal feed
insolens
EcopulpX-200 Primalco Trichoderma Cellulose pulp bleaching
reesei
Ecosane Biotec Trichoderma Animal feed
reesei
Grindazym GP, Danisco ingredients A. niger Bird and pig feed
GV
Irgazyme 40 Nalco-Genencor, Ciba, T. Paper industry and
Geigy longibrachiatum animal feed
Solvay Solvay Enzymes T. reesei Starch and bread making
pentonase
Xylanase Seikagaku Trichoderma sp. Carbohydrate structural
studies
Xylanase GS35 Iogen T. reesei Pulp bleaching
strategies. Therefore, optimizations of production and purification processes are the

key to obtain maximum xylanase yield either by solid-state fermentation (SSF) or
submerged fermentation (SmF) (Narang and Satyanarayana 2001). Nowadays, the
most of commercial xylanases production processes are carried out by SmF as
shown in Table 7.15. Cultivation media plays important role in the production of
xylanases in industrial scale. A suitable and economically viable media formulation
which supports the maximum xylanase production is usually required for
large-scale production. Therefore, abundantly available natural agriculture residues
are widely used for xylanase production. Agricultural wastes such as corn cob, con
stover, and wheat straw were successfully used as suitable substrates for fungal
xylanases production (Damiano et al. 2003; Gupta et al. 2000).
7.10.1 Physical and Nutritional Parameters Affecting

Endo-1,4-b-xylanase Production
In SSF, biomass and xylanase productions depend on several factors such as the
type of microorganism, inoculums size, temperature, oxygen diffusion, the period
of cultivation, the substrate used and its pretreatment, size, water activity, relative
humidity, pH, and many other factors. On the other hand, for SmF, the temperature,
pH, agitation, and dissolved O2 are the most critical physical parameters affecting
xylanase production in SmF. Most of the xylanase-producing microorganisms
reported so far are either mesophilic or thermophilic (Coughlan and Hazlewood
1993). It was observed that these microbes show highest xylanase activity and
optimum growth at temperature between 40 and 70 °C. However, those extremo-
philic such as Thermotoga sp., Dictyoglomus, and Geobacillus exhibit even higher
optimum temperature range of up to 80 °C (Ko et al. 2011; Sharma et al. 2007;
Qureshy et al. 2002). Xylanases of fungal origin usually are found less thermophilic
in nature. Even though some exceptional cases have been reported, the optimum
temperature for most fungal xylanases is below 55 °C. This has been one of the
major drawbacks of fungal xylanase in industrial application. On the other hand,
bacterial xylanases exhibit higher thermostability. The pH of the fermentation
media is another major factor effecting the xylanase production as well as the
enzyme stability. Fungal xylanases usually are highly alkaliphilic as they can be
active at wide ranges of pH, usually between 5.0 and 10.0, whereas the bacterial
xylanases have quite narrow pH spectrum (Dhillon et al. 2000). Aeration and
agitation rates during fermentation are the other crucial factors in case of xylanase
production process. Studies show that xylanase production by Bacillus sp. is
enhanced at an agitation rate of 200–250 rpm and 30 % and above dissolved O2
saturation (Anuradha et al. 2007; Sanghi et al. 2009). In cretin fungal species which
exhibit mycelia growth pattern, higher agitation rate results in poor growth as well
as less xylanase production due to the high sheer force. Studies made by Archana
and Satyanarayana (1998) and Sharma et al. (2007) deal with the optimization of
physical parameters effecting xylanase production.
In addition, carbon and nitrogen sources and their concentration in the fer-
mentation media are found to be not only effecting the xylanase production but also
the localization of enzymes. The fungal xylanase producers are able to utilize wide
verity lignocellulosic-derived substrates such as corn cob, corn straw, wheat bran,
rice straw, and sugarcane bagasse. For production in SSF system, these substrates
are used as they are cheaper and easily available raw materials (Chauhan et al.
2007). However, in bacterial cells in SmF, simpler and readily utilizable nutrients
are used. Organic nitrogen sources such as tryptone, peptone, yeast extract, and
soybean meal are widely used as they enhance the xylanase production as well as
biomass. Kapoor et al. (2008) observed a significant increase in xylanase produc-
tion which was observed when the Bacillus sp. SSP-34 was provided with yeast
extract in combination with peptone. In similar study, xylanase production by
Bacillus sam-3 was increased up to 25-folds higher when the inorganic nitrogen
source was replaced by soybean meal. Use of corn steep powder also significantly
enhanced xylanase production by T. reesei (Lappalainen et al. 2000). Apart from
these major nutrients, the incorporation of micronutrients such as trace elements,
vitamins, and amino acids improved the xylanase production to great extend in
cases of thermo-anaerobe-mediated xylanase fermentation. In cases of recombinant
strains, the xylanase expression greatly depends on the gene inducers present in the
fermentation media. IPTG is the most widely used chemical inducer for ‘lac’-based
expression system (Mamo et al. 2007). However, in large-scale production, IPTG is
not applicable due to its high cost.
In cases of recombinant xylanase, the expression of xylanase genes usually
depends on several aspects such as biosynthesis mechanism, gene regulation, vector
construction, expression host used, expression media, and other growth parameters
during the fermentation. For commercial applications, xylanases have to be ideally
produced quickly and in large quantities. The conventional production strategies
have been followed are found to be less effective to meet the growing need in the
present xylanase market. Therefore, many researches are going on for developing
recombinant strains for xylanase production. However, a very limited number of
these recombinant strains are studied further for the scaling up of xylanase pro-
duction in pilot scale. Hence, more investigations on this area are required and it is
very significant to have more efficient and economically viable process to reduce
the cost of xylanase production.
7.10.2 Optimization of Endo-1,4-b-xylanase Production
Media screening and medium composition optimization are usually the preliminary
steps involved in bioprocess optimization which are always carried out in shake
flask level to select the most suitable medium formulation. Both physical and
chemical parameters effecting the cell growth as well as xylanases production are
monitored and optimized at this stage with a number of experiments. Basar et al.
(2010) observed that the recombinant xylanase production in E. coli is a
growth-associated process. They observed a rapid growth of E. coli, reaching the
stationary phase after 16 h and the maximal xylanase production up to 324.72
U mL−1 was obtained in defined medium. They also reported the effects of different
concentrations of glucose in growth media on cell growth and xylanase production.
Farliahati et al. (2009) reported the xylanase expression in recombinant E. coli
DH5a in different media compositions (defined, semi-defined, and complex).
Optimization study of pH and agitation ranges also carried out in shake flask
cultivation mode. From these studies, they observed the highest xylanase produc-
tion (up to 2122 U mL−1) in defined media at initial pH 7 and agitation speed of
200 rpm. A suitable cultivation strategy for the production of two truncated ther-
mostable recombinant xylanases (XynlAN and XynlANC), isolated from
Rhodothermus marinas, was developed by Karlsson et al. (1998). They found that
the fed-batch cultivations of E. coli strain BL21 (DE3) with a controlled expo-
nential glucose feed led to high specific production of the recombinant proteins and
addition of complex nutrients such as Tryptone Soya Broth (TSB) to the media
exhibits positive impact on cell growth rate and cell productivity. Single addition of
either lactose or isopropyl-thio-go-galactoside (IPTG) was used for induction. In
lactose-induced cells, the production of recombinant xylanase was delayed for
approximately 30 min compared to those induced with IPTG, but the specific
product levels were comparable at 3 h after induction. Huang et al. (2006) cloned a
xylanase gene from B. subtilis into E. coli and found that the xylanase distributions
in extracellular, intracellular, and periplasmic fractions were in ratio 22.4, 28.0, and
49.6 %, respectively, with an optimal enzyme production at pH 6.0 and 50 °C. The
superiority of defined medium for xylanase production using recombinant bacteria

was also reported by Farliahati et al. (2009).
Catabolic repression of the xylanases biosynthesis is found to be a common
phenomenon usually in the presence of glucose in culture medium. When glucose is
abundant in the grown medium, the repression of catabolic enzyme synthesis takes
place due to the gene regulation. When the microbe can easily absorb the readily
available glucose in the medium, they will not utilize for other complex nutrients by
spending energy to metabolize complex nutrients. Xylanase production reported to
be reduced due to the catabolic repression that makes the xylanase coding gene in
inactive mode. In another study by Basar et al. (2010) they enhanced xylanase
production using recombinant E. coli by optimizing the media composition and
other growth factors. Two basal mediums (defined media and complex media) have
been used and found out that the defined media gives the highest growth as well as
the xylanase production under experimental conditions. At 20 % enhanced dis-
solved oxygen tension (DOT) in lab scale bioreactor, they increased xylanase
production up to 1784 mL−1.
7.10.3 Application of Response Surface Methodology

(RSM) in Optimization of Xylanase Production
The conventional media optimization method based on step-by-step process vary-

ing one variable at a time (OFAT) while keeping the other variables constant is a
time-consuming process and less reliable in many cases. To overcome this problem,
statistical approach using experimental designs was applied. Response surface
methodology (RSM) is the most reliable statistical application that has been used
for optimization of fermentation media. Optimizations of physiological and nutri-
tional growth factors are found to be very important procedures to reduce the
production cost of xylanases in industrial scale. Many researches have been carried
out based on RSM which used to understand and evaluate the significant interaction
between the physiological and nutritional factors effecting the cell growth and
product formation. A prior knowledge about these growth factors and their inter-
actions is necessary for the designing of a more realistic model. Hence, many of the
RSM studies are found to be based on the results obtained from OFAT method.
Selection of these growth parameters has been done by checking their significance
level using a 2FFD Pareto chart before proposing an experimental design. Most
significant factors were then used as variables in central composite design
(CCD) (Farliahati et al. 2010). The effect of various growth parameters in pro-
duction of xylanase enzyme was studied by Mullai et al. (2010). In his study,
statistical experimental designs (CCD) were constructed with the help of statistical
application MINITAB 14. Plackett–Burman (PB) design for seven different vari-
ables, NH4NO3, KH2PO4, CaCl2, MgSO47H2O, FeSO47H2O, MnSO47H2O, and
NaCl at two levels +1 and −1 (higher and lower level, respectively) and the
statistical significance were studied by determining the F-value. The optimum

levels of selected variables from the Plackett–Burman design were investigated
using central composite design (CCD). The response was measured in terms of
xylanase activity. Counter plots and response surface 3D graphical representations
generated could show the interactions of different variables under experimental
conditions. Garai and Kumar (2012) optimized the xylanase production by A.
candidus in SSF. They studied the effect of various nitrogen sources in the pro-
duction media and they found that xylanase production was enhanced when the
ammonium nitrate was used as sole nitrogen source. In the same study, applying
RSM-based BoxBehnken design, the researcher also optimized the physical
parameters such as time of incubation, temperature, and moisture content which
effects xylanase activity. The crude xylanase recovered at the end of solid-state
fermentation was used for the scarification of ammonia-treated lignocellulose
materials to study the substrate-binding assay. Among the various substrates used,
corn comb was found releasing the maximum reduced sugar (438.47 mg g−1) when
it was treated with crude xylanase enzyme. In RSM-based optimization, xylanase
production was optimized by Box–Behnken design. Initially, Planket–Berman
design was employed to identify the key ingredients and process conditions which
exhibit the most significant effect on the xylanase expression. In the later stage,
using Box–Behnken design, the optimal level of the identified key ingredients in
production media as well as the optimum range of process parameters such as
cultivation time, pH, temperature, and agitation are identified. Using this approach
6.83-fold increase in xylanase production was obtained under optimized conditions
when compared to that of initial un-optimized conditions.
Solid-state fermentation (SSF) is also widely used for large-scale production of
xylanase enzymes. It is basically designed to mimic the natural habitat of microbes
that are less likely to grow in submerged fermentation (SMF) conditions. In SSF,
the solid bed is used as nutrient source as well as the mechanical support for cell
growth. A. niger, T. reesei, and T. koningii are the major fungal species used to
produce xylanase under SSF conditions (Dhiman et al. 2008; Garg et al. 1998).
However, xylanase production by bacteria is commonly carried out in submerged
fermentation conditions where the physical parameters such as temperature, pH,
and dissolved O2 level involved are controlled to the optimum levels with more
ease, and therefore an enhanced production can be achieved. Esteban et al. (1982)
reported that the large-scale production of xylanase in SmF is the growth-associated
process. However, several exceptional cases were also been reported such as
xylanase production using Bacillus sp. (Dey et al. 1992). Later study by Bocchini
et al. (2002) reported the production of highly thermostable cellulase-free xylanase
in SMF using B. circulans. Similar study was also carried out by Sharma et al.
(2007) who used Geobacillus thermoleovorans AP7 to produce highly
thermo-alkali-stable xylanase. The switch to xylanase-producing recombinant
E. coli is seen as an economic strategy toward higher productivity and easier
downstream purification. Sandhu and Kennedy (1984) reported a large-scale pro-
duction of xylanase by transforming E. coli with recombinant plasmids carrying a
1,4-b,D-xylanase gene from B. polymyxa. In another study, xylanase gene isolated
from A. usmani was cloned in pET-28a expression vector and when it is expressed
in E. coli BL21, the maximum xylanase activity was found to be 49.4 U mg−1. The
gene expression was induced by IPTG and the xylanase was found expressed with
6-X histidine tag that facilitates an easy purification of expressed protein (Zhou
et al. 2008b). A very successful cultivation strategy for the production of two
truncated thermostable recombinant xylanases (XynIAN and XynIANC), isolated
from Rhodothermus marinas, was developed by Karlsson et al. (1999). They found
that the fed-batch cultivations of E. coli strain BL21 (DE3) with a controlled
exponential glucose feed led to high specific production of the recombinant proteins
with additional complex medium. Highest xylanase production in defined medium
was achieved by Farliahati et al. (2009). In this study, xylanase production reached
up to 2122.5 U mL−1 at pH 7.4 and when (NH4)2HPO4 was used as the main
nitrogen source. They also reported that the xylanase production was a
growth-associated process as the enzyme secretion was greatly dependent on bio-
mass concentration for strain E. coli DH5a.
Biswas et al. (2010) used mutant IITD3A of Melanocarpus albomyces for the
production of high titer cellulose-free xylanase in 14-L stirred tank bioreactor where
an optimized (RSM based) production media and process parameters are used. The
authors reported the xylanase activity reached up to 415 U mL−1 after 24 h by pH
recycling strategy (between 7.8 and 8.2) during the production phase. They also
reported that the xylanase production is significantly influenced by aeration and
agitation. A 5.2-fold increase (overall volumetric productivity of 22,000 U L−1 h−1)
in activity was observed when the cells exhibited pellet form at an agitation speed
of 600 RPM.
Kumar et al. (2009) optimized the xylanase production in lab scale stirred tank
bioreactor using mycelial fungal strain T. lanuginosus MC 134. Fermentation media
contains corn cob as a sole source of carbon which was inoculated with T.
lanuginosus MC 134 and observed the production in terms of xylanase activity over
a period of 9 days at various process parameters. A highest xylanase activity, 2009
U mL−1, was obtained after 6 days of fermentation. However, a significant decrease
(40 %) in productivity was observed in bioreactors when compared to shake flask
culture. Similarly, xylanase production by several microbial strains using corn cob
was reported and from these studies it was clear that the production largely depends
on rheological factors in bioreactors and the type of extraction methods used.
Ruanglek et al. (2007) successfully cloned and expressed XylB (564 bp) encoding
endo-1,4-b-xylanase obtained from A. niger BCC14405 in Pichia pastoris under
AOX1 promoter. A maximum of 7352 U mg−1 of xylanase-specific activity was
obtained in 2 L lab scale bioreactor with BMGY/BSM medium under exponential
feeding strategy. During the batch phase which was aimed to achieve HCDC, the
temperature and pH were maintained at 30 °C and 5.0, respectively. Agitation was
maintained in the range of 600–1000 rpm. Fed-batch phase was started after 16–
20 h by feeding 67 % (v/v) glycerol plus 1 % (v/v) trace mineral solution in
exponential manner. Table 7.16 shows examples for xylanase production in lab and
pilot-scale submerged fermentation by different fungal strains.
Table 7.16 A comparison of xylanase production in lab and pilot-scale submerged fermentation
using fungal strains
Name of the Substrate Mode of Xylanase Productivity References
organism used operation activity (IU L−1 h−1)
(IU L−1)
Aspergillus niger Xylan (from Batch (20 L) 290,000 3820 Yuan et al.
corncob) (2005)
Aspergillus oryzae Spent sulfite Batch (15 L) 199,000 13,100 Chipeta
liquor et al.
(2008)
Thermomyces Oat husk Batch (2 L) 210,000 4380 Xiong
lanuginosus DSM hydrolysate et al.
10635 (2004)
Thermomyces Coarse Batch (5 L) 2,010,000 13,950 Kumar
lanuginosus MC corncob et al.
134 (2009)
Trichoderma Oat husk Fed-batch (5 1,350,000 14,060 Xiong
reesei hydrolysate L) et al.
(2005)
Melanocarpus Soluble Batch with 550,000 22,000 Biswas
albomyces wheat straw pH cycling et al.
extract (14 L) (2010)
7.10.4 pH Optima and Stability of Endo-1,4-b-xylanase

Enzymes
In general, endo-1,4-b-xylanases are active at wide pH ranges and their pH optima

depend on the producer organism and the physiochemical properties of the substrate
molecules. Fungal xylanases are usually acidophilic in nature. However, the opti-
mal pH for bacterial xylanases is usually above 5.5. Endo-1,4-b-xylanase from A.
kawachii reported as the most acidophilic xylanase which has a very low pH
optimum of 2. On the other hand, xylanase from Bacillus sp. 41 M1 reported as the
most alkalophilic xylanase which has pH optimum 9.0 (Nakamura et al. 1993). In
certain cases, different pH optimums were observed in different xylanases secreted
by same organism. For example, XynI and XynII produced by H. jecorina were
reported to have pH optimums 3–4 and 5–5.5, respectively. This showed that the pI
values of the enzymes are correlated with the amino acid residues at various
positions on xylanase. However, among Xyl-11 family, those are acidophilic in
nature possess an Asp residues at position 33 even though there are very rare
exceptions such as A. kawachii IFO 4308 xylanase which has pH optimum close to
5.0 (Zhu et al. 1994). However, many alkalophilic xylanases such as Xyn11 from A.
versicolor (Carmona et al. 1998), XynC from Uccinogenes sp. (Zhu et al. 1994)
also displayed an Asp at the same position. Similarly, studies on P. griseofulvum
Xyl-11 reported that it displays a Ser residue at position 44 and mutating this
residue resulted significant changes in pH optimum of the enzyme (Andre-Leroux

et al. 2008).
There have been many different explanations for the factors determining the
optimal pH value of xylanases. However, it believes that certain amino acid resi-
dues of xylanase structure play key role in the determination of the optimal pH
value of the enzyme. In case of acidic pH optima, the Asp/Glu residues near the
catalytic cleft become protonated and it decreases the electrostatic repulsion
between the substrate and enzyme, whereas in alkaline xylanases, instead of acidic
residues more alkaline residues such as arginine are seen. de Lemos Esteves et al.
(2005) experimented various mutations in Streptomyces sp. S38, and observed a pH
optimum shift from 6.0 to 7.5, which enhanced the xylanase enzyme activity
several folds higher than the enzyme from the wild type. The pI values of catalytic
domain present on xylanase play a key role in its pH activity profile. Many recent
studies reveals that, among GH11 xylanase, the residues those contribute positive
charges and hydrogen bonds to the catalytic domain help in increasing or
decreasing the pI values depending upon the electrostatic interaction between the
substrate molecules (Joshi et al. 1997). Apart from these residues, factors such as
occurrence of salt bridges are also reported to have significant effects on pH optima
(Hakulinen et al. 1998).
7.10.5 Endo-1,4-b-xylanase of Extremophilic Origin
Considerable progress has been made in the isolation of extremophilic microor-

ganisms and their successful cultivation in the laboratory. Commercial applications
of the xylanases demand highly thermostable enzyme which can work effectively
under elevated temperature. Many advantages such as reduced contamination risk
and faster reaction rates have been proposed for the use of thermophiles in
biotechnology processes. In general, parameters such as temperature, pH, and
chemical and enzymatic stability are important for the industrial applicability of any
enzyme. Recently, a simple model, based on the thermodynamic and kinetic
parameters for inactivation of the enzyme, has been presented for predicting
(1) thermostabilities in the presence of substrate and (2) residual activities of
enzymes in harsh processing environments. The study of extremophiles and their
enzymes can extend the present understanding of protein chemistry in addition to
expanding the potential applications of biocatalysts. Studies of alkaliphiles have led
to the discovery of a variety of enzymes which exhibit some unique properties.
Biological detergents contain enzymes such as alkaline proteases and/or alkaline
cellulases from alkaliphiles. Commercial production became economical only after
the discovery of alkaliphilic Bacillus-producing CGT with enhanced pH stability.
Alkaline xylanases have gained importance due to their application for the devel-
opment of eco-friendly technologies used in paper and pulp industries. The
enzymes are able to hydrolyze xylan which is soluble in alkaline solutions.
7.10.5.1 Alkaliphilic and Acidophilic Endo-1,4-b-xylanase
The pH tolerance of microbial endo-1,4-b-xylanase enzymes depends on the natural

growing environment of the microbial species. Endo-1,4-b-xylanase-producing
alkalophilic microbes grow optimally at higher pH above 9.0 and those produce
acidophilic xylanases grow between pH 1.0–5.0 (Kimura et al. 2000). These
organisms are usually isolated from various natural environments such as soil,
decomposing organic matters, paper pulp industry wastes, and many other sources.
Bacillus sp. C-59-2 was the first reported xylanase-producing alkaliphilic
microorganism (Nakamura et al. 1993). Since then, there have been a number of
reports on xylanases production using alkaliphilic and acidophilic microbes.
However, the studies indicated that the intracellular xylanase produced by many of
these microbes may not be adapted to extreme environmental growth conditions of
their hosts. A large number of alkaliphilic microbes found to produce xylanase at
optimal pH close to neutral although their activity can be retained in alkaline
conditions. Many exceptional cases are also reported such as XylB from Bacillus
sp. which has pH optima of 9.0–10 (Gessesse and Mamo 1998). Many acidophilic
xylanases of fungal and bacterial origin have also been reported in last two decades.
Cryptococcus sp. S-2 and Penicillium sp. 40 (Kimura et al. 2000) are the most
studied extremely acidophilic (pH optima 2–3) xylanase producers.
7.10.5.2 Psychrophilic Endo-1,4-b-xylanase
Even though the xylanase-producing microorganisms are abundant in nature, only a

very less number of endo-1,4-b-xylanase-producing psychrophilies have been
identified so far. Gram-negative bacteria such as Pseudoalteromonas haloplanktis
TAH3a (Collins et al. 2005a) and Flavobacterium frigidarium (Humphry et al.
2001); gram-positive Clostridium sp. PXYL1 (Akila and Chandra 2002); fungal
species such as Alternaria alternate, Penicillium sp., and Phoma sp.; and yeast sp
Cryptococcus adeliae (Petrescu et al. 2000) are the major psychrophilic organisms
reported to be endo-1,4-b-xylanase producers. Almost all these microbial species
are isolated from Antarctic regions. The best studied two psychrophilic xylanases,
which are from Pseudoalteromonas haloplanktis and Cryptococcus adeliae, were
classified under GH 8 and GH10, respectively. The (a/a) 6 barrel structure of these
structures is described by Van Petegem et al. (2003). The limited studies revealed
that these enzyme groups have very poor stability due to the reduced number of salt
bridges present on it but higher catalytic activity even at very low temperature.
Georlette et al. (2004) also observed that psychrophilic xylanases are characterized
by up to 10-folds higher activity even at mild cool temperature of 5 °C when
compared to mesophilic xylanase activity. Moreover, the psychrophilic xylanase
displayed higher catalytic and retained 60 % of maximum activity at very low
temperature. However, the psychrophilic xylanases are totally unstable at high
temperature (temperature 55 °C) and exhibited 12 times shorter half-life compared
to mesophilic xylanases (Collins et al. 2005b).
7.10.5.3 Thermophilic Endo-1,4-b-xylanase
A large number of thermophilic and hyper-thermophilic (optimal growth temper-

ature 50–80 °C and more than 80 °C, respectively) microbes which are able to
produce xylanases have been isolated from various sources such as thermal hot
springs, deep marine fields, self-heating organic debris, etc. Almost all of these
enzymes were grouped under families 10 and 11. Extremophile microbial species
such as Caldicellulosiruptor sp. (Luthi et al. 1990), Rhodothermus marinus (Abou
Hachem et al. 2000), and Bacillus stearothermophilus (Khasin et al. 1993) are the
major producers of thermophilic family 10 xylanases, whereas family 11 ther-
mophilic xylanases are less common and have been isolated from extremophiles
such as Bacillus strain D3 (Connerton et al. 1999), Chaetomium thermophilum
growing medicinal mushrooms in Egypt as a new strategy for cancer treatment,
dictyoglomus thermophilum (McCarthy et al. 2000), paecilomyces variotii (Kumar
et al. 2000), and thermomyces lanuginosus (Schlacher et al. 1996).
Optimum activity range of these thermophilic xylanases was found between 65
and 80 °C, whereas the xylanases from Thermotoga sp. exhibit optimal activity at
105 °C and pH 5.5. X-ray crystallographic structure and amino acid sequential
analysis of many of these thermophilic xylanase indicated very close similarities
with mesophilic xylanase and probable reason for the higher thermostability could
be the minor changes in amino acid alignment. Many mutagenic studies also have
indicated that minor modifications in mesophilic aminoacid array enhanced the
thermostability to many folds (Winterhalter and Liebl 1995).
7.10.5.4 Features Responsible for Endo-1,4-b-xylanase

Thermostability
Thermostability comparison made between the enzymes are usually misleading

since in most cases, the enzymes compared do not share the same experimental
conditions such as incubation time, concentration of protein, nature, and concen-
tration of the substrate. Many researchers believe that the high thermostability of
xylanases is due to the presence of a network of certain charged amino acid residues
near to the catalytic sites. These residues are usually found coupled with the
disulfide bridges between b-strand and the a-helix. However, many recombinant
thermostable xylanases are expressed in E. coli and are found to have thermosta-
bility ranging from 70 to 80 °C. When analyzing their protein structure, compar-
atively longer N-terminal ends are observed which may account for its higher
thermostability (Morris et al. 1996). Many other analyzes also propose that the
thermostability is due to the presence of highly flexible a-helix and C-terminal
regions where the protein unfolding is generally initiated (Kumar et al. 2000).
However, several other factors have also been identified that explain the xylanase
thermostability such as follows:
• Presence of disulfide bridges which may strengthen the whole protein frame-
work would give more stability to the enzyme.
• Presence of ion and aromatic pairs and their interactions may favor to more
stable framework.
• Strategic position of water molecules on the framework, since the readily
available water bindings are required for the catalytic activity even at higher
temperature.
• Complex folding nature of enzyme. The higher the folding complexity, the
higher the stability.
• Occurrence of oligomerization and the presence of certain aromatic residues
result in the hydrophobic patches which allows stronger monomeric interactions
enhancing the enzyme stability.
Certain GH11 endo-1,4-b-xylanases display disulfide bridges at position
between b-strand and a-helix. However, these enzymes do not exhibit higher
thermostability, whereas endo-1,4-b-xylanase produced by Dictyoglomus ther-
mophilum exhibits thermostability even though that do not have any disulfide
bridges on their structure. Therefore, the presence of disulfide bridges does not
necessarily account for the thermophilic characters of the enzymes (Table 7.17).
Table 7.17 Techniques used for purification of endo-1,4-b-xylanase enzymes

Source Purification Buffer solution References
Acrophialophora Gel filtration and ion 50 mM acetate Salles et al.
nainiana exchange (2000)
chromatographies
Bacteroides Metal affinity resin and 50 mM phosphate Mirande
xylanisolvens batch/gravity-flow et al. (2010)
XB1A column
Sulfolobus Ultrafiltration and 50 mM Tris–HCl Cannio et al.
solfataricus AKTA Fast Protein (2004)
Liquid Chromatography
Streptomyces Filtration and overnight 50 mM sodium citrate Nascimento
sp. AMT-3 dialysis et al. (2002)
Glaciecola Precipitation with 60 % 50 mM citrate Guo et al.
mesophila KMM saturation of ammonium (2009)
241 sulfate and His-Tag
Ni-affinity column
Bacillus sp. YJ6 Sephacryl S-100 h 20 mM citrate (pH 3.0– Yin et al.
chromatography 6.0), sodium phosphate (2010)
(pH 6.0–8.0), Tris–HCl
(pH 8.0–9.0), sodium
carbonate (pH 8.0–11.0)
Paenibacillus Precipitation with 20 % 50 mM phosphate Valenzuela
barcinonensis ammonium sulfate and et al. (2010)
cation exchange
chromatography
Aspergillus DEAE–Sephadex and McIlvaine (pH 4.0–8.0), Carmona
versicolor HPLC GF-510 gel Tris–HCl (pH 8.0–9.0), et al. (2005)
filtration glycine–NaOH (9.0–9.5)
7.10.5.5 Improvement of Endo-1,4-b-xylanase Thermostability
In last two decades, thermostability of xylanase enzymes was continuously

improved through the protein engineering approaches. In most of these studies, the
researches considered to modify the mesophilic xylanase enzyme structure to
design a new structural model similar to the thermophilic xylanase. Arase et al.
(1993) used chemical random mutagenesis in XYNA from B. pumilus and this
strategy was successful to increasing the enzymes’ half-life from 4 to 12 min at
57 °C. In a similar study, the specific activity and optimum temperature of Xyl1
from Streptomyces sp. S38 was enhanced from 2100 to 2500 IU mg−1 with the shift
of the optimal temperature from 57 to 66 °C (Georis et al. 2000). Other study
showed that the replacement of the N-terminal end of mesophilic xylanase xlnB
from Streptomyces lividans with those from thermostable xylanases TfxA from T.
fusca by random gene shuffling and observed a significant increase in thermosta-
bility (Shibuya et al. 2000). Introduction of two disulfide bridges, one between B9
strand and the a-helix and another between N and C-terminal ends, also enhanced
the thermostability of Tx-xyl (Paës and O’Donohue 2006). The authors also
reported that the occurrence of residues that met at N-terminal end brought negative
impact on xylanase thermostability and temperature optimum which are reduced by
10-folds and 5 °C, respectively. From these engineering strategies applied in
xylanases protein engineering, researchers concluded that:
• The a-helix on xylanase structure seems very sensitive to thermal unfolding—
making these are more rigid and may enhance the thermostability of the enzyme.
However, it may also diminish the catalytic activity (Muilu et al. 1998).
• N-terminal end plays key role in thermostabilization in GH11 xylanase—linking
the N-terminal extremity to A2 strand or C-terminal extremity enhanced the
thermostability of xylanase. Introduction of an additional complete b strand by
mutagenesis also beneficial for improved thermostability.
• Presence of disulfide bridges does not necessarily enhance the thermophilic
behavior of the enzymes. Therefore, addition of these bridges does not always
bring positive impact on enzyme thermostability.
7.11 Industrial Applications of Endo-1,4-b-xylanase
Industrial production of xylanases has been grown significantly during last few
decades based on their increased application. Xylan can be converted into many
useful products such as xylitol and furfurol through enzymatic hydrolysis
(Subramaniyan and Prema 2002). The hydrolysis could be also carried out through
acid treatment process which is relatively easy, fast, and cheap. However, the
application of this method in industries is limited due to the formation of toxic end
substances that may have cross reactions with product of interest (Beg et al. 2001).
Therefore, enzymatic hydrolysis method is widely applied even though it is a slow
process but more efficient and eco-friendly. During industrial application, the
xylolytic enzymes are not commonly applied in pure form but in combination with
other hydrolytic enzymes to achieve better results. The industrial applications of
xylanases cover different areas and the list of applications increased by time and
thus increases the market demand of xylanases accordingly.
7.11.1 Animal Feed Industry
Xylanases are widely used in animal feed manufacturing. They are usually applied
in combination of other hydrolytic enzymes such as pectinase, protease, cellulose,
lipase, glucanase, alginase, and phytase. These enzymes are used to reduce the
viscosity of raw materials used in animal feeds. The complex arabinoxylan that is
present on the cell wall of most grains may reduce the effective digestion and
absorption of nutrients in poultry. Studies on this area have reported that the
xylanase-treated feed has better digestibility and nutrient absorption rate when
compared to raw feeds (Bedford and Classen 1992). In animal feed industries, the
feed conversion rate (FCR) is defined as the ratio of feed consumed to the body
weight gained by the animal. A lower FCR means improved feed digestibility and
nutrient absorption. Xylanase enzymes are widely incorporated in poultry feed to
reduce the FCR. It have been reported that the addition of endo-xylanase is isolated
from Acidothermus cellulolyticus in poultry feed and observed a significant
decrease in FCR, indicating an improved digestibility of feed (Clarkson et al. 2001).
Similar results were observed when endo-xylanase is isolated from Neocallimastix
patriciarum added to canola seed after oil extraction and applied for cattle feeding.
The digestibility and stability of nutritive value of silage for cattle are found to be
enhanced when it was treated with xylanase along with probiotic lactobacillus. The
xylose formed by the de-polymerization of hemicellulose was fermented by the
probiotic lactobacillus and thus enhanced the feed nutritive value. It was also
reported that xylanase supplementation in the animal diet improved the animal
growth through the better nutrition absorbability and enhancement of immune
response as well as the gut micro-flora (Gao et al. 2008). In other study, Vandeplas
et al. (2010) reported that when T. viride xylanase is incorporated in poultry feed, it
increased nutrient digestion by 60 % and resulted in a significant increase in
chicken weight. Pretreatment of forage crops with xylanase leads to better digestion
in cattle and these enzymes are often found in synergy with other cellulolytic
enzymes. The assimilation of ruminant feed was also improved many folds by
xylanases addition (Yang and Xie 2010).
7.11.2 Food and Beverages Industry
Food and beverages industries use also xylanases in wide range of applications.
Xylanases are capable of hydrolyzing the hemicellulose in wheat that enhances the
water absorption of dough at leavening level in bread making process. This could
possibly enhance the fermentation rate and as a result the softness of the bread and
volume was increased (Rouau 1993). Xylanase cocktails are often used to reduce
the stickiness of the dough thus enhance the crumb (Goesaert et al. 2005; Butt et al.
2008). Other study showed also that under specific process condition, incorporation
of xylanase in the dough increased the shelf life of bread and also improved the
volume up to 24 % (Verjans et al. 2010). However, enzyme selection is critical in
these conditions since the cereals used for bread making often exhibit the xylanase
inhibition by TAXI, XIP, or TLXI (Gebruers et al. 2004). Pasta processing is
another area where xylanases are widely used as solubilizing agent of durum wheat
semolina without changing its rheological properties (Ingelbrecht et al. 2001; Brijs
et al. 2004). It was also reported that xylanase enzymes can improve the separation
of wheat flour into starch and gluten (Frederix et al. 2004). However, in certain
cases, action of Xyl-11 does not favor the separation due to high viscosity caused
by the un-extractable arabino xylanase hydrolysis. In such conditions, synergetic
dosages of Xyl-10 and Xyl-11 are used as it can mediate-improved agglomeration
of gluten protein even though the wheat quality is very poor (Van Der Borght et al.
2005). Table 7.18 shows some examples of commonly used xylanase preparations
for the baking industry. Xylanases are widely used also in juice, beer, and wine
making industries. The enzymatic breakdown of fruits and vegetables hemicellulose
can increase the clarity and stability of juice and wine. Xylanases can be used along
with several other enzymes such as pectinase and amylase to improve the quality of
beverage products in terms of aroma, taste, clarity, and stability (Wong et al. 1988).
It was reported that up to 27 % decrease in insoluble fibers was achieved in juice
clarification by addition of xylanase enzyme isolated from Sclerotinia sclerotiorum
(Olfa et al. 2007). In beer production, partial hydrolysis of arabinoxylan found in
the barley cell wall gives a middy viscous appearance to the fermented beer. In such
Table 7.18 Examples of commonly used xylanase preparations for the baking industry (Collins
et al. 2005a, b)
Supplier Brand Enzyme source
Beldem-Puratos Bel’ase B210 Bacillus subtilis
Bel’ase F25 Aspergillus niger
Genencor Intl. Multifectw Trichoderma reesei
Danisco Grindamyle H Aspergillus niger
DSM Bakezymew HS, BXP Aspergillus niger
Novozymes Pentopan Mono Thermomyces lanuginosus
AB enzymes Veronw 191 Aspergillus niger
Veronw Special Bacillus subtilis
cases, various xylanase enzyme groups are used to increase the filtration perfor-
mance of beer wort by hydrolysis of viscous AXs fractions. Xylanase inhibitors
present in the malt are the major hinderance to xylanase action. The xylanase
activity is retained with the help of other synergetic enzymes used in the process
(Debyser et al. 1997).
7.11.3 Paper and Pulp Industries
Endo-1,4-b-xylanase enzymes play significant role in paper and pulp industry since
they facilitate an eco-friendly bio-bleaching of wood pulp by reducing the use of
chemical bleaching agents such as chlorine and chlorine dioxide. For the past few
decades, kraft pulp bleaching industry is recognized as the biggest area of xylanase
application. Several studies reported that the removal of lignin content can be
achieved by xylanase enzymes far better than by other lignin-degrading enzymes
(Lundgren et al. 1994). Use of chemical bleaching agents in conventional pulp
bleaching methods cause serious environmental pollution issues and this drove the
researcher’s attention on xylanase enzymes (Subramaniyam and Prema 2002).
Table 7.19 shows major endo-1,4-b-xylanase supplier in pulp bleaching industry.
Application of endo-1,4-b-xylanase enzymes in pulp bleaching can reduce using
chlorine as well as the total energy requirement for this process. Kulkarni et al.
(1999) observed that xylanase pretreatment of kraft pulp lowers the usage of
chemicals by 10–20 %. The concept of TCF (total chlorine free) bleaching tech-
nologies was also recently introduced to make the process more eco-friendly.
However, cellulase-free endo-1,4-b-xylanase is required in this process since the
cellulose enzyme may degrade the cellulose which is the main target of the industry
Table 7.19 Major xylanase supplier in pulp bleaching industry (Beg et al. 2001)
Supplier Product trade name
Alko Rajamaki, Finland Ecopulp
Sandoz, Charlotte, N.C. and Basle, Switzerland Cartazyme
Clarient, UK Cartazyme HS, HT and SR 10
Genercor, Finland; Ciba Giegy, Switzerland Irgazyme 40–4X/Albazyme 40–4X
Novo Nordisk, Denmark Pulpzyme HA, HB and HC
Biocon India, Bangalore Bleachzyme F
Rohn Enzyme 0Y; Primalco, Finland Ecopulp X-100, X-200, and X-200/4
Solvay Interox, USA Optipulp L-8000
Thomas Swan, UK Ecozyme
Iogen, Canada GS-35, HS70
(Bajpai and Bajpai 2001). Relatively lower molecular weight of xylanase enzymes
helps them to penetrate more deep into the lignocelluloses substrates and facilitate
better hydrolytic actions (Beaugrand et al. 2005; Beg et al. 2001). Higher ther-
mostability and alkaline stability are other key features that make xylanase more
favorable for bio-bleaching industry (Buchert et al. 1995). The mechanisms of pulp
bleaching by xylanase have been studied and many researchers believe that xyla-
nase hydrolysis takes place at lignin xylan interaction and thus results in a loosely
packed cellulosic framework (Shatalov and Pereira 2007). However, it was recently
reported the potential use of Aspergillus oryzae MDU-4-based fungal xylanase for
effective deinking of newspaper pulp where a significant improvement in optical
properties such as brightness up to 57.9 % ISO with decreased (211 ppm) residual
ink concentration (Chutani and Sharma 2015).
7.11.4 Biofuel Production
The next-generation fuel production is based on renewable sources such as ligno-

cellulosic biomass. As first step, the complex lignocellulosic biomass needs to be
breakdown to a mixture of relatively smaller units made of hexoses and pentoses or
their monosaccharides that are further fermented to ethanol or other biofuels
(Margeot et al. 2009). The process of converting the lignocellulosic feedstocks into
biofuels usually involves the following steps:
• Enzyme hydrolysis: the critical step involved where the hydrolysis of ligno-
celluloses into various sugar monomers by the action of cocktail of hydrolytic
enzymes.
• Fermentation: production of biofuel by the utilization of sugar monomers
formed by various microbial species, usually carried out by different bacterial
and yeast strains depends on the desired type of biofuel.
• Distillation: separation and purification of biofuel produced.
The controlled hydrolysis of lignocelluloses using enzymatic cocktail to produce
the desirable products is the challenge involved in this process. Xylolytic enzymes,
such as Endo-1,4-b-xylanase in particular, are very essential in biomass hydrolysis
as they initiate progressive cell wall in the assembly and thus mediate the cell wall
penetration by other cellulolytic enzymes (Beaugrand et al. 2005).
7.11.5 Other Applications
Apart from the traditional applications in pulp beaching industry, xylanases have
been used in several other areas as well. The broad spectrum of xylanase application
includes the saccharification of xylan to xylose, plant cell protoplasting, coffee
mucilage liquefaction, vegetable softening, extraction of plant pigments and oils,

etc. (Kulkarni et al. 1999). In laundry industries, endo-1,4-b-xylanase was effec-
tively used to remove the plant-based stains. The stain-removing capacity of family
11 endo-xylanase was improved when it chemically linked with cellulose-binding
domain of cellulases. In textile industries, xylanase enzymes can be used to process
the plant fiber as they can decrease the lignin content and improve the color and
quality of fibers (Csiszár et al. 2001; Battan et al. 2012). Until now, xylanase
applications in pharmaceutical industries are very limited. However, it is used in
some dietary supplements in combination with other enzymes (Polizeli et al. 2005).
Commercial products such as chewing gum contain xylitol and it can be easily
produced by hydrolysis of xylan using xylanase enzymes.
7.11.6 Future Perspectives
The current list of highly diversified applications of xylanases clearly demonstrates

the increased demand of this type of enzymes. In addition, the continuous
improvement of the enzyme catalytic properties increases the potential uses of this
enzyme by adding many new industrial applications. The improvement of catalytic
properties of the xylanases by shifting the optimal operation conditions of the
enzyme and by isolation of new potential xylanases from extremophilic microbes
are further expressed in suitable mesophilic host to industrialize the process. In
addition, with the increased knowledge about protein engineering, it become pos-
sible also to increase enzyme stability and kinetic properties using different
approaches such as changing of amino acid sequence, creation of sulfide bridge
within the molecule, and changing the enzyme 3D configuration. All these will help
to increase the potential application of xylanases under extreme operation
conditions.
However, some recent studies were skeptic about the future application of
xylanases in biofuel sector based on the current dramatic drop of oil price which
make biofuel non-competitive alternative to fossil fuel in economic point of view.
However, xylan biorefinery is not limited to fuel market but the biohydrolysis
products of xylan such as pentose oligomers and monomers are also used as
intermediate in the production of many biochemicals. Moreover, the increased
public awareness about the negative impacts of chemicals on the environment,
ecosystem, and human and animal health increased the pressure on chemical
industries to replace the current chemical-based catalytic processes to a biobased
and eco-friendly processes especially in paper/pulp, leather, and detergent indus-
tries. In addition, there is significant increased demand for xylanases application in
food and feed industries for the production of safe and high-quality product. These
all together support our current estimation of the continuous increase of xylanases
demand in many industrial applications.
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Chapter 8
Mannanase
Suttipun Keawsompong
Abstract β-Mannanases (endo-1,4-β-D-mannanase) is endohydrolase that catalyze

the random hydrolysis of the β-1,4-D-mannopyranosyl linkage within the main
chain of various mannan-based polysaccharides to yield mannooligosaccharides
products. β-Mannanase have been isolated and characterized from different sources
including bacteria, fungi, higher plants, and animals. However, microbial man-
nanases are wildly used in the industrial application. β-Mannanases was classified
based on the amino acid sequence similarity into glycoside hydrolase (GH) families
5 and 26 and a few member of family 113. These enzymes from different organisms
have different properties such as enzyme activity, optimal pH, and optimal tem-
perature. So, β-mannanases with high specific activity and remarkable enzymatic
properties are required for the application. β-Mannanase is very useful enzyme that
has been used in several industrial applications including food, feed, pulp, and
paper industries. This enzyme can be used to improve the bleaching of pulp by
facilitating the release of lignin from paper pulp leading to the reduction of
chemical reagents. It can be used to reduce the viscosity of instant coffee and to
clarify fruit juices and wines in food industry. β-Mannanase also have been used as
animal feed additive enzyme to increase the nutritional value of animal feed
components. Moreover, there is increasing interest in using β-mannanase to pro-
duce mannooligosaccharides (MOS) which have the prebiotic properties from
natural mannan-based substrates. This enzyme is also used for the pretreatment of
biomass in the bioethanol production.
S. Keawsompong (&)
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University,
Bangkok, Thailand

216 S. Keawsompong
8.1 Mannans
Mannans and heteromannans are widely distributed in nature as part of the hemi-
cellulose fraction in hardwoods and softwoods, seeds of leguminous plants
(Handford et al. 2003; Buckeridge et al. 2000), and in beans (Lundqvist et al. 2003).
Hemicelluloses are copolymers of both hexose and pentose sugars. The branched
structure allows hemicellulose to exist in an amorphous form that is more sus-
ceptible to hydrolysis. Within biomass, mannans or the hemicelluloses are situated
between the lignin and the collection of cellulose fibers underneath. Consistent with
their structure and side group substitutions, mannans seem to be interspersed and
covalently linked with lignins at various points while producing a coat around
underlying cellulose strands via hydrogen bonds, but as few H-bonds are involved
they are much more easily broken down than cellulose. The mannan layer with its
covalent linkage to lignin and its non-covalent interaction with cellulose maybe
important in maintaining the integrity of the cellulose in situ and in helping to
protect the fibers against degradation to cellulases (Puls and Schuseil 1993).
Mannan is the predominant hemicellulosic polysaccharide in softwoods from
gymnosperms, but is the minor hemicellulose in hardwood from angiosperms (Puls
and Schuseil 1993). Unsubstituted β-1,4-mannan, composed of a main chain of
β-mannose residues (Fig. 8.1), is an important structural component of some marine
algae and terrestrial plants such as ivory nut (Chanzy et al. 2004) and coffee bean
(Nunes et al. 2006). It resembles cellulose in the conformation of the individual
polysaccharide chains, and is water insoluble.
The galactomannans are mainly found in the seeds of the family of
Leguminoseae and are located in the endospermic part of the seeds (Dea and
Morrison 1975). Galactomannans consist of water-soluble 1,4-linked β-D-manno-
pyranosyl residues with side chains of single 1,6-linked α-D-galactopyranosyl
groups (Shobha et al. 2005).
Glucomannan is a water-soluble polysaccharide that is considered a dietary fiber.
It is a hemicellulose component in the cell walls of some plant species (Gonzáles
et al. 2004). Glucomannan is a food additive used as an emulsifier and thickener.
The polysaccharide consists of glucose and mannose in a proportion of 1:3 joined
by β-(1,4) linkages (Popa and Spiridon 1998). Hardwoods consist of glucomannan
with a glucose/mannose ratio of 1:1.5–2 (Hongshu et al. 2002).
Galactoglucomannan is a water-soluble hemicellulose, consisting of galactose,
glucose, and mannose. Many softwood species, e.g., Norway spruce are rich of
galactoglucomannans and can contain it up to 10–20 %. Galactoglucomannan
consists of a backbone of randomly distributed (1 → 4)-linked mannose and glu-
cose units with (1 → 6)-linked galactose units attached to mannose units. The
hydroxyl groups in locations C2 and C3 in mannose are partially substituted by
acetyl groups (Willför et al. 2007).
8 Mannanase 217
Fig. 8.1 Structure of a mannan, b galactomannan, c glucomannan, and d galactoglucomannan.

Source: Dhawan and Kaur (2007)
8.2 Mannan-Degrading Enzymes
The mannan-degrading enzymes, a variety of hydrolytic enzymes involves in the

degradation of mannan polysaccharides, are consisted of β-mannanase,
β-mannosidase, β-glucosidase, and other addition enzymes such as acetyl mannan
esterase and α-galactosidase (Moreira and Filho 2008). The overall mechanism of
mannan-degrading enzymes is shown in Fig. 8.2. β-Mannanase, endo acting enzyme,
randomly hydrolyzes the β-1,4-linked internal linkage of the mannan backbone to
produce mannooligosacharides and new chain ends. β-Mannosidase, exo acting
218 S. Keawsompong
Fig. 8.2 Mechanism of mannan-degrading enzymes in the galactoglucomannan degradation.

Source Puls and Schuseil (1993)
enzyme, cleaves β-1,4-linked mannosides and releases mannose from the nonre-
ducing ends of mannan and mannooligosacharides. β-Glucosidases, exo acting
enzyme, hydrolyzes β-1,4-glucopyranose at the nonreducing ends of oligosaccharides
that released from glucomannan and galactoglucomannan degradation. In addition,
the side chain sugars that attached at various points on mannan are removed by
α-galactosidase and acetyl mannan esterase. α-Galactosidase hydrolyzes α-1,6-linked
D-galactopyranose at side chain of galactomannan and galactoglucomannan. Acetyl
mannan esterase releases acetyl groups from galactoglucomannan.
However, mannan degradation by these enzymes is affected by the degree and
pattern of substitution of glucose and glucose residues in backbone of these
polysaccharides (Van Zyl et al. 2010). In addition, pattern of distribution of
O-acetyl group in glucomannan also affects the hydrolysis by the enzymes.
8.2.1 β-Mannanase
β-Mannanase or endo-1,4-β-D-mannanases (EC 3.2.1.78) is the endo hydrolase

enzyme that catalyze the random hydrolysis of internal β-1,4-mannosidic linkages
in backbone chain of mannan polysaccharides, producing various size of man-
nooligosacharides (Puls 1997). β-Mannanase requires at least four sugar residues of
backbone chain for enzyme binding and efficient hydrolysis (Davies et al. 1997).
β-Mannanase hydrolyzes mannooligosaccharides up to a degree of polymerization
(DP) of 4 (Chauhan et al. 2012).
8 Mannanase 219
β-Mannanase is classified into glycoside hydrolases (GH) based on the sequence

similarity. Glycoside hydrolase is a group of enzymes that hydrolyze the glycosidic
bond between molecules in polysaccharides. Based on the amino acid sequence
similarity, glycoside hydrolases are classified into 131 families. This classification
is available on The Carbohydrate-Active Enzymes database (CAZy). Moreover,
each glycoside hydrolase families can be classified into clan based on the catalytic
domains and the protein structure. The different glycoside hydrolase families have
been found to have different folds but, some enzymes from different families have
related folds. Based on these criteria, β-mannanase is classified into glycoside
hydrolase family 5, 26, and a few members in 113 (Dhawan and Kaur 2007; Zhang
et al. 2008). Both of these families are classified into clan A which have a typical
structure of (β/α)8-barrel fold (TIM barrel fold). The crystal structure of
β-mannanases from both of GH family 5 and 26 showed that glutamic acid is
conserved catalytic modules (nucleophiles and acid/base) that are positioned on the
C-terminal of β4 and β7 strands, respectively. In addition, β-mannanase also has an
open cleft with at least four subsites on the active site (Hogg et al. 2003; Zhang
et al. 2008; Chauhan et al. 2012).
Glycosyl hydrolases consist of two hydrolysis mechanisms, retaining and
inverting, leading to either overall retention or inversion of the anomeric configu-
ration at the hydrolysis site (Henrissat et al. 1995). β-Mannanase hydrolyzes the
mannan substrates by retaining mechanism by double displacement reaction
(Fig. 8.3). For the retaining mechanism, two carboxylic acid residues on the active
Fig. 8.3 The retaining mechanism of β-mannanase. Source Chauhan et al. (2012)
220 S. Keawsompong
site of enzyme involve in the hydrolysis as nucleophiles and acid/base catalytic

module. In the double displacement reaction, β-mannanase enzyme attacks by a
nucleophilic carboxylate on the anomeric carbon with concomitant releases of the
aglycon, resulting in a covalent intermediate. Then, the covalent intermediate is
attacked by a nucleophilic water lead to releasing of the glycoside from the enzyme
(Chauhan et al. 2012).
Although, β-mannanases have the similar structure including active site, but
there are difference in the presence and position of catalytic carbohydrate binding
modules (CBMs) that may contain in the structure (Ximenes et al. 2005). CBMs is
use for enhance β-mannanase activity for degrading of cellulose-conjugated man-
nan polysaccharide (Benech et al. 2007). β-Mannanases from GH family 5 contain
CBMs that bind crystalline polysaccharides. For example, β-mannanase from
Cellulomonas fimi, GH family 5, has a mannan-binding module that binds in a
reversible way to soluble mannan, cellulose, chitin, and xylan (Stoll et al. 2000).
Moreover, overexpression of mannanase gene that contains CBMs in Escherichia
coli showed the activity of enzyme for several cellulose substrates (Ximenes et al.
2005). But, β-mannanases from GH family 26 lack of CBMs, so there are no
enzyme activity in crystalline polysaccharides substrate (Hogg et al. 2003). So, this
refers to the different substrate target of β-mannanase in GH family 5 and 26 in
nature.
β-Mannanse enzymes have been isolated and characterized from several different
sources such as bacteria, fungi, plants, and animals. Although the β-mannanases are
widely distributed in microorganisms, higher plants, and animals but the microbial
mannanases are regarded to be useful for their applications. Numerous bacterial and
fungal β-mannanases have been identified and characterized from various strains in
20 different genuses. Among bacteria, most of β-mannanases producing strains are
gram positive bacteria such as Bacillus species and Clostridia species (Dhawan and
Kaur 2007). However, some gram negative bacterial strains have been reported to
produce β-mannanases such as Klebiella sp. (Titapoka et al. 2008) and Vibrio
sp. (Tamamura et al. 1995). The most of β-mannanases producing strains among
fungi are in the genus Aspergillus while Penicillium sp. and Trichoderma sp. have
also been reported (Chauhan et al. 2012). Moreover, some actinomycetes such as
Streptomyces sp. and Cellulomonas sp. have been reported to be β-mannanases
producing strains (Stoll et al. 2000; Bhoria et al. 2009).
Microbial manannanase are extracellular enzyme which could be induced by
mannan polysaccharide (Moreira and Filho 2008; Chauhan et al. 2012). Many
nutritional and physiochemical factors involve and influence in the β-mannanase
production such as carbon and nitrogen sources, inorganic salts, temperature, pH,
time, and dissolved oxygen concentration (Moreira and Filho 2008; Chauhan et al.
2012). There are differences in the incubation time and optimum temperature and
pH for microbial β-mannanase production. For the incubation time, it ranges from
24 to 96 h in bacteria and 3–11 days in fungi. The optimum temperature for
β-mannanase production is in the mesophilic range that corresponds with temper-
ature for microbial growth. For the optimum pH, neutral to alkaline pH is the best
8 Mannanase 221
condition for bacterial growth, and production and acidic pH for fungi (Chauhan
et al. 2012). However, bacterial β-mannanases are more thermostable than fungal
β-mannanases that are important for industrial application, such as β-mannanases
from Bacillus species have the advantages of high activity and convenient isolation
and thus have been used in research and industry (Araujo and Ward 1990).
8.3 Application of β-Mannanase
β-Mannanase has been used in several industrial applications because of its broad
substrate specificity.
8.3.1 Pulp and Paper Industry
β-Mannanase can be used in the enzymatic bleaching of softwood pulps to digest

the mannan component without affecting the cellulose component. The use of
β-mannanase with other enzymes is the alternative method that can equally facil-
itate lignin removal in pulp bleaching and give results comparable to alkaline
pretreatment without the environmental pollution problems (Dhawan and Kaur
2007).
8.3.2 Detergent Industry
Alkaline β-mannanase with stable in detergents has been used in laundry segments
as stain removal boosters. Mannans are generally used as thickening agents in
several products such as hair gel, shampoo, conditioner, and toothpaste. The stains
containing mannan are difficult to remove so this enzyme can cleave into smaller
carbohydrate fragments that can reduce the stain cleaning process and remove
during the washing. Moreover, β-mannanase can be formulated as sanitization
products, contact lens cleanser and hard surface cleansers (Chauhan et al. 2012).
8.3.3 Food Industry
β-Mannanase has been used in the viscosity reduction of coffee bean extracts by
hydrolyzing the mannan component in the coffee extract (Chauhan et al. 2012). It
has been used in the maceration of fruit and vegetable materials and clarification of
222 S. Keawsompong
fruit juices (Moreira and Filho 2008). In addition, β-mannanase can be used in
enzymatic oil extraction of coconut. The enzymatic process can eliminate the
problems of alfatoxin contamination and oxidative rancidity of products (Chauhan
et al. 2012).
8.3.4 Feed Industry
β-Mannanase can be used to improve the nutritional value of animal feed especially
poultry. Mannan polysaccharides are commonly found in feed ingredients such as
soyabean meal, guar meal, copra meal, palm kernel meal, and sesame meal. All
these meals have some common properties such as high fiber content, low
palatability, lack of several essential amino acids, and high viscosity coupled with
several anti-nutritional factors such as mannan, galactomannan, xylan, and arabi-
noxylan that limit the utilization in the animal intestine (Chauhan et al. 2012).
Moreover, they have been found to be highly deleterious to animal performance,
severely compromising weight gain and feed conversion as well as glucose and
water absorption (Dhawan and Kaur 2007). Therefore, incorporation of
β-mannanase in feed can help to cleave mannan and release of nutrients results in
increased villus height in duodenum and jejunum that leads to increase in surface
area and adsorption and decreased intestinal viscosity. So, it can improve both the
weight gain and feed conversion efficiency (Adibmoradi and Mehri 2007). Hemicell
supplied by ChemGen, USA is a fermentation product of B. lentus containing high
amount of β mannanases that degrade mannan in feed (Daskiran et al. 2004).
8.3.5 Mannooligosaccharide Production
Mannooligosaccharide (MOS) is non-oligosaccharides digestibles (NODs) that can

be produced from mannan polysaccharides. MOS is widely used in nutrition as a
natural additive which can improve gastrointestinal health and also overall health
by supporting the microflora in the digestive system.
MOS affects bacterial attachment in the intestinal tract leading to the reduction in
the prevalence and concentration of different strains of Salmonella, as well as
E. coli. Moreover, it effects on promoting beneficial bacteria, such as Lactobacillus
and Bifidobacteria (Spring et al. 2000). MOS helps to limit the pathogenic
microorganisms bacteria by blocking the colonization on the intestinal mucosa lead
so they cannot physically reach or adhere to the intestinal cells and are eliminated
from body.
Large surface area is a key for optimal digestive function of small intestine so the
surface of the small intestine should be covered with long healthy villi. Several
studies of MOS in poultry have looked at the intestinal structure and discovered
8 Mannanase 223
longer villi and a more shallow crypt (Yang et al. 2008; Baurhoo et al. 2009). These
studies showed the results that MOS could increase the energy of digestion and
production of digestive enzymes such as alkaline phosphatase, maltase, and leucine
aminopeptidase. Moreover, MOS could increase the goblet cells, mucus-producing
cells so the villi and intestinal surface could be more protected.
8.3.6 Pharmaceutical Applications
Mannose has been used as a component of medicine because of its properties such
as fast dissolving and structure forming (Chauhan et al. 2012). Mannose also has
been used as a remedy for urinary tract infection (Van Zyl et al. 2010). There is a
significant increase in using this sugar, so β-mannanase and other enzymes can be
used for the economical production of mannose from low-cost mannan substrates
such as palm kernel cake and copra meal. Guar gum has the positive effects on
some physiological functions like reducing plasma cholesterol and body fat without
reducing protein utilization and increased fecal excretion volume (Takeno et al.
1990). Therefore, a partially hydrolyzed guar gum (PHGG) with β-mannanase is
used in beverage form for treatment of several diseases such as irritable bowel
syndrome (IBS). PHGG can increase stool weight and decrease colon transit time
by providing non-digestible bulk, retaining water, and serving as a substrate for
microbial growth in the colon (Parisi et al. 2002). In addition, PHGG supplemented
with oral rehydration solution is also used for the treatment of acute diarrhea in
children by providing short chain fatty acids in large intestine and maintaining the
balance of salt and water (Alam et al. 2000).
8.3.7 Pretreatment of Biomass in the Bioethanol Production
The molecules in wood and soft wood are linked together in complexes known as
cellulose and hemicelluloses and together can represent up to 75 % of dried wood
weight. Moreover, sugar and nutrient is found in wood tissue and soft wood.
Getting access to these complexes is not easy. Surrounding the cellulose and
hemicelluloses is lignin which serves as the glue and protects the polysaccharides
from enzyme and microbial deconstruction. The lignin, cellulose, and hemicellulose
form strong bonds and their combined structure is named lignocellulose. The
process to overcome lignocellulosic recalcitrance and expose the cellulose and
hemicellulose, so that individual sugars can be released, is called pretreatment
shown in Fig. 8.4.
Pretreatment represents the necessary steps to convert the raw substrate into a
suitable form and includes size reduction by grinding, physical, chemical, or
enzymatic hydrolysis to increase substrate availability to release nutrient and sugar.
224 S. Keawsompong
Fig. 8.4 Effect of pretreatment on lignocellulose. Source Mosier et al. 2005
8.3.7.1 Chemical Treatment
Acid Pretreatment
Concentrated acid hydrolysis disrupts the hydrogen bonding of the cellulose chains,
converting the cellulose from a crystalline state to an amorphous state with little
glucose liberation (Kosaric and Vardar-Sukan 2001; Orozco et al. 2007). In 1965,
Oshima showed that crystalline cellulose achieves complete solubility at 72 %
H2SO4 or 42 % HCl at the relatively low temperatures (10–45 °C) (Oshima 1965;
Kosaric and Vardar-Sukan 2001). In the process of concentrated acidic hydrolysis,
dilute acid is used initially in the prehydrolysis digestion column for hemicellulose
removal prior to passing to the secondary counter current column to contact a strong
acidic solution With the concentrated acid method, glucose yields greater than 90 %
have been achieved with a rapid production rates whereas the increased acid
concentration of HCl, a reduction of xylose yield has been showed by the work of
Orozco et al. (2007).
Alkaline Hydrolysis
Alkali is seen as a swelling agent where the alkali acts indirectly with water being
the lysis agent (Kosaric and Vardar-Sukan 2001). By swelling the biomass, the
surface area is increased opening up the structure for water to migrate into the
material. Once inside the biomass, the water disrupts the hydrogen bonding
between the hemicellulose and the lignin carbohydrates (Balat et al. 2008). The
effect is the decrease in crystal and lignin disruption. The advantages of using alkali
over acidic methods are the removal of the lignin fraction without the degradation
8 Mannanase 225
of the other major constituents. The compromise is the increased length of reaction
times [hours or days in comparison to minutes for other processing methods (Balat
et al. 2008)].
Balat et al. (2008) pretreated corn stover with NaOH (2 %) and combined with
irradiation (500 gGy). They found that the glucose yields to increase from 20 % for
NaOH pretreatment to 43 % in combination (Balat et al. 2008). Although NaOH is
the dominant alkali approach, lime [calcium hydroxide, Ca(OH)2] can also be
employed as an alternative method. Ca(OH)2 has been used in the pretreatment of
several feedstocks in pilot scale applications, such as wheat straw (358 K for 3 h
and corn stover (373 K for 13 h). The attraction of lime/alkali pretreatment is the
removal of acetyl and various uronic acid substitutions on hemicellulose that lowers
the accessibility of the enzymes to the hemicellulose and cellulose surface (Sun and
Cheng 2002; Balat et al. 2008).
8.3.7.2 Physical Treatment
Steam Pretreatment
Steam pretreatment, also known as “steam explosion,” has been extensively

investigated and tested in several pilot plants and demo plants worldwide (Galbe
and Zacchi 2012). An additional acid catalyst can be used to increase the effec-
tiveness of the steam pretreatment, in which case hemicellulose recovery and the
enzymatic hydrolysis of the solids both increase (Galbe et al. 2005). The effects of
initial moisture content on steam consumption, mechanism and rate of heat transfer,
pentosan solubilization, and subsequent glucose yield were summarized. Treatment
at 190 °C with complete bleed-down (no explosion), when compared with that at
240 °C with explosion from full pressure, showed at least as good solubilization of
pentosan and enzymatic hydrolysis but showed greater pentosan destruction for the
same degree of pentosan removal. Neither the explosion nor the high temperatures
(above 190 °C) are necessary (Brownell and Saddle 1987).
8.3.7.3 Enzymatic Hydrolysis
During hydrolysis, the polymers of hemicellulose are degraded into their mono-
meric subunits. Complete hemicellulose hydrolysis results in several hexoses and
pentoses. Main sugar in hemicellulose derived from soft wood is mannose
(Taherzadeh and Karimi 2007). The enzyme hydrolysis is catalyzed by hemicel-
lulose enzymes; without any pretreatment the conversion of native hemicellulose to
sugar is extremely slow, since hemicellulose is well protected by the lignin.
Therefore, pretreatment of these materials is necessary to increase the rate of
hydrolysis of hemicellulose (Galbe et al. 2007).
Efficiently releasing all sugars in biomass therefore requires the combined action
of a large number of different enzymes. Furthermore, some of these enzymes could
226 S. Keawsompong
work synergistically and increase overall amount of monosaccharides released.

Cervero et al. (2010) suggested that binary mixtures of commercial enzymes, 1:1
mixture of Mannaway and Gammanase, were used to increase the conversion of
cell-wall material present in biomass to obtain monosaccharides. The binary mix-
tures of commercial enzymes showed good synergistic effect releasing 30 % more
mannose than the sum obtained using these enzymes individually.
8.3.8 Other Applications
β-Mannanase can use as slime control agent in water purification system, waste
water treatment, and cooling water treatment system. It has been used in the
enzymatic hydrolysis of galactomannan to enhance the flow of oil and gas in
drilling operation in the oil and gas industries (Chauhan et al. 2012).
8.4 Conclusions
β-Mannanase (endo-1,4-β-D-mannanase) is an endohydrolase that catalyzes the

random hydrolysis of the β-1,4-D-mannopyranosyl linkage within the main chain of
mannans and various mannan-based polysaccharides to yield mannooligosaccha-
rides products. It can effectively remove mannan (hemicellulose) from biomass
structure in pretreatment, resulting in better digestion of cellulosic materials.
β-mannanases enzyme has been isolated and characterized from several different
sources including bacteria, fungi, higher plants, and animals. It can be widely
applied in industry, especially in the pretreatment of biomass conversion to bio-
sugar for fermentation.
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Chapter 9
The Role and Applications of Xyloglucan
Hydrolase in Biomass
Degradation/Bioconversion
M. Saritha, Anju Arora, Jairam Choudhary, Vijaya Rani,

Surender Singh, Anamika Sharma, Shalley Sharma and Lata Nain
Abstract Lignocellulosic biomass is currently the most promising alternative

energy source for realizing sustainable demands of agrarian economies. Its natural
recalcitrance to degradation necessitates a detailed study on the complex bio-
chemistry involved in bioconversion of this lignin–carbohydrate complex.
A comprehension of the enzymology and role of principal and accessory glycosyl
hydrolases involved in biomass degradation are, hence, noteworthy in this context
and the xyloglucan-active hydrolases warrant special mention. These are enzymes
which carry out hydrolysis and transglucosylation of xyloglucan, the major hemi-
cellulosic polysaccharide in plant biomass. The structurally complex xyloglucans
cover and cross-link the cellulosic microfibrils in plant cell walls and make cellu-
lose inaccessible to saccharification by cellulases. Solubilisation of biomass
polysaccharides and release of sugars are central to the biomass-to-bioethanol
process. Complete conversion of biomass carbohydrates requires a suite of
hydrolytic enzymes, which may be designed specifically to accommodate the
predominant and subsidiary biomass-cleaving enzymes. Xyloglucan hydrolases
which are known to act synergistically with cellulases and xylanases in loosening
the plant cell wall are vital enzymes to be deployed for successful bioconversion
processes. This chapter is an insight into the capacity of these accessory, but
indispensable, hydrolytic enzymes in unlocking the inaccessible biomass polysac-
charides for increased sugar recovery and thereby, in drafting the fuels of future.
9.1 Introduction
Biofuel production employs renewable agricultural waste and dedicated energy

crops, which also provide opportunity for alleviation of green house gas release.
Various plants and parts of plants are used for biofuel production such as grains for
M. Saritha A. Arora J. Choudhary V. Rani S. Singh A. Sharma S. Sharma

L. Nain (&)
Division of Microbiology, Indian Agricultural Research Institute,
New Delhi 110012, India

232 M. Saritha et al.
25
Ethanol production (Billion Gallons)

20
Rest of World
15 Canada
China
10 Europe
Brazil
5 USA
0
2007 2008 2009 2010 2011 2012 2013
Fig. 9.1 Country-wise global ethanol production (2007–2013). Adapted from www.afdc.energy.
gov/data/
the first-generation and lignocellulosic biomass for the second-generation of bio-

fuels. The United States is the world’s largest producer of bioethanol, having
produced over 13 billion gallons in 2013 alone (Ethanol Industry Outlook Report
2013). Together, the U.S. and Brazil produce 84 % of the world’s ethanol, which
amounted to 23,429 million gallons in 2013 (Fig. 9.1). Driven by the growth in use
of biofuels and natural gas, nonpetroleum energy makes up the highest percentage
of total fuel consumption for transport since 1954. According to a new report of the
U.S. Energy Information Administration (EIA), 8.5 % of the fuel used by trans-
portation sector in 2014 came from nonpetroleum sources (AEO 2015). Today,
most of the gasoline available throughout the United States is a blend of 90 %
gasoline and up to 10 % ethanol, known as ‘E10’.
The vast majority of the U.S. ethanol is produced from corn, while Brazil
primarily uses sugarcane. The current production of first-generation bioethanol from
sugars or starch has, but raised a worldwide “food vs fuel” debate putting a question
mark on food security. The diversion of food crops for biofuel production also leads
to food price hikes. Advanced generation biofuels are, therefore, imperative as they
are based on inexpensive and plentiful resources which do not compete with food
crops and do not threaten food security.
As a result, the center of attention in development of technology for ethanol
production has shifted toward the use of nonedible residual lignocellulosic materials
as well as to lower the cost of production. Lignocellulosic biomass is the most
abundant source of renewable energy on the planet. The sugars present in the
lignocellulosic biomass can be converted to biofuel molecules such as ethanol and
butanol. Production of biofuels from lignocellulosic biomass requires the efficient
deconstruction of carbohydrate polymers to fermentable sugars by a cocktail of
9 The Role and Applications of Xyloglucan Hydrolase … 233
enzymes containing cellulases, xylanases, and other glycosyl hydrolases. The

economic feasibility of production of bioethanol from lignocellulose and its com-
petition with petrol is, but, a main challenge. The main bottlenecks in this tech-
nology are low efficiency due to the natural recalcitrance of lignocellulose to
deconstruction and high cost of enzymatic conversion (Mohanram et al. 2013). To
address these, significant research has to be directed toward the identification of
efficient enzyme systems and process conditions for the improved enzymatic uti-
lization of biomass feedstocks. This warrants a detailed understanding of the
enzymology involved in biomass bioconversion.
9.2 Polysaccharides of Lignocellulosic Biomass
Lignocellulose is the main structural constituent of woody plants, herbs such as

grasses and crop biomass. It is the most abundant biopolymer on the planet and
comprises about 50 % of world’s biomass (Claassen et al. 1999). About 686 MT of
crop residues are generated every year in India, as predicted by The Ministry of
Agriculture, Govt. of India (Hiloidhari et al. 2014), out of which 234 MT can be
used as substrate for production of many value-added products like biofuel.
Lignocellulose consists of 40–60 % cellulose, 20–40 % hemicellulose, and
10–25 % lignin (Wyman 1996). Lignocellulosic biomass is a substrate of massive
biotechnological value because of the chemical attributes of its components
(Malherbe and Cloete 2003). The average composition of some lignocellulosic
materials that can be used as substrates for biofuel production is given in Table 9.1.
Plant biomass is mainly composed of the polysaccharides, cellulose, and
hemicellulose with pentose and hexose sugars as the main building blocks.
Cellulose is a D-glucose polymer with organized microfibrillar forms, with each
microfibril containing up to 36 glucan chains having thousands of glucose residues
joined by β-1,4-glycosidic bonds. Hemicelluloses are complex homopolysaccha-
rides (glucan, xylan, mannan) or heteropolysaccharides (xyloglucan, galactoglu-
comannans, arabinoxylan, glucomannan) with both hexose (glucose, mannose,
galactose) and pentose sugars (arabinose, xylose) along with their uronic acid
derivatives (glucouronic acid, galatouronic acid, mannuronic acid) (Mohanram
et al. 2013).
Thus, plant cell walls are built from a mixture of polysaccharides, proteins, and
lignin, whereas the noncrystalline matrix of plant cell walls is built of one or more
of the different types of hemicelluloses such as galactoglucomannan, glucomannan,
arabinoglucuronoxylan, glucuronoxylan, and xyloglucan (Timell 1967). Some of
the common hemicelluloses of plant origin are given in Table 9.2.
Among the hemicelluloses, xyloglucans are widely distributed throughout the
plant kingdom as a major structural and storage polysaccharide. The Type I plant
cell wall, predominant in dicotyledonous plants and non-commelinoid mono-
cotyledons, is characterized by equal proportions of cellulose and xyloglucans. On
Table 9.1 Average composition of biomass expressed as percentage on dry weight basis
Biomass Cellulose (%) Hemicellulose (%) Lignin (%)
Barley wood 40 20 15
Bermuda grass 25 35.7 6.4
Birch wood 40 33 21
Corn cobs 42 39 14
Corn stalks 35 15 19
Corn stover 38 26 19
Cotton seed hair 80–95 5–20 0–5
Flax sheaves 35 24 22
Forage sorghum 34 17 16
Grasses 25–40 35–50 10–30
Groundnut shells 38 36 16
Hardwood stem 40–55 34–40 18–25
Leaves 15–20 80–85 0–5
Miscanthus 43 24 19
Oat straw 41 16 11
Paper 85–99 0–5 0–15
Pine 41 10 27
Rice straw 32 24 13
Rice husk 36 15 19
Rye straw 31 25 7
Saw dust 55 14 21
Sorghum straw 33 18 15
Soybean stalks 34 25 20
Sugarcane 33 30 29
Sugarcane bagasse 42 25 20
Sweet sorghum 23 14 11
Switch grass 37 29 19
Salix 41.5 22–25 25
Softwood stem 45–50 25–35 25–35
Spruce 45 26 28
Wheat straw 30 24 18
Adapted from Jørgensen et al. (2007) and Kumar et al. (2009)
the other hand, Type II cell wall, present in commelinoid monocots like rice,
contains less of xyloglucans and shows predominance of glucuronoarabinoxylan
along with cellulose microfibrils (Yokoyama et al. 2004). Whatever the plant type,
the considerable amount of xyloglucan present is always around 20–25 % of dry
weight in dicots and 2–5 % in grasses (Hayashi 1989; Benko et al. 2008).
Table 9.2 Common hemicelluloses of plant origin

Polysaccharide type Biological Amount Type of linkage between monomeric
origin (%) units
Glucuronoxylans Hardwood 15–35 β-D-xylopyranosyl units linked by β-
(1,4) glycosidic bonds
Galactoglucomannans Softwood 20–25 β-D-glucopyranosyl and β-D-
mannopyranosyl units, linked by β-
(1,4) glycosidic bonds
Arabinoglucuronoxylan Agricultural 5–10 β-(1,4)-D-xylopyranose backbone
crops containing 4-O-methyl-α-D-
(nonwoody glucopyranosyluronic acid and α-L-
material) arabinofuranosyl linked by α-(1,2) and
α-(1,3) glycosidic bonds
Xyloglucan Hardwood 2–25 β-1,4-linked D-glucose backbone with
(mainly 75 % of these residues substituted at
dicotyledons) O-6 with D-xylose. L-arabinose and D-
galactose residues can be attached to
the xylose residues forming di-, or
triglycosyl side chains.
Glucomannan Softwood and 2–5 β-(1 → 4)-linked D-mannose and D-
hard woods glucose
Galactoglucomannan Softwood 10–25 β-(1 → 4)-linked mannose and
glucose units with α-(1 → 6)-linked
galactose units attached to mannose
units
Arabinoxylan Cereal grain 0.15–30 β-(1,4)-D-xylopyranose backbone is
cell walls substituted by a-L-arabinofuranosyl
units in the positions 2-O and/or 3-
O and by α-D-glucopyranosyl uronic
unit or its 4-O-methyl derivative in the
position 2-O
Adapted from Girio et al. (2010)
9.2.1 Xyloglucan: A Predominant Cell Wall Hemicellulose
Xyloglucan is one of the most widespread hemicellulosic polysaccharides in a

lignocellulosic biomass. Xyloglucans form a sheath around the cellulose
microfibrils preventing them from direct aggregation and cross-link different cel-
lulose microfibrils via hydrogen bonds and other non-covalent interactions, thereby
providing structural strength to the plant cell wall (Fry 1989; Hayashi 1989;
Baumann 2007a). These polymers consist of a linear backbone of β-1,4-glucan
linkages and are structurally related to cellulose, but are distinguished by having up
to 75 % of β-D-glucopyranose (β-D-Glcp) residues covalently linked to α-D-xylo-
pyranose (α-D-Xylp) at the O-6 position (Carpita and McCann 2000). Some of the
xylosyl residues are further substituted by galactosyl, arabinosyl, or fucosyl resi-
dues, forming a complex structure (Enkhbaatar et al. 2012; Damásio et al. 2012).
Different xyloglucan side chains are depicted using a single-letter nomenclature, as
Fig. 9.2 The typical structures of the subunits in XXXG-type xyloglucans. Adapted from http://
www.ccrc.uga.edu/
proposed by Fry et al. (1993), in order to overcome the complexity in their

structural description. According to it, the unsubstituted and substituted
Glcp residues are represented as G and X, respectively. The substituted
Glcp residue may be further denoted as L/F/U/S, depending upon the substituted
saccharide residue. For example, F represents a Glcp residue that is substituted with
a fucose-containing trisaccharide. Most xyloglucans consist of repeating units of
either XXXG (XXXG-type), or XXGG (XXGG-type) (Vincken et al. 1997). The
primary walls of a wide range of dicots, non-graminaceous monocots, and gym-
nosperms contain fucosylated xyloglucans with an XXXG-type structure, the typ-
ical subunit structures of which are shown in the Fig. 9.2.
The side chains confer several properties on the polymer. The high solubility of
xyloglucan in water and its rheological properties make it a useful food additive,
thickener, stabilizer, and gelling agent (Yamatoya and Shirakawa 2003; Baumann
2007b). Xyloglucan can also replace starch and galactomanan used in papermaking
(Shankaracharya 1998). However, it cannot form ordered crystalline microfibrils
like cellulose (Fry 1989). Pauly et al. (1999) described three structural domains of
xyloglucans: the xyloglucan cross-links that are susceptible to enzymatic cleavage,
a xyloglucan fraction that is tightly bound to the microfibril surface and a third
component that is trapped within the microfibril periphery. This structural com-
plexity suggests the importance of understanding the mechanism of xyloglucan
metabolism and biochemical characterization of the enzymes involved.
9.3 Enzymology of the Biomass Bioconversion Process
Due to high carbohydrate content, lignocellulose biomass like agricultural waste

and plant residues, dedicated energy crops, municipal, and industrial wastes hold
great potential for large scale production of bioethanol (Farrell et al. 2006). The
cellulose and hemicellulose components of biomass can be converted into sugar
monomers from which a range of biomaterials viz. ethanol or other important
chemical intermediates can be synthesized via fermentation or other processes.
The classical model for hydrolysis of cellulose to glucose involves the synergistic
action of endocellulases (EC 3.2.1.4), exocellulases (cellobiohydrolases, CBH, EC
3.2.1.91; glucanohydrolases, EC 3.2.1.74), and β-glucosidases (EC 3.2.1.21).
Endocellulases cleave glucan chains internally in a random fashion, which results in

a faster decrease in length of polymer and a gradual increase in the reducing sugar
concentration. Exocellulases hydrolyze cellulose mainly by removing cellobiose
either from the reducing or non reducing ends, which leads to a rapid release of
reducing sugars but a small change in length of polymer. By cooperative action of
endocellulases and exocellulases on cellulose, cello-oligosaccharides and cellobiose
are produced, which are further hydrolyzed by β-glucosidase to glucose (Mohanram
et al. 2013). Though cellulases are the most important hydrolytic enzymes for sac-
charification of complex polymers, they can only unlock the sugars entrapped in
cellulose leaving behind the unused hemicellulose. Thus, the hemicellulose portion
of biomass is the largest polysaccharide fraction that gets wasted in most lignocel-
lulosic biomass-associated ethanol production processes (Girio et al. 2010). As the
cellulose polymers are entangled with hemicellulose which gives them structural
strength, the accessibility of the cellulose polymers to hydrolytic enzymes is hin-
dered, which thereby interferes with the saccharification process.
Hemicelluloses are cleaved by enzymes like carbohydrate esterases, glycoside
hydrolases, polysaccharide lyases, xyloglucan hydrolases, endohemicellulases, and
others, the concerted action of which hydrolyze ester bonds, glycosidic bonds, and
remove the chain substituents or side chains (van den Brink and de Vries 2011;
Sweeney and Xu 2012). The various hydrolytic enzymes which target the linkages
present in the hemicellulose polymers are detailed in Table 9.3.
Table 9.3 List of hemicellulases that can be supplemented to cellulase enzyme to enhance
saccharification efficiency of lignocellulosic biomass
Enzyme GH family Enzyme Site of action
classification
number (EC)
β-Xylanase GH 10, GH EC 3.2.1.8 Hydrolyses β-1,4 glycosidic bond
11 in xylan
β-Xylosidase GH 52 EC 3.2.1.37 Hydrolyses xylobioses
Glucanase GH 16 EC 3.2.1.- Hydrolyses β-1,3; β-1,6; α-1,4; α-1,6
glucans
Mannanase GH 26 EC 3.2.1.78 Hydrolyses β-1,4-mannosidic linkage in
the main chain of mannan,
galactomannan, glucomannan and
galactoglucomannan
Xyloglucan GH 12, GH EC Hydrolyses xyloglucan (β-1,4 glucan
hydrolase 16 3.2.1.150,151,155 with α-1,6 linked xylose)
Arabinofuranosidase GH 43, GH EC 3.2.1.55 Removes arabinose substituents from
51, GH 54, α-arabinofuranoside, arabinoxylans,
GH 127 arabinogalactans
Glucouronidase GH 67, GH EC 3.2.1.139 Removes α-1,2 linked glucuronoyl or its
79 methyl ester in xylan
Acetyl xylan CE 1, CE 5, EC 3.1.1.72 Removes acetyl group from xylans or
esterase CE 16 other xylooligosaccharides
Feruloyl esterase CE 10 EC 3.1.1.73 Hydrolyses feruloyl esters
Glucuronoyl CE 15 EC 3.1.1.- Demethylates methyl glucuronoyl α-1,2
esterase linked to backbone xylose
Adapted from Sweeney and Xu (2012)
Depending upon the type and abundance of hemicellulose polymer present in the
biomass used as substrate, different combinations of hydrolytic enzymes listed in
Table 9.3 can be supplemented to cellulases. Presence of hemicellulases in
hydrolytic enzyme cocktail has been reported to enhance the saccharification yield
by releasing the sugars entrapped in hemicelluloses and also by increasing the
accessibility of the cellulase enzyme to the cellulose complex (Singh et al. 2014).
9.4 Xyloglucan-Active Enzymes in Modification of Cell

Wall Polysaccharides
The hydrogen bonding present between cellulose and xyloglucan makes the
extraction of latter difficult. In order to access and modify the cellulose fibers
embedded and cross-linked by xyloglucans in the plant cell walls, remodeling of
xyloglucan is required. In nature, the enzymatic hydrolysis of xyloglucan and the
resulting xyloglucan solubilization is important in controlling wall strength during
cell growth (Takahashi et al. 2015). The enzymatic machinery comprising of
specific xyloglucanases that can digest xyloglucans to xyloglucan-oligosaccharides
is, thus, of utmost importance in improving accessibility of cellulose to hydrolytic
enzymes and in enhancing the conversion rate of polymers to sugars in order to
economize the saccharification process. In contrast to the cell wall degrading
enzymes, they cause cell wall modifications that are usually associated with the
weakening of extracellular barriers.
Xyloglucan depolymerization was earlier thought to be due to the action of
endoglucanases and other undefined hydrolases. Later, xyloglucan endotransgly-
colases (XETs) and xyloglucan-specific endo-β-1,4-glucanase were identified from
germinating Nasturtium seeds, where it catalyzed the seed storage xyloglucan
depolymerization (Rose et al. 2003). Subsequently, several proteins belonging to
the same class and with sufficient sequence homology were discovered. Presently,
xyloglucan hydrolases which carry out hydrolysis of xyloglucan are classified in the
xyloglucan endotransglucosylase (XET)/hydrolase (XEH) superfamily, known as
the XTH superfamily (Rose et al. 2003). The modes of action of these enzymes are
illustrated in the Fig. 9.3.
Several xyloglucan-active degrading enzymes of the XTH superfamily have been
reported by various researchers. These include xyloglucan-specific
endo-β-1,4-glucanase (EC 3.2.1.151), xyloglucan-specific exo-β-1,4-glucanase (EC
3.2.1.155), oligoxyloglucan reducing-end-specific cellobiohydrolase (EC 3.2.1.150),
xyloglucan endotransferase (EC 2.4.1.207), xyloglucan 4-glucosyltransferase (EC
2.4.1.168), xyloglucan 6-xylosyltransferase (EC 2.4.2.39), and xyloglucan
2-galactose transferase (EC 2.4.1.-), belonging to several glycosyl hydrolase
(GH) families: GH5, GH12, GH16, GH 26, GH44, and GH74 (Hayashi et al. 1980;
Fry et al. 1992; Pauly et al. 1999; Faik et al. 2002; Madson et al. 2003; Grishutin et al.
Fig. 9.3 Modes of xyloglucan depolymerization by the XTH family of enzymes
Table 9.4 Nomenclature and enzyme characteristics of xyloglucan hydrolase

Particulars Xyloglucan hydrolase
Oligoxyloglucan β-glycosidase Xyloglucosyl transferase
EC number 3.2.1.120 2.4.1.207
Systemic name Oligoxyloglucanxyloglucohydrolase Xyloglucan:
Xyloglucosyltransferase
Reaction type Hydrolysis of O-glycosyl bond Transglycosylation
pH optimaa 4.5–5.0 5.0–6.0
Temp. optimaa 55–60 °C 25–30 °C
Km value Varies according to substrate 1.4–2.2 (xyloglucan)
Tamarind xyloglucan: 0.0024
Xyoglucan oligosaccharide: 0.966
Thermostabilitya Stable below 35 °C Stable below 35 °C
Inhibitors Cu2+,Fe2+,Fe3+, Hg2+, SDS Ammonium sulfate, 4-O-methyl
glucuronoxylan, Ag+, AlCl3,
alginate, ATP, CaCl2, La3+,
NaN3, etc.
a
Varies according to the organism from which enzyme is produced
Adapted from BRENDA enzyme database (http://www.brenda-enzymes.org/)
2004; Yaoi et al. 2004; Baumann 2007b). The particulars of a few enzymes involved
in xyloglucan hydrolysis are given in Table 9.4.
As stated previously, xyloglucan hydrolases are believed to be cell
wall-loosening enzymes that are responsible for cell elongation by continuous
splicing of existing polymers and linking of new xyloglucan residues (Fry et al.
1992; Miedes et al. 2011). High expression of the enzyme in growing parts like
elongating tissues in Cicer arientinum (Romo et al., 2005), hypocotyls in pine
(Lorences 2004), germinating Nasturtium seeds (Stratilova et al. 2010), fiber
elongation in cotton (Michailidis et al. 2009), and even ripening of tomatoes
(Miedes and Lorences 2009) have been reported. Enhanced expression of the
enzyme in plants has also been reported on contact with pathogenic or beneficial
microorganisms. Mycorrhizal association in Medicago truncatula caused enhanced
expression of enzyme in roots both intimately and distantly associated with the
fungal partner (Maldonado-Mendoza et al. 2005) and Botryosphaeria dothidea
caused enhanced expression of the enzyme in apple tree (Bai et al. 2015).
However, due to their role in maintaining the cell wall structure, xyloglucan-
active enzymes may also be considered potential hemicellulose-repairing enzymes.
The XTHs produced during infection of apple fruit by Penicillium expansum has a
dual role of integrating newly secreted xyloglucan chains into an existing
wall-bound xyloglucan and restructuring existing cell wall material by catalyzing
transglucosylation between previously wall-bound xyloglucan molecules
(Muñoz-Bertomeu and Lorences 2014). Their activity gets inhibited as the infection
progresses, leading to hemicellulose degradation.
9.4.1 Mechanism of Action of Xyloglucan Hydrolases
All glycosyl hydrolase enzymes act by causing splits in glycosidic bonds and
presumably, utilize either the inverting or the retaining mechanism. The inverting
mechanism is a direct displacement mechanism, resulting in the inverted configu-
ration on the anomeric carbon. Enzymes employing this reaction mechanism belong
to either GH 44 or GH 74 family and generally exhibit a distance of approximately
11 Å between the catalytic residues (Baumann 2007b). The cellulosomes of
Clostridium thermocellum are found to produce extracellular cellulases, along with
an inverting xyloglucanase CtXGH74A (Martinez-Fleites et al. 2006). Retaining
xyloglucan hydrolases utilize the double displacement mechanism for catalysis. The
cell wall active xyloglucan hydrolases belonging to the GH 16 family employ this
mechanism that involves the formation of a covalent glycosyl-enzyme intermediate
(Baumann 2007b). The only xyloglucan hydrolyzing enzyme from GH16 charac-
terized in detail is NXG1, originally isolated from Nasturtium cotyledons (Edwards
et al. 1985). Recently, close homologs of NXG1 analyzed by expression profiling
have been shown to be expressed during different stages of plant development.
These include the OsXTH19 from rice leaves (Yokoyama et al. 2004) and the
SlXTH6 from tomato shoot, seeds, and roots (Saladie et al. 2006). The GH16
transglycosylases which catalyze endotransglycosylation of xyloglucan that enables
cell expansion also employ the retaining mechanism (Sinnott 1990). The modes of
action of xyloglucanases have also been variously classified into the endo-mode
involving cleavage at multiple sites along the backbone, and the exo-mode
involving reducing end-specific cleavage (Feng et al. 2014).
9.4.2 Role of Xyloglucan-Active Enzymes in Biomass

Saccharification for Biofuel Production
Xyloglucan hydrolase is a ubiquitous enzyme produced by both plants and

microorganisms. Plants, being more complex systems, express a great diversity of
these hydrolytic enzymes, but cannot be exploited for their commercial production.
Microorganisms offer more congenial culturing conditions for greater production in
less space, while allowing for manipulation of culture conditions or gene expression
to a greater extent.
The first described xyloglucanase of fungal origin was the xyloglucan-specific
endo-β-1,4-glucanase of Aspergillus aculeatus (XEG; 34 kDa, pI 3.4, GH12)
(Pauly et al. 1999). Since then, several enzymes involved in the transformation of
xyloglucan have been identified from microbial sources. Table 9.5 gives a list of
various microorganisms known to produce xyloglucan-hydrolysing enzymes.
There have also been several reports on gene cloning of xyloglucanases from
bacteria and fungi. Two GH74 genes from Streptomyces avermitilis (sav_1856 and
sav_2574 genes) have been cloned and expressed using a Streptomyces expression
system (Ichinose et al. 2012). The C-terminal of sav_1856 gene product
(SaGH74A) was found to be similar to CBM2, which showed similarity with the
sequences of CBMs (Carbohydrate Binding Molecules) from Cellulomonas fimi
and Clostridium cellulovorans, while the sav_2574 gene product (SaGH74B), in
contrast showed relatively low similarity with the other GH74 enzymes. Song et al.
(2013) cloned two GH family 12 xyloglucanase genes (designated RmXEG12A and
RmXEG12B) from the thermophilic fungus Rhizomucor miehei and expressed in
Escherichia coli. The xyloglucanases had the ability to hydrolyze tamarind
xyloglucan into oligomers, mainly XXXG, XXLG or XLXG, and XLLG, and
showed no activity toward other linear polymers. In another study, Aspergillus
nidulans A773 (pyrG89) recombinant secretion of a xyloglucan-specific
endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus was studied
(Damásio et al. 2012). XegA was found to generate a xyloglucan-oligosaccharides
pattern similar to that observed for cellulases from family GH12, demonstrating that
its mode of action includes hydrolysis of the glycosidic linkages between glucosyl
residues that are not branched with xylose.
Various endoglucanases (non-xyloglucan-specific endo-β-1,4-glucanases) have
been reported to hydrolyze xyloglucan as a substrate analogue (Vlasenko et al.
2010). The Trichoderma reesei endoglucanase (Cel12) hydrolyzes 1,4-β-glucans
such as cellulose, 1,3-1,4-β-glucan and xyloglucan (Yuan et al. 2001). However,
xyloglucan-specific endo-β-1,4-glucanases which display high activity toward
xyloglucan, with little or no activity toward cellulose or cellulose derivatives, have
also been reported (Song et al. 2013). Also, enzymes annotated as 1,4-β-D-glucan
glucohydrolases or β-glucosidases of GH3 family have greater activities toward
xyloglucan oligosaccharide than toward cello-oligosaccharide (Yaoi and Miyazaki
2012). Xyloglucanases from the GH74 family have been frequently reported to
have high specificity toward xyloglucan (Enkhbaatar et al. 2012; Feng et al. 2014).
242
Table 9.5 Characteristics of xyloglucan hydrolases produced by different microorganisms

Microorganism Molecular weight Isoelectric point Optimum Optimum Km for xyloglucan References
(kDa) (pI) temperature pH (mg/ml)
Bacteria
Ruminococcus 78 – 25 5.0 2.79 Warner et al. (2011)
flavefaciens
Bacillus licheniformis 26 – – – – Gloster et al. (2007)
Paenibacillus pabuli 40 – – – – Gloster et al. (2007)
Fungi
Aspergillus japonicus 32 2.8 – 5.0 0.67 Grishutin et al.
(2004)
Aspergillus niger – – – – 0.54 Powlowski et al.
(2009)
Chrysosporium 78 3.8 – 6.0 0.31 Grishutin et al.
lucknowense (2004)
Trichoderma reesei 75–105 4.1–4.3 – 5.3 0.30 Grishutin et al.
(2004)
Geotrichum sp. 80 4.8 55 °C 5.5 – Yaoi and Mitsuishi
(2004)
M. Saritha et al.
These cleave the ‘X–X’ and ‘X–G’ motifs and have been characterized as having
endo-processive modes of action, i.e., the enzymes act by sliding along the back-
bone during the catalytic action (Matsuzawa et al. 2014). These include the
endo-xyloglucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae
(Feng et al. 2014), and SaGH74A and SaGH74B from Streptomyces avermitilis
(Ichinose et al. 2012). In contrast, the OXG-RCBH from Geotrichum sp. M128
recognizes the reducing end of various xyloglucan-derived oligosaccharides and
releases two glycosyl residues from this site in the exo-mode (Ichinose et al. 2012).
A growing interest in xyloglucanases is mainly due to the possibility of their
application in a number of biotechnological processes, such as conversion of plant
waste, modification of xyloglucans for use in food and feed industries as well as
pulp and paper industry, production of novel surfactants from oligoxyloglucans,
and thermally reversible xyloglucan gels for drug delivery (Sinitsyna et al. 2010;
Damásio et al. 2012). During the conversion of biomass to value-added products, a
suitable cocktail of various hydrolytic enzymes is required to release all the sugars
present in the biomass. Enzymatic saccharification has renewed and centralized the
focus on different aspects of hemicellulases as they play an important role in
improving the economics of the overall process. The barriers, which include
development of robust enzyme formulations containing all accessory enzymes in
commercial cellulases, have to be alleviated for successful commercialization of
biofuels. An economically feasible enzyme technology for complete hydrolysis of
biomass polysaccharides into sugar monomers is very important for cost-effective
biofuel production. To achieve this, there is need to search for organisms with
hypercellulase activity for developing better quality cellulase formulations with
superior attributes such as higher efficiencies, thermostability, and lowered feed-
back inhibition and tolerance to inhibition by toxic byproducts from pretreatments,
using sophisticated biotechnological tools (Bon and Ferrara 2007).
Another feasible option is to supplement cellulase preparations with hemicel-
lulases and other accessory enzymes to enhance the sugar recovery from pretreated
lignocellulosic biomass. It has been reported that Trichoderma reesei, a widely
known cellulolytic filamentous fungus and the main industrial source of commer-
cially available cellulases has only one GH74 gene that codes for xyloglucanase,
while several other fungi have more than two genes for the same (Ichinose et al.
2012). In this context, microbial xyloglucan hydrolase preparations may be
important for designing enzyme cocktails for industrial biomass conversion,
because the degradation of xyloglucan could enhance cellulase accessibility to
cellulose (Kaida et al. 2009). There are several reports on improvement in sugar
yields during biomass bioconversion when cellulases were supplemented with
xyloglucan hydrolase. Supplementation of α xylosidase with commercial cellulases
enhanced the release of free glucose (82–88 %) and xylose (55–60 %) from
hydrogen peroxide pretreated corn stover (Jabbour et al. 2013). Xyloglucanase
addition has also been shown to enhance the hydrolysability of various lignocel-
lulosic substrates when added to a cellulase mixture (Nishitani 1995; Benko et al.
2008). Hydrolysis of glycosidic linkage of xyloglucans resulted in swelling of
cellulose microfibrils which enhanced cellulose accessibility and, subsequently,
efficiency of enzymatic hydrolysis (Vincken et al. 1995; Chanliaud et al. 2004).

Hydrolysis of various steam pretreated lignocellulosic substrates was greatly
enhanced by the supplementation of family 10 and 11 endo-xylanases (GH10 EX
and GH11 EX) and family 5 xyloglucanase (GH5 XG) with cellulose monocom-
ponents (Hu 2014). However, the extent of enhancement caused by xyloglucan
hydrolase depends on the relative concentration of hemicellulose in the biomass
(Benko et al. 2008). In other words, the accessibility of polymers to hydrolytic
enzymes is affected by the method of pretreatment adopted in the conversion
process. Higher conversion rate is observed when large amount of hemicellulose
remains in the raw material after pretreatment signifying the need of xyloglucan
hydrolases to loosen the complex network of sugars (Benko et al. 2008).
Supplementations of other hemicellulases like xylanase (Choudhary et al. 2014) and
xylosidase (Jabbour et al. 2013) has also caused significant enhancement in sugar
production, increasing the efficiency of the overall bioconversion process. It is quite
evident from the above reports that supplementation of hemicellulases to enzymatic
cocktails can cause significant enhancement in conversion rate of polymers to
sugars but its role in economy of the overall bioconversion process cannot be
clearly envisaged. It is well known that enzyme production and purification is a
costly affair. It cannot be clearly said whether enhancement in sugar yield would
compensate the cost involved in the production of the hydrolytic enzyme.
Development of transgenic microbes capable of producing the entire hydrolytic
enzyme required for biomass conversion in appropriate ratio is one way to save the
cost incurred in the process (Rani et al. 2014). Hyper-xyloglucan hydrolase pro-
ducing microorganisms may be developed to produce xyloglucan-active enzyme
preparation to design cellulase cocktails of the future. Another recent approach has
been the creation of engineered chimeras by fusing xyloglucan-specific CBMs to
xyloglucanases to assist polysaccharide recognition and binding, increasing the
enzyme concentration on the substrate surface and thereby improving the hydrol-
ysis rate (Guillen et al. 2010). Recently, the efficiency of a xyloglucan-specific
endo-β-1,4-glucanase from Aspergillus niveus (XegA) was enhanced by fusion of
the xyloglucan-specific CBM44 on XegA leading to the creation of an engineered
CBM44-XegA chimera (Furtado et al. 2015). This fusion conferred superior cat-
alytic properties and thermal stability on the XegA. Further detailed enzymatic
studies at the molecular level are required to provide the fundamental understanding
required to engineer these proteins toward new applications.
9.5 Conclusion
Breakdown of lignocellulosic biomass by microorganism is a highly complex

process, requiring multiple types of synergistic catalytic activities concurrently
acting upon a variety of both soluble and insoluble polymeric substrates. Industrial
biomass utilization processes will also require a combination of hydrolytic enzymes
in order to degrade lignocellulosic feedstocks. Structural complexity of
lignocellulosic biomass makes the enzymatic saccharification of polysaccharides

present in biomass more difficult and costlier. As xyloglucan is a major hemicel-
lulosic polysaccharide present in the cell wall of dicotyledonous plants which
provides structural rigidity to cell wall, xyloglucan hydrolases are of special
interest. Commercial cellulases in combination with accessory enzymes like xyla-
nases and xyloglucanases enhance the yields of glucose and xylose from biomass,
and may prove beneficial in industrial biofuel production. This is because cellulases
interact synergistically with endo-xylanases and xyloglucanases to improve sugar
recovery from various pretreated lignocellulosic substrates. The future efforts will
have to focus on detailed enzyme structure–function analysis of the various
xyloglucanases used by microorganisms during plant cell wall degradation to be
used as accessory enzymes for effective saccharification of lignocellulosic biomass.
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Part III
Lignocellulose Oxidureductases
Chapter 10
Role of Mushroom Mn-Oxidizing
Peroxidases in Biomass Conversion
Mirjana Stajić, Jelena Vukojević, Ivan Milovanović,

Jasmina Ćilerdžić and Aleksandar Knežević
Abstract Intensive agricultural and industrial production followed by increased

production of lignocellulosic wastes, disruption of environment, and depletion of
natural resources are features of the modern society. However, these wastes present
sustainable resources of fibers and energy and can be useful raw materials for many
industries. Therefore, development of the optimal ways for their environmental and
economical friendly biological pretreatment where the main participants will be
fungi, owing to their ability to produce lignocellulolytic enzymes, preoccupies
scientists. Mn-oxidizing peroxidases play crucial role in the process and based on
the substrate specificity, this group is divided into Mn-dependent- and versatile
peroxidases. However, delignification capacity depends on fungal species and
strain, namely on their potential of lignocellulolytic enzyme production and
degradation selectivity, type and composition of lignocellulosic wastes, and fer-
mentation conditions. Species which predominantly degrade lignin and significantly
weaker cellulose could have important role in processes of food, feed, paper, and
biofuel production.
10.1 Introduction
Steady growth of the world population accompanied by increase of agricultural and

industrial production leads to enhancement of types and amount of plant residues,
on the one hand, and to disruption of environment and rapid depletion of natural
energy and fiber sources, on the other hand. Dominant crops are different at the
continents, for example rice is the most common in Asia, maize in America, and
wheat in Europe, and result of that is accumulation of different lignocellulosic
wastes. In 2010, rice residues were the most abundant, with annual production of
104,507 × 104 tons, and wheat and maize ones followed them with production of
98,104 × 104 tons and 84,031 × 104 tons, respectively (Gupta and Verma 2015).
M. Stajić (&) J. Vukojević I. Milovanović J. Ćilerdžić A. Knežević

Faculty of Biology, University of Belgrade, Takovska 43, Belgrade 11000, Serbia

252 M. Stajić et al.
According to Asim et al. (2015), world production of agricultural waste in 2013

was 150 billion tons. The municipal waste also takes significant place in total
annual waste production, approximately 10 %, and it also contains certain amount
of lignocellulosic residues in the form of paper products, food scraps, and garden
waste (Blumenthal 2011). Nowadays, management of the wastes presents real
challenge because major part is improperly deposited and fewer amounts is burned
producing not only CO2 but also other pollutants. However, lignocellulosic biomass
can be useful raw materials for many industries as well as potential source of
renewable energy. Currently, special attention is given to regular disposal and
recycling of the enormous quantity of plant wastes, energy recovering, as well as to
raising awareness of the importance of these processes.
Plant cell wall is lignocellulosic in nature and composed of cellulose (40–50 %)
and hemicellulose (20–30 %) impregnated with lignin (10–25 %) (Kirk and Cullen
1998; Anwar et al. 2014). Cellulose is the most abundant insoluble and resistant
carbohydrate in biosphere with crystalline and amorphous regions. It is composed
of chains built of (1,4)-D-glucopyranose units connected by β-1,4 linkages that have
tendency of forming intra- and intermolecular hydrogen bounds and in such a way
micro-fibrils (Iqbal et al. 2011). This polymer links by non-covalent linkages with
second complex and heterogeneous polymer, i.e., hemicellulose. The heterogeneity
originates from the presence of numerous sugars, among which xylan is the most
frequent in grasses and straws and mannan in hard- and softwood (Anwar et al.
2014).
Lignin is the most recalcitrant natural aromatic compound on Earth, which
provides strength and rigidity to plant cell walls, protection of structural polysac-
charides from hydrolytic enzymes, and barrier against microbial attack. This
heterogeneous polymer is composed of oxygenated phenylpropanoid units (con-
iferyl, sinapyl, and p-coumaryl alcohols), which are bounded by aryl–ether, aryl–
aryl, and carbon–carbon bonds (Guerriero et al. 2015). However, composition,
branching level, type of linkages with carbohydrates, as well as content of lignin
vary from species to species and also depend on plant development stage. Thus,
composition variation depending on plant species was demonstrated by Lapierre
(1993) who showed that ratio among lignin units, p-hydroxyphenyl, guaiacyl, and
syringyl, in spruce was 2:98:0, in birch 0:24:76 and in wheat straw 5:49:46.
10.2 Lignocellulose Degradation
Nowadays, special attention is given to possibility of degradation of lignin from

lignocellulosic biomass and making access to cellulose and hemicellulose that can
be converted into feeds, paper, biofuels, and other valued products. Regarding the
complex structure, degradation of lignocellulose is very demanding process that can
be realized in four ways: physical, chemical, physico-chemical, and biological,
which differ one from another in efficiency and drawbacks (Yang and Wyman
2008; Asgher et al. 2013). Although special equipments and industrial processes are
10 Role of Mushroom Mn-Oxidizing Peroxidases … 253
needed for physical treatments, they are useful because they lead to reduction of
particle size, commonly by grinding, pelleting or heating, and in such a way
improve digestibility of lignocellulosic biomass (Kuijk et al. 2015). Chemical
hydrolysis is based on usage of either acids (hydrochloric or sulphuric acid in high
concentrations) or bases (sodium or ammonium hydroxide) or not selective
chemicals (H2O2), and physico-chemical hydrolysis combine heat, moisture,
pressure, and chemicals (Hendriks and Zeeman 2009). However, numerous studies
have shown that these degradation processes are inefficient, very expensive, and
hazardous for the environment. Therefore preference is given to an alternative
environmental and economical friendly biological delignification, which unique
participants are bacteria and fungi owing to their ability to produce lignocellulolytic
enzymes. The main advantages of the process are energy saving and environmental
protection (Kuijk et al. 2015). Nevertheless, the process has specific demands and
some flaws. Namely, it can be realized only under aerobic conditions and effec-
tiveness depends on lignin structure, i.e. type and ratio of its constituents, branching
level, as well as lignin amount. Grabber (2005) showed that highly branched lignin
is less degradable than mainly linear lignin, Skyba et al. (2013) that syringyl-rich
lignin is more resistant to degradation by fungal enzymes, and Arora and Sharma
(2009) that negative correlation exists between lignin amount and dry matter
digestibility. Loss of dry matter by consumption of cellulose and hemicellulose by
fungi, slowness, and requirement for sterile conditions are the main drawbacks of
the process (Kuijk et al. 2015).
Filamentous fungi, among which Basidiomycetes stand out specially, are good
degraders of lignocellulose. According to lignocellulose decay pattern, Eriksson
et al. (1990) separated three groups of fungi: white rot, brown rot and soft rot, and
their activities depend on environmental conditions, plant species, and cell type.
White rot fungi are common in hardwood forests and include several hundred
species of Basidiomycetes and some Ascomycetes, which breakdown lignin
especially during the early phase of colonization, make access to the cellulose and
hemicellulose, and depolimerize them in fruiting stage (Hammel 1997; Kirk and
Cullen 1998; Sánchez 2009; Kuijk et al. 2015). Ginterová and Lazarová (1987)
showed that ratio among degraded lignin, cellulose, and hemicellulose at the end of
wheat straw colonization by Pleurotus ostreatus was 37.4:20:32.3 % and at the end
of fructification 41.8: 62.1: 60.8 %. Some species, such as Phanerochaete
chrysosporium, degrade lignin, cellulose, and hemicellulose simultaneously and
other species, for example Ceriporiopsis subvermispora, attack lignin first. White
rot fungi are the most efficient lignin mineralizators which have ability to cleave
ether bounds in non-phenolic lignin substructures whose proportion in the woody
biomass is 90 %. Contrary to these fungi, brown-rot species that are mainly present
in coniferous forests only modify lignin and their main activity is cellulose and
hemicellulose depolymerisation, while soft rot species degrade only cellulose under
conditions of high moisture and low lignin content (Sánchez 2009).
According to Leonowicz et al. (1999) and Sánchez (2009), white rot fungi
produce three groups of enzymes: (A) lignocellulolytic enzymes, which include
ligninases (lignin peroxidase, Mn-oxidizing peroxidases, laccase, horseradish
peroxidase, protocatechuate 3,4-dioxygenase, catechol 1,2-dioxygenase and

1,2,4-trihydroxybenzene 1,2-dioxygenase), celullases (endo-1,4-β-glucanase, cel-
lobiohydrolase, β-glucosidase), and hemicellulases (endo-1,4-β-xylanase, xylan
1,4-β-xylosidase, endo-1,4-β-mannanase, β-mannosidase, β-glucosidase,
α-galactosidase); (B) enzymes which cooperate with the first group of enzymes but
never attack wood independently (superoxide dismutase and glyoxal oxidase);
(C) feedback type enzymes that include glucose 1-oxidase, aryl-alcohol oxidase,
pyranose 2-oxidase, cellobiose dehydrogenase, and cellobiose quinone oxidore-
ductase, which are involved both in lignin depolymerization (indirectly) and cel-
lulose degradation.
Due to unique lignin properties, such as molecule size and branching level,
specific bounds and insolubility in water, delignification should be extracellular,
oxidative, less specific, and slow process where non-specific oxidoreductases have
the main role (Hammel 1997). Essential enzymes for delignification beginning are
fungal peroxidases, which produce certain radicals that can penetrate cell wall, and
enzymes producing H2O2 (aryl-alcohol oxidases, glucose 1-oxidase, pyranose
2-oxidase, glyoxal oxidase, and cellobiose quinone oxidoreductase) that is required
for peroxidase activity (Hammel and Cullen 2008). Additionally, in that stage of
lignin mineralization superoxide dismutase also has important role because of its
ability to catalyze conversion of superoxide anion into O2 and H2O2 and in such a
way protects fungal degrader from radicals.
Lignin peroxidases (EC 1.11.1.14) are H2O2 dependent heme-containing
enzymes which oxidize aromatic compounds, such as phenols and anilines that
cannot react directly with heme. These enzymes are strong oxidants because iron
ion from porphyrin ring is electron-deficient, and the presence of tryptophan
enables direct oxidation of non-phenolic substructures contrary to Mn-dependent
peroxidases, which indirectly degrade them by production of free radicals (Hammel
and Cullen 2008). Laccases (EC 1.10.3.2) are polyphenol oxidases that contain one
to four copper ions (Cu2+) and catalyze the one-electron oxidation of a broad
number of substrates (phenols, methoxyphenols, some non-phenolic aromatic
compounds, aromatic amines, some aliphatic alcohols, and lignin) using molecular
oxygen as the electron acceptor. During the process, Cu2+ reduces to Cu+ and
oxygen to water (Thurston 1994; Muñoz et al. 1997a, b; Medeiros et al. 1999).
Three more enzymes are important for lignin degradation process. First of them is
aryl-alcohol oxidases (EC 1.1.3.7), flavoenzymes that oxidize various aromatic
compounds as well as aliphatic polyunsaturated alcohols providing H2O2 required
for activity of lignin- and Mn-oxidizing peroxidases, and generates OH that ini-
tiates attack on lignocellulose (Evans et al. 1991; Gutiérrez et al. 1994; Varela et al.
2001). These enzymes act synergistically with laccases and prevent repolymer-
ization of products generated during degradation of lignin and other aromatic
compounds (Guillén et al. 2000). Aryl-alcohol dehydrogenases (EC 1.1.1.90),
which catalyze conversion of aromatic alcohol to aromatic aldehyde, and quinone
reductases (EC 1.6.5.5) that catalyze obtaining semiquinone from quinone, are also
involved in delignification.
Degradation of hemocellulose requires the presence of more enzymes, known as

hemicellulases, due to more complex structure of the polysaccharide. Among
numerous hemicellulases, xylan 1,4-β-xylosidases and endo-1,4-β-xylanases have
the main role in depolymerization. Cleavage of xylan to oligosaccharides is cat-
alyzed by endo-1,4-β-xylanases (EC 3.2.1.8) and hemicellulose mineralization is
finished by action of 1,4-β-xylosidases (EC 3.2.1.37), which hydrolyze oligosac-
charides to xylose (Sánchez 2009). However, some accessory enzymes (xylan
esterases, ferulic and p-coumaric esterases, α-1-arabinofuranosidases, and α-4-O-
methyl glucuronosidases) are necessary for completing the reaction.
Three cellulases catalyze synchronously hydrolysis of cellulose β-1,4-glycosidic
linkages. First, endo-1,4-β-glucanases (EC 3.2.1.4) randomly attacks numerous
sites inside the molecule. Further, cellobiohydrolases (EC 3.2.1.91), also known as
exoglucanases, hydrolyze removing monomers and dimers from the end of the
chain and act synergistically with the first enzymes. At the end, β-glucosidases (EC
3.2.1.21) cleave bounds inside glucose dimers, sometimes within cellulose
oligosaccharides, releasing glucose (Sánchez 2009).
10.2.1 Mn-Oxidizing Peroxidases
Wood-decaying and soil litter decomposing Basidiomycetes (members of families

Meruliaceae, Coriolaceae, Polyporaceae, Strophariaceae, and Tricholomataceae)
have ability to synthesize Mn-oxidizing peroxidases that belong to class II of
superfamily of plant-fungal-prokaryotic peroxidases, and which divided into the
Mn-dependent peroxidases (EC 1.11.1.13; MnPs) and versatile peroxidases (EC
1.11.1.16; VPs) based on the substrate specificity (Fernández-Fueyo et al. 2014).
MnPs posses a Mn2+ oxidation site that binds the cations, and VPs both Mn2+
oxidation site and site for direct oxidation of low- and high-redox-potential
lignin-related compounds (Perez-Boada et al. 2005). These enzymes are produced
in numerous isoforms characterized by certain structure and properties, especially
by isoelectric points and molecular weights (Martinez et al. 1996; Camarero et al.
1999; Hofrichter 2002; Stajic et al. 2006a, 2010; Lanfermann et al. 2015). Thus,
Ganoderma applanatum, Ceriporiopsis subvermispora, Phanerochaete
chrysosporium, and Phlebia chrysocreas synthetise 2, 4, 5, and 7 MnP isoforms,
respectively, which are encoded by separate genes (Martinez et al. 2004; Salame
et al. 2012; Lanfermann et al. 2015). Pleurotus ostreatus besides 6 MnPs also
produce 3 VPs, which structures, stability, and catalytic properties were charac-
terized by Fernández-Fueyo et al. (2014). However, expression of the genes
depends on cultivation conditions, substrate composition, and nutrient availability,
as well as concentration of Mn2+ ions (Urzúa et al. 1995; Ruiz-Dueñas et al. 1999;
Stajić et al. 2006a, b; 2009; Salame et al. 2012). Thus, Urzúa et al. (1995) obtained
7 isoforms of C. subvermispora MnP by cultivation in salt medium and only 4
isoenzymes on wood chips. In the case of Ph. chrysosporium, Datta et al. (1991)
noted expression of mnp5 gene during its cultivation on wood pulp, mnp4 gene was
transcribed in growth on wood-containing soil (Stuardo et al. 2004), while nitrogen

poor medium but enriched with Mn2+ expressed mnp1 and mnp2 genes (Hammel
and Cullen 2008). Letter, Salame et al. (2012) showed that Mn2+-deficient medium
caused increase of expression of mnp3 and mnp9 P. ostreatus genes and in such a
way activity of the MnP izoforms raised in 200-fold, while expression of mnp4
gene, responsible for VP synthesis, was down-regulated in the medium enriched
with this ions. Repression of VP activities in the Mn2+ presence, though the
enzymes have ability to oxidize the ions, Salame et al. (2012) explained by the fact
that VPs are expressed during the later stages of lignin degradation when majority
of Mn2+ presented in the biomass are exhausted. However, Urzúa et al. (1995)
showed that requirement for Mn2+ also depends on nature of the aromatic substrate.
The main environmental factors which affect Mn-oxidizing peroxidase activities
are pH and temperature. According to Fernández-Fueyo et al. (2014), the most
appropriate pH for wood lignin degradation by P. ostreatus is 3.0 due to numerous
lysine and other alkaline residues that increase positive charge and stability at acidic
pH values. However, pH values below 3.0 as well as pH 9.0 inactivate all perox-
idases in very short time. The peroxidase instability in alkali environment the
authors explained by loss of the Ca2+ ions. P. ostreatus VPs are more stable on high
temperature than MnPs, and some of these enzymes can retain about 80 % of
activity after incubation at 60 °C for 10 min.
10.2.1.1 Structure
Mn-oxidizing peroxidases are H2O2-dependent heme glycoproteins with an iron

protoporphyrin IX prosthetic group in the active site, which generate Mn3+ that
initiates delignification (Wariishi et al. 1988). Heme iron is Fe3+ located at an
internal cavity and connects with the protein through two histidine residues which
situate proximally and distally to the iron ion. The central cavity is delimited with
two approximately equal domains built of 10–12 predominantly α-helices com-
posed of conserved amino acid residues and located proximally and distally to the
heme (Martinez 2002; Fernández-Fueyo et al. 2014). Heme pocket bounds with two
Ca2+ ions (one above and one below the heme) which link with oxygen of amino
acid residues and form Ca2+ binding sites that stabilize the protein structure. Amino
acid residues in distal Ca2+ binding sites are conserved in all fungal Mn-oxidizing
peroxidases, while proximal site differs among fungal species by two residues. Four
or five disulfide bridges, composed of cysteine residues, also stabilize the protein
structure (Martinez 2002). Heme connects with enzyme surface by two channels.
One of them is located in front of heme propionate and delimited by three acidic
residues which form Mn2+ binding site. When Mn2+ enters in the channel it oxi-
dizes to Mn3+ which is released from the active site in the presence of chelators that
are extracellular metabolites of white rot fungi. Second, narrow, channel is the place
where H2O2 reacts with heme Fe3+ and has important role in protection of the
enzyme from substrate radicals which could inactivate it (Ruiz-Dueñas et al. 2009).
Mn-oxidizing peroxidases posses multiple substrate sites at both sides of the heme
pocket and could oxidize different substrates.
However, Fernández-Fueyo et al. (2014) demonstrated significant differences
between Pleurotus ostreatus MnPs and VPs in amino acid sequence length, amino
acid composition and distribution, as well as in substrate specificity and catalytic
efficiency on Mn2+. Thus, MnPs contains sequences composed of 331 and VPs of
339 residues mainly located in C-terminal region. In MnPs number of proline
residues varies from 25 to 26 and in VPs from 30 to 31, while number of lysine
residues is ranged from 7 to 10 in all isoforms except MnP4 where the number is
20. In the case of substrate and ability of Mn2+ oxidation, VPs have wider speci-
ficity and higher efficiency than MnPs. This could be explained by the fact that VPs,
besides Mn2+ oxidation site composing of three conserved acidic residues near the
internal heme propionate, also posses lignin oxidation site near tryptophan that is
substituted by an alanine or aspartic acid in MnPs. Exceptions are P. ostreatus
MnP1 and P. pulmonarius peroxidase which posses tryptophan but not activity
characterized for VPs, that Fernández-Fueyo et al. (2014) explained by change of
shape and charge of surface around tryptophan and in such a way substrate binding.
Analyzing the structures of P. ostreatus MnP4 and VP1 these authors noted a few
differences: (i) in domain close to the Ca2+ ion; (ii) in the main heme access
channel, which is wider in VP1 than in MnP4 due to the presence of three substrate
oxidation sites; (iii) in shape and charge of lower channel lip, as wide reservoir with
negative charge in VP1 and as narrow groove of less charged surface in MnP4;
(iv) in number of lysine residues, MnP4 has the minimum one unit more than VP1,
which influences isoelectic points, and (v) in number of hydrogen bonds and salt
bridges that are more numerous in MnP4 than in VP1.
10.2.1.2 Catalytic Properties
Mn-dependent peroxidases play a crucial role in the process of delignification and

characterized by classical peroxidase cycle and inability to oxidize directly
non-phenolic lignin-related structures (Hammel and Cullen 2008). H2O2 or organic
peroxide donate one electron to MnP heme producing Compound I (Fe4+-
oxo-porphyrin-radical complex) and one water molecule is expelled (Fig. 10.1).
Compound I is reduced to Compound II (Fe4+-oxo-porphyrin complex) by per-
oxidase substrate, phenols or Mn2+. However, efficient completion of MnP catalytic
cycle, i.e. reduction of Compound II to the native enzyme requires the presence of
only Mn2+ that is commonly presented in lignocellulose and oxidizes to Mn3+ with
release of the second water molecule (Martinez 2002). Mn3+ is small quite unstable
oxidizer, which is stabilized by organic acids such as oxalate, malonate, tartrate,
and lactate, and can diffuse into the plant cell wall and catalyze cleavage of bounds
in phenolic lignin substructures producing a phenoxyl radical intermediate
(Fig. 10.1). Chelates of Mn3+ with the acids can also oxidize other phenolic, var-
ious non-phenolic aromatic, amino-aromatic, thiol compounds, and unsaturated
fatty acids to phenoxyl, aryl, amino, thiyl, and peroxyl radicals, respectively. These
Fig. 10.1 Mechanism of action of Mn-oxidizing peroxidases
radicals can cleave Cα–Cβ and β-aryl ether bonds in abundant non-phenolic lignin
substructures. Contrary to fatty acids, which are normally presented in the fungal
environment, thiols are not produced by fungi and SH-groups are released in small
amount by protein degradation during cell lyses (Hofrichter 2002). Likewise, Mn3+
can oxidize the oxalate from chelate generating CO2 and anion radical, which in the
presence of O2 forms another CO2 molecule and superoxide radical (O– 2 ) that at low
wood pH values transforms into perhydroxyl radical (HOO) leading to lipid per-
oxidation and releasing of ligninolytic peroxyl radicals (Fig. 10.1). However, more
efficient mechanism of mineralization of non-phenolic lignin moieties means their
modification by cellobiose dehydrogenase to structures which are accessible to
MnPs. Chelates of Mn3+ with the acids can also react with each other and convert to
alkyl radicals which then react spontaneously with oxygen and form some other
radicals, for example superoxide, that could be used by native enzyme in the
absence of H2O2 (Hofrichter 2002). This author also emphasized that high con-
centrations of H2O2 or O2 lead to formation of Compound III, catalytically inactive
form, which cause reversible inactivation of MnPs.
Martinez et al. (1996) first purified and characterized two VPs after submerged
cultivation of Pleurotus eryngii in glucose/peptone/yeast extract medium. Later
Martinez (2002) showed that these enzymes can oxidize both Mn2+ and aromatic
compounds, and that present hybrids of MnPs and lignin peroxidases. These
enzymes occur naturally and until now it is shown that various species of the genera
Pleurotus and Bjerkandera, Lentinus edodes, Panus tigrinus, etc. can synthesize
them (Hofrichter 2002; Hammel and Cullen 2008).
10.3 Plant Residue Conversion
At the beginning of twenty-first century, humankind faces with high rate of world
population growth and in such a way with food insufficient, environmental pollu-
tion, and climate change. It should be emphasized that 70 % of agricultural prod-
ucts are not utilized and become ballast and potential pollutant of environment.
Numerous fungi have essential role in conversion of this enormous amount of
lignocellulosic waste in food, feed, paper pulp, biofuels, and various chemicals.
However, all of the conversions require delignification without or with minimal loss
of carbohydrates, which could be achieved by selection of the most efficient fungal
species and strains, the optimal lignocellulosic biomass, as well as by optimization
of cultivation conditions (Kuijk et al. 2015). Nutritional value and availability are
the main criteria for selection of lignocellulosic residues. However, composition of
plant waste of the same origin and consequently its degradation can vary because of
existence of numerous cultivars of the same crop, for example number of wheat
cultivars is still grown. It was demonstrated by Arora and Sharma (2009) who noted
that Phlebia floridensis more efficiently delignificated wheat straw from north-
eastern India than straw from other climatic regions. As efficiency of fungal
enzymatic system depends on substrate type, selection of degrader is conditioned
by lignocellulosic residue. Thus, Ceriporiopsis subvermispora, Pleurotus eryngii,
P. ostreatus, Lentinus edodes, Hericium clathroides, and Trametes versicolor are
excellent selective degraders, namely they mineralize more than 20 % of lignin and
less than 5 % of cellulose in wheat straw (Tuyen et al. 2012; Knežević et al. 2013a,
b; Stajić et al. 2013; Kuijk et al. 2015). Numerous studies confirmed that C.
subvermispora and P. eryngii are the most effective mineralizers of wheat straw
lignin and showed that Mn-oxidizing peroxidases have the essential role in the
process of delignification (Fernandez-Fueyo et al. 2012; Tuyen et al. 2012).
However, Asiegbu et al. (1996) and Chi et al. (2007) demonstrated that combi-
nation of few fungal species can be more effective in depolymerization of lignin in
some plant wastes than their monocultures. Thus, extent of spruce sawdust delig-
nification by co-culture of Pleurotus sajor-caju, T. versicolor, and Phanerochaete
chrysosporium was significantly higher than by each monoculture separately (16
and 0–5 %, respectively), and co-culture of C. subvermispora and P. ostreatus
caused higher loss of lignin from aspen wood than in its colonization with the
monocultures. However, due to divergence in genotype, significant differences in
lignocellulolytic enzyme production and selectivity of lignocellulose degradation
exist among strains of the same mushroom species (Simonić et al. 2010; Knežević
et al. 2013b). For example, percents of degraded wheat straw lignin and cellulose
by some P. ostreatus strains were almost the same (Salvachua et al. 2011), while
other strains predominantly depolymerized lignin (Adamović et al. 1998). Solid
state fermentation, small lignocellulosic particles of suitable shape, high oxygen
level, moderate water content, certain inoculum type (spores or spawn) in moderate
amount, and the presence of copper, manganese, linoleic acid, and veratryl alcohol
are good conditions for biological treatment of lignocellulose by white rot fungi
(Kuijk et al. 2015).
10.3.1 Food
Ability of mushrooms to produce lignocellulolytic enzymes has double benefit,

degradation of plant residues, and production of high valued food. Therefore,
numerous species are cultivated commercially and cheap and quality food, rich in
proteins, fibers, vitamins, minerals as well as biologically active compounds, is
obtained. Their consumption could compensate insufficient proteins in regions
where famine presents serious problem and enhance immune system and resistance
to diseases.
According to enzymatic system, mushrooms are divided into two groups, pri-
mary degraders-species with strong system and high degradation ability, and sec-
ondary degraders-species with poor enzymatic system that need help of other
degraders. For example, species of the genera Pleurotus, Lentinus, Grifola,
Auricularia, Flammulina, etc. belong to the first group, while Agaricus species
make the second group.
Frequently cultivation in the plastic bags enables usage of various lignocellulosic
wastes from agriculture, forestry, and food industries for compost preparation. If it
bears in mind that production of cereal residues in Europe is high (55,585 × 104
tons in 2010) and if wastes from sugar crops and tuber vegetables are added to the
value, it comes to 61,484 × 104 tons and it can be concluded that amount of
potential substrates for mushroom production is enormous (Gupta and Verma
2015). Nowadays, mushroom producers could obtain high biological efficiency,
from 60 to 75 %, depending on species. Additionally, considering that only third of
world cereal straw production is used in the processes, it can be estimated potential
of mushroom production.
Nowadays, three species of the genus Agaricus (A. bisporus, A. blazei, and A.
bitorquis) are produced in the highest amounts and the first of them is leader
(Chang and Miles 2004). Based on annual world production, Lentinus edodes takes
the second place and numerous members of the genus Pleurotus (P. ostreatus,
P. ostreatus var. florida, P. eryngii, P. eryngii var. nebrodensis, P. sajor-caju,
P. cystidiosus, P. citrinopileatus, P. djamor, P. ferulae, P. tuber-regium, and
P. sapidus) take third place. Efficiently usage of wide range of plant residues (straw,
sawdust, maize, sugar cane pulp, wastes from cotton production, etc.), without any
treatment and enrichment, by Pleurotus species (they can convert 100 g of dry
lignocellulosic wastes in 50–70 g of fresh fruiting bodies in only few weeks) is the
result of possession of strong ligninolytic enzymes (especially Mn-oxidizing per-
oxidases) characterized by well selectivity (Ginterová and Lazarová 1987).
However, biological efficiency depends on substrate composition and strain. Thus,
depending on wheat cultivar, content of reducing sugar differs considerably,
between 13.1 and 40.7 mg/g and yield of various P. ostreatus strains can vary
between 123 and 262 kg of fruiting bodies per ton of wheat straw (Kuijk et al.
2015).
Nowadays, number of cultivated mushroom species is relatively small, 100
species are cultivated economically, 60 commercially, and only 10 in industrial
scale, but current trend is growth of world production in favor of new species, such
as Agaricus blazei, Agrocybe aegerita, Armillaria mellea, Auricularia fuscosuc-
cinea, Coprinus comatus, Dictyophora duplicata, Lepista nuda, Stropharia
rugosaannulata, Tremella cinnabarina, T. aurantialba, and Tricholoma giganteum
(Chang and Miles 2004).
10.3.2 Feed
Most agricultural and industrial wastes that are used for animal feeding are rich in
low digestible fibers and poor in nutrients, especially in proteins and vitamins.
Besides high nutritionally valued food, result of mushroom cultivation is also
production of enormous amount of spent substrate that can be an important feed
ingredient. Mushroom mycelium is rich in protein, essential amino acids, and chitin
and could be important nitrogen sources, while β-glucans and other extra- and
intracellular polysaccharides could be an additional glucose sources and
immunostimulators for ruminants (Reis et al. 2012; Cheung 2013). Thus, protein
content in wheat straw after P. ostreatus cultivation can be increased by 89 % that
leads to overcoming of the main limitation of straw feed, i.e. nitrogen lack (Kuijk
et al. 2015). The authors also emphasized that the protein increases rumen
microorganism population and in such a way cellulose and hemicellulose
digestibility and energy releasing. Spend mushroom compost is also rich in vita-
mins, minerals, antimicrobial agents, probiotics, and other biologically active
compounds that modify animal metabolism, enhance growth, improve immune
system, and increase resistance to various diseases (Enshasy and Hatti-Kaul 2013).
Mushroom contribution to feed production is also transformation of natural
recalcitrant lignin into digestible compounds owing to well developed lignocellu-
lolytic enzyme system, especially Mn-oxidizing peroxidases. Although ruminants
posses cellulolytic microorganisms in rumen that can depolymerize cellulose and
hemicellulose from plant cell wall, connection of these carbohydrates for lignin by
covalent or other linkages is negatively correlated with their digestibility under
rumen anaerobic conditions. Rodrigues et al. (2008) showed that cultivation of
various white rot mushrooms on wheat straw could increase degradability of neutral
detergent fibers in rumen in even 13 % due to their strong ligninolytic enzymes
which act to lignin. Contrary to wheat straw that was not biologically treated, spent
wheat straw composts of Pleurotus ostreatus and P. sajor-caju were well consumed
and digested by sheep (Calzada et al. 1987; Fazaeli et al. 2006). Similar efficient
consumption and decomposition of crude protein, hemicellulose, and cellulose from
spend Coprinus fimetarius rice straw compost and spent Ganoderma sp. wheat
straw compost by goats were reported by Rai et al. (1989) and Shrivastava et al.
(2012). However, cows are more selective and their feed can be enriched with
maximum 17 % of spend P. ostreatus wheat straw substrate (Adamović et al.
1998).
Kuijk et al. (2015) reviewed results of numerous studies and also reported that
addition of selected microelements and inducers (copper, manganese, linoleic acid,
di-rhamnolipid, and veratryl alcohol) to lignocellulosic residues enhance activity of
fungal Mn-oxidizing peroxidases and indirectly fiber digestibility by animals.
However, some mushroom metabolites could be toxic for animals and humans and
special attention has to be given to that aspect of plant waste bioconversion by
fungi.
10.3.3 Paper Pulp
Since nowadays forest preservation is compulsory, plant residues that are produced
in enormous amount, but not used, can be important natural sources for paper-
making. Good sample for extraordinary production of some agricultural wastes was
given by González Alriols et al. (2009). Namely, they noted huge quantity of
unutilized Elaeis guineensis residues after oil extraction in Malaysia, even 15
million tons per year, which present serious ballast. In a few last decades, white rot
fungi or their enzymes have the main role in the process, known as biopulping. The
main reasons for that are its healthy, environmental, and economical friendly
relationships contrary to traditional usage of alkaline sulfide, as well as improve-
ment of pulp quality and quantity (Bajpai et al. 2001). Yang et al. (2008) showed
that pretreatment of eucalyptus woody biomass with Trametes hirsuta led to
increasing fiber internal bond strength in 32 %. Fungal ligninolytic enzymes, pri-
marily peroxidases, catalyze one-electron oxidation of lignin phenolic groups
producing phenoxy radicals on fiber surfaces and in such a way enhancing inter
fiber adhesion (Singh and Singh 2014). Thus, this modified lignin could replace
traditionally harmful adhesives that is current trend and one more possible appli-
cation of fungal enzymatic system.
Besides enhancement of the bond strength and fiber adhesion, wood pretreat-
ment with white rot fungi removes pitches rich in lipophylic compounds (resin and
fatty acids and triglycerides), which can reduce paper and effluent quality and cause
technical problems (Martinez-Inigo et al. 2000; Dorado et al. 2001; Gutiérrez et al.
2006; Van Beek et al. 2007). Martinez-Inigo et al. (2000) reported that Phlebia
radiate and Poria subvermispora were the most active in the removal of lipophylic
extractives and in such a way pitch from eucalypt wood, and Dorado et al. (2001)
that Bjerkandera sp. and Trametes versicolor eliminated up to 90 % of these
compounds from Pinus sylvestris wooden biomass. Letter Van Beek et al. (2007)
showed that T. versicolor was also effective in removal of resin acids and
triglycerides from spruce wood chips, which content was reduced in 40 and 100 %,
respectively, and that Pycnoporus cinnabarinus caused losses of sterols, resin acids,
and triglycerides from various pulp systems in 65–100 %. Further study showed
that this ability of white rot fungi is based on production of lipase, resinase,
lipoxigenases, and laccases (Gutiérrez et al. 2009). As pitch control is combined
with lignin removal from the pulp, white rot fungi play important role in pulp
bleaching (bio-bleaching) and thus in improving paper brightness, environment
protection, and energy saving (Bajpai et al. 2001; Jerusik 2010). However, the
process should be controlled in order to avoid cellulose degradation.
Besides papermaking, white rot fungi and their enzymes have one more role,
bioremediation of waste water. Namely, huge amount of water, approximately 45–
227 × 103 litters, is used for production of a ton of product resulting in the
releasing of the same quantity of effluent and further in zooplankton and fish dying,
slime and scum formation, esthetic problems, and disruption of the environment
(Pokhrel and Viraraghavan 2004). Fungi are characterized by better survival in
effluents and production of strong extracellular ligninolytic enzymes, especially
lignin- and Mn-oxidizing peroxidases that degrade various phenolic compounds
among which colors are the most resistant. Thus, numerous studies showed high
effectiveness of Trametes pubescens, Phanerochaete chrysosporium, and Pleurotus
ostreatus in the process (Prasad and Gupta 1997; Choudhury et al. 1998; González
et al. 2010; Zhang et al. 2012). Prasad and Gupta (1997), Choudhury et al. (1998),
and Saxena and Gupta (1998) noted high level of color degradation, even 80–
93.8 %, in effluents from paper industry treated by T. versicolor, Ph. chrysosporium
(sole or in combination with Pycnoporus sanguineus), Pleurotus ostreatus, and
Heterobasidion annosum.
10.3.4 Biofuels
Intensive world industrialization and motorization result in enormous rise of

demands for fuels, and nowadays 80 % needed energy is met by use of fossil fuels
that have numerous harmful effects on environment. If rapid depletion of the fuels is
added to the negative effects, current trend of finding new, alternative, renewable,
and sustainable energy sources is justified. One of the most environmental friendly
are biofuels, the consumption of which in 2020 should be about 20 % of overall
energy usage (Nigam and Singh 2011). Biofuels are classified into two groups,
primary (wood, wood chips and pellets, etc.) and secondary which are produced by
biomass processing (ethanol, biodiesel, etc.), and secondary ones are further clas-
sified based on sources (forest, agricultural or fishery products, municipal wastes
and wastes from food industry and services) and type (solid, liquid or gaseous). The
main advantages of biofuel utilization are usage of broad distributed sustainable
natural bioresources that could be serious environmental ballast, as well as lower
CO2 and polycyclic aromatic hydrocarbons emissions (Demirbas 2009).
Although crops rich in sugars or compounds that can be converted into the
sugars (starch or cellulose) present an essential bioresources for bioethanol pro-
duction, current trend is development of techniques for its production from inedible
fast-growing biomass (certain trees and grasses), for example from grass
Miscanthus sinensis, which production is 5–6 times higher than cereal one, or from
dried citrus waste that amount is about 800,000 tons only in USA (Zechendorf
1999; Tabka et al. 2006). However, in order to cellulose to be broken down to the
glucose and further ferment, depolymerization of recalcitrant network around cel-
lulose fibers, built of lignin, is necessary. Thus, it comes again to white rot fungi
and their peroxidases which are the main participants in the first step of bioethanol
production known as pretreatment, i.e. the essential catalyzers of beginning phase
of lignin degradation. This pretreatment also depends on fungal species and strain,
cultivation conditions, enzyme production and activity, and oxidative mechanisms
(Dias et al. 2010; Wan and Li 2010; Salvachua et al. 2011). Dias et al. (2010)
reported that various white rot fungi degraded between 2 and 65 % of wheat straw
lignin during its solid state fermentation and in such a way increased
cellulose/lignin ratio. However, Salvachua et al. (2011) emphasized that mode of
lignocellulosic mineralization is different from species to species, for example
contrary to Panus tigrinus and Phlebia radiata that degraded lignin and polysac-
charides simultaneously, Pleurotus eryngii and Phellinus robustus removed lignin
selectively and faster. Likewise, according to these authors, delignification extent is
not always in correlation with ligninolytic enzymes production, as well as with fiber
digestibility and sugar production. Thus, contrary to P. radiata and P. robustus,
characterized both by high Mn-oxidizing peroxidase activities and effective lignin
removal, good lignin degraders Bjerkandera adusta and Coriolopsis rigida had low
active enzymes. In the case of effectiveness of cellulose and hemicellulose enzy-
matic hydrolysis in the second step of bioethanol production, Pycnoporus coccineus
was high effective hemicellulose and weak cellulose degrader (98 and 31 %,
respectively), Bjerkandera adusta was almost equal depolymerizer of these poly-
mers (43 and 54 %, respectively), Stereum hirsutum mineralized only significant
portion of cellulose (43 %), the highest glucose yield was obtained by cultivation of
Poria subvermispora (69 %) and Irpex lacteus (66 %) and the lowest by P. eryngii
and P. robustus.
10.4 Future Trends
Importance of fungi and their enzymes in various biotechnological processes

stimulates serious investment for study of genomes of numerous fungal species, i.e.
sequencing, characterization of ligninolytic enzyme genes, their expression in
appropriate organisms, improvement of product properties, and production of
recombinants in significant amount under non-natural conditions. As Mn-oxidizing
peroxidases are secreted under conditions characterized by high moisture level and
acidic environment (pH about 3.0), which are not typical in biotechnological pro-
cesses, it needs to adapt the enzymes to the conditions. According to Guerriero et al.
(2015), the adaptation could be reached either by directed evolution of certain
ancestral enzyme, i.e. mutation of its gene, or by enzyme engineering, and result in
the both cases is similar enzyme form but with improved and desired property.
Namely, one enzyme rarely possesses all catalytic properties required for effective
lignocellulosic conversion. Therefore recombination can be used for unification of
several beneficial characteristics from a few enzymes into superior one with high
stability and broad substrate promiscuity. Finding of appropriate organism for
expression of the gene leads to obtaining of significant amount of the recombinant
(Alcalde 2015). Thus, Ogawa et al. (1998) showed that expression of mnp genes of
Pleurotus ostreatus, excellent Mn-oxidizing peroxidase producer, in Coprinus
cinereus, characterized with fast growth, led to increase of lignin degradation level
in only 16 days, and Pérez-Boada et al. (2002) and Salame et al. (2012) obtained
active versatile peroxidase by expression of mnp4 gene in Escherichia coli but in
lower yield. Considering that lignocellulolytic enzymes act synergistically, genetic
engineering could be used for fusion of certain enzymes and also for generation of
strain deficient in some enzyme. With this aim, Chalamcherla et al. (2010) getting
cellulase-deficient P. ostreatus strain characterized by low cellulose and high lignin
degradation, as well as by high capacity of application in various biotechnological
processes. Nowadays, special attention is also given to optimization of cultivation
conditions for production of certain lignocellulolytic enzyme and study of enzy-
matic system of fungal species living in extreme environment as well as their genes
encoding proteins with unknown function among which some can be lignocellu-
lolytic enzyme (Guerriero et al. 2015). Everything mentioned show great possi-
bilities of usage and managing of fungi and their enzymes with the aim of biomass
conversion into high-valued products and environment protection.
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Chapter 11
Role and Application of Versatile
Peroxidase (VP) for Utilizing
Lignocellulose in Biorefineries
Nadine Busse and Peter Czermak
Abstract Within the last few years, the relatively new heme peroxidase, versatile
peroxidase (VP), attracted some attention as a model enzyme and as an industrial
biocatalyst. VPs are interesting for efficient delignification processes due to their
ability to degrade a broad spectrum of recalcitrant substrates without using medi-
ators. Lignin is very complex, highly aromatic, and one of the major compounds of
lignocellulose, which is an important feedstock for biorefineries, particularly when
originated from wood. In utilizing lignocellulose (e.g., second generation biofuels),
lignin modification, and removal must first be addressed. Moreover, lignin degra-
dation for value-added bio-products is also of great interest. An industrial imple-
mentation of VPs would be beneficial in this context, but involves several scientific
and technical challenges.
11.1 Introduction
Lignin modification and removal plays a key role in utilizing lignocellulose/

lignocellulosic biomass for second generation biofuels, and other bio-based prod-
ucts, and therefore must be addressed first. Lignocellulose from wood is of partic-
ularly high interest for this process due to the following reasons: Wood is
inexpensive, available in large amounts (Stöcker 2008), CO2 neutral (Lange 2007),
and rich in lignin (18–35 %), as well as the carbohydrates cellulose (40–55 %) and
hemicellulose (24–40 %) (Sun and Cheng 2002; Howard et al. 2003).
Due to the fact that lignin is the only naturally synthesized aromatic biopolymer
(Dashtban et al. 2010) its effective use and degradation to value-added macro-
molecules (e.g., low cost carbon fibers, polymer modifiers) and/or aromatic
chemicals (e.g., phenols, vanillin, quinone, guaiacol, BTX chemicals) is of crucial
N. Busse P. Czermak (&)

Institute of Bioprocess Engineering and Pharmaceutical Technology,
Mittelhessen University of Applied Sciences, Wiesenstrasse 14,
35390 Giessen, Germany
e-mail: [email protected]; [email protected]

272 N. Busse and P. Czermak
importance, with the latter of these being of high economic potential and therefore
particularly interesting (Holladay et al. 2007; Huang and Ramaswamy 2013).
However, the contributing factors affecting the probability of their successful
implementation include the structural complexity of native lignin, its recalcitrance,
and its uniqueness (the woody plant’s fingerprint) leading to inconsistent, poly-
disperse product streams. These facts still constitute a major scientific and technical
challenge (Holladay et al. 2007). Consequently, further fundamental research and
development is needed for lignin degradation in both general and targeted usage.
Prerequisites for efficient lignin modification and degradation in biorefineries
are:
(i) Conservation of important lignin related aromatic structures.
(ii) Subsequent processes like saccharification of cellulose by cellulases should
not be negatively influenced.
(iii) Mild conditions.
(iv) Moderate waste streams/effluent treatment and moderate operating costs.
Ligninolytic heme peroxidases (POXs) have gained increased importance in this
context, with particular attention on the use of versatile peroxidases (VPs). VPs are
relatively new POXs and were first identified as MnPs in the late nineties. Within
the last few years they have attracted attention “as a model enzyme and as a source
of industrial/environmental biocatalyst” (Ruiz-Dueñas et al. 2009a). The main
reason for this is their capability to digest/convert a broad spectrum of complex
substrates, ranging from azo dyes like RB5 to (non)phenolic lignin, without the
necessity of mediators (Pogni et al. 2005; Martínez et al. 2009). Nonetheless,
several factors hinder their short-term commercialization and industrial imple-
mentation. This chapter outlines the main issues for industrial applications, starting
first with a brief overview of the involvement of POXs in natural delignification.
11.2 Involvement of Heme Peroxidases

in the Lignocellulose Degradation Mechanism
In nature, lignocellulose (LC) degradation involves several factors as follows:

(i) Lignocellulose composition (e.g., lignin content, extractables).
(ii) Environmental conditions (e.g., T, pH, moisture, N2, O2) (Blanchette 1991;
Wong 2009).
(iii) Biological community.
Biological community includes microbial population, fungi, and insects, and
determines the degradation process. In order to enable the use of LC, lignin modi-
fication and removal must first be addressed. Basidiomycetes, white-rot fungi, are
well known to be the most efficient lignin degraders due to their extracellular
ligninolytic enzymes (Kirk and Farrell 1987), the H2O2-dependent heme peroxidases
11 Role and Application of Versatile Peroxidase (VP) for Utilizing… 273
Fig. 11.1 Summary of the natural degradation process of lignocellulosic biomass and the
importance of ligninolytic heme peroxidases (modified based on Dashtban et al. 2010). Here, the
reactions of the first POX intermediates are demonstrated. Lac laccase, LiP lignin peroxidase, MnP
manganese peroxidase, VP versatile peroxidase; Lac-I, LiP-I, MnP-I, and VP-I are corresponding
protein radicals, generally known as compound I. S stands for an appropriate substrate of low
molecular weight resulting in relatively reactive radicals which ultimately mediate the cleavage of
lignin bonds and lignin modifications
(POX) and O2-dependent phenol oxidases lacasses (Lac, EC 1.10.3.2) as seen in

Fig. 11.1. Figure 11.1 demonstrates a part of the full enzymatic reaction mechanism
for better clarity (specifically the reactions of the first enzyme intermediate). More
details on this will follow in subsequent chapters. The ligninolytic enzyme system
varies individually in composition. In addition, it must be further differentiated into
selective and nonselective lignin degraders (Table 11.1). The former selectively
remove lignin without considerable cellulose degradation, whereas the latter also
attack cellulose (Blanchette 1991; Hatakka 2005; Hatakka and Hammel 2011).
Some can however produce both types, such as Heterobasidion annosum (in
Table 11.1) (Eriksson et al. 1990).
Concerning lignin degradation, two pathways must be differentiated (Fig. 11.1):
(i) Direct (black dashed lines).
(ii) Indirect (black solid lines).
Table 11.1 A selection of Species Secreted enzymes

POX and Lac producing
white-rot fungi (modified Lignin-selective
based on Hatakka (1994), Bjerkandera adusta LiP, MnP, VPa
Ayala et al. (2008), Sánchez Bjerkandera species VPb
(2009) and Lundell et al. Ceriporiopsis subvermispora MnP, Lac
(2010))
Dichomitus squalens MnP, Lac
Heterobasidion annosum MnP
Phanerochaete chrysosporium LiP, MnP
Phellinus pini LiP, MnP
Phlebia radiata LiP, MnP, Lac
Phlebia subserialis MnP
Phlebia tremellosus LiP, MnP, Lac
Pleurotus eryngii MnP, VPc, Lac
Pleurotus ostreatus MnP, VPd, Lac
Pleurotus sapidus VPe
Pycnoporus cinnabarinus LiP, MnP, Lac
Nonselective
Ganoderma applanatum LiP
Heterobasidion annosum MnP
Trametes versicolor LiP, MnP, VPf, Lac
Bold indicates VP producing white-rot fungi
a
Wang et al. (2003) and Busse et al. (2013)
b
Mester and Field (1998) and Moreira et al. (2006)
c
Martínez et al. (1996) and Camarero et al. (1999)
d
Hatakka and Hammel (2011)
e
Zorn et al. (2005) and Schüttmann et al. (2014)
f
Carabajal et al. (2013)
Direct degradation refers to when the ligninolytic enzyme is in its active state
(characterized by an appended suffix-I) and directly attacks native lignin linkages.
However, when low molecular weight (LMW) substrate (S) is present/provided,
this interposes an intermediate step where enzyme action results first in
substrate-related diffusible radicals mediating nonenzymatic reactions, which then
cause the cleavage of lignin bonds. This is called indirect lignin degradation,
because the LMW substrates serve as so-called mediators. Lac rely on phenolic
mediators for promoting their oxidation capabilities “towards more recalcitrant
non-phenolic lignin compounds” (Cañas and Camarero 2010). For oxidative ability,
the enzyme redox potential is a limited criterion (Ayala 2010). Lac exhibits lower
redox potential properties (≤ 0.8 V) than POX (> 1 V) (Bourbonnais and Paice
1990; Cañas and Camarero 2010). Lignin comprises 80–90 % non-phenolic moi-
eties, where just 10–20 % are phenolics (Kawai et al. 1999). The cleavage of
non-phenolics is thus essential for efficient lignin modification and degradation. The
redox potential of non-phenolic lignin compounds is also > 1 V (Cañas and
Camarero 2010). Consequently, POX have recently gained increased attention for
delignification purposes (Cañas and Camarero 2010) and received higher interest as
industrial biocatalysts (Martínez 2007). Nevertheless, manganese peroxidases

(MnP, EC 1.11.1.13) principally need manganese (Mn2+) as a substrate and
mediator to form Mn3+ ions. These ions will be subsequently chelated by organic
acids (e.g., oxalate, malonate, lactate) to powerful oxidants. As a result, MnPs are
able to oxidize certain non-phenolic aromatics (e.g., carboxylic acids, methoxy-
benzenes, lipids) and are not restricted to phenolics as usual. Recalcitrant
non-phenolic lignin units still remain unaffected. Therefore, it is supposed that MnP
activity rather causes phenoxyl radical formation (as in case of Lac) which in turn is
responsible for subsequent bond cleavages in the biopolymer lignin (Hatakka
2005). Contrary to this, lignin peroxidases (LiP, EC 1.11.1.14) prefer to react with
non-phenolic lignin substructures, interestingly, without the need for mediators.
White-rot fungi, however, simultaneously produces the non-phenolic monomer
veratryl alcohol (VA) as a second metabolite (Schoemaker and Piontek 1996). VA
is well known to be an essential substrate S for LiP, elevating its activity, as well as
increasing lignin degradation. Reaction with VA again causes radical formation
(veratryl alcohol radicals VA•), which additionally enables phenolic compound
oxidation.
Versatile peroxidases (VP, EC 1.11.1.16) exemplify catalytic properties of both
LiP and MnP, and thus initiate similar reaction pathways. VPs, however, are less
specific in facilitating degradation among a broad spectrum of substrates, irre-
spective of phenolic or non-phenolic, and do not require the presence of mediators
(Pogni et al. 2005).
In conclusion, lignin degradation in nature is not performed by just one enzyme,
but rather a synergistic process of an array of ligninolytic enzymes and their iso-
zymes, accessory enzymes (e.g., H2O2-generating oxidases (aryl-alcohol oxidase,
glyoxal oxidase)), aryl-alcohol oxidases, and quinone reductases) and
non-enzymatic reactions (Fig. 11.1). The latter mainly produces hydroxyl radicals
(•OH) which support lignin modification and degradation, as well as the degradation
of cellulose and hemicellulose in a non-specific manner. The involvement of such
radicals results in easier penetration of:
(i) POX, since they are unable to penetrate through the lignified cell wall and
therefore first erosion is necessary (Hammel et al. 1993).
(ii) Lignocellulolytic enzymes, such as (hemi)cellulases for subsequent (hemi)-
cellulose digestion (Dashtban et al. 2010).
11.3 Catalytic Mechanism of VP
As initially mentioned, VPs are relative new POXs and are attractive (model)
biocatalysts (Ruiz-Dueñas et al. 2009a). One key advantage is their independence
from mediators. For industrial application, mediators are used in high amounts.
Therefore, mediators must be (i) cheap, (ii) non-toxic, (iii) readily available,
(iv) in large quantities, and/or (v) easy recyclable (Rodríguez Couto and Toca
Herrera 2006; Cañas and Camarero 2010).
Research work concerning delignification processes by VPs are limited, particu-
larly in kinetic studies with representative lignin model compounds (LMCs)
like adlerol (1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)-1,3-propanediol), a
non-phenolic β-O-4 lignin model dimer. The reason for this is the underlying, highly
complex, POX reaction mechanism which will be demonstrated in the following, prior
to focusing on mediator-independent adlerol degradation.
11.3.1 The General Peroxidase Reaction Mechanism
The catalytic peroxidase cycle is expected to follow a ping–pong mechanism.

Contrary to the classical mechanism, peroxidase reaction schemes are further
assumed to be irreversible, as sketched in Fig. 11.2 in simplified terms. Enzyme–
substrate complex formation (e.g., ES) prior to reaction product generation can thus
be neglected, since they are considered of fleeting non-detectable existence
(Dunford 1991). This is valid for most heme peroxidases, (Dunford 2010) with iron
protoporphyrin (P) IX and iron/heme (Fe3+) as the catalytic center and the pros-
thetic group, respectively (de Montellano 2010).
During the catalytic cycle (Fig. 11.2), the enzyme undergoes two fundamental
structural changes. First (pathway 1), the reaction is initiated by H2O2 through a
pH-independent, two-electron oxidation (pH range is *2–7.5 for LiP (Andrawis
et al. 1988) or 3.5 and 7 for VP (Pérez-Boada et al. 2005)). As a result, the enzyme
will be converted from its native resting ferric/ground state (E0) to the so-called
compound I (EI, first intermediate) (Eq. 11.1), a protein cation radical
(de Montellano 2010) of strong oxidative power (Arnao et al. 1990b).
! E ½Fe
k1
E0 ½Fe3 þ þ H2 O2 I 4þ
¼ OP þ þ H2 O ð11:1Þ
with k as the reaction rate constant and the index for describing the pathway
number.
Fig. 11.2 Simplified “POX ping–pong” mechanism. E0–EII represents the three main peroxidase
oxidation states. The symbols S and S•+ stand for an appropriate substrate (e.g., lignin, lignin
model compounds such as adlerol) and its corresponding radical cation. The reaction will be
initiated by H2O2 (pathway 1), and followed by two consecutive, one-electron reduction steps
(pathway 2–3). Reprinted from Busse et al. (2013) with slight modification
Subsequently, EI returns back to E0 via a second enzyme intermediate (EII,

compound II) due to two consecutive one-electron reduction steps triggered by
suitable reducing substrates (S, the actual electron donor “preferentially
electron-rich aromatic compounds” (Lundell et al. 1993a)). Both reactions are
pH-dependent (Pérez-Boada et al. 2005; Wong 2009) resulting in the release of
radical cation intermediates (S•+) (Palmer et al. 1987) (pathway 2 and 3 in
Fig. 11.2; Eqs. 11.2, 11.3):
! E ½Fe
k2
EI ½Fe4 þ ¼ OP þ þ S II 4þ
¼ O þ S þ ð11:2Þ
! E ½Fe
k3
EII ½Fe4 þ ¼ O þ S 0 3þ
þ S þ þ H2 O ð11:3Þ
In Eq. (11.2), substrates causing EI reduction can also reduce EII (Eq. 11.3) as
described by Dunford (2010) for horseradish peroxidase (HRP). The same applies
for ligninolytic enzymes, such as LiP, and phenolic substrates (Schoemaker 1990).
Here, the overall net enzyme reaction can be simply expressed by Eq. (11.4).
! 2S
r
þ
2S + H2 O2 þ 2H2 O ð11:4Þ
Corresponding consumption rates r for S and H2O2 result in Eq. (11.5).
1 d[S] d[H2 O2 1 d[S þ

r¼ ¼ ¼ ð11:5Þ
2 dt dt 2 dt
Assuming the POX cycle is not inhibited, and considering k3 is rate limiting
(k3 = kcat because of k2 ≫ k3), r results in Eq. (11.6) (Rasmussen et al. 1995).
2k3 ½ET ½S2 ½S1

r¼ ð11:6Þ
k1 ½S2 þ ½S1
k3
X
II
½ET ¼ ½Ei ð11:7Þ
i¼0
ET means total enzyme.

When non-phenolic substrates are involved, caution should be exercised
regarding generalization. EII of LiP, for instance, can be reduced just by
di-methoxylated (minimum amount of alkoxy substituents), non-phenolic aromatic
compounds, whereas EI also accepts mono-methoxylated aromatic components
(Schoemaker 1990).
All produced radical cation intermediates (Fig. 11.2) will further pass through
diffusion-controlled, non-enzymatic reactions as schematically illustrated in
Fig. 11.3. Such radical reactions yield complex product mixtures. Depending on the
Fig. 11.3 Schematic drawing of nonenzymatic radical reactions–radical chain reactions. The
reaction is initiated by the cation radical intermediate S•+, a product of enzyme activity
nature of S•+, subsequent non-enzymatic reactions are the following (recently

summarized by Busse et al. (2013) based on Palmer et al. (1987) and Schoemaker
(1990)):
(a) One-electron oxidation of another appropriate substrate molecule RH (R also
stands for aromatic residuals, e.g., lignin or lignin derivatives), which in turn is
transferred to R•+ (Fig. 11.3). S•+ is reduced back to its ground state (Palmer
et al. 1987). This kind of reaction is referred to as mediator behavior.
(b) S•+ can also immediately be degraded to free radicals R•1 (R1 stands again for
aromatic residual) (Palmer et al. 1987), which is caused by rapid deprotonation
processes (Schoemaker et al. 1994a) (Fig. 11.3). Note, index 1 is just used for
differentiation purposes.
(c) Side-chain cleavage, e.g., Cα–Cβ bond cleavage (Fig. 11.4, section 11.3.2),
and C–H bond cleavage.
(d) Demethoxylation.
(e) Ether-bond cleavage [e.g., addition of solvent (H2O)] (Palmer et al. 1987).
(f) Hydroxylation of benzylic methylene groups, e.g., through oxygen incorpo-
ration via O2 or H2O in the solvent (Fig. 11.3).
(g) Phenol formation by nucleophilic attack, e.g., the addition of a solvent such as
H2O (Palmer et al. 1987).
(h) Aromatic ring cleavage caused by reactions with perhydroxy radicals HOO•/
HO•2 (Fig. 11.3); details will follow in the next section (Palmer et al. 1987).
The highly reactive free-radical species (in Fig. 11.3 denoted as R• and R•1) can bind
to molecular oxygen (O2), forming final products as well as superoxide anion
radicals (O•−2 ) (Schoemaker 1990) via degradation of organic peroxy radical
intermediates (ROO• or R1OO• in Fig. 11.3) (Palmer et al. 1987). O•− 2 ultimately
undergoes rapid disproportionation forming H2O2 and O2 (Harman et al. 1986;
Schoemaker 1990). Here, the O•− 2 is in a pH-dependent equilibrium with its pro-
tonated counterpart HOO• (Bielski et al. 1985). At low pH conditions, HOO•
predominates, existing as a powerful oxidant (Halliwell and Gutteridge 1985) and
thus contributing to the substrate degradation processes. This occurs because
additional bond cleavage is initiated as a consequence of the substrate (S) oxidizing
to its radical cation, where HOO• is reduced to H2O2 (Palmer et al. 1987).
Alternatively, R1OO• can further react with substrate molecules RH, resulting in
new free radical R• and organic peroxide R1OOR′ formation. If R′ represents
hydrogen, organic hydroperoxide (R1OOH) will instead be produced. As shown in
Fig. 11.3, R1OOH competes with the co-substrate H2O2 for reaction with E0–EI and
with S for EI conversion, depending on their relative concentrations. Reactions of
R1OOH with EI lead to undesired enzyme inactivations. In a low-oxygen atmo-
sphere, R1OOH formation diminishes (Acosta et al. 1988), but polymerization
increases (Palmer et al. 1987). R• products may also attack the peroxidase, again
leading to inactivation (Nicell et al. 1993). However, these radicals can also
undergo dimerization and polymerization processes (e.g., reaction with other free
radicals such as R•1) (Palmer et al. 1987), or can act as suitable substrates S for
maintaining the catalytic cycle. The latter is, however, limited in applicability for
radical cation intermediates S•+, as demonstrated later.
With consideration of further enzymatic reactions, both O•− •
2 and HOO (de-
pending on pH) can additionally serve as electron donors, instead of reducing
substrates (competitive reaction). Consequently, all three enzyme states, EI, EII, and
EIII may be, for instance, reduced by O•−2 under O2 production (Bielski et al. 1985;
Kettle et al. 2007) according to Eqs. (11.8)–(11.11), based on Kettle et al. (2007).
The reaction in Eq. (11.9) is supposed to prevent enzyme inhibition caused by poor
reducing substrates, in order to maintain activity (Kettle et al. 2007).
EI þ O
2 ! E þ O2
II
ð11:8Þ
EII þ O
2 þH
þ
! E0 þ O2 þ H2 O ð11:9Þ
EIII þ O
2 þ 2H
þ
! E0 þ H2 O2 þ O2 ð11:10Þ
or
EIII þ O
2 þ 2H
þ
! EI þ H2 O þ O2 ð11:11Þ
At this point, it becomes obvious that peroxidase activity at least leads to radical
cation formation by reacting with a suitable substrate, which triggers several
non-enzymatic reactions, similar to a “domino effect.” Moreover, the complexity of
the peroxidase reaction mechanism is evident, which explains why even the
degradation of simple aromatic monomers (e.g., VA), except pathway I in Fig. 11.2
(Dunford 2010), is not fully understood, and that further fundamental research is
still needed. Indeed, in a recent review, Busse et al. (2013) found that the mech-
anism is far more complex and strongly depends on (i) reaction conditions, (ii) POX
type, (iii) the presence of inhibitors, (iv) the used substrate.
11.3.2 Adlerol Degradation
Adlerol is a non-phenolic lignin model dimer with a β-O-4 linkage (structure I in

Fig. 11.4). Such LMCs are the most abundant units in lignin polymers, with con-
centrations ranging from 50 to 65 % (Adler 1977). However, the question arises if
adlerol is a representative model compound, since this kind of systems does not
include linkages to wood polysaccharides. Studying fungal lignin-degrading ability
based on 14C-lignins, by contrast, is suggested as the most reliable method (Hatakka
and Hammel 2011). From this perspective, and due to the fact that natural lignin is a
very complex, water-insoluble biopolymer, low molecular weight LMCs like
adlerol, which is also less soluble in water, do not initially seem to appropriately
mimic reality. Not even industrial/technical lignins (e.g., high polydisperse
ligninsulfonates) or dimeric LMCs attached to PEG (polyethylene glycol) would
ever result in realistic test scenarios. Woody biomass is exclusively the most rep-
resentative substrate, which complicates the situation even more. Major drawbacks
include: (i) variations in quality from batch to batch, and the fact that (ii) high
enzyme loads are required just to achieve satisfactory reaction rates.
Alternatively, the choice of an appropriate model system depends on the research
subject(s). Upon the examination of the fundamental issues, including studying
reaction mechanisms, enzyme (deactivation) kinetics, and establishing monitoring
systems for process control, it can be determined that LMCs are in fact very useful.
β-O-4 cleavage, for instance, is considered as a substantial indicator for both enzy-
matic lignin depolymerization and modification (Tien 1987; Wong 2009), making
adlerol a suitable LMC for studying delignification mechanisms. Moreover, the β-O-4
bond is stable at an acidic pH (Glasser et al. 1993) simulating the most challenging
scenario, because a low pH is generally needed for optimal POX activity.
Fig. 11.4 Graphical illustration of the adlerol (β-O-4 model compound, structure I) biocatalysis
based on LiP. Several degradation variants (partly in a summarized manner) are shown derived
from proposed reaction schemes by Tien and Kirk (1984), Kirk and Farrell (1987), Lundell et al.
(1993b) and Schoemaker et al. (1994b). I adlerol, II adlerol-derived radical cation, III veratryl
alcohol radical (VA), IV veratraldehyde (VAld) with 60–70 % major product, V Cβ-cation, VI
glycoaldehyde, VII guaiacol, VIII Cβ-centered radical, IX hemiacetal intermediate (unstable),
X keton, a second major product (15 %). Reprinted from Busse et al. (2013) with slight
modifications
In Fig. 11.4, the basic adlerol degradation steps, induced by (fungal) heme per-
oxidases, are schematically explained in conjunction to the previously described,
(non)enzymatic reactions. The catalytic cycle is initiated by H2O2 to form EI as usual.
EI further reacts with adlerol (structure I) to EII, resulting in the production of an
adlerol-derived cation radical intermediate (structure II) (Lundell et al. 1993b). Once
the radical cation is generated, it will be rapidly fragmented, non-enzymatically
(Hatakka et al. 1991). For this, various theories have been under consideration, as
summarized in Fig. 11.4. The reason for this is most likely due to both (i) S•+
chemistry (as already mentioned above), and (ii) pH-dependent product distribution
(Schoemaker 1990). Despite the non-enzymatic reaction, veratraldehyde (VAld,
structure IV) is the main fission product in the end. This same procedure is repeated
when another adlerol molecule is oxidized by EII (refer to pathway 3, Fig. 11.4).
In case adlerol will not serve as the reducing substrate for some reason, com-
ponent II (S•+), III (VA•), and VII (guaiacol) could act as substrates for driving the
cycle back to E0. The latter causes polymerization of its radical products (Chance
and Maehly 1955), which are phenoxy radicals (Lundell et al. 1993b). A reduction
of EII–E0 by component III also results in veratraldehyde (VAld) formation
(Schoemaker 1990; Schoemaker et al. 1994b). However, this is only an occurance
under low-oxygen conditions (Schoemaker et al. 1994b), since the existence of III
is supposed to be short-lived by reacting with O2 immediately. It is important here
to remember that the mediator function of radical products of component III can
cause co-oxidation (thus degradation) of adlerol as well. It is believed that efficient
mediation can only be realized if the degrading substrate will bind close to the
active site of the enzyme (Schoemaker et al. 1994b).
Under substrate (adlerol) limited conditions, Tien and Kirk (1984) demonstrated
a non-stereospecifity of their used LiP, due to complete cleavage of the β-O-4 lignin
model compound. This is an important feature, because lignin has a highly irregular
structure (Schoemaker 1990). Conversely, the involvement of nonenzymatic radical
(chain) reactions is obvious in assisting enzymatic degradation.
11.3.3 VP Reaction and Deactivation Kinetics
Up until now, the normal POX cycle has been introduced (pathway 1–3 in
Figs. 11.2, 11.4). In Fig. 11.5, the supposed VP reaction mechanism is summa-
rized. EI and EII in Fig. 11.5 are expected to be tryptophan radical (Trp•) containing
proteins. Furthermore, this tryptophan radical is suggested to be located on the
enzyme surface (≈10–11 Å to the heme center (Pogni et al. 2005; Ruiz-Dueñas
et al. 2009a)) for substrate oxidation purposes. This means there is no direct contact
between the heme (Fe3+) co-factor and the reducing aromatic substrates (e.g., VA
(non-phenolic monomer) or complex lignin-derived polymers (macromolecules))
avoiding steric hindrance. Interactions between substrate and heme group are
Fig. 11.5 Supposed H2O2-dependent reaction mechanism of the crude VP from B. adusta for
adlerol degradation. E0–EIII are enzyme intermediates. The symbols S and S•+ stand for an
appropriate substrate (e.g., adlerol) and the corresponding radical cation. O•−
2 denotes superoxide
radical anions. Pathway 1–3 depicts the usual POX reaction cycle with reversible steps, as
described by equation (a) and (b) (Ruiz-Dueñas et al. 2009b; Busse et al. 2013). The cycle will be
initiated by H2O2 (pathway 1). Pathway 4, 5, and 7 show important side reactions with H2O2. The
latter is in competition with pathway 6. Enzyme deactivation (Ei: inactivated enzyme) is found to
occur via EIII, as sketched in pathway 7. Pathway 8 illustrates a spontaneous unimolecular decay of
EIII. Reprinted from Busse et al. (2013)
enabled via long-range electron transfer (LRET) (Ruiz-Dueñas et al. 2009a). The
involvement of LRET in lignin degradation processes through ligninolytic enzymes
is generally accepted (Busse et al. 2013).
Pathway 1 is the only reaction which has been understood in the past (Dunford
2010), and therefore particular evidence for pathway 3 is still pending (Busse et al.
2013). Thus, for the adlerol bioconversion by a VP type from B. adusta, pathway 3
in Fig. 11.5 was assumed based on transient state kinetics made through VA oxi-
dation studies using a recombinant VP from E. coli (Ruiz-Dueñas et al. 2009b).
Moreover, reversible binary enzyme–substrate (E–S) complex (precursor complex
(Dunford 2010)) formation that occur before irreversible product release are cer-
tainly possible during the catalytic cycle of VP (concerns pathway 2 and 3,
Fig. 11.5) (Ruiz-Dueñas et al. 2009b). Both EI and EII exhibit reactions described
by Eqs. (11.12, 11.13) (Ruiz-Dueñas and Martínez 2009; Ruiz-Dueñas et al.
2009b).
$ E S ! E ½Fe
Km2 k2
EI ½Fe4 þ ¼ OTrp þ S I II 3þ
Trp þ S þ ð11:12Þ
Trp þ S $ E S ! E ½Fe
Km3 k3
EII ½Fe3 þ II 0 3þ
þ S þ ð11:13Þ
Kmi and ki (i = 2, 3) are the corresponding equilibrium dissociation constant and

rate constant, respectively. As a result, the kinetics of VP form a two substrate
ping–pong bi bi mechanism with the reaction rate r explained in Eq. (11.14) (Busse
et al. 2013).
vmax ½S1 ½S2

r¼ ð11:14Þ
KmS1 ½S2 þ KmS2 ½S1 þ ½S1 ½S2
vmax, S1, S2, KmS1 , and KmS2 stand for maximum reaction velocity, co-substrate H2O2,
substrate S (e.g., adlerol), dissociation constant for S1 and dissociation constant
for S2.
Maintaining constant S2 concentration, while varying S1, Eq. (11.14) can be
simplified to Eq. (11.15).
max ½S1
vapp
r¼ ð11:15Þ
Kmapp þ ½S1
vmax ½S2
max ¼
vapp ð11:16Þ
KmS2 þ ½S2
KmS1 ½S2
Kmapp ¼ ð11:17Þ
KmS2 þ ½S2
vapp app
max and Km are the apparent kinetic parameters of maximum reaction velocity and
dissociation constant, respectively. Equations (11.14)–(11.17) are valid in the
absence of inhibitions/deactivation phenomena and product(s) (Bisswanger 2008;
Cornish-Bowden 2012). Due to the well-known H2O2-sensitivity of POX,
inactivation/deactivation reactions are substantial side effects of crucial importance.
Figure 11.5 concerns the major reaction pathways that have a co-substrate H2O2 in
addition to substrate S. Both substrates compete for reactions with EI and EII (refer
to Fig. 11.5 pathway 2, 4, and 3, 5, respectively). When a certain S/H2O2/E ratio is
unbalanced, POX deviate from their normal catalytic cycle, and instead promote
reactions via compound III (EIII), diminishing enzyme activity (pathway 5, 7)
(Arnao et al. 1990a, b; Wariishi and Gold 1990; Busse et al. 2013). EIII is another
third enzyme intermediate and exhibits non, or sometimes barley any catalytic
activity (Schoemaker 1990). Subsequently, EIII can react further with H2O2,
resulting in an enzyme deactivation Ei (pathway 7 (Wariishi and Gold 1990;
Goodwin et al. 1994; Busse et al. 2013)), also known as suicide inactivation
(Valderrama et al. 2002). Research investigations have shown that VP deactivation
follows a time-dependent irrversible mechanism (Vlasits et al. 2010; Busse et al.

2013). Assuming first order kinetics (Eq. 11.18), and that no substrate will be
present, the observed deactivation constant kd(obs) can be determined according to
Eq. (11.19) (Busse et al. 2013).
!E
kdðobsÞ
E i ð11:18Þ
kiapp ½S1
kdðobsÞ ¼ ð11:19Þ
Kiapp ½S1
kiapp and Kiapp denote the apparent maximal rate of enzyme inactivation and the
apparent concentration of inhibitor for reaching half-maximal rate of inactivation,
respectively.
In the presence of substrate S, another competition with H2O2 occurs, since EIII
may also react with S by returning back to E0 (pathway 6, Fig. 11.5) under radical
cation intermediate and H2O2 formation (assumed based on Yokota and Yamazaki
(1965) and Acosta et al. (1988)). Alternatively, radical cation products S•+ are able to
revert EIII–E0. However, it does not necessarily mean that all potential substrates are
suitable for such EIII conversion. On the contrary, for a LiP H2 isozyme, for instance,
neither phenolics nor their corresponding phenoxyl radical products nor VA were
capable of returning EIII back to the native state E0, but VA•+ worked fine (Chung
and Aust 1995). The validity of VP and adlerol and its radical cation products still
needs to be clarified. Whatever the case, once an appropriate substrate like adlerol is
added, H2O2-dependent enzyme deactivation is competitively inhibited, and
Eq. (11.19) yields Eq. (11.20). This implies that the substrate does in fact possess
protective properties (Busse et al. 2013). For illustration refer to Fig. 11.6.
kapp ½S1
kdðobsÞ ¼ i ð11:20Þ
Kiapp 1 þ ½SS22 þ ½S1
Km
Depending on the environmental circumstances, e.g., the used buffer system, the
right term in Eqs. (11.19) and (11.20) must be extended by summation of k0i
(Copeland 2002). Thus, k0i denotes the enzyme inactivation rate caused by other
factors apart from H2O2. Furthermore, reaction pathway 5 and 7 (Fig. 11.5), and thus
enzyme deactivation, are pH-dependent and will be favored by an acidic environ-
ment (Cai and Tien 1992; Busse et al. 2013). VPs, and all other POX, commonly
have their reaction optimum around pH 3–4 (Wong 2009; Hofrichter et al. 2010;
Liers et al. 2010; Busse et al. 2013) and are consequently quite susceptible to H2O2
(catalytic amounts of > 30–50 µM (Goodwin et al. 1994; Busse et al. 2013)). This is
a major drawback of POX compared to Lac, particularly when attempting to reach
industrial applications, as will be discussed next. Spontaneous decay of EI (is
unstable (Dunford 2010)) to EII contributes to additional loss in activity (reaction
path is omitted in Fig. 11.5). The unimolecular reaction described by pathway 8
Fig. 11.6 H2O2-dependent deactivation kinetics in the absence of the substrate adlerol
(experimental data (squares), computer simulation (red solid line)) and in the presence of
4 mM adlerol (experimental data (triangles), computer simulation (black solid line)). Reaction
conditions: T: 30 ± 1 °C, pH: 4.0 (100 mM sodium tartrate buffer), n: 375 rpm, enzyme
concentration: *0.06 mg mL−1, V: 10 mL or 50 mL. Each examination was carried out in
duplicates (parallel) with a maximal standard deviation of 10 % from the mean. The inset plot
illustrates the corresponding half-life (t1/2) course as a function of initial H2O2 concentration.
Reprinted from Busse et al. (2013)
(Fig. 11.5), EIII decay to E0 along with O•−2 release, (Nakajima and Yamazaki 1987;
Arnao et al. 1990b) would imply enzyme regeneration or loss of activity.
Finally, it is obvious that balancing the S/E/H2O2 ratio is an important task for
minimizing enzyme deactivations, and thus avoiding excessive H2O2 concentra-
tions. It is in fact impossible to fully eliminate enzyme deactivation, but it can be
delayed. Maintaining enzyme consumption as low as possible is therefore a major
tasks, since POX are currently still costly.
11.4 Technical Challenges for VP Applicability on Large

Scale
11.4.1 Ensuring Cost-Effective Availability and Stability
As already stated, POX and especially VP have received a lot of attention as

biocatalysts. Unfortunately, an industrial implementation has not yet be realized.
This progress is hampered by (i) low availability of active POX in sufficient

amounts, (ii) H2O2 related instabilities (Ayala et al. 2008), and (iii) high enzyme
costs (Casella et al. 2010). Therefore, major efforts are directed toward protein
engineering for improving enzyme performance, particularly stability with respect
to H2O2, catalytic activity by redox potential enhancement, and ensuring avail-
ability by efficient homologous and heterologous (over)production (Conesa et al.
2000; Ayala et al. 2008; Longoria et al. 2010). As potential expression systems,
bacteria (E. coli), yeast (S. cerevisiae, Pichia) and fungi (white-rot fungi,
Aspergillus) are all under consideration (Conesa et al. 2000; de Weert and Lokman
2010). Moreover, active POX production in insect cells using recombinant bac-
ulovirus has been examined (Pease et al. 1991; Johnson et al. 1992). However, to
date, none of the investigated homologous and heterologous production approaches
have matched industrial requirements (de Weert and Lokman 2010). Although
homologous expression shows promising results, the most frequent cause of failure
is the instability in productivity, which is not yet fully understood (Singh and Chen
2008). Further research and development is still needed. An efficient POX-specific
production is dependent on (i) choice of production strain (Conesa et al. 2000; de
Weert and Lokman 2010), (ii) vector system (de Weert and Lokman 2010), and
(iii) culture/operating conditions (Conesa et al. 2000; Rühl et al. 2008) and inclu-
sive production plant configuration (Rühl et al. 2007).
MnP overproduction in heterologous systems might generally be easier than for
LiP and VP. It is conceivable that due to their high reactivity to a broader substrate
spectrum, production of active LiP and VP, is comparatively more harmful to host
organisms (Tsukihara et al. 2006). Table 11.2 lists host organisms for VP pro-
duction that have been studied. Here, the homologous expression system is believed
to provide a promising platform for large-scale production, resulting in recombinant
enzymes exhibiting structural and kinetic properties identical to the native one
(Tsukihara et al. 2006). In general, P. ostreatus strains are well suited for industrial
production of ligninolytic enzymes (Rühl et al. 2008).
Table 11.2 Hosts for homologous and heterologous VP production (modified based on de Weert
and Lokman (2010))
Organism Host strain Yield References
Homologous
P. ostreatus P. ostreatus TM2-18 21 mg L-1 Tsukihara et al.
7300 U L-1 (2006)
Heterologous
B. adusta E. coli strain BL21 *12 mg L−1 with max. Mohorčič et al.
(DE3)pLysS 0.25 mU mg−1a (2009)
P. eryngii Aspergillus nidulans 466 U L−1 Eibes et al.
(2009)
a
Specific activity was determined based on VA degradation
11.4.2 Supply of Lignocellulosic Biomass
In using lignocellulosic biomass as a substrate, an appropriate pretreatment is

mandatory before an enzymatic bioconversion can be addressed. For reaching
satisfactory reaction rates with the smallest possible enzyme amount, or in other
words, for reaching high turnover numbers, wood substrates with high substrate
specific surfaces (high surface to volume ratios) are favored. The minimal
requirement is that native lignin must be readily accessible for enzymatic attack.
Ground wood/wood flour provides the best preconditions, although a relative high
energy demand is required depending on the particle size. Particle size is limiting
for mass transfer (Viamajala et al. 2010). Furthermore, native lignin structures have
to be conserved. Since lignin is an amorphous thermoplastic polymer (Saake and
Lehnen 2012), the grinding temperature should be maintained below its glass
transition temperature (Tg) (range hard-/soft-wood: 65–105 °C (Saake and Lehnen
2012)). Melting lignin would result in undesired and uncontrolled modifications
(structural changes, lignin relocalization) influencing subsequent enzymatic con-
version (Donohoe et al. 2008; Viamajala et al. 2010). The same is also valid for
pretreating methods reaching temperatures above Tg.
Inconsistency of wood substrate, particularly lignocellulose composition, is
another variable that interferes with enzyme activity and product quantity as well as
quality. Hybrid poplar (e.g., made of Populus maximowiczii and Populus nigra)
from short-rotation forestry is a future based raw material since it is fast-growing.
Its use means variations in wood composition will be severely minimized.
11.4.3 Supply of Lignin-Containing Product Streams
Liquid lignin product streams (technical lignins) generated via chemical pretreat-
ment of lignocellulosic biomass, for instance, is an alternative substrate for further
processing and finishing by POX. The initial chemical oxidation should preferably
lead to lower molecular weight and more biodegradable intermediates (Mantzavinos
and Psillakis 2004). However, it has to be taken into account that such a pretreatment
can also generate inhibitory products. Moreover, the pH may have to be adjusted.
This might be difficult to achieve. Depending on the isolation method, the resulting
technical lignin may not be soluble at an acidic pH. In general, most technical lignins
are soluble in organic solvents (e.g., dimethyl sulfoxide (DMSO), dioxane,
dimethylformamide, and pyridine) (Saake and Lehnen 2012). VP remains active in
organic solvents like DMSO, at least in the presence of an inorganic substrate
(Rodakiewicz-Nowak et al. 2006). Nonetheless, with the goal of reaching industrial
application, further addition of organic solvents may be uneconomic and in fact
problematic, relating to the recovery of the organic solvent as well as resulting
ecological aspects.
11.4.4 Bioprocess Engineering for Delignification
For the provision of optimal operating conditions (e.g., T, buffer system, S/H2O2/E
ratio, presence of additives, operation mode), the choice of an appropriate reactor
design is an important issue of bioprocess engineering in order to guarantee the
economic implementation of VP and POX for delignification purposes.
11.4.4.1 Reactor Design
For a rational reactor design, several aspects must be taken into account. Due to
complex and rather uncontrollable radical reactions as seen above, such aspects
include first and foremost an efficient maximization of catalytic turnover numbers
(kcat = vmax/[ET]) in order to avoid an excess of disruptive factors like increased H2O2
and phenol concentrations. Phenols cause polymerization and formation of phenoxyl
radicals affecting the catalytic cycle. A continuous removal of such compounds is
therefore highly recommended. Moreover, reuse of the enzyme is also beneficial.
Combining the enzymatic conversion process by connecting an external cross-
flow ultrafiltration (UF) unit to an enzyme membrane reactor system, as sketched in
Fig. 11.7, represents a promising technological approach for delignification
(Flaschel et al. 1983; Lema et al. 2010; Huang and Ramaswamy 2013). Such
configurations provide several important advantages:
(i) Continuous operation mode.
(ii) Complete biocatalyst retention and recycling.
(iii) Application of free/native enzyme is possible (homogeneous catalysis),
avoiding time-consuming enzyme immobilization accompanied by mass
transfer limitations (Busse et al. 2016; López et al. 2007).
(iv) Replacement of fresh active enzyme depending on deactivation in order to
maintain specified substrate conversion levels (Busse et al. 2016).
(v) Physical biocatalyst immobilization.
(vi) Rapid fractionation of biocatalyst and unreacted high molecular weight
substrates from desired product.
(vii) Continuous removal of undesired by-products and enzyme inhibitors, shift-
ing reaction toward product yield maximization (Busse et al. 2016; López
et al. 2007).
Membrane filtration processes like tubular UF membranes are already well estab-
lished for lignin recovery and separation in biorefineries (Jönsson 2013) and in the
pulp and paper industry (Ebrahimi et al. 2009). This is due to the following eco-
nomic aspects (Huang and Ramaswamy 2013; Jönsson 2013):
(i) Low chemical consumption.
(ii) Fractionation is controllable by pore size.
(iii) Feed volume reduction for further processing (Jönsson and Wimmerstedt 1985).
(iv) Any T and pH adjustments are superfluous.
Despite the fact that tubular UF membranes exhibit low packing densities and high
energy demands (Leiknes 2009), they have “the largest foot-print” (Jönsson 2013).
Moreover, these membranes require low pretreatment (Jönsson 2013), tolerate feed
streams of high fouling potential and of high pollution (e.g., solids) and are easy to
clean (Melin and Rautenbach 2007; Leiknes 2009). Nevertheless, proteins are
serious membrane foulants (Blatt et al. 1970; Porter 1972; Field 1996), besides
lignin-containing feed streams (Sridhar and Bhattacharya 1991; Pfromm and
Watkins 1997; Ebrahimi et al. 2016; Busse et al. 2016). Consequently, constant
enzyme addition, and/or an increased amount of lignin-derived fragments, result in
secondary layer formation on the membrane surface (membrane fouling) limiting
filtration performance, e.g., permeability or throughput, solute retention (Busse
et al. 2016), and membrane service life. Fouling is a common issue in membrane
filtration processes and is mainly affected by (i) membrane properties (e.g., selected
pore size, material, configuration), (ii) hydrodynamic conditions, and (iii) feed
composition (Mulder 1996; Cheryan 1998; Melin and Rautenbach 2007). Filtration
performance is a crucial parameter, since it determines the efficiency of enzymatic
conversion, protein load, and reactor size, along with hydraulic retention time
Fig. 11.7 Schematic illustration of a promising enzyme membrane reactor configuration

(continuous stirred tank reactor (CSTR) equipped with an external ultrafiltration (UF) module)
for delignification purposes. Enzymatic reaction can be assumed to take place primarily in the
CSTR at constant reaction volume VR. F volumetric flow rate; c, ci concentration, concentration of
component i; VR reaction volume; t process time; ri reaction rate; J(t) time-dependent permeate
flux; AM membrane surface; ΔPTM transmembrane pressure; η dynamic viscosity of the permeate
depending on temperature T and concentration c; RT(t) total hydraulic resistance as a function of
time t due to membrane fouling
(refer to Fig. 11.7) (Busse et al. 2016). Thus, the maintenance of constant high
volumetric flow rates (or fluxes) is a major task throughout filtration processes.
Using ceramic membranes is advantageous in comparison to polymer mem-
branes. Major aspects are:
(i) High thermal and chemical stability which is beneficial for cleaning.
(ii) High mechanical stability, particularly when filtering abrasive media.
(iii) Less susceptible to fouling and low adhesion potential for molecular organic
substances (Ebrahimi et al. 2015; Fan et al. 2015).
(iv) High membrane service life/lifetime (6 years for ceramic membranes and 1.5
for polymeric) (Arkell et al. 2014).
Conversely, polymeric membranes are less expensive, easy to configure and scal-
able (Van der Bruggen 2009).
Several applications have already been reported using POX in membrane biore-
actor systems. However, these have just been for treating xenobiotics and pollutions
(e.g., azo dye Orange II by MnP, nonylphenol by VP), which use mostly polymeric
UF membranes (López et al. 2007; Méndez-Hernández et al. 2015). The choice for
the optimal material ultimately depends on individual needs (Arkell et al. 2014).
11.4.4.2 Process Control
Key prerequisite for implementing an effective process control, and thus ensuring
optimal operation condition, is an efficient and reliable online monitoring system
(except T and pH, since they are standard). Although H2O2 concentration is such a
crucial parameter, the availability of commercial H2O2 online sensors, with suffi-
cient sensitivity over a wide pH and T range, is limited. Contrarily, there are several
fluorescence probes, such as some based on oxidation of N-
acetyl-3,7-dihydroxy-phenoxazine (e.g., Amplex Red®) by HRP, for measuring
H2O2 within a range of 10 nM to 100 µM (Gomes et al. 2005). Disadvantages of
using such probes are: (i) sample preparation is required, (ii) HRP inactivation at c
(H2O2) > 100 µM (Towne et al. 2004), thus first requiring the finding of an
appropriate pre-dilution, (iii) incubation times of 15–30 min before receiving first
results, and (iv) pH is also limited, ranging from 7.5 to 8.5. Similar conditions are
true concerning any HRP/POX-related biosensor for H2O2 measurements. In the
current state, amperometric sensors equipped with platinum electrodes appear rather
suitable, since they are not susceptible to H2O2. Sample preparations are also
superfluous, among other things. Particularly, the membrane protected PEROX
H2O2 online sensors from ProMinent Dosiertechnik in Heidelberg, Germany, meet
useful requirements, including aspects such as a wide pH-/T- and c(H2O2)-range
with a comparatively low response time. First successful applications of this have
already been reported for chloroperoxidase catalyzed oxidations (Seelbach et al.
1997; van Deurzen et al. 1997).
Another important parameter is the actual enzyme activity, which could be
detected by simply monitoring the production of certain fission products and/or
substrate consumption. When complex substrates such as lignin are involved and
product formation is hardly manageable, as already illustrated above, lignin degra-
dation is usually monitored by UV absorption measurements at λ = 280 nm. Highly
concentrated lignin solutions appear dark brown. The color intensity is an indirect
indicator of lignin amount (Kinstre 1993). Conversely, the release of lignin-derived
high molecular weight fragments from LC biomass would result in an intensification
of color in the culture medium. A precise assignment of these fragments is not
possible, and further analytical methods will be required which are limited in
applicability, partly very costly, and not yet sufficiently standardized. An extensive
overview will be given by Lin and Dence (1992). Moreover, a fundamental expertise
is mandatory for process optimization and data interpretation. At present, there is no
simple, standalone analytical method. Based on these facts, alternative (in)direct
measurement techniques must be found. Such techniques should be (i) cheap,
(ii) easy to implement, online, (iii) robust, (iv) quick and precise and (v) low main-
tenance. Measuring dissolved oxygen (DO), especially via optical sensors, is cur-
rently the most promising parameter for process monitoring due to pseudo-catalase
activity of POX at H2O2 excess (or low c(S)/c(H2O2) ratios) resulting in O2 formation
(Acosta et al. 1988; Wariishi and Gold 1989; Vlasits et al. 2010). Excessive c(H2O2)
causes considerable enzyme deactivation (Busse et al. 2013). On the contrary, O2
consumption is an indicator for high c(S)/c(H2O2) ratios (López et al. 2007). Thus, O2
consumption is also indicative for substrate degradation processes as seen in
Figs. 11.3 and 11.4, whereas O2 can also be generated simultaneously concerning
HO•2/O•− 2 release (Figs. 11.3 and 11.5). Recent studies by López et al. (2007), treating
xenobiotic compounds such as Organe II by MnP, have shown that DO could be
valuable for implementing process control strategies (e.g., feedback control).
However, based on the variety of radical reactions, O2 formation/consumption is not
trivial (Palmer et al. 1987). Further research is therefore needed for a better (process)
understanding, particularly in the field of delignification. Moreover, the presence of
substrates (e.g., VA and adlerol) capable of bond cleavage seems to be a precondition
for significant O2 consumption (Palmer et al. 1987).
11.5 Conclusion and Outlook
The high susceptibility to H2O2 of VP (POX in general) is an important issue which

needs to be overcome, as they are currently making wild type less attractive for
industrial use. In vivo relative low H2O2 levels are rather assumed due to H2O2
assimilation by the fungus avoiding unfavorable high concentrations (Hammel et al.
1993; Böckle et al. 1999). Moreover, VPs are regrettably limited in availability. Thus,
numerous efforts are being carried out for recombinant VP production, in order to
elevate H2O2 tolerance and availability. Alternatively, enzyme immobilization could
be another technique to overcome premature inactivation (Ei formation). However,
immobilization may not prevent EIII formations at H2O2 excess. Enzyme recondi-
tioning (EIII → E0, pathway 6 in Fig. 11.5) in turn is again influenced by the
underlying reaction conditions and not by the immobilization method. Despite sev-
eral studies, the proof-of-concept is still outstanding. Additionally, in the case of
woody biomass, good enzyme penetration must be further guaranteed, affected by
porosity and the pore size, of the substrate to be treated.
Another promising/future based strategy may be enzymes from insects and
insect-associated microorganisms. Insects are the most diverse taxonomic animal
class, colonizing almost every ecological niche. To succeed in these sometimes
extreme niches, insects have established diverse biological and chemical systems,
e.g., the production of defense molecules (Gross et al. 2008; Schlipalius et al. 2012),
protein stabilization (Bale 2002), or lytic enzymes (Landureau and Jolles 1970; Mika
et al. 2013). Insects also house symbiotic microorganisms as digestive helpers,
fodder, or both (Scharf et al. 2011). This is possible due to the special metabolic
systems, or exogenous enzymes, of the associated organisms. For instance, insects
like bark beetles, ambrosia beetles and termites, are able to feed on wood, making
them xylophagous, and are well known as wood pests. The function of their
digestive systems is not yet completely clarified. The enzymatic apparatus for the
oxidation of lignin and the hydrolysis of cellulose is only marginally understood.
One reason might be the diverse sources of the key enzymes, which may be pro-
duced either by gut-inhabiting microorganisms (Morrison et al. 2009), by symbiotic
fungi cultivated by the insects (Currie 2001), or by the insects themselves as
endogenous enzymes (Pauchet et al. 2010). Nevertheless, a lot of work has been
done in attempts to understand the lignocellulolytic system of the insects and the
degree of participation by symbionts (Geib et al. 2008; Scharf et al. 2011).
Currently, POX are still costly for industrial use. Nonetheless, there is no doubt
that once limitations are resolved, particularly H2O2 instability and efficient pro-
duction, the potential for large-scale application of VPs raises significantly (Casella
et al. 2010). However, many questions with respect to the enzymatic delignification
mechanism still remain in place, i.e., substrate preparation/pretreatment, E0
recovery, control of inhibitory reactions/factors besides H2O2, involvement of
accessory factors and enzymes, etc. Moreover, for better process control, devel-
opments focusing on simple and fast online monitoring systems will also be nec-
essary. For these purposes, further fundamental research is needed.
Acknowledgments The researchers would like to thank the Hessen State Ministry of Higher
Education, Research and the Arts for the financial support within the Hessen initiative for scientific
and economic excellence (LOEWE).
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Chapter 12
Fungal Aryl-Alcohol Oxidase
in Lignocellulose Degradation
and Bioconversion
Juan Carro, Ana Serrano, Patricia Ferreira and Angel T. Martínez
Abstract Bioconversion of lignocellulosic materials draws much interest as they

are regarded as a renewable source of energy and platform chemicals. The enzyme
aryl-alcohol oxidase (AAO) has been extensively studied, revealing its involvement
in biodegradation of lignocellulose by several well-known white-rot fungi. Its
physiological role is to supply hydrogen peroxide from the oxidation of aromatic
substrates derived from fungal secondary metabolism or lignin degradation, which
can: (i) be used by peroxidases to oxidise lignin; or (ii) give rise, through Fenton
reaction, to hydroxyl radical that is able (itself) to depolymerise cellulose and
oxidise lignin. Several features make this enzyme an appealing biocatalyst that has
shown its potential for industrial applications: AAO has a broad range of substrates
that it oxidises by stereoselective hydride transfer reaction mechanism, and reduces
atmospheric molecular oxygen as a co-substrate producing hydrogen peroxide.
Abbreviations
AAO Aryl-alcohol oxidase
DFF 2,5-Diformylfuran
FDCA 2,5-Furandicarboxylic acid
FFCA 5-Formylfurancarboxylic acid
GMC Glucose-methanol-choline oxidase/dehydrogenase superfamily
HMF 5-Hydroxymethylfurfural
HMFCA 5-Hydroxymethylfurancarboxylic acid
LiP Lignin peroxidase
MnP Manganese peroxidase
J. Carro A. Serrano A.T. Martínez (&)

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
P. Ferreira
Department of Biochemistry and BIFI, University of Zaragoza, Pedro Cerbuna 12, 50009
Zaragoza, Spain

302 J. Carro et al.
PEF Poly(ethylene furandicarbolylate)

UPO Unspecific peroxygenase
VP Versatile peroxidase
12.1 Introduction to AAO
Aryl-alcohol oxidase (AAO) is a fungal enzyme secreted by different fungi,

including basidiomycetes involved in the degradation of lignocellulose. It belongs
to the glucose-methanol-choline oxidase/dehydrogenase (GMC) protein super-
family (Cavener 1992) that includes oxidoreductases, although there are exceptions
such as hydroxynitrile lyase, with different substrate specificities. They all share a
common fold in two subunits (the cofactor-binding and substrate-binding ones) and
the presence of FAD as a cofactor.
AAO activity was reported during the 1960s in cultures of the lignicolous fungus
Trametes versicolor by Farmer et al. (1960), although this enzyme was not further
characterised. It was during the late 1980s and early 1990s that AAO was isolated
from several Agaricales species, such as Pleurotus sajor-caju (Bourbonnais and
Paice 1988), Pleurotus eryngii (Guillén et al. 1990) and Pleurotus ostreatus (Sannia
et al. 1991), and identified as the source of the H2O2 (Guillén et al. 1994) and
aromatic aldehydes found in cultures of the above fungi (Guillén and Evans 1994;
Gutiérrez et al. 1994). During subsequent years, its involvement in the ligninolytic
process was demonstrated. AAO is responsible, along with other GMC oxidore-
ductases and copper-radical oxidases, for the H2O2 production that, as described
below, is required by: (i) the activity of ligninolytic peroxidases; and (ii) the for-
mation of oxygen radicals that exert oxidative and depolymerising activities on
polysaccharides and also on lignin.
Some AAOs have been exhaustively characterized, being those of P. eryngii
(Ferreira et al. 2005; Guillén et al. 1992) and Bjerkandera adusta (de Jong et al.
1994; Romero et al. 2009) two remarkable examples. Their substrate specificity
showed to be broad, probably due to its extracellular and ligninolytic nature, since it
proved to use secondary fungal metabolites, p-methoxybenzyl alcohol and other
benzylic alcohols, as substrates. In fact, it is currently known that AAO can oxidise
both phenolic and non-phenolic aryl-alcohols, together with other polyunsaturated
(aliphatic) primary alcohols, to their corresponding aldehydes, as well as aromatic
secondary alcohols, albeit with much lower efficiency (Table 12.1). Moreover, the
presence of low amounts of acids attributed to its activity in some fungal cultures
(and in vitro reactions) paved the way for the demonstration of its activity on aryl
aldehydes (Ferreira et al. 2010).
This plethora of AAO substrates (Table 12.1), among which several reduced
species derived from lignin decay products are noticeable, makes it a promising
biocatalyst. The lignocellulosic materials are the main renewable resource on Earth
12 Fungal Aryl-Alcohol Oxidase … 303
Table 12.1 Comparison of the catalytic efficiencies of P. eryngii and B. adusta AAO oxidising
representative alcohol and aldehyde substrates
kcat/Km (s−1 mM−1)
P. eryngii B. adusta
Benzyl alcohol 47 ± 9 18 ± 1
OH
p-Fluorobenzyl alcohol F 59 ± 6 22 ± 2
OH
m-Fluorobenzyl alcohol 13 ± 1 47 ± 2
OH
F
p-Chlorobenzyl alcohol Cl 398 ± 32 361 ± 15

OH
m-Chlorobenzyl alcohol 203 ± 4 1050 ± 30

OH
Cl
p-Methoxybenzyl alcohol O 5230 ± 620 646 ± 45

OH
m-Methoxybenzyl alcohol 65 ± 24 349 ± 8

OH
O
Veratryl alcohol O 210 ± 5 22 ± 1

OH
O
Isovanillyl alcohol O 152 ± 5 51 ± 1

OH
HO
Vanillyl alcohol HO 0 31 ± 1
OH
O
3-Chloro-p-methoxybenzyl O 4090 ± 200 1480 ± 110

alcohol OH
Cl
2-4-Hexadien-1-ol HO 1270 ± 60 186 ± 7

Cinnamyl alcohol 78 ± 11 305 ± 11
OH
Coniferyl alcohol HO 0 5 ± 0.2

OH
O
m-Chlorobenzaldehyde 0.64 ± 0.05 1 ± 0.09

O
Cl
3-Chloro-p-methoxy-benzaldehyde O 0.085 ± 0.006 0.050 ± 0.002

O
Cl
m-Fluorobenzaldehyde 0.40 ± 0.02 0.05 ± 0.005

O
F
p-Nitrobenzaldehyde O O 0.31 ± 0.006 0.010 ± 0.001

N+
-
O
(continued)
304 J. Carro et al.

kcat/Km (s−1 mM−1)
P. eryngii B. adusta
5-Hydroxymethyl-2-furfural HO O 0.21 ± 0.02 n.d.
O
2,5-Diformylfuran O O 0.150 ± 0.008 n.d.

O
n.d. not determined
because they are widespread and abundant (forests cover 27 % of world’s area) and,
thus, they are cheap and can be easily stored. As a consequence, the conversion of
these materials into biofuels in biorefineries is of great interest. However, not only
biofuels, that is heat and power, are important, but other by-products obtained
during the biorefinery processes are also being carefully examined aiming at using
them for the production of valorised chemicals (Bozell and Petersen 2010). AAO is
a candidate for the enzymatic delignification of plant biomass (in synergy with
other oxidoreductases) and for the production of aromatic aldehydes and acids that
can originate from this renewable resource.
In this chapter, AAO involvement in lignocellulose decay owing to its capacity
to produce H2O2, as well as its potential applications in different industrial pro-
cesses, are discussed.
12.2 AAO and Lignocellulose Decay
Lignocellulosic biomass (in woody and nonwoody vascular plants) accounts for the
60 % of the total carbon fixed by land photosynthesis, and is constituted of three
main polymers: cellulose, hemicellulose and lignin (Higuchi 1997). Both cellulose
and lignin are the two most abundant polymers on Earth. Lignin is located in the
middle lamella, where it attains its highest concentration, and the secondary wall of
vascular plants, together with the above polysaccharides. Its main functions are
conferring rigidity to the plants, waterproofing vessels and providing protection
against desiccation, pathogens and irradiation (Gellerstedt and Henriksson 2008).
Since lignin is very recalcitrant, it constitutes the main sink of carbon in land
ecosystems and, as a consequence, its degradation is a key step to complete the
carbon cycle. In fact, before it started to be biodegraded and mineralised, at the end
of the Carboniferous period (*300 million year ago), the carbon that the plants
fixed accumulated and therefore gave rise to the coal deposits we currently use as
source of fossil fuels (Floudas et al. 2012).
The only organisms capable of degrading wood are some saprotrophic agari-
comycetes, feeding on the simple sugars produced when cellulose and
hemicelluloses are hydrolysed. However, to accomplish this task, fungi must

overcome a very recalcitrant barrier: lignin. Lignin is an amorphous polymer
including a variety of bonds established among the phenylpropanoid units that form
its structure. Although these fungi do not use lignin as a nutrient, they have to
remove or modify lignin to gain access to carbohydrates. The development of an
extracellular system capable of altering the structure of lignin was, thus, a great
achievement in evolutionary and ecological terms, since the polymer is mineralised
and the carbon “sequestered” in it may go back to the atmosphere as CO2. It is
generally accepted that the first lignin-degrading organism must have been a
basidiomycete (Floudas et al. 2012), which developed some strategies to act on the
recalcitrant lignin polymer oxidising its subunits, causing bond breakages and
releasing the carbohydrates embedded within its matrix.
The role of H2O2 in lignocellulose decay was studied as it proved to be produced
simultaneously with the ligninolytic system. It was seen that the addition of cata-
lase, an enzyme that degrades H2O2, to cultures of the white-rot fungus
Phanerochaete chrysosporium diminished its lignin-degrading ability (Faison and
Kirk 1983). Two families of oxidoreductases include oxidases being able to pro-
duce this strong oxidant: copper-radical oxidases and GMC oxidoreductases.
The copper-radical oxidases are proteins that have one copper ion and a protein
radical involved in catalysis, such as extracellular glyoxal oxidase (Kersten and
Kirk 1987) and the related enzymes recently identified from sequenced genomes
(Kersten and Cullen 2014), among others. GMC oxidoreductases are a superfamily
of enzymes that share common structural patterns, including the FAD cofactor and
a histidine catalytic base, although they differ in their substrate specificities.
Extracellular GMC enzymes involved in the lignocellulose-degradation process are
cellobiose dehydrogenase (Ayers et al. 1978; Bao et al. 1993), pyranose 2-oxidase
(Daniel et al. 1994), aryl-alcohol oxidase (Guillén et al. 1990) and methanol oxi-
dase (Nishida and Eriksson 1987). The latter enzyme lacks a signal peptide to
transport the protein to the extracellular space, but its presence out of the hyphae
has been revealed, and it is supposed to be secreted by alternative secretion path-
ways not known yet (Daniel et al. 2007). Glucose oxidase (Eriksson et al. 1986) is
an intracellular enzyme, so its role in lignocellulose degradation is controversial,
since a transport system for the H2O2 formed would be required in order that it
could carry out functions in the extracellular medium.
AAO activity has been reported in different fungi including T. versicolor (Farmer
et al. 1960), Fusarium solani (Iwahara et al. 1980), Rigidoporus microporus
(Waldner et al. 1988), Pleurotus species (Bourbonnais and Paice 1988; Guillén et al.
1992; Sannia et al. 1991), B. adusta (Kimura et al. 1990; Muheim et al. 1990b;
Romero et al. 2009, 2010) and Botrytis cinerea (Goetghebeur et al. 1993); and the
corresponding genes have been identified in many basidiomycete genomes (Ferreira
et al. 2015; Floudas et al. 2012; Hernández-Ortega et al. 2012a), as described below.
Further evidence of the simultaneous expression of AAO along with enzymes
typically expressed under ligninolytic condition such as lignin peroxidase (LiP) in B.
adusta (Muheim et al. 1990a) and manganese-oxidising peroxidases in Pleurotus
cultures (Camarero et al. 1996), supported the involvement of this oxidase in lignin
306 J. Carro et al.
degradation. Moreover, the enzyme was located in the hyphal sheath (which is
formed by secreted fungal polysaccharide) during lignocellulose degradation
(Barrasa et al. 1998), as it had been previously reported for ligninolytic peroxidases
and laccases (Gallagher et al. 1989; Green et al. 1992).
The activity of AAO is supported by both non-phenolic and phenolic aryl
alcohols that can derive from fungal metabolism (de Jong et al. 1994; Gutiérrez
et al. 1994) and from lignin degradation (Kirk and Farrell 1987; Shimada and
Higuchi 1991) and are substrates for this enzyme in cooperation with related
dehydrogenases. It was seen that H2O2 is produced when adding aromatic alcohols,
as well as the corresponding aldehydes and acids, to the mycelium of P. eryngii
(Guillén et al. 1994). Thus, it was postulated that the redox cycling of these
compounds results in a continuous supply of H2O2. The product of the reaction with
AAO (aldehyde or acid) needs to be reduced in order to be re-used by the enzyme.
Therefore, it is hypothesised that the oxidised species is transported to the intra-
cellular space, where aryl-alcohol or aryl-aldehyde dehydrogenases, both
NADPH-dependent enzymes, convert it into alcohol again (de Jong et al. 1994;
Guillén and Evans 1994). Supply of reduced NADPH must be supported by the
high carbon availability for a fungus under ligninolytic conditions (Fig. 12.1).
P. eryngii AAO’s preferential substrate is p-methoxybenzyl alcohol
(Table 12.1), which is a secondary metabolite detected in fungal cultures grown
+
Fungal NADP NADPH
hypha Secondary Aryl-alcohol Catabolism
metabolism
t b li dehydrogenase
p-methoxybenzyl
alcohol H2O
+
glucose CO2
p-anisaldehyde AAO +
O2
H2O2 + Fe2+ vanillin
H2O Fenton reaction
Peroxidase OH·
Lignin Lignin Cellulose

OH Breakdown
O of linkages
OH
O Anabolism
Plant
lignin subunit lignin cation radical
cell-wall
Fig. 12.1 Scheme of the natural role of AAO in decay of plant cell-wall producing H2O2 for:
(i) activation of lignin-degrading peroxidases; and (ii) formation of cellulose-depolymerising
hydroxyl radical (also causing lignin oxidation)
both in glucose and lignin media (Gutiérrez et al. 1994). It is known that aromatic
alcohols, aldehydes and acids are derived from the shikimic acid pathway of fungal
secondary metabolism (Turner and Aldridge 1983). It was seen that the oxidised
product, p-methoxybenzaldehyde (also known as p-anisaldehyde), was much more
abundant in the Pleurotus cultures than its alcoholic counterpart (Gutiérrez et al.
1994). High levels of related compounds, such as 3-chloro-p-anisaldehyde, were
also found to be abundant in B. adusta cultures (de Jong et al. 1992) and agree with
catalytic efficiencies towards the corresponding alcohol for B. adusta AAO, for
which seems to be the preferential substrate (Table 12.1). These chlorinated
derivatives are also minor extracellular metabolites in P. eryngii and P. ostreatus
(Gutiérrez et al. 1994; Okamoto et al. 2002).
These findings further supported the hypothesis postulating that AAO was acting
in the extracellular space involved in the said redox-cycling process for H2O2
production.
Based on macroscopic and chemical composition features, the wood-degrading
processes were split into two different types involving different decay mechanisms.
Some fungi leave a whitish residue, and thus were named white-rot fungi, while
others produce a brown residue and were called brown-rot fungi (Martínez et al.
2005; Schwarze et al. 2000; Zabel and Morrell 1992). Owing to genomic and
enzymatic studies, it is now thought that AAO could act as an auxiliary enzyme in
both processes due to its H2O2-producing activity and presence in the sequenced
genomes of Agaricomycotina responsible for the two types of wood-decay
processes.
12.2.1 White-Rot Decay
White-rot fungi developed an enzymatic machinery to degrade lignin and, hence, be

able to use cellulose and hemicellulose as a source of carbon and energy. The main
enzymes involved are metalloproteins including LiP, manganese peroxidase
(MnP) and versatile peroxidase (VP), as well as laccases (Ruiz-Dueñas and
Martínez 2009). Ligninolytic peroxidases are heme proteins that use H2O2 as the
electron-accepting substrate to oxidise the lignin units. In contrast, laccases have
copper as a cofactor, use O2 as electron acceptor, and are thought to often act
through small intermediate compounds, redox mediators, which in turn oxidise
lignin. Since most enzymes cannot penetrate the intricate and compact structure of
sound wood, small chemical oxidisers, activated oxygen species (as hydroxyl
radical), metal cations (as Mn3+ produced by MnP and VP) and aromatic radicals
(formed by different oxidoreductases) are probably responsible for the first stages of
lignin decay (Evans et al. 1994). In white-rot decay there is a need of a continuous
H2O2 flow in the extracellular environment in order that peroxidases be able to act
on lignin. Moreover, hydroxyl radical can be produced through Fenton reaction
between Fe2+ and H2O2, and participate in the oxidative modification of cellulose
308 J. Carro et al.
(as described below for brown-rot decay) and also of lignin (Fig. 12.1) (Bes et al.
1983; Forney et al. 1982; Gómez-Toribio et al. 2009).
White-rot fungi can be classified into two groups according to their gross
degradation patterns. Some of them degrade lignin and cellulose simultaneously as
it is the case for P. chrysosporium, one of the most studied lignin-degrading
organisms. Instead, others, such as Ceriporiopsis subvermispora, degrade lignin
before cellulose (Otjen and Blanchette 1986). The main differences between the
sequenced genomes of these two fungi appear to be related to (i) the peroxidase
repertoire, and (ii) the genes involved in lipid metabolism (Fernández-Fueyo et al.
2012). The latter is related to the fact that free radicals from unsaturated lipids are
supposed to also play a role in lignin attack (Bao et al. 1994). Selective white-rot
fungi are the most interesting for industrial (biotechnological) applications in which
carbohydrates are the raw material, since they release cellulose from the lignin
matrix without significantly consuming it.
The analysis of genomes of white-rot fungi in which AAO appeared to be
produced, along with peroxidases, further confirmed its involvement in the
white-rot process as an auxiliary enzyme producing H2O2. For instance, Floudas
et al. (2012) analysed 24 basidiomycete genomes to search for enzymes involved in
the degradation of lignin. On the one hand, all white-rot genomes studied possessed
AAO genes with the only exception of Auricularia delicata. On the other hand,
AAO appears to be the most common H2O2-producing GMC, since methanol
oxidase genes are not as abundant, glucose oxidase genes are absent, and pyranose
2-oxidase genes are only found in two of the genomes studied (Table 12.2).
Table 12.2 Inventory of peroxidase and GMC genes in eleven white-rot Agaricomycotina
genomes
AD PST FM DS TV SH BA PB PC GS CS
Peroxidases 21 21 33 21 39 11 34 16 18 14 25
GMCs AAO 0 4 2 9 3 14 11 3 3 9 4
CDH 1 1 1 1 1 1 1 1 1 1 1
MOX 3 3 2 4 4 7 5 6 3 4 1
P2O 2 0 0 0 2 0 1 1 1 0 0
From Floudas et al. (2012) and Ruiz-Dueñas et al. (2013) and Ferreira et al. (2015)
AAO, aryl-alcohol oxidase; CDH, cellobiose dehydrogenase; MOX, methanol oxidase and P2O,
pyranose 2-oxidase
AD, Auricularia delicata; PST, Punctularia strigosozonata; FM, Fomitiporia mediterranea; DS,
Dichomitus squalens; TV, Trametes versicolor; SH, Stereum hirsutum; BA, Bjerkandera adusta;
PB, Phlebia brevispora; PC, Phanerochaete chrysosporium; GS, Ganoderma sp. in the
Ganoderma lucidum complex and CS, Ceriporiopsis subvermispora
12.2.2 Brown-Rot Decay
The brown-rot decay is based on a non-enzymatic attack on wood that leads to

lignin modification, instead of degradation/mineralisation and cellulose depoly-
merisation. On having no longer the machinery for degrading lignocellulose
(Table 12.3), brown-rot fungi are the only organisms known to be able to nearly
completely remove all polysaccharides from wood without causing substantial
lignin degradation (Arantes et al. 2012). Nevertheless, it has been demonstrated that
they do alter lignin in order to gain access to the carbohydrates leaving it partially
modified but still polymeric and with a recognisable chemical structure (Martínez
et al. 2011). Genomic studies have proved that these fungi stem from the white-rot
lineage, and that they have lost most of the energy-consuming apparatus their
white-rot ancestors used to have, which means that peroxidases are absent or
residual in their genomes (only one generic peroxidase gene in some species)
(Ruiz-Dueñas et al. 2013). They have originated independently in several lineages
and have representatives in five basidiomycete orders (Floudas et al. 2012).
The initial oxidative step alters the plant cell-wall structure in order to make it
more accessible to enzymes involved in subsequent decay (Goodell et al. 1997).
Reactive oxygen species, such as hydroxyl radical, generated by Fenton chemistry,
play a key role in this oxidative process (Fig. 12.1) (Baldrian and Valaskova 2008;
Halliwell 1965). Fenton chemistry is based on reactions among H2O2 and Fe2+ ions
giving rise to hydroxyl free radical. This is a radical-chain mechanism where Fe2+ is
generated from Fe3+ in a cycle that involves fungal enzymes. The hydroxyl radical
is the most powerful (non-specific) oxidant in biological systems but it has an
extremely short half-life (10−9 s) and, as a consequence, Fenton reactions need
occur immediately adjacent to the site of oxidative action within the plant cell wall
because of spatial diffusion limitations (Arantes et al. 2012).
Since one of the most remarkable chemical alterations that brown-rot decay
cause to lignin is demethoxylation (Niemenmaa et al. 2008; Yelle et al. 2008),
which ultimately gives rise to methanol, methanol oxidase is thought to be the most
important H2O2-producing enzyme in brown-rot decay. Nevertheless, genomic
Table 12.3 Inventory of peroxidase and GMC genes (see Table 12.2 for enzyme abbreviations)
in six brown-rot Agaricomycotina genomes
CP GT FP WC DSP RP
Peroxidases 0 0 1 1 0 1
GMCs AAO 0 2 1 0 0 2
CDH 1 1 0 0 0 0
MOX 2 1 4 4 1 4
P2O 0 0 0 0 0 0
From Floudas et al. (2012) and Ferreira et al. (2015)
CP, Coniophora puteana; GT, Gloeophyllum trabeum; FP, Fomitopsis pinicola; WC, Wolfiporia
cocos; DSP, Dacryopinax sp. and RP, Rhodonia placenta
310 J. Carro et al.
studies have shown that brown-rot fungi have AAO too (Martinez et al. 2009),
suggesting that the latter oxidase might play a role in Fenton chemistry. Floudas
et al. (2012) analysed seven brown-rot fungal genomes and found AAO genes in
several of them, although they were not as abundant as those of methanol oxidases
(Table 12.3).
12.3 Paper Pulp Industry: Biopulping and Biobleaching
One of the main steps of pulp and paper industry is the treatment of wood chips to
separate cellulosic fibres (the actual raw material used) from the lignin forming the
middle lamella, a process called pulping. The concern about environment and
energy wasting has stimulated studies on the use of microorganisms to accomplish
this task. The resulting biopulping process is thought to be energetically and
environmentally more favourable than chemical and/or mechanical treatments
(Blanchette et al. 1992; Rasmussen et al. 2010).
The biopulping process starts with the colonisation of wood xylem and par-
enchyma by the fungi. After hyphae have grown on the substrate, the fungus will
start producing its ligninolytic enzymatic system in order to degrade the middle
lamellae and separate fibres (Breen and Singleton 1999). Since the loss of cellulose
is undesirable, the organisms of choice are white-rot fungi showing preference for
lignin degradation rather than cellulose degradation, whose task is to make cellu-
losic fibres accessible for the papermaking process (Scott and Swaney 1998).
Several selective ligninolytic fungi, such as C. subvermispora and P. eryngii,
together with the model white-rot fungus P. chrysosporium, and brown-rot fungi
such as Postia placenta, among others, have been investigated for biopulping of
wood and annual plants (Akhtar et al. 1997; Camarero et al. 1998; Ferraz et al.
2008; Giles et al. 2014; Masarin et al. 2009; Vicentim et al. 2009). AAO is sup-
posed to act as an auxiliary enzyme providing H2O2 in these processes, as it is the
case of natural lignin degradation.
Pulp bleaching, which is the removal of chromophores in order to obtain white
paper pulp, is another process in which AAO has shown to contribute. In a study in
which two flax pulps were treated with fungal enzymes—laccases, peroxidases and
feruroyl esterases—the ability of AAO from Pleurotus pulmonarius CBS 507.85,
which is a natural hyperproducer of this enzyme, to aid in this process was tested
(Sigoillot et al. 2005). The results showed that the presence of AAO along with
laccase improved the bleaching process probably due to the ability of AAO to
prevent the repolymerisation of the phenoxy radicals released by laccases by using
them as electron acceptors (as an alternative for O2). In a similar way, AAO can be
combined with ligninolytic peroxidases, in the presence of a substrate enabling it to
release the H2O2 required by the former enzymes.
12.4 Bioconversions
12.4.1 Flavour Synthesis
White-rot fungi are among the most versatile flavour and aroma producers in nature
(Fraatz and Zorn 2011; Lapadatescu et al. 2000). These compounds are mainly of
aromatic nature and synthesised through biotransformations by plant, enzymatic or
microbial processes (Serra et al. 2005). Since there exists demand for naturally
produced compounds, the biotechnological production of flavours and aromas
attracts much attention (Krings and Berger 1998) due to the great economic
importance this industry has.
One of the most important flavours is vanillin, naturally produced by orchids of
the Vanilla genus, but this source only represents the 1 % of the commercial vanilla
flavour. As a consequence, several methods of obtaining vanillin have been
developed (Priefert et al. 2001) that use bioconversion of lignin and phenyl-
propanoids, such as eugenol (Overhage et al. 2003). The ability of B. adusta AAO
to oxidise vanillyl alcohol reported by Romero et al. (2009), could be exploited in
the biotechnological production of this flavour. It has been suggested that AAO
may be used to avoid formation of vanillyl alcohol as a by-product diminishing the
yield of vanillin formation (Fig. 12.2a) by the fungi of the genus Pycnoporus
(Lomascolo et al. 2011).
Fig. 12.2 Catalytic (a)

efficiencies of P. eryngii and
B. adusta AAOs in B. adusta AAO
B
bioconversion of some 31±1 s-1 mM-1
flavours and aromas.
a Oxidation of vanillyl
alcohol into vanillin.
b Oxidation of benzyl alcohol
into benzaldehyde.
c Oxidation of (b)
p-methoxybenzyl alcohol into
p-methoxybenzaldehyde B. adusta AAO
18±1 s-11 mM-11
P. eryngii AAO
47±9 s-1 mM-1
(c) HO O
d t AAO
B adusta
B.
646±45 s-1 mM-1
y g AAO
P. eryngii
5230±620 s-1 mM-1
O O
312 J. Carro et al.
Another very remarkable commercial aromatic compound is benzaldehyde.

Some research has been carried out using the fungus B. adusta for the production of
this chemical from L-phenylalanine (Lapadatescu et al. 2000; Lapadatescu and
Bonnarme 1999). In this reaction of L-phenylalanine for flavour production,
aryl-alcohols are the main products obtained, as reported for B. adusta
(Lapadatescu et al. 2000) and P. chrysosporium (Jensen et al. 1994). Consequently,
the application of AAO to such biotransformations could result in the obtention of
higher levels of aromatic aldehydes, since their alcohol counterparts are substrates
of the enzyme (Fig. 12.2b). Finally, using AAO plus an unspecific peroxygenase
(UPO) from the fungus Agrocybe aegerita (Ullrich et al. 2004), an oxidoreductase
cascade can be used for toluene conversion into benzaldehyde, with the second
enzyme using the peroxide generated by AAO.
Another flavour that AAO produces as a consequence of its auxiliary role in
lignin degradation is p-anisaldehyde (4-methoxybenzaldehyde) (Fig. 12.2c). It was
shown that the corresponding alcohol is the physiological and preferential substrate
of the P. eryngii enzyme (Ferreira et al. 2005; Guillén et al. 1992) and that AAO
and mycelium-associated aromatic dehydrogenases establish a concerted anisalde-
hyde redox cycle to produce H2O2 continuously as described above (Fig. 12.1).
Therefore, AAO can be used for biotransformations aiming at the production of
vanillin, benzaldehyde, anisaldehyde and other aromas.
12.4.2 Deracemization of Chiral Secondary Alcohols
Many of the drugs and potential drug candidates possess chiral centres and most of
them need to be commercialised as enantiomers rather than racemates given that
enantiomers often carry out different activities within biological systems (Carey
et al. 2006). Therefore, chiral intermediates for pharmaceuticals are synthesised
through enantioselective asymmetric reactions (Patel 2013). As an alternative,
deracemization of chiral mixtures is used by the pharmaceutical industry to obtain
pure enantiomers. Among chiral compounds, some secondary alcohols are used as
chiral intermediates and analytical reagents, and the development of synthesis
procedures for the production of enantiomerically enriched (enantioenriched)
alcohols has gained importance in the pharmaceutical industry.
Biological systems are generally chiral and, as a consequence, many enzymes
are regio- and enantioselective. These properties are regarded to be a consequence
of the active sites’ architecture and the enzyme’s mechanism. Therefore, many
microorganisms and enzymes offer attractive alternatives for easy production
(asymmetrical synthesis or deracemization) of enantiomeric compounds of interest
in the fine chemicals and pharmaceutical sectors (de Albuquerque et al. 2015;
Matsuda et al. 2009). The AAO catalytic mechanism consists in a hydride
abstraction from the benzylic position of the alcohol by the oxidised flavin, in a
reaction aided by an active-site histidine acting as a catalytic base to form the
alkoxide intermediate (Hernández-Ortega et al. 2012a). Due to active site
architecture, and the simultaneous nature of the hydride and proton abstractions,
hydride transfer by AAO is stereoselective (only takes place from the pro-
R position) as shown using the two α-monodeuterated enantiomers of p-methox-
ybenzyl alcohol (Hernández-Ortega et al. 2012b). Taking advantage of this infor-
mation, Hernández-Ortega et al. (2012b) assayed the transformation of racemic
secondary alcohols using P. eryngii AAO. They saw that the enzyme was able to
oxidise the racemic 1-(p-methoxyphenyl)-ethanol, although it showed an apparent
efficiency orders of magnitude smaller than the one for p-methoxybenzyl alcohol. In
this reaction, AAO enantioselectivity towards the (S) isomer was shown using
chiral HPLC, which allows for the isolation of (R) isomer from chiral mixtures
(Fig. 12.3). Moreover, the AAO enantioselectivity towards another secondary
alcohol 1-(p-fluorophenyl)-ethanol was estimated as a S/R ratio of 21, in reactions
using the individual enantiomers.
Therefore, it is plausible that AAO could be used for the isolation of isomers
from racemates taking advantage of its kinetic behaviour. However, AAO activity
on secondary alcohols is low, due to some hindrances among such substrates and
the residues forming the active site (Fernández et al. 2009). A mutated variant
(F501A), in which the side chain of a bulky aromatic residue was removed to make
room in the cavity, was created with the purpose of facilitating oxidation of sec-
ondary alcohols. The removal of this side chain resulted in a stereoselectivity S/
R ratio on 1-(p-fluorophenyl)-ethanol three-fold higher than that of the wild-type
enzyme. Hence, improved variants by directed evolution, as it has been done with
galactose oxidase for these purposes (Escalettes and Turner 2008), or further
site-directed mutagenesis would result in better deracemization reactions.
(a) (b)
(R)-isomer (S)-isomer (R)-isomer
A225
A225
30 35 40 45 50 55 30 35 40 45 50 55
Retention time (min) Retention time (min)
Fig. 12.3 Chiral HPLC chromatograms showing the deracemization of 1-(p-methoxyphenyl)-

ethanol by P. eryngii AAO. a Chromatogram of the untreated racemate. b Chromatogram after
24-h reaction with AAO, where only the peak of the (R) isomer is detected (the peak of the p-
methoxyacetophenone, formed from oxidation of the (S) isomer, eluted in a different region of the
chromatogram)
314 J. Carro et al.
12.4.3 Oxidation of Furfurals
Another remarkable class of chemicals derived from lignocellulosics, which are

substrates of AAO, is furfurals. They are formed as by-products of the pretreatment
of lignocellulosics in the paper pulp and the bioethanol industries. In spite of the
fact that they imply the loss of raw material in such processes, they are now
regarded as valorised by-products in biorefinery processes. Moreover,
5-hydroxymethylfurfural (HMF hereinafter) is commercially produced from the
dehydration of hexoses, mainly fructose, (Karinen et al. 2011) but direct production
from glucose is also possible (Zhao et al. 2007).
HMF is regarded as a chemical building block due to the presence of several
functionalities within the molecule (furan ring and hydroxyl and carbonyl groups)
(Rosatella et al. 2011), which may undergo chemical reactions that give rise to a
bunch of compounds: phenolic resins, bioplastics, Schiff bases, polyurethane foams
of epoxy resins, as well as disubstituted furan derivatives (Moreau et al. 2004). The
obtention of 2,5-furandicarboxylic acid (FDCA hereinafter) from HMF (Fig. 12.4)
is of great interest since FDCA can polymerise to give rise to polyesters that may
substitute for those produced from fossil fuels. Such polymers are called poly
(ethylene furandicarbolylate) polyesters (PEFs) and show good mechanical and
barrier properties, are biodegradable and come from renewable resources
(Papageorgiou et al. 2014).
Several processes involving enzymes and organisms have been reported aiming
at the obtention of FDCA from HMF (Carro et al. 2015; Dijkman et al. 2014, 2015;
Dijkman and Fraaije 2014; Hanke 2012; Koopman et al. 2010; van Deurzen et al.
1997). Regarding the use of AAO with this purpose, Hanke (2012) screened for
available AAOs capable of oxidising HMF with variable results. With the same
purpose, Carro et al. (2015) analysed the ability of P. eryngii AAO to oxidise HMF.
Fig. 12.4 Oxidative pathways from HMF (1) to FDCA (5). FFCA (4) formation can take place
through two alternative intermediate compounds: DFF (2) or HMFCA (3)
In this way, it proved to be catalytically active towards HMF and to be able to

catalyse two subsequent oxidations in its molecule giving rise to
5-formylfurancarboxylic acid (FFCA, hereinafter; 84 %) and FDCA (6 %) in 24 h.
When they tested the ability to oxidise the intermediates between HMF and FDCA,
AAO proved to be catalytically active on diformylfuran (DFF), producing 86 % of
conversion into FFCA and a 14 % into FDCA. However, it did not react either with
5-hydroxymethylfurancarboxylic acid (HMFCA) or with FFCA. NMR studies of
the compounds involved in this chemical pathway demonstrated that carbonyl
groups of both DFF and FFCA were hydrated in aqueous medium giving rise to
diols in 53 and 8 % of abundance, respectively. These gem-diols resulting from the
hydration of aldehydes had been reported to be substrates of the P. eryngii AAO
(Ferreira et al. 2010). However, HMF underwent no hydration, which agrees with
AAO being active towards DFF (formed by oxidation of the HMF hydroxyl group),
but not towards HMFCA, which would be formed by a prior oxidation of the HMF
carbonyl group. Such results suggest that the oxidative pathway for the oxidation of
HMF to FFCA catalysed by this AAO proceeded via DFF. Although this enzyme
was not able to oxidise FFCA, this constraint was overcome through the application
of a sequential enzymatic cascade together with UPO (Ullrich et al. 2004). This
enzyme used the H2O2 produced by AAO to introduce a hydroxyl group in the
formyl group of FFCA, thus producing FDCA, attaining 91 % of conversion after
120 h (Fig. 12.5).
These findings paved the way to implement AAO together with the peroxyge-
nase to obtain a valorised by-product originated from lignocellulosic biomass.
Furthermore, they widened the spectrum of its aromatic substrates, which is now
thought to range from benzylic carbocycles to heterocycles, as HMF and DFF.
AAO
13±1 s-1 mM-1
1 2
O2 H2O2 47%
AAO
9±1 s-1 mM-1 UPO
2’ 4 5
53% O2 H2O2 H2O2 H2O
Fig. 12.5 Sequential enzymatic cascade for the production of FDCA (5) from HMF (1) using
P. eryngii AAO and A. aegerita UPO via DFF (2) and its hydrated gem-diol counterpart (2′)
(abundance of DFF and its gem-diol at equilibrium shown as percentage), and FFCA (4). AAO
catalytic efficiencies are indicated
316 J. Carro et al.
12.5 Conclusions
The finite character of fossil fuels makes it necessary to find new resources,
preferentially renewable ones, which allow for the substitution of the classical
resources. In this way, lignocelluloses are a remarkable material due to their
ubiquitous and renewable character. Furthermore, the concern about the environ-
ment impels us to search for new catalytic procedures that take advantage from
natural processes, instead of the chemical environmentally polluting and
energy-wasting ones. Hence, the use of organisms or enzymes to carry out catalytic
industrial processes is gaining importance.
AAO is a very promiscuous enzyme that catalyses the oxidation of a great deal
of polyunsaturated alcohols (and hydrated aldehydes). Its applicability on some
biotechnology-based industrial processes has been reviewed here and it has showed
to be promising as a biocatalyst in several conversions. AAO, because of its
involvement in the lignocellulose decay process, has potential to be applied to
lignocellulose biorefineries. AAO has so far shown its potential applications in
paper pulp manufacture and bioconversion of lignocellulose-derived compounds
(such as HMF), synthesis of flavours, and deracemization of chiral alcohols
(Fig. 12.6). In spite of the fact that the use of enzymes (among which AAO) for
these purposes is not yet sufficiently developed, much progress has been done the
recent years in this field as one can infer from the huge amount of publications
available on enzymatic biocatalysis.
Oxidation
O id ti off D
Deracemization
i ti Fl
Flavour and
d C ll l
Cellulose
furfurals of chiral alcohols aroma synthesis industries
Applications
pp cat o s
AAO
Physiology
H2O2 production
Substrate of Production of
peroxidases hydroxyl radical
Cellulose
depolymerisation
and lignin oxidation
Fig. 12.6 Scheme depicting the physiological role of AAO in lignocellulose degradation and its
potential applications in the cellulose and other chemical sectors
Acknowledgments This work was supported by the HIPOP (BIO2011-26694) and the NOESIS
(BIO2014-56388-R) projects of the Spanish Ministry of Economy and Competitiveness, and the
INDOX (KBBE-2013-7-613549) and EnzOx2 (H2020-BBI-PPP-2015-RIA-720297) European
projects. J.C. acknowledges a FPU fellowship of the Spanish Ministry of Education, Culture and
Sport.
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Chapter 13
Monosaccharide Oxygenase
Avnish Kumar, Monika Asthana, Hirawati Deval

and Sarika Amdekar
Abstract Oxygenases are enzymes that catalyze the insertion of oxygen atom into
an organic substrate. With the intention to perform this kind of reactions, those
enzymes want to prompt molecular oxygen to conquer its spin-forbidden response
with the organic substrate. In most cases, oxygenases make use of organic cofactors
to transfer electron to molecular oxygen for its activation. Due to chemo-, regio-,
and/or enantio-selective activity both mono- or dioxygenases are attractive bio-
catalysts. Oxygenases having many organic substrates, of these monosaccharide
reactive oxygenases are focused in this chapter. Most abundant protein on earth
known as Rubisco monooxygenase and 5-carbon monosaccharide, Sialic acid
monooxygenase is well studied but any dioxygenase capable to incorporate O2
atom in monosachharide is still unknown.
13.1 Introduction of Oxygenases and Monosaccharides
Biochemical reactions in which electrons are exchanged starting with one molecule
then onto the next are catalyzed by a wide assortment of enzymes, alluded to as
oxidoreductases or redox catalysts (EC 1.x.x.x) (Dixon and Webb 1979). Taking
into account the kind of reactions they catalyze, oxidoreductases have been sepa-
rated into 22 diverse EC-subclasses. A more broad order was proposed by Xu
(2005), in which these proteins were separated into four subgroups: (i) oxidases,
A. Kumar M. Asthana (&)

Department of Biotechnology, School of Life Sciences,
Dr. Bhim Rao Ambedkar University, Agra, U.P. 282004, India
H. Deval
National Institute of Virology (NIV), Indian Council of Medical Research,
Ministry of Health and Family Welfare, Govt. of India, Gorakhpur Unit,
B.R.D. Medical College Campus, Gorakhpur 273013, India
S. Amdekar
Department of Microbiology, Barkatullah University, Bhopal, M.P 462026, India

324 A. Kumar et al.
Table 13.1 Dioxygenases and their cofactors

Enzyme Type of reaction Cofactors
Metapyrocatechase Ring cleavage Fe2+
Pyrocatechase Ring cleavage Fe3+
Prostaglandin Formation of a cyclic Heme, Tryptophane,
cyclooxygenase peroxide glutathione
Lipoxygenase Formation of an open Fe2+
peroxide
Cysteamine dioxygenase Oxygenation of sulfur Fe2+, sulfides
Tryptophane Ring cleavage Heme
2,3-dioxygenase
4-Hydroxyphenyl-pyruvate Hydroxylation, oxidative Fe2+, ascorbic acid, required
dioxygenase decarboxylation a-ketoglutarate
Monosaccharide dioxgenases are not found to be explained so far in our search study
Oxydizing enzymes
(Oxygen serve as electron acceptor)
Oxygenase Oxidases Peroxidases Dehydrogenase/

Use O2 as acceptor Use H2O2 to Reductases
produce H 2 O or H 2O2 produce H2O
Di-oxygenase
Mono-Oxygenase
Diverse Iron, Copper, or

flavin dependent
Cytochrome P- Flavin dependent Other

450 dependent Mono-oxygenases
Phenol oxygenase Lipoxygenase
Fig. 13.1 Classification of oxidative enzymes

13 Monosaccharide Oxygenase 325
(ii) peroxidases, (iii) oxygenases/hydroxylases and (iv) dehydrogenases/reductases

(Table 13.1; Fig. 13.1).
13.1.1 Oxygenases
Catalyze the incorporation of oxygen atom into the substrate. They are classified
into two groups: monooxygenase and dioxygenases. Dioxygenases consolidate
both oxygen atoms into the substrate, while monooxygenases incorporate a single
oxygen atom as a hydroxyl group into the substrate and the second oxygen atom is
reduced to water. So as to do this sort of reactions, these catalysts need to actuate
molecular oxygen to overcome its spin-forbidden reaction with the organic sub-
strate. Mostly, monooxygenases use (in)organic cofactors to exchange electrons to
molecular oxygen for its activation.
13.1.2 Monosaccharides
Monosaccharides are a class of carbohydrates that cannot be separated to less

complex sugars by hydrolysis and that constitute the building blocks of oligosac-
charides and polysaccharides. Monosaccharide comprise of no less than three
carbon molecules, one of which is connected to an oxygen atom to shape an
aldehyde group (CHO) or a ketone, and the others of which are each appended to a
hydroxyl group (OH), e.g., glucose, fructose, mannose, glucosamine, man-
nosamine, gluconic acid, sialic acid, uronic acid, N-acetyl glucosamine, N-acetyl
muramic acid, and so forth. Monosaccharide can be occurred as chains or rings.
Six-carbon sugars (hexoses) and five-carbon sugars (pentoses) are the most fre-
quently encountered monosaccharide in nature.
13.2 Monooxygenase Reactions
Oxidoreductases are getting more attention these days for selective oxygenation of
aromatic compounds and a wide range of reactions have been documented using
these enzymes from various microbial sources. Monooxygenases are an emerging
class of biotechnologically relevant enzymes due to their chemo-, regio-, and/or
enantio-selective, making those appealing biocatalysts. Monooxygenases incorpo-
rate one atom from O2 into a substrate that accepts oxygen and using another
substrate that furnishes the two H atoms to reduce the other oxygen to water.
Monooxygenases responds with O2 and supplement one atom of oxygen into the
substrate and second oxygen atom forms water with a reduced co-substrate.
326 A. Kumar et al.
S þ O2 þ RH2 ! SO þ H2 O þ R
Here, RH2 is reduced co-substrate and R is oxidized co-substrate.

If monooxygenase split hydrogen from the substrate itself then it is known as
internal monooxygenase otherwise it is called external monooxygenase.
In monooxygenation reactions, monooxygenases require electrons rather than
O2. Reactions in which organic compounds are oxygenated or hydroxylated are of
great value for organic synthesis. In any case, specific oxyfunctionalization of
organic substrates can be a specific issue in organic synthesis. These reactions are
frequently completed with solid oxidizing operators and they additionally happen
with little chemo-, regio-, and enantio selectivity (Loughlin 2000). These issues are
taken away by managing the use of oxygenases. Under optimum conditions of pH
scale and temperature, aqueous solvent, etc. these enzymes catalyze a specific
oxyfunctionalization of organic substrates. For instance, styrene monooxygenase
was demonstrated enantio specifically catalyze the epoxidation of styrene subor-
dinates, yielding the comparing (S)-epoxides with >99 % e.e. (Panke et al. 1998;
van Hellemond et al. 2007).
Chemoselectivity refers to the selective reactivity of one functional group in the
presence of others; often this process in convoluted and protecting groups are on
the molecular connectivity alone.
Regio selectivity is the preference of one direction of chemical bond making or
breaking of over all other possible directions, e.g., selectively generation of one
constitutional isomer rather than the other.
Enantio selectivity is the production of a compound by a method that selectively
preferred the formation of a specific enantiomer or diastereomer.
All styrene monooxygenases (Fig. 13.2) perform enantio-selective epoxidations
of styrene and chemically analogous compounds, which makes them interesting for
biotechnological applications (Montersino et al. 2011).
In the reaction, monooxygenases (EC 1.13.x.x and EC1.14.x.x) need to enact
molecular oxygen, as no reaction will proceed without actuation due to the spin
state of O2. This actuation endless supply of electrons to sub-atomic oxygen, after
which oxygenation of the natural substrate can happen. The formation of reactive
oxygen intermediate could be identified according to the cofactor present in the
+ +
NAD(P)H+H +O2 NAD (P) + H2O
(Styrene monooxygenase)
Styrene Styrene oxide
Styrene + FADH2 + O2 (S)-2-phenyloxirane + FAD + H2O
Fig. 13.2 Styrene oxygenase reaction

monooxygenase. Sometimes, no cofactor at all is available. internal monooxyge-

nases get these electrons from the substrate itself, though outside monooxygenases
are subject to outer electron contributors, e.g., coenzyme NAD(P)H (van Berkel
et al. 2006a). Torres et al. (2010) has reviewed a brief discourses of the distinctive
groups of monooxygenases, in light of the types of cofactor they require for
catalysis are as take after. Monooxygenases is presented, based on the type of
cofactor that these enzymes require. This includes not only the cytochrome P450
and flavin-dependent monooxygenases, but also enzymes that utilize pterin, metal
ions (copper or iron) or no cofactor at all. As most of these monooxygenases require
nicotinamide coenzymes as electron donors.
Heme-dependent Monooxygenases Additionally alluded to as cytochrome P450
monooxygenases or CYPs (EC 1.14.13.x, EC 1.14.14.x and EC 1.14.15.x) can be
found in numerous life shapes: eukaryotes (warm blooded animals, plants and
organisms) and microbes express a wide assortment of these catalysts (Nelson et al.
1996). CYPs are most inexhaustible in eukaryotes. They are b-sort
heme-subordinate outer monooxygenases and they owe their name to the excep-
tional light assimilation at 450 nm of the lessened CO-bound heme-complex. This
high retention is utilized to assess the centralization of the monooxygenase (Omura
and Sato 1964).
Rather than CYPs, qualities encoding Flavin-dependent monooxygenases have
all the earmarks of being moderately bottomless in prokaryotic genomes. Likewise
eukaryotes have been appeared to contain large portions of these qualities (e.g., the
genome of Arabidopsis thaliana contains 29 quality homologs of purported
‘flavin-containing monooxygenases’) (Schlaich 2007). The flavins used by these
catalysts are either FMN or FAD and are either bound firmly to the chemical
(prosthetic gathering) or capacity as a substrate (coenzyme) (van Berkel et al.
2006b). Flavin-subordinate monooxygenases can catalyze different responses, for
instance epoxidations, Baeyer–Villiger oxidations (Fig. 13.3) and halogenations.
Keeping in mind the end goal to perform these responses, flavin-subordinate
monooxygenases create a receptive transitional by shaping a covalent bond between
atomic oxygen and the C4a of the flavin (Ghisla and Massey 1989). Depending
upon the protonation state, this intermediate is proposed to lead to either
Fig. 13.3 Asymmetric chemical synthetic routes that catalyze selective oxygen insertion are
usually mediated by heavy metals with complex ligands and/or explosive peroxidic oxidizing
agents. A way to circumvent the use of these hazardous materials is by applying the
flavin-dependent Type I Baeyer–Villiger monooxygenases. These enzymes are able to catalyze
a broad range of oxidative reactions such as Baeyer–Villiger and heteroatom oxidations
328 A. Kumar et al.
nucleophilic or electrophilic oxygenations. Although this intermediate is well sta-

blized within the enzyme. (Entsch and van Berkel 1995), it will rot to the oxidized
flavin without a suitable natural substrate or halide, yielding hydrogen peroxide
(H2O2) as an item (Massey 1994).
Copper-dependent Monooxygenases (EC 1.14.17.x) constitute a comparatively
tiny family of enzymes that need copper ions for hydroxylation of their substrates.
Enzymes belonging to this family have primarily been found in organism organ-
isms. The best-studied member of this family is dopamine-monooxygenase (DbM),
a protein that hydroxylates the carbon of Dopastat at the expense of one equivalent
of molecular element and 2 equivalents of ascorbate, thereby yielding nore-
pinephrine as the main product (Levin et al. 1960). In Drosophila melanogaster, the
reaction of Dopastat is administered by aminoalkanoic acid monooxygenase (TbM)
(Monastirioti et al. 1996). Besides DbM or TbM, these organisms contain a pep-
tidylglycine–hydroxylating monooxygenase (PHM) and a monooxygenase X
(MOX) (Xin et al. 2004). The latter monooxygenase was identified for the 1st time
throughout a hunt for genes whose expression is altered in aging human fibroblasts
(Chambers et al. 1998). Furthermore, copper-dependent monooxygenases have
been identified that do not need ascorbate as molecule. An example of these
monooxygenases is that the membrane-associated methane series monooxygenase
(pMMO) from Methylococcus capsulatus. The monooxygenase utilizes two
ascorbate molecules to scale back each copper cofactor from Cu2+ to Cu+.
Thereafter, the enzyme binds the organic substrate and the reduced copper particle
reacts with molecular element to create a copper-peroxide (Aboelella et al. 2004;
Prigge et al. 2004). This reactive intermediate then oxygenates the substrate,
yielding water and the hydroxylated substrate.
Non-heme Iron-dependent Monooxygenases Utilize two iron atoms as chemical
compound for his or her aerophilic activity (Fig. 13.4). These di-iron-dependent
enzymes are conjointly stated as microorganism multicomponent monooxygenases
Fig. 13.4 Proposed catalytic cycle of non-heme iron-dependent monooxygenases (Valentine et al.
1999)
(BMMs), catalyze hydroxylation and epoxidation reactions and consist of 3 com-

ponents; a monooxygenase, a reductase and a tiny restrictive macromolecule
(Wallar and Lipscomb 1996; Leahy et al. 2003). The best-characterized member of
this family is the soluble methane monooxygenase (sMMO) from M. capsulatus
(Murrell et al. 2000).
Other members of this family include alkene monooxygenase (Gallagher et al.
1997), phenol hydroxylases (Whited and Gibson 1991), membrane-bound alkane
hydroxylases (van Beilen and Funhoff 2007) and four-component alkene/aromatic
monooxygenases (e.g., toluene 4-monooxygenase).
Pterin-dependent Monooxygenases (EC 1.14.16.x) is a family of monooxyge-
nases known to hydroxylate the amino acids essential amino acid, tyrosine and
tryptophane at their aromatic ring. Although some of these enzymes are found in
bacterium, they are primarily from organism origin. These monooxygenases bind
one iron atom in their situation with two histidines and a salt and utilize tetrahy-
drobiopterin (BH4) as a molecule. Upon hydroxylation of the substrate, this
coenzyme is regenerate to 4-hydroxybiopterin that, after dehydration, forms qui-
nonoid dihydrobiopterin. In vivo, a NAD(P)H-dependent dihydropteridine reduc-
tase recycles its latter product to BH4 (Fitzpatrick 1999). One of the best-studied
members of this monooxygenase-family is phenylalanine 4-monooxygenase (PheH,
EC 1.14.16.1). However, during chemical change no accumulation of the ferrous
protein has been determined. Therefore, the ferrous protein is thought-about to be
the resting type in absence of substrates. In this state, the enzyme is projected to
bind molecular element in its active site. It has been suggested that O2 is activated
by each the ferrous-ion and therefore the sure reduced tetrahydrobiopterin, after that
the side-product 4-hydroxybiopterin is fashioned additionally to the reactive
FeIVO-intermediate. This intermediate then hydroxylates the substrate, yielding the
product and ferrous protein. However, it is important to notice that the existence of
the reactive intermediates has not been confirmed, however (Fitzpatrick 2003).
Cofactor-independent Monooxygenases These are in majority of monooxyge-
nases. It requires an organic or bronze chemical compound for their chemical
change activity. However, several monooxygenases have been found that do not
need any cofactors. In 1993, tetracenomycin F1 monooxygenase (TcmH) from
Streptomyces glauscens was refined and characterized. This enzyme was shown to
be an internal cofactor-independent monooxygenase, as it required solely molecular
element for its activity and used the substrate as chemical agent. Furthermore,
inhibition studies suggested that sulfhydryl teams and essential amino acid residues
were essential for its aerophilic activity (Shen and Hutchinson 1993). The finding of
new members of this monooxygenase-family showed that a lot of those enzymes
are primarily gift in streptomycetes. For example, ActVA-orf6 from Streptomyces
coelicolorA3(2) is a monooxygenase that is thought to turn the formation of
dihydrokalafungin, an actinorhodin precursor. An exception to these findings could
be a recently known quinolmonooxygenase (YgiN) from E. coli (Adams and Jia
2005). In streptomycetes, the cofactor-independent enzymes appear to be concerned
330 A. Kumar et al.
in the synthesis of polyketide antibiotics, such as the antitumor antibiotic tetra-

cenomycin C. Another role was suggested for a theoretic monooxygenase from
eubacteria T.B. that features a similar crystal structure as ActVA-orf6. This protein
is steered to neutralize reactive element and chemical element species, which are
generated by host cells that are attacked by M. tuberculosis (Lemieux et al. 2005).
Other cofactor-dependent monooxygenases—In 2005, a methyltransferase
homolog was found to hydroxylate the C-10 carbon atom of 15-demethoxy–
rhodomycin. This aclacinomycin 10-hydroxylase from Streptomyces purpurascens
was found to insert an element atom from molecular element into the substrate.
Surprisingly, this enzyme needed S-adenosyl-L-methionine as a cofactor (Jansson
et al. 2003, 2005). It is proposed that the charge of the chemical compound
facilitates the delocalization of electrons upon chemical change of the substrate.
Thereafter, molecular oxygen is activated by the substrate, yielding a hydrox-
yperoxide intermediate. Thus far, this is the sole monooxygenase that has been
identified that does not belong to at least one of the antecedently mentioned
monooxygenase-families.
13.3 Dioxygenase Reactions
Dioxygenases introduce both gas atom of associate gas molecule into a substrate. In
most instances, the two oxygen atoms react with one substrate molecule (in-
tramolecular dioxygenase)
S þ O2 ! SO2
Some dioxygenase, however, incorporate one atom into every of the oxygen
molecule into completely different molecules of the same substrate.
S2 þ O2 ! 2SO
Or into two completely different substrate (intermolecular dioxygenase)
S þ S0 þ O2 ! SO þ S0 O
Dioxygenases include two major classes: haem-dependent iron sulfur dioxyge-

nases and Rieske iron–sulfur non-haem dioxygenases the majority of that is NADH
dependent (Wackett and Hershberger 2001; Burton 2003). The reactions catalyzed
by these enzymes include aromatic ring cleavage and hydroperoxidation or cis-
dihydroxylation (Fig. 13.5). Rieske non-heme iron-dependent oxygenases are
important enzymes that catalyze a wide variety of reactions in the biodegradation of
xenobiotics and the biosynthesis of bioactive natural products.
Pseudomonas putida UV4

Toluene Dioxygenase
Toluene
Industrial synthesis of
Toluene 2,3-cis-dihydrodiol
07 steps to end product from cis-dihydrodiol
6C-Methyl-D-mannose
Fig. 13.5 Toluene Dioxygenase reaction
In result of an expansion in the accessibility of catalysts that can perform par-

ticular oxygenations; an increasing enthusiasm is demonstrated by the enzyme
industries for oxidative biocatalysts.
Rieske oxygenases were first identified as enzymes involved in the degradation
of aromatic compounds by Pseudomonas putida (Fig. 13.6) (Axcell and Geary
1975; Gibson et al. 1968). These enzymes were ultimately characterized as the three
component systems naphthalene dioxygenase and toluene dioxygenase (Yeh et al.
1977; Ensley and Gibson 1983; Ensley et al. 1982). Nowadays, such enzymes are
best known as catalysts of the first step in the oxidative degradation of aromatic
compounds via regio—and stereospecific cis-dihydroxylation to produce dihydro-
diols (Jerina et al. 1971). As regards their substrate specificity some compounds
such as arenes (toluene, naphthalene) and carboxylates (benzoate, phthalate) are
substrate of this type of enzymes.
Nitropropane Dioxygenase FMN Dependent Reaction
2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme
that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl
compounds and nitrite, with oxygen as the electron acceptor (Fig. 13.6). Although
nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in
chemical industry for a quick and effective way of synthesizing common reagents.
Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxy-
ganase has a potential for bioremediation purposes.
332 A. Kumar et al.
Fig. 13.6 Nitropropane

Dioxygenase reaction
Nearly all dioxygenases contains iron or copper, which in most instances is a

right away constituent of the enzyme protein. Iron will also be component or
ferroprotoporphyrin IX (Heme). The metallic turns on oxygens connected to the
enzyme, a manner that is associated with polarization of the complex. Inside the
case of the iron containing enzyme this will be formulated as below:
EFe2 þ þ O2 , EFe2 þ OO
The enzyme oxygen complex adds to the substrate with the elimination of a
proton and the E−Fe2+ complex is regenerated.
13.4 Monosaccharides Monooxygenase
Monooxygenases are much more common than dioxygenases. The oxygenases if

utilize monosaccharides to produce reactive oxygen-intermediate are termed here as
monosacharride oxygenases.
Ribulose 1,5-Bisphosphate (RuBP) Carboxylase/Oxygenase (RubisCO)
Rubisco, the most abundant enzyme in the biosphere (Ellis 1979), fixes CO2 into
organic carbon that supports nearly all life on Earth. In today’s atmosphere, O2
competes with CO2 at Rubisco’s catalytic site, producing the toxic compound
phosphoglycolate (Somerville and Ogren 1979). Phosphoglycolate must be
metabolized at the expense of energy and loss of fixed carbon and nitrogen (Bauwe
et al. 2010). To overcome Rubisco’s limitations, many photosynthetic organisms
have evolved carbon-concentrating mechanisms (CCMs) (Sage et al. 2012;
Giordano et al. 2005). CCMs increase the CO2 concentration around Rubisco,
decreasing O2 competition and enhancing carbon fixation. Ribulose is a ketopen-
tose (monosaccharide) that comprise 5 carbon atoms, and together with a ketone
functional group. Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase
(RubisCO) is one of the precise enzymes of the reductive pentose phosphate
pathway, or Calvin–Bassham–Benson cycle on this pathway, RubisCO capabilities

to catalyze the real CO2 assimilatory step to convert RuBP and CO2 into two
molecules of 3-phosphoglyceric acid via a six-carbon carboxylated intermediate.
The unique activity of oxygenase relative to carboxylase became extraordinarily
small and the most beneficial pH changed into about 9, at which pH the carboxylase
activity was nil. Kinetics and immunological studies confirmed that the large
subunit moiety share the catalytic web site for both carboxylase and oxygenase
response (Nishimura and Akazawa 1974; Takabe and Akazawa 1975). As a con-
sequence RuBP carboxylase is unique in its bifunctional nature and additionally
due to the fact, in contrast to most different oxygenase it lacks characteristics
oxygenase prosthetic groups, i.e., flavin, Cu, and Fe (Chollet et al. 1975); this
activity of the enzyme may be characterized as an internal monooxygenase.
Under conditions of high O2 and low CO2, however, rubisco has oxygenase
activity, initiating the phenomenon called photorespiration (Fig. 13.7).
Rubisco-carboxylase-oxygenase: within the oxygenase reaction, O2 reacts with
rubisco’s 2nd substrate, RuBP, to shape 3 PG and 2 phosphoglycolate.
The charge of carboxylase reaction is four times that of the oxygenase reaction
below everyday atmospheric situations at 25 °C; the stromal awareness of carbon
dioxide is then 10 µM and that of oxygen is 250 µM. The oxygenase response, just
like the carboxylase reaction, requires that lysine 201 be in the carbamate shape.
Due to the fact this carbamate forms only inside the presence of carbon dioxide, this
assets might save you rubisco from catalyzing the oxygenase reaction exclusively
whilst carbon dioxide is absent.
Fig. 13.7 The likely mechanism of oxygenase reactions catalyzed by way of RuBP carboxylase–
oxygenase reactions
334 A. Kumar et al.
CO2 is likewise the sole carbon source for the predominant life-forms on this
planet, and its efficient incorporation into organic matter is directly related to the
productivity of important ecosystems, including agriculturally significant plants.
For maximum organisms, ribulose 1,5-bisphosphate (RuBP)
carboxylase/oxygenase (RubisCO) catalyzes the number one step of CO2 fixation
(Gutteridge and Gatenby 1995; Hartman and Harpel 1994; Tabita 1995); in spite of
the reality that it’s miles the most considerable protein located on earth (Ellis 1979),
RubisCO’s catalytic performance is seriously limited by way of the ability to
catalyze a competing O2 fixation response. This ends in inefficient CO2 fixation and
occasional productiveness. Right now, the molecular foundation for CO2/O2 dis-
crimination is not completely understood; however, the relative capacity of this
enzyme to choose either carboxylation or oxygenation is not immutable, but, varies
for different source of RubisCO (Jordan and Ogren 1981; Read and Tabita 1994).
The pyrenoid is a spherical structure in the chloroplast stroma, discovered more
than 130 years ago (Schmitz 1882; Vaucher 1803; Brown 1967). Pyrenoids have
been found in nearly all of the major oceanic eukaryotic primary producers and
mediate *28–44 % of global carbon fixation (Field et al. 1998; Behrenfeld et al.
2001; Rousseaux and Gregg 2013; Mann 1996; Thierstein and Young 2004; Meyer
and Griffiths 2013). A pyrenoid typically consists of a matrix surrounded by a
starch sheath and traversed by membrane tubules continuous with the photosyn-
thetic thylakoid membranes (Engel et al. 2015). This matrix is thought to consist
primarily of tightly packed Rubisco and its chaperone, Rubisco activase (McKay
and Gibbs 1991). In higher plants and non-pyrenoid-containing photosynthetic
eukaryotes, Rubisco is instead soluble throughout the chloroplast stroma. The
molecular mechanism by which Rubisco aggregates to form the pyrenoid matrix
remains enigmatic.
Two mechanisms for Rubisco accumulation in the pyrenoid have been proposed:
(i) Rubisco holoenzymes could bind each other directly through hydrophobic
residues (Meyer et al. 2012), or (ii) a linker protein may link Rubisco holoenzymes
together (Engel et al. 2015; Meyer et al. 2012). The second model is based on
analogy to the well-characterized prokaryotic carbon-concentrating organelle, the
b-carboxysome, where Rubisco aggregation is mediated by a linker protein con-
sisting of repeats of a domain resembling the Rubisco small subunit (Long et al.
2007).
The reaction, which occurs in C4 plants and bacteria, uses the high energy
phosphate of PEP to drive the incorporation of a carboxyl group in order to form
oxaloacetate. In C4 plants, PEP carboxylase is located in the mesophyll cells on the
external surfaces of the plant. Incorporation of CO2 in this manner helps to shuttle
CO2 to the bundle sheath cells, where the Calvin cycle enzymes are concentrated. It
also helps to avoid the wasteful photorespiration cycle due to the oxygenase activity
of the CO2 fixing enzyme, rubisco.
By catalyzing carbon dioxide fixation during photosynthesis, this enzyme is
responsible for virtually all of the reduced carbon found in living organisms.
Rubisco couples the inorganic and organic carbon pools on earth and is the most
significant route for linking these pools together by the synthesis of carbohydrate. It
Photosynthesis :
CO2 + H2O + ribulose-1,5-P2 2(3-phospho-D-glycerate) -----(Eq.1)
Photorespiration :
O2 + ribulose-1,5-P2 3-phospho-D-glycerate + 2- phosphoglycolate
-----(Eq.2)
Fig. 13.8 Equation of rubisco carboxylase/oxygenase catalytic mechanism during photosynthesis

and photorespiration
is, however, a poor catalyst, having both a low affinity for carbon dioxide and a
small turnover number. Autotrophic organisms must devote a major part of their
synthetic capacity to produce sufficient enzyme to sustain life.
Rubisco catalyzes the first reaction in the pathways of both photosynthesis and
photorespiration, which are, respectively, its carboxylase and oxygenase activities
(Fig. 13.8).
Photosynthesis and photorespiration are interlocking metabolic cycles, with
Rubisco determining the relative rates of carbon flow through the two pathways
(Fig. 13.9). In the dark, plants are carrying out mitochondrial respiration by the
oxidation of substrates to CO2 and the conversion of O2 to H2O. On top of that,
there is another process in plants that, like mitochondrial respiration, consumes O2
and produces CO2 and, like photosynthesis, is driven by light. This process is called
photorespiration and is a costly side reaction of photosynthesis. Rubisco requires
both CO2 and Mg as obligatory cofactors, resulting in carbamylation of an active
site lysine residue by CO2 and coordination of Mg2+.
In the Oxygenase Reaction (Eq. 2) of Photorespiration However, the enediol
intermediate reacts with molecular oxygen to form a hydroperoxy derivative that
breaks down to 2-phosphoglycolate and 3-phosphoglycerate. Photorespiration
consumes ribulose bisphosphate, which results in no net gain of carbon, and also
requires the consumption of energy to recycle the lost carbon. Partitioning of
ribulose bisphosphate between the two reactions of photosynthesis and photores-
piration can vary significantly between different photosynthetic organisms, which
has stimulated interest in improving the carboxylation efficiency of crop plants.
Rubisco activity is modulated in plants by an inhibitor and an activator. The
inhibitor 2′-carboxy arabinitol 1-phosphate (2CAIP) accumulates in some plants
during darkness and binds to the active site of Rubisco. 2CAIP is degraded by a
specific phosphatase, which presumably allows Rubisco to function during pho-
tosynthesis in the light. Rubisco can be severely inhibited by a range of sugar
bisphosphates, including substrate analogs. The enzyme Rubisco activase has the
ability to relieve the inhibition caused by sugar bisphosphates, possibly by inter-
acting with Rubisco and altering the affinity of the enzyme for bisphosphates.
336 A. Kumar et al.
Pyruvate
+
Pi – elimination
3P D-Glycerate
2P-Glycolate
Addition &
Hydration
H2O
abstraction
cis – enediol Xylulose 1, 5 – P2

Ribulose 1, 5 – P2
3-keto arabinitol 1,5 – P2
Fig. 13.9 The reactions catalyzed by Rubisco. The first intermediate of catalysis is the C2, C3 cis-
enediol form of ribulose bisphosphate (ribulose 1,5-P2) after abstraction of the C3 proton. The
enediol can partition a number of ways, the majority into the products of carboxylation (upper
reactions) or oxygenation (lower reactions). However, a number of misprotonated isomers of
ribulose 1,5-P2, for example, xylulose-bisphosphate, have been detected with the wild-type
enzyme that are produced in quantity by mutations of specific amino acid residues involved in
proton transfer. Phosphate elimination of the carbanion forms of intermediates are also produced
by some mutants. R,—CHOH—CH20P02 v 3P D-glycerate, 3-phospho glycerate; 2P glycolate,
2-phosphoglycolate. Five discrete partial reactions have been described for the carboxylation
reaction (Eq. 1) of ribulose bisphosphate. The initial formation of a C2, C3-enediol intermediate of
the ribulose bisphosphate substrate is followed by its carboxylation, where the enediol reacts with
CO2 at the C2 position. The resulting six-carbon intermediate is hydrolytically cleaved to two
molecules of 3-phosphoglycerate, resulting in an overall gain of carbon during photosynthesis
Regulation of Its Enzymatic Activity

RuBisCO is usually only active throughout the day as ribulose 1,5-bisphosphate is
not regenerated within the dark. This can be as a result of the regulation of many
alternative enzymes within the Calvin cycle. Additionally, the activity of RuBisCO is
coordinated thereupon of the opposite enzymes of the Calvin cycle in several ways.
Regulation by Ions
Upon illumination of the chloroplasts, the pH of the stroma rises from 7.0 to 8.0 as
a result of the proton (hydrogen particle, H+) gradient created across the thylakoid
membrane. Simultaneously, magnesium ions (Mg2+) transported outside the thy-
lakoids to raise the concentration level of magnesium within the stroma of the
chloroplasts. RuBisCO includes a high optimum hydrogen ion concentration (can
be >9.0, counting on the metal particle concentration) and, thus, becomes “acti-
vated” by the addition of CO2 and magnesium to the active sites as delineated
above.
Regulation by RuBisCO Activase
In plants and a few alga, another enzyme, RuBisCO activase, is needed to permit the
speedy formation of the crucial carbamate within the site of RuBisCO. RuBisCO
Activase is needed as a result of the ribulose 1,5-bisphosphate (RuBP) substrate
binds more powerfully to the active sites lacking the carbamate and markedly slows
down the “activation” method. During Day time light, RuBisCO activase promotes
the discharge of the repressive, or—in some views—storage RuBP from the catalytic
sites. Activase is also needed in some plants (e.g., tobacco and lots of beans) as a
result of, darkly, RuBisCO is suppressed (or protected against hydrolysis) by a
competitive inhibitor synthesized by these plants, a substrate analog 2-carboxy-D-
arabitinol 1-phosphate (CA1P) (Andralojc et al. 1994). CA1P binds tightly to the site
of carbamylated RuBisCO and inhibits catalytic activity. Within the light, RuBisCO
Activase conjointly promotes the release of CA1P from the catalytic sites. Once the
CA1P is released from RuBisCO, it is speedily converted to a non-inhibitory type by
a light-activated CA1P-phosphatase. Finally, once each many hundred reactions, the
traditional reactions with CO2 or oxygen are not completed, and other repressive
substrate analogs square measure shaped within the site. Once again, RuBisCO
Activase will promote the discharge of those analogs from the chemical action sites
and maintain the catalyst in an exceedingly catalytically active type. The properties
of Activase limit the chemical action potential of plants at high temperatures
(Crafts-Brandner and Salvucci 2000). CA1P has conjointly been shown to stay
RuBisCO in an exceedingly conformation that is protected against chemical action
(Khan et al. 1999). At high temperatures, RuBisCO Activase aggregates and might
now not activate RuBisCO. This contributes to the attenuated carboxylating capa-
bility discovered throughout heat stress (Salvucci et al. 2001).
Regulation by ATP/ADP and Stromal Reduction/Oxidation State Through the
Activase
The removal of the inhibitory RuBP, CA1P, and the other inhibitory substrate
analogs by activase requires the consumption of ATP. This reaction is inhibited by
338 A. Kumar et al.
the presence of ADP, and, thus, activase activity depends on the ratio of these
compounds in the chloroplast stroma. Furthermore, in most plants, the sensitivity of
activase to the ratio of ATP/ADP is modified by the stromal reduction/oxidation
(redox) state through another small regulatory protein, thioredoxin. In this manner,
the activity of activase and the activation state of RuBisCO can be modulated in
response to light intensity and, thus, the rate of formation of the ribulose
1,5-bisphosphate substrate (Zhang et al. 2002).
Regulation by Phosphate
In cyanobacteria, inorganic phosphate (Pi) participates in the coordinated regulation
of photosynthesis. Pi binds to the RuBisCO active site and to another site on the
large chain where it can influence transitions between activated and less active
conformations of the enzyme. Activation of bacterial RuBisCO might be particu-
larly sensitive to Pi levels, which can act in the same way as RuBisCO activase in
higher plants (Marcus and Gurevitz 2000).
Regulation by Carbon Dioxide
Since carbon dioxide and oxygen compete at the active site of RuBisCO, carbon
fixation by RuBisCO can be enhanced by increasing the carbon dioxide level in the
compartment containing RuBisCO (chloroplast stroma). Several times during the
evolution of plants, mechanisms have evolved for increasing the level of carbon
dioxide in the stroma (see C4 carbon fixation). The use of oxygen as a substrate
appears to be a puzzling process, since it seems to throw away captured energy.
However, it may be a mechanism for preventing overload during periods of high light
flux. This weakness in the enzyme is the cause of photorespiration, such that healthy
leaves in bright light may have zero net carbon fixations when the ratio of O2 to CO2
reaches a threshold at which oxygen is fixed instead of carbon. This phenomenon is
primarily temperature-dependent. High temperature decreases the concentration of
CO2 dissolved in the moisture in the leaf tissues. This phenomenon is also related to
water stress. Since plant leaves are evaporatively cooled, limited water causes high
leaf temperatures. C4 plants use the enzyme PEP carboxylase initially, which has a
higher affinity for CO2. The process first makes a 4-carbon intermediate compound,
which is shuttled into a site of C3 photosynthesis then de-carboxylated, releasing
CO2 to boost the concentration of CO2, hence the name C4 plants.
Crassulacean acid metabolism (CAM) plants keep their stomata (on the under-
side of the leaf) closed during the day, which conserves water but prevents the
light-independent reactions (i.e. the Calvin Cycle) from taking place, since these
reactions require CO2 to pass by gas exchange through these openings. Evaporation
through the upper side of a leaf is prevented by a layer of wax.
Sialic Acid Monooxygenase
Sialic acids are a family of nine-carbon acidic monosaccharides that occur obvi-
ously at the cease of sugar chains attached to the surfaces of cells and soluble
proteins. Sialic acids are sugars usually discovered on the outer terminal role of
glycan chains that cover the surface of all vertebrate cells inside the human body,
the very best concentration of sialic acid (as N-acetylneuraminic acid) takes place
within the brain in which it participates as an necessary a part of ganglioside

structure in synaptogenesis and neural transmission.
The maximum generally expressed sialic acid is N-acetylneuraminic acid
(Neu5Ac) that is the precursor for N-glycolylneuraminic acid (Neu5Gc) synthesis—
a conversion mediated by means of motion of the CMP-N-acetylneuraminic acid
hydroxylase (CMAH) enzyme.
Various reactions involving the sialic acids has been given in figure 13.10.
Cells from higher animals and various microorganisms produce sialic acid in a
protracted pathway beginning from glucose.
The CMP-N-acetylneuraminate monooxygenase (CMAH) enzyme converts
Neu5Ac to the Neu5Gc “non-human” form of this sugar, (Fig. 13.10) (Irie et al.
1998). However, the Varki group has proposed that the functional loss of
CMP-N-acetylneuraminate monooxygenase (CMAH) during evolution of humans
from the great apes leads to several possible implications for its role in human
development, the most notable of which being less constrained brain growth (Varki
1992). This premise is supported by evidence that the mutation that abrogated the
activity of CMAH transcripts found in present-day humans occurred after the
Homo-Pan divergence (Chou et al. 1998) but prior to brain expansion during
human evolution (Chou et al. 2002). Unlike most primate brains that stop growing
relatively soon after birth, human brains continue to grow postnatally at a rate
similar to the whole body, presumably because they were freed from the constraints
imposed by Neu5Gc in other mammals that limit brain expansion after birth (Chou
et al. 2002).
In the human body, the highest concentration of sialic acid (as N-acetylneur-
aminic acid) occurs in the brain where it participates as an integral part of gan-
glioside structure in synaptogenesis and neural transmission. Human milk also
contains a high concentration of sialic acid attached to the terminal end of free
oligosaccharides, but its metabolic fate and biological role are currently unknown.
Sialic acids are components of the carbohydrate chains of glycoconjugates and
are involved in cell–cell recognition (Rutishauser et al. 1988; Kelm et al. 1996) and
cell-pathogen interactions (Smit et al. 1984; Suzuki et al. 1987). Sialic acid is a
generic designation used for N-acylneuraminic acids and their derivatives (Varki
1992). N-Acetylneuraminic acid (NeuAc)1 and N-glycolylneuraminic acid (NeuGc)
are two of the most abundant derivatives.
N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals,
but it is not detectable in humans. The expression of NeuGc is controlled by
cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase
activity (Irie et al. 1998).
Monosaccharide monooxygenases are noticeably specialized enzymes that have
evolved to catalyze the insertion of single oxygen atom from molecular oxygen
(O2) into a monosacchride. These enzymes activate molecular oxygen through
generation of a peroxy-intermediate that is capable to carry out the monooxy-
genation reaction. In most cases, monooxygenases utilize bound cofactor(s) which
include flavin, or metal ions to facilitate the activation of molecular oxygen.
340 A. Kumar et al.
Neu5Ac
Neu5Ac
LYSOSOME
Neu5Ac
CMP
CYTOSOL
CMP-Neu5Ac
CMP GOLGI
ManNAc
+
Pyruvate
Glucose
ATP N-Acetylneuramic Acid
9
ADP (Neu5Ac)
Fructose -6- PO4 8 PO4
Glutamine H2 O CMP-N-Acetylneuramic Acid + PPi
1
NH3
Glucosamine -6- PO4 +Glutamic acid N-Acetylneuramic Acid -9- PO4 +Pi
Acetyl CoA
2 7 10
PEP NADPH
N-Acetylglucosamine -6- PO4 N-Acetylmannosamine -6- PO4
3
ADP
N-Acetylglucosamine -1- PO4
CMP-N-glycolylneuramic Acid
UTP ATP
4 O2 & H 2 O
6
UDP-N-Acetylglucosamine
N-glycolylneuraminic Acid
5*
(Neu5Gc)
ManNAc
GlcNAc
Fig. 13.10 Schema displaying the metabolism of sialic acids modified from Varki et al. (1999):
1 Glycosamine-6-phoshate synthase; 2 Glucosamine-6-phosphate N-acetyltransferase; 3
N-acetylglucosamine-!6-phosphate mutase; 4 UDP-N-acetylglucosaminepyrophos-phorylase; 5 *key
enzyme: UDP-N-acetylglucosamine-2-epimerase; activity is initially low in young rats; 6
N-acetylmannosamine kinase; 7 N-acetylneuraminate-9-phosphate synthase; 8 N-acetylneuraminate-9-
phosphatephatase; 9 CMP-N-acetylneuraminatesynthetase; 10 monooxygenase
Examples were described in which monooxygenases utilize the substrate as a

“cofactor” to generate the active peroxy-intermediate. By formation of a
peroxy-intermediate, these enzymes are capable of catalyze an extensive range of
oxidative reactions. The oxidative power of monooxygenases enables to catalyze
wide range of oxidation reactions; e.g., hydroxylations, epoxidations,

heteroatom-dealkylations, dehalogenations, dehydrogenations and oxidation of
heteroatoms. Monooxygenases containing other (or no) cofactors are more limited
of their reactivity scope. Besides the formation of the peroxy-intermediate,
cofactor-dependent monooxygenases have some common characteristic; the elec-
trons which can be required for the reduction of cofactor and thus for the activation
of molecular oxygen are (in)directly provided by nicotinamide coenzymes NADH
or NADPH. Therefore, cost-effective regeneration of these coenzymes is imperative
for successful industrial applications of monooxygenases. Of the various possible
approaches which are described in this chapter. The enzymatic regeneration of
nicotinamide coenzymes is the most advanced and successful so far. Unfortunately,
this confined substrate acceptance limits the applicability of these monooxygenases
as biocatalyst.
13.5 Future Recommendations
A few unidentified monosaccharide monooxygenases and dioxygenses can be

existed in nature. With the identification of new members of cofactor-independent
monooxygenase-family, various studies had been accomplished in order to
understand how these monooxygenases perform their oxidative reactions in the
absence/presence of cofactors. This is indicated by the proposed catalytic mecha-
nism of these enzymes; in contrast to other monooxygenases, molecular oxygen is
activated by the substrate, after which the oxygenation reaction occurs. This sug-
gests that the nature of the substrate is important for the catalytic activity of these
monooxygenases. Internal cofactor-independent monooxygenases are interesting
enzymes for biotechnological applications as they do not require expensive
cofactors/coenzymes.
Appendix 1: Some Reactions of Oxidoreductases
1. Higher plants incorporate large amounts of D-GluA residues into cell wall
glucuronoarabinoxylans. D-GluA forms irreversibly from UDP-D-Glc by UDP-
D-glucose dehydrogenase (EC 1.1.1.22)
+ +
2NAD 2NADH + H
UDP-D-glucose UDP-D-glucuronic acid

(EC1.1.1.22)
342 A. Kumar et al.
2. GDP-L-fucose formation requires subsequent activities of GDP-D-mannose

4,6-dehydratase (EC 4.2.1.47) and a bifunctional GDP-L-fucose synthase (EC
1.1.1.271).
NADPH + H+ NADP+
GDP-6-deoxy-D-lyxo-hexos-4-ulose GDP-L-fucose
(EC 1.1.1.271)
3. N-acetyl-D-glucosamine can be further converted to UDP-N-acetyl-D-glucuronic

acid (UDP-N-acetylglucosamine 6-dehydrogenase, EC 1.1.1.136).
2NAD+ +H2O 2NADH + 2H+

UDP-N-acetyl-2-
UDP-N-acetyl-D-
amino-2-deoxy-D-
glucosamine
glucuronic acid
(EC 1.1.1.136)
4. The peptidoglycan building stone, UDP-N-MurAc, forms from UDP-N-acetyl-

D-glucosamine and phosphoenolpyruvic acid via the intermediate UDP-N-
acetyl-3-O-(1-carboxyvinyl)-D-glucosamine (UDP-N-acetyl-D-glucosamine
1-carboxyvinyl transferase, EC 2.5.1.7), which is reduced to UDP-N-MurAc by
UDP-N-acetylmuramate dehydrogenase (EC 1.1.1.158)
NADH + H+ NAD+
UDP-N-acetyl-3-O- UDP-N-
(1-carboxyvinyl)-D- acetylmuramic
glucosamine (EC 1.1.1.158) acid
5. Biogenesis of UDP-D-GlcA can also occur via an alternative pathway from

myo-inositol, which involves the action of myo-inositol oxygenase (EC
1.13.99.1)
O2 H2O
myo-inositol D-glucuronic
acid
(EC 1.13.99.1)
Appendix 2
Table 13.2 List of oxedoreductases as per their EC number—the enzymes oxidizes a substrate by
transferring the oxygen from molecular oxygen O2 (as in air) to it are called as oxygenases (EC
1.13 or EC 1.14.)
EC 1.1 Acting on the CH–OH group of donors
EC 1.2 Acting on the aldehyde or oxo group of donors
EC 1.3 Acting on the CH–CH group of donors
EC 1.4 Acting on the CH–NH2 group of donors
EC 1.5 Acting on the CH–NH group of donors
EC 1.6 Acting on NADH or NADPH
EC 1.7 Acting on other nitrogenous compounds as donors
EC 1.8 Acting on a sulfur group of donors
EC 1.9 Acting on a heme group of donors
EC 1.10 Acting on diphenols and related substances as donors
EC 1.11 Acting on a peroxide as acceptor
EC 1.12 Acting on hydrogen as donor
EC 1.13 Acting on single donors with incorporation of molecular oxygen (oxygenases)
EC 1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
EC 1.15 Acting on superoxide radicals as acceptor
EC 1.16 Oxidizing metal ions
EC 1.17 Acting on CH or CH2 groups
EC 1.18 Acting on iron-sulfur proteins as donors
EC 1.19 Acting on reduced flavodoxin as donor
EC 1.20 Acting on phosphorus or arsenic in donors
EC 1.21 Acting on the reaction X–H + Y–H = X–Y
EC 1.22 Acting on halogen in donors
EC 1.23 Reducing C–O–C group as acceptor
EC 1.97 Other oxidoreductases
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Biofuel and Biorefinery Technologies 3

ISSN 2363-7609 ISSN 2363-7617 (electronic)

Library of Congress Control Number: 2016947366

© Springer International Publishing Switzerland 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

This book summarizes the information on different biomass converting enzymes

invaluable reference for undergraduates, post-graduates, researchers and practi-

Galway, Ireland Vijai Kumar Gupta

1 Microbial Enzymes for Conversion of Biomass to Bioenergy . . . . . . 1

Part III Lignocellulose Oxidureductases

M.P. Raghavendra, S. Chandra Nayaka and Vijai Kumar Gupta

Abstract Microbial enzymes are capable of degrading a wide range of complex

© Springer International Publishing Switzerland 2016 1

1.2 Importance of Biofuel Research

Proteins and Secondary

Pretreatment, hydrolysis Microbial degradation

Lignocellulose and other carbohydrates

Xylose, mannose, glucose,

Gaseous fuels: H2, methane

Fig. 1.1 Conversion of carbohydrate biomass into biofuel

1.3 Lignocelluloses and Its Structural Complexity

in which the lattice is disordered or paracrystalline (Ranjan 2014). Hence, microbes

1.4 Enzymatic Hydrolysis of Lignocelluloses

Even though lignocellulose is considered as the most abundant and renewable

1. Adsorption of cellulase enzymes onto the surface of the cellulose,

1.5 Microbial Enzymes for Degradation of Cellulose

The isolation and characterization of novel glycoside hydrolases from eubacteria

1.6 Novel Enzymes for Cellulose Degradation

In addition to enzymes that act directly on polysaccharides, lignocelluloses

glycoside hydrolases) that catalyze oxidative cleavage of polysaccharides have been

Table 1.1 (continued)

Table 1.1 (continued)

1.7 Swollenins: A Novel Protein for Cellulose Degradation

Expansins in plants have been proposed to disrupt hydrogen bonding between

phosphoric acid swollen cellulose degradation activity, which corresponded to

1.8 CIP1 and CIP2

Cellulose-induced protein 1 (Cip1) protein consists of a glycoside hydrolase family

1.9 Consolidated Bioprocessing (CBP) for Enhanced

CBP can be achieved by two ways by modifying cellulase producer to ferment

1.10 Recycling of Enzymes

A key factor that prevents the commercialization of enzymatic cellulose hydrolysis

1.11 Potential Strategies for Stabilizing Cellulases

1.12 Enzyme Cocktails

endoglucanases have been shown to be active in 20 % [C2mim][OAc], suggesting

1.13 Enzyme Engineering

1.14 Feruloyl Esterases (Faes)

1.15 Future Scope

modeling capabilities for metabolic and regulatory networks of biomass degrading

Aksel T, Barrick D (2009) Analysis of repeat-protein folding using nearest-neighbor statistical

Hehemann J, Correc G, Barbeyron T, Helbert W, Czjzek M, Michel G (2010) Transfer of

Neelamegam Annamalai, Mayavan Veeramuthu Rajeswari

Abstract Cellobiohydrolases or exoglucanases are produced by various bacteria

© Springer International Publishing Switzerland 2016 29

2.2 Exoglucanases or Cellobiohydrolases (CBH)

Exoglucanases, also known as cellobiohydrolases (CBHs) that act at the end of

2.3 CBH I and II

2.4 Structure and Mode of Action of CBHs

2.5 Substrates and Assay Methods

In general, enzymes in cellulase mixture which show relatively high activity on