Hindawi Publishing Corporation

Enzyme Research
Volume 2012, Article ID 921362, 14 pages
doi:10.1155/2012/921362

Review Article
Pullulanase: Role in Starch Hydrolysis and
Potential Industrial Applications

Siew Ling Hii,1 Joo Shun Tan,2 Tau Chuan Ling,3 and Arbakariya Bin Ariff4
1 Department of Chemical Engineering, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman,
53300 Kuala Lumpur, Malaysia
2 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
3 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
4 Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Correspondence should be addressed to Arbakariya Bin Ariff, [email protected]

Received 26 March 2012; Revised 12 June 2012; Accepted 12 June 2012

Academic Editor: Joaquim Cabral

Copyright © 2012 Siew Ling Hii et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The use of pullulanase (EC 3.2.1.41) has recently been the subject of increased applications in starch-based industries especially
those aimed for glucose production. Pullulanase, an important debranching enzyme, has been widely utilised to hydrolyse
the α-1,6 glucosidic linkages in starch, amylopectin, pullulan, and related oligosaccharides, which enables a complete and
efficient conversion of the branched polysaccharides into small fermentable sugars during saccharification process. The industrial
manufacturing of glucose involves two successive enzymatic steps: liquefaction, carried out after gelatinisation by the action of α-
amylase; saccharification, which results in further transformation of maltodextrins into glucose. During saccharification process,
pullulanase has been used to increase the final glucose concentration with reduced amount of glucoamylase. Therefore, the
reversion reaction that involves resynthesis of saccharides from glucose molecules is prevented. To date, five groups of pullulanase
enzymes have been reported, that is, (i) pullulanase type I, (ii) amylopullulanase, (iii) neopullulanase, (iv) isopullulanase, and (v)
pullulan hydrolase type III. The current paper extensively reviews each category of pullulanase, properties of pullulanase, merits
of applying pullulanase during starch bioprocessing, current genetic engineering works related to pullulanase genes, and possible
industrial applications of pullulanase.

1. Introduction enzymes breaks the polymer into its constituent glucose

molecules. The end unit of the polymeric chain has a latent
Starch is a major industrial raw material and is chemically aldehyde group and is known as the reducing end group.
and/or enzymatically processed into variety of products for Most starches are mixture of two polymers with high
subsequent use in various industries, ranging from food molecular weight: (i) a linear chain molecule—amylose, and
(especially high-fructose and glucose syrups) to washing (ii) a branch polymer of glucose—amylopectin. Starches
detergent industries [1–3]. Starch is, after cellulose, one of of different origins have different amylose and amylopectin
the most abundant heterogeneous polysaccharide produced ratios which differ significantly in many physical properties
by plants in the form of water insoluble granules. It is a [4, 5]. In addition, the ratio of amylose to amylopectin in
polymeric carbohydrate, composed of C, H, and O atoms starch varies considerably depending on the origin, plant
in the ratio of 6 : 10 : 5, (C6 H10 O5 )n . Molecules of starch are species, variety within plants, plants organs, age of organ,
made of hundreds or thousands of glucose, corresponding and growth conditions. This results in various crystalline
to values of n that range from 50 to several thousands. organization of starch in the granules and henceforth
Glucose units are linked to one another through C1 oxygen as different sensitivities of starch to enzymatic hydrolysis [6].
glucosidic bond. Glucosidic bonds are stable under alkaline Amylose is the constituent of starch with anhydroglucose
conditions while treatment of starch with acids or certain units linked by α-D-1,4 glucosidic bonds to form linear
2 Enzyme Research

6 6 6 the manufacture of glucose syrups contain 75% to 85% of

CH2 OH CH2 OH CH2 OH amylopectin [11] and in most plants, ranging from 60% up
to 90%, and reached about 100% in waxy cultivars of rice,
5 O 5 O 5 O maize, sorghum, barley, pea, and potato [5].
4 1 4 1 4 1
2 2 2 2. Starch-Converting Enzymes
3 O 3 O 3
The starch polymer, because of its complex structure,
requires a combination of enzymes (endoamylases, and
exoamylases) for the depolymerization of starch into
n oligosaccharides and smaller sugars, or to transform starch
by transferring oligoglucosidic linkages and residues by the
creation of new bonds (debranching enzymes and glycosyl-
α-(1, 4) linkage transferases) [3, 12, 13]. The enzymes commonly used
for starch processing are generically classified as amylases
Figure 1: Section of the amylose molecule showing the repeating
anhydroglucose unit (modified from [8]).
[12, 14]. The process of enzymatic starch conversion is
displayed in Figure 4. Basically, there are four types of starch-
converting enzymes: (i) endoamylases; (ii) exoamylases; (iii)
debranching enzymes; (iv) transferases.
chains (Figure 1). A trace amount of branching is also
present in the amylose molecule, although the side chains 2.1. Endoamylases. Endo-acting enzymes or endoamylases
through branches are shorter in amylose than those in are able to cleave α-1,4 glucosidic bonds present in the
amylopectin [5]. The level of amylose and its molecular inner part (endo-) of the amylose or amylopectin chain.
weight varies with types of starch. In addition, degree Enzyme α-amylase (EC 3.2.1.1) is a well-known endoamylase
of polymerization (DP) of amylose covers a wide range, that hydrolyses α-1,4 linkages in the interior part of starch
depending upon the starch source and origin. Aqueous polymer in a random fashion, which leads to the formation
solutions of amylose are very unstable due to intermolecular of linear and branched oligosaccharides, or α-limit dextrins.
attraction and association of neighboring amylose molecules, These enzymes are found in a wide variety of microorgan-
which leads to increase in viscosity [7]. Retrogradation and isms, belonging to Bacteria and Archaea [12, 15].
precipitation of amylose particles may occur under certain
specific conditions.
Amylopectin is one of the largest molecules in nature 2.2. Exoamylases. Exoamylases such as glucoamylases (EC
with an average DP of about 2 million. The molecular 3.2.1.3) and α-glucosidases (EC 3.2.1.20) cleave both α-1,4
weight of amylopectin is about 1000 times as high as and α-1,6 bonds on the external glucose residues of amylose
the molecular weight of amylose [8]. In addition to α- or amylopectin from the nonreducing end and thus produce
D-1,4 bonds which are present in amylose and the linear only glucose (glucoamylase and α-glucosidases). On the
segments of amylopectin, amylopectin molecule has α-D- other hand, β-amylases (EC 3.2.1.2) that cleave exclusively
1,6 bonds which occur every 20 to 30 anhydroglucose units α-1,4 bonds produce maltose and β-limit dextrin [12, 13, 16,
(Figure 2). Glucose units with an α-1,6 glucosidic linkages 17].
are the branching points which cause interlinkages of glucose
residue that give rise to a ramified of bush-like structure of 2.3. Transferases. Transferases is another group of starch-
amylopectin molecule [9]. The branches in the amylopectin converting enzymes that cleave an α-1,4 glucosidic bond
are not randomly organized but are clustered of 7 nm to of the donor molecule and transfer part of the donor to a
10 nm intervals (Figure 3), which are the universal features glucosidic acceptor with the formation of a new glucosidic
in structure of starch, regardless the source of botanical bond. Enzymes such as amylomaltase (EC 2.4.1.25) and
plant [10]. The branches themselves form an organized 3D cyclodextrin glycosyltransferase (EC 2.4.1.19) form a new α-
structure: A-chains are not substituted at the C6 positions 1,4 glucosidic bond while branching enzyme (EC 2.4.1.18)
and inner B-chains are α-(1,6)-branches at one point or forms a new α-1, 6 glucosidic bond [3, 12]. The third group
several points. The branching points make up about 5% of starch-converting enzymes is the debranching enzymes
glucose unit in amylopectin [7]. Generally, most cereal and is described in details in the following section.
starches possess A-chains pattern, while cereal starches rich
in amylase, tuber such as potato and rhizome such as canna 3. Starch-Debranching Enzymes
yield the B-chains pattern, where C-chains patterns are
normally found in legume starches [10]. Debranching enzymes catalyse the hydrolysis of α-1,6-
Aqueous solutions of amylopectin are characterised by glucosidic bonds in amylopectin and/or glycogen and related
high viscosity, clarity, stability, and resistance to gelling. polymers. The affinity of debranching enzymes for the α-
Amylopectin binds weakly with iodine and the complex 1,6-bond distinguishes these enzymes from other amylases
typically gives a red/brown color. The level of amylopectin which have primary affinity for α-1,4-glucosidic linkages.
varies with starch types. The majority of starches used in Debranching enzymes are classified into two major groups,
Enzyme Research 3

OH CH2 OH
HO
O O
O
O 1 H α-1, 6-glycosidic bond
HO
CH2 OH
HO
CH2 OH
O O
O 6
HO OH CH2
HO HO O
O O
HO OH
O
CH2 OH HO HO
O O
O
α-1, 4-glycosidic bond CH2 OH

Figure 2: Section of the amylopectin molecule showing the α-1, 4 and α-1,6 chain linkages in starch (modified from [8]).

Reducing chain end glucose residue bound to a tetrasaccharide through a 1,6-

bond [5, 14, 18]. Amylo-1,6-glucosidase will only hydrolyse
C chain a 1,6-α-branch point if the side chain consists of a single
α-1, 6-linkage
glucose unit. The smallest known substrate that amylo-1,6-
Nonreducing glucosidase can hydrolyze is the branched pentasaccharide
chain end 63 -α-glucosyl maltotetraose, which is hydrolyzed to glucose
and maltotetraose (Figure 5). Amylo-1,6-glucosidases are
not of industrial importance at present, so their properties
and function are not described further in this paper.

3.2. Direct Debranching Enzymes—Pullulanase and Isoamy-

lase. Direct-acting debranching enzymes which are present
in plants and bacteria can directly hydrolyse α-1, 6-glucosidic
bonds of unmodified substrate and classified into pullu-
lanases, isoamylases, or R-enzymes on the basis of substrate
specificity.
B chain
A chain
Pullulanase. Pullulanase with EC 3.2.1.41 or also known as
α-dextrin 6-glucanohydrolase, pullulan 6-glucanohydrolase,
Cluster limit dextrinase, and amylopectin 6-glucanohydrolase is
derived from various microorganisms such as Bacillus aci-
Figure 3: A diagram showing how the side-branching chains are dopullulyticus, Klebsiella planticola [21], Bacillus deramificans
clustered together within the amylopectin molecule (modified from [22], thermophilic Bacillus sp. AN-7 [23], Bacillus cereus
[7]). FDTA-13 [24], and Geobacillus stearothermophilus [25].
Microbial pullulanase attracts more interest because of its
specific action on α-1,6 linkages in pullulan, a linear α-glucan
that is, direct and indirect [19]. As shown in Table 1, these consisting essentially of maltotriosyl units connected by 1,6-
enzymes may be divided into three major groups according α-bonds. The structure of pullulan produced by the fungus,
to their substrate specificity: (a) microbial pullulanases Aureobasidium pullulans, is shown in Figure 6.
and plant R-enzymes (pullulan-6-glucanohydrolases), (b) To date, five groups of pullulan-hydrolyzing enzyme
isoamylases (glycogen-6-glucanohydrolases), and (c) amylo- have been reported in the literature and the details are
1,6-glucosidases (dextrin 6-α-D-glucosidases) found in summarised in Table 2. Enzymes hydrolysing pullulan are
higher microorganisms [5, 14, 18, 19]. classified into groups based on the substrate specificities and
reaction products. Pullulanases type I, which are able to
3.1. Indirect Debranching Enzyme. The indirect debranching hydrolyse efficiently the α-(1,6) glucosidic bonds in pullulan
enzyme, that is, amylo-1,6-glucosidase, requires a modifica- and branched polysaccharides, have been extensively studied
tion of the substrate by another enzyme or enzymes prior [26–30]. Pullulanases type II, also called amylopullulanases,
to their debranching action [18, 19] and it is normally are prominent in starch processing industry due to the
present in animal and yeast [5]. The amylo-1,6-glucosidase specific debranching capacity of hydrolysing either α-(1,6) or
first requires the removal of an oligosaccharide by trans- α-(1,4) glucosidic linkages. This enzyme debranch pullulan
glucosylase (4-α-glucanotransferase, EC 2.4.1.25), leaving a and gives maltotriose as final product and it also attacks
4 Enzyme Research

C
α-, β-, γ-
cyclodextrins
C
A
Cyclodextrin
glycosyltransferase
A

B
C Panose
Endoamylase Maltose
(C) Debranching
Isopanose
enzyme
(A) α-amylase Pullulan hydrolase
α-(1, 4) links Exoamylase types I, II, and III
Maltotriose
Branching Pullulanase II
(B) β-amylase Glucoamylase enzyme
α-(1, 4) links α-glucosidase Pullulanase I
Maltotriose
α-(1, 4) and α-(1, 6) links
α-(1, 6) links
Maltose

α-limit dextrin Isoamylase Glucose

Maltose and Glucose
β-limit
dextrin
Linear oligosaccharides
Linear oligosaccharides

Glucose Maltose

Figure 4: Schematic presentation of the action of amylases. Black circles indicate reducing sugars (modified from [17]).

Glycogen enzymes whereas most enzymes involved in starch conver-

sion are extracellular [35]. These two types of pullulan-
Phosphorylase
hydrolysing enzyme (neopullulanase and isopullulanase)
Glu-1-P have practically no action on starch [36]. As these enzymes
Transferase
Amylo-1, can precisely recognise the structural differences between α-
6-glucosidase
1,4 and α-1,6 glucosidic linkages, they are widely applied in
+ Glucose
structural analysis of oligo- and polysaccharides [31].
Unlike all pullulan-hydrolysing enzymes as described
above, pullulan hydrolase type III detected by Niehaus et al.
[37] has ability to attack α-(1,6) as well as α-(1, 4)-glucosidic
Figure 5: Action of amylo-1,6-glucosidase (modified from [18]). linkages in pullulan leading to the formation of a mixture
of maltotriose, panose, maltose, and glucose. The enzyme
is also able to degrade starch, amylose, and amylopectin
forming maltotriose and maltose as main products.
α-(1,4) bonds in starch, amylose, and amylopectin [3, 12,
31]. Both pullulanases type I and type II attach α-1,6 Isoamylase. Isoamylase (EC 3.2.1.68) (glycogen 6-glucano-
glucosidic linkages in pullulan, producing maltotriose while hydrolase) is the only known enzyme that debranches
these enzymes are unable to degrade cyclodextrin [32]. glycogen completely. This enzyme contains two subunits
Very few reports are available on other pullulanases. with a molecular weight of 45 000, pI of pH 4.4 and requires
Pullulan hydrolase type I (neopullulanase) and type II no metal cofactor. This enzyme is most active at pH ranging
(isopullulanase) are only able to cleave α-1,4 glucosidic from pH 3 to pH 4 and has maximum temperature stability
linkages in pullulan, releasing panose and isopanose, and at 45◦ C to 55◦ C [1].
are highly active on cyclodextrins [32–34]. The enzymes that
degrade cyclodextrins faster than starch are sometimes des- 3.3. Comparisons between Pullulanase and Isoamylase. The
ignated cyclodextrinases (EC 3.2.1.54, cyclomaltodextrinase) major difference between pullulanase and isoamylase is that
[3, 34]. Interestingly, cyclomaltodextrinases are intracellular pullulanases hydrolyse the α-1,6 glucosidic bond in pullulan
Enzyme Research 5

CH2 OH CH2 OH CH2 OH

O O O

O O
O
CH2 OH CH2 OH

O O O

O O
O
CH2 OH CH2 OH

O O O

O O O

Figure 6: Generalized structure of pullulan from Aureobasidium pullulans strain CH-1 (modified from [2]).

Table 1: Carbohydrate structure requirements for hydrolysis of 1,6- The debranching enzyme from yeast and mold is known
bonds by debranching enzymes. Symbols: ↓: cleavage by enzyme as isoamylase, and the enzymes produced by bacteria such
mentioned; ∅: reducing end. as Aerobacter aerogenes or Klebsiella aerogenes are called
pullulanase [14]. A comparison of the action of Klebsiella
Carbohydrate structure Debranching enzyme involved
pneumoniae pullulanase and Pseudomonas amyloderamosa
isoamylase on various substrates is given in Table 3. Although
Amylo-1,6-glucosidase differing in their specificity, both types of enzymes directly
hydrolyse α-1,6 linkages in amylopectin, removing side
branches of various lengths from the main polymer chain.
The main chain and the side branches are then completely
Pullulanase available for hydrolysis by other hydrolytic enzymes [10, 38].
During starch saccharification processes, formation of α-
limit and β-limit dextrins is the common phenomenon when
R-enzyme amylopectin is treated with either α-amylase or β-amylase,
respectively. The concentrations of final glucose obtained in
the saccharification are greatly reduced due to the present
of these limit dextrins [38]. The bacterial pullulanases are
Isoamylase able to hydrolyse amylopectin and its β-limit dextrins and
(Source: [18, 19]).
attack the partially degraded polymer (Table 3). Therefore,
pullulanases from either Klebsiella planticola or Bacillus
acidopullulyticus are more commonly used than isoamylase
(e.g., from Pseudomonas amylodermosa), mainly because
and amylopectin, while isoamylase can only hydrolyse the of their greater temperature stability and more β-amylase-
α-1,6 bond in amylopectin and glycogen [12]. Pullulanase compatible pH range [10]. Another disadvantage of using
requires each of the two chains linked by an α-1,6- glucosidic isoamylase during saccharification process is the inability to
bond contains at least two α-1,4-linked glucose units. Thus, hydrolyse 2- and 3-glucose unit side chains in β-limit and α-
the smallest substrate for pullulanase is the tetrasaccha- limit dextrins. Thus, simultaneous action of β-amylase and
ride 62 -α-maltosylmaltose. In contrast, isoamylase prefers isoamylase cannot quantitatively converts amylopectin to
substrates with high-molecular-weight oligosaccharides and maltose [19]. The β-amylases are also found to be a potential
the smallest substrate meeting this requirement is the inhibitor for the isoamylases activity [22]. Pullulanase, on
pentasaccharide 63 -α-maltosyl maltotriose such as glycogen the other hand, is generally used in combination with
from animal sources [38]. The fine structure of amylopectin amyloglucosidase, α-amylase, and β-amylase. Furthermore,
is distinct from that of glycogen in animals and bacteria the presence of maltotriose or maltotetraose in the fluid
as glycogen is randomly branched. The branches are more competitively inhibits isoamylase action [21].
numerous, and the chains are shorter as compared to The most established advantage of using pullulanase
amylopectin [39]. instead of isoamylase during starch saccharification process
6 Enzyme Research

Table 2: Reaction specificities of pullulan-degrading enzymes.

Enzyme EC number Bonds processed Preferred substrate End products References

Pullulanase type I Oligo- and
3.2.1.41 α-(1,6) Trimer (maltotriose) [17, 27]
polysaccharides, Pullulan
α-(1,6) Pullulan, Trimer (maltotriose)
Pullulanase type II [31, 32,
3.2.1.41 Mixture of glucose,
(amylopullulanase) Poly- and oligosaccharide 40]
α-(1,4) maltose, and
(starch)
maltotriose
Pullulan hydrolase type I
3.2.1.135 α-(1,4) Pullulan Panose [41–43]
(neopullulanase)
Pullulan hydrolase type II
3.2.1.57 α-(1,4) Pullulan Isopanose [12]
(isopullulanase)
Mixture of panose,
α-(1,4) and
Pullulan maltose, and
Pullulan hydrolase type III 3.2.1.— α-(1,6) [37]
maltotriose
Starch, amylose, and Maltotriose and
amylopectin maltose

3.4. Advantages of Using Pullulanase in Starch Saccharification

Processes. The majority of starches which are of indus-
trial importance contain approximately 80% amylopectin
Amylose (Table 4). The branch points occur on average every 20 to
25 D-glucose units, so that amylopectin contains 4% to 5%
of α-1,6 glucosidic linkages [11]. During starch conversion
process, starch is first gelatinised and solubilised at high
temperatures and the long-chain molecules broken down
Amylopectin
into smaller units (maltodextrins) which can be either in the
form of branch or linear (Figure 7).
α-amylase The α-1, 6-glucosidic linkages present in starch
molecules act as a kind of barrier to the action of various
starch hydrolysing enzymes during the saccharification
process that followed after gelatinisation process. When
starch is subjected to hydrolysis by α-amylases, this endo-
acting enzyme is able to bypass the branch points, but in
general is not capable of hydrolyzing the α-1,6 glucosidic
linkage. As a result, the amylopectin fraction is only partially
Figure 7: Partial hydrolysis of starch by α-amylase (modified from
degraded [11, 18]. The branch points containing α-1,6
[11]). glucosidic linkages are resistant to attack and their presence
also imposes a certain degree of resistance on neighbouring
α-1,4 linkages. Thus, the prolonged action of α-amylase on
amylopectin results in the formation of “α-limit dextrins,”
Table 3: Action of pullulanase and isoamylase on various sub- which are not susceptible to further hydrolysis by the
strates.
α-amylase (Figure 7). Similarly, when amylopectin is treated
Relative rate of hydrolysis with β-amylase, hydrolysis stops as a 1,6-α branch point is
Substrate
Isoamylase Pullulanase approached, resulting in the formation of β-limit dextrins
Pullulan Very low 100 [18].
Pure amylopectin 100 15 During saccharification, partially hydrolysed amylose
Glycogen (oyster) 124 1
and amylopectin molecules are depolymerised by the action
of glucoamylase which removes glucose units in a stepwise
Rabbit liver 111 0.6
manner from the nonreducing chain ends [11, 45]. The
rate of hydrolysis by glucoamylase depends on the particular
linkage, size of molecule, and the order in which α-1,4 and α-
is that the time of addition of isoamylase to the hydrolysis 1,6 linkages are arranged [45]. Glucoamylase hydrolyses the
system is very critical. For example, when isoamylase is α-1,4 links very efficiently, and at a much slower rate for α-
used before amyloglucosidase, the amylopectin fraction is 1,6 links. For example, the rates of hydrolysing 1,4-α, 1,6-
depolymerised too rapidly and therefore is highly susceptible α and 1,3-α-links in tetrasaccharides are in the proportion
to retrogradation [44]. of 300 : 6 : 1 [11, 46]. As shown in Figure 8, glucoamylases
Enzyme Research 7

Amylose

Amylopectin

Glucoamylase

(Slow) (Fast)

Glucose

Figure 8: Action of glucoamylase on liquefied starch (modified from [11]).

Table 4: Properties and composition of starch granules.

Starch Type Size range (diameter μm) Shape Amylose (%) Amylopectin (%)
Sago Pith 5–65 Oval, truncated 27 73
Corn Cereal 3–26 Round, polygonal 28 72
Potato Tuber 5–100 Oval, spherical 21 79
Wheat Cereal 2–35 Round, reticular 28 72
Tapioca Root 4–35 Oval, truncated 17 83
Rice Cereal 3–8 Polygonal, angular 17 83
(Source: [4, 5]).

slowly hydrolyse 1,6-α-glucosidic linkages in amylopectin final glucose concentration due to continuous formation
and partially hydrolyse amylopectin, but the action of this of isomaltose [12, 46]. Hence, minimising this reaction is
maltogenic exoamylases ceases or stops as a branch point is important for increment of the final glucose yield in indus-
approached [11]. trial process [45]. The efficiency of saccharification reaction
Glucoamylase also catalyses the reverse reaction (rever- could be improved by incorporating a specific amylopectin-
sion), in which dextrose molecules are combined to form debranching enzyme in the system. If a debranching enzyme,
maltose and isomaltose [11, 38, 47]. When α-amylase and such as pullulanase and glucoamylase, is simultaneously used
glucoamylase are used in sequence to saccharify sago starch, during saccharification, the pullulanase would specifically
the isomaltose is produced [48]. However, treatment with hydrolyse the branch points in the amylopectin residues, fol-
a mixture of glucoamylase and pullulanase during the lowed by the hydrolysis of linear 1,4-α-glucosidic linkages by
saccharification of sago starch resulted in the production glucoamylase (Figure 10). As a result, the maximum dextrose
of glucose but no isomaltose. The reversion of dextrose levels that could be achieved are higher (Figure 9) [20, 38].
specifically involves the condensation of a β-anomer of D- The use of a debranching enzyme would increase the
glucopyranose with either an α- or a β-D-glucose molecule rate of overall saccharification process and reduce the total
in the presence of glucoamylase, as shown below: amount of glucoamylase that is required for complete con-
version process. The practical advantage of using pullulanase
D-glucose + β-D-glucose −→ Dissacharide + H2 O. (1)
together with glucoamylase is that less glucoamylase activity
Isomaltose cannot be enzymatically hydrolysed into is needed. Reduction in the use of up to 60% glucoamylase
smaller molecule [22]. In industry, isomaltose is byproducts has been reported [11, 50]. This does not in itself give any
that decrease overall glucose yield and are unacceptable cost advantage. However, less glucoamylase is used and fewer
in the final high-fructose syrup used as sweetener [49]. branched oligosaccharides accumulated toward the end of
Figure 9 shows the incubation of maltodextrin with high the saccharification and this is the point at which isomaltose
loading of glucoamylase which results in reduction of production becomes significant [20].
8 Enzyme Research

97 98

96 C
97.5
95

D-glucose (%)
A
Glucose (%)

94 97
93
B
92 96.5

91
96
90 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
0 50 100 150 Pullulanase dosage (unit/g dry substance)
Time (h)
Figure 11: The effect of pullulanase activity on maximum D-
Figure 9: The percentage (%) of glucose formed from maltodextrin glucose concentration (modified from [11]).
using various enzyme solutions. Symbols: A, 200 U/kg Aspergillus
niger glucoamylase; B, 400 U/kg Aspergillus niger glucoamylase; C,
200 U/kg Aspergillus niger glucoamylase plus 200 U/kg Bacillus 98
acidopullulyticus pullulanase (modified from [20]).

D-glucose concentration (%)

97
b
Pullulanase 96
a
(Fast)
95

Amylopectin 94
20 25 30 35 40 45 50
Glucoamylase Substrate concentration (% w/v)
(Slow) (Faster)
Figure 12: The effect of substrate concentration on maximum D-
glucose concentration. Symbols: a, without pullulanase; b, with
pullulanase (modified from [11, 18]).

by up to about 50% in the production of 55% fructose syrup

[11].
The effect of pullulanase on the amount of substrate that
Figure 10: Effect of pullulanase during saccharification (modified can be treated to produce maximum D-glucose is shown
from [11]). in Figure 12. Saccharification may be carried out at higher
substrate concentrations (30% to 40%) than in the normal
process (25% to 30%) if a combination of a debranching
enzyme and glucoamylase is used [20, 50]. Furthermore, the
The effect of pullulanase activity on the increment of D- extra cost of using pullulanase is recouped by saving the cost
glucose production is illustrated in Figure 11. In the standard of evaporation and glucoamylase. For high-fructose syrups
saccharification process, without pullulanase, the maximum production, cost of further processing could also be reduced
D-glucose that could be obtained under the given conditions [20].
is about 96%. By using pullulanase, substantially lower The capacity of syrup production plant could be
glucoamylase dosage is possible to increase the D-glucose increased by reducing the starch saccharification time.
level by about 2%. In addition, using pullulanase together In a conventional process, saccharification time could be
with β-amylase in starch saccharification process, maltose increased by using high glucoamylase dosage and the time
yield could be increased by about 20 to 25% [11, 50]. By could be reduced from 45 to 30 h by doubling the dosage.
using pullulanase in the system, increase in crystallization Unfortunately, the reversion reaction (isomaltose formation
yield could also be achieved for dextrose production. In order from D-glucose) is very significant, and its therefore difficult
to obtain the targeted fructose concentration, pullulanase to stop the saccharification at, or close to, maximum D-
has been employed in fructose syrup production process, glucose. If normal glucoamylase dosage with a combination
which in turn reduced the isomerization costs. For example, of a debranching enzyme is used, the reaction time could
polysaccharide content in the final product can be reduced be reduced without the problems of overconversion [11]. As
Enzyme Research 9

97 type II has not been yet in commercial production due to

low yield and limited activity at high starch concentrations
D-glucose concentration (%)

96 (>30%) [74]. Several alkaline pullulanases type II, which

d were active at pH ranging from 8.5 to 12, have been isolated
95 c
and characterized from Bacillus spp. [41, 59, 75, 76].
Neopullulanases have been reported from many types
b of microorganisms such as Bacillus stearothermophilus
94
[42], Bacteroides thetaiotaomicron 95-1 [57], and Bacillus
polymyxa CECT 155 [58]. Neopullulanases hydrolyse pul-
93 lulan to produce panose as the main product with the
a final molar ratio of panose, maltose, and glucose of 3 : 1 : 1
92 [77]. Neopullulanase is also closely related to cyclomal-
0 24 48 72 96 120 todextrinases based on similarity of their specific reaction
Reaction time (h) towards pullulan [34]. Acidic isopullulanases have been
Figure 13: The effect of pullulanase dosage on reaction time. Sym- characterised from Aspergillus niger ATCC 9642 [36] and
bols: a, without pullulanase addition; b, 0.04 unit of pullulanase/g Bacillus sp. US 149 [31]. Recently, pullulan hydrolase type III
dry solid; c, 0.08 unit of pullulanase/g dry solid; d, 0.16 unit of has been isolated and characterised from hyperthermophilic
pullulanase/g dry solid (modified from [11]). archaeon, Thermococcus aggregans [37]. This is the only
enzyme presently known that attacks α-1,4- as well as α-1,6-
glucosidic linkages in pullulan and is active at above 100◦ C.
illustrated in Figure 13, for process without the debranching
enzyme, maximum D-glucose was obtained after about 80 h 4. Genetic Engineering of Pullulanase
of reaction. When the pullulanase loading is increased, the
same level of D-glucose can be attained at shorter incubation Wild-type microorganisms normally produce low activity
time (30 h). of pullulanase enzymes, which are not enough to meet
the demand for biotechnological applications. Molecular
cloning of the corresponding genes and their expression
3.5. Properties of Pullulanases. As mentioned earlier,
in heterologous hosts is one of the possible approaches
enzymes which hydrolyse pullulan have been classified
to circumvent this problem. Large quantities of specific
into five major groups. A number of pullulanases have
gene can be isolated in pure form by molecular cloning
been purified and characterised from different bacterial
and the target DNA can be produced in large amounts
sources (Table 5). Pullulanase type I has been characterised
under the control of the expression vector. Overexpression
from mesophilic bacteria such as Aerobacter aerogenes [69],
following the cloning step can significantly increase the
Bacillus acidopullulyticus [11], Klebsiella pneumonia [70, 71],
enzyme yield by subcloning the target gene into a suitable
and Streptomyces sp. [52]. Moderate thermophilic gram-
expression vector. Another approach is to insert a signal
positive bacteria such as Bacillus flavocaldarius [51], Bacillus
sequence into the host bacteria for the transfer of ligated
thermoleovorans [30], Clostridium sp. [72], and Thermos
gene product to the periplasmic region [78]. Research related
caldophilus [27] also have ability to secrete pullulanase type I
to pullulanase gene has been carried out since 1980s. Most
while pullulanase type I from hyperthermophilic bacterium,
of the pullulanase genes were isolated from Bacillus spp.
Fervidobacterium pennavorans, has also been reported [28].
(Table 6). In addition, pullulanase genes from Anaerobranca
Unlike pullulanase type I, pullulanase type II is widely
gottschalkii, Desulfurococcus mucosus, and Klebsiella aerogenes
distributed among extreme thermophilic Bacteria and hyper-
have also been reported [24, 29, 60]; Escherichia coli and
thermophilic Archaea [43]. The most thermostable and
Bacillus subtilis were the two commonly used host strains for
thermoactive pullulanase type II reported to date was derived
the expression of pullulanase gene.
from the hyperthermophilic archaeon Pyrococcus woesei [26]
and Pyrococcus furiosus [55, 73].The temperature optima for
these thermoactive pullulanase were ranged from 85◦ C to 5. Industrial Applications of Pullulanase
105◦ C and maintained its thermostability even in the absent
of substrate and calcium ions [17]. The gene encoding the 5.1. Saccharification of Starch. Since 1960s, almost all pro-
pullulanase of Pyrococcus woesei was cloned and expressed cesses to convert starch to glucose have changed to enzymatic
in E. coli, which was the first archaeal pullulanase that was hydrolysis from traditional acid method [79]. Today, much of
cloned and expressed in a mesophilic host [26]. With the the starch hydrolysates available in the market are enzyme-
present of 5 mM Ca2+ , the activity of pullulanase type II converted products of higher DE. Dextrose equivalent (DE)
produced by Pyrococcus furiosus and Thermococcus litoralis is a measure of reducing sugar on a dry basis, pure dextrose
was active and stable at temperature ranging from 130◦ C (glucose) being DE 100 and starch being close to DE 0. The
to 140◦ C [55]. Thermoactive pullulanase type II from enzymatic conversion of starch into glucose, maltose, and
Desulfurococcus mucosus, Thermococcus celer, Thermococcus fructose for use as food sweeteners represents an important
strain TYS, and Thermococcus strain TY has also been growth area in industrial enzyme usage. The primary
reported [56]. However, this hyperthermophilic pullulanase application of pullulanase is in starch saccharification and
10 Enzyme Research

Table 5: Physicochemical properties of selected pullulan-hydrolyzing enzymes from various microorganisms.

Organism Mr a (kDa) pI Optimal pH Optimal temperature (◦ C) Reference

Type I pullulanase
Bacillus flavocaldarius KP 1228 55 n.d.b 7.0 75–80 [51]
Streptomyces sp. No. 27 n.d. n.d. 4–7 55–60 [52]
Thermus caldophilus GK-24 65 6.1 5.5 75 [27]
Fervidobacterium pennavorans Ven 5 240 n.d. 6.0 85 [28]
Bacillus thermoleovorans US 105 (expressed in E. coli) n.d. n.d. 5.6–6.0 75 [30]
Type II pullulanase
Thermoanaerobacter strain B6A 450 4.5 5.0 75 [53]
Clostridium thermosulfurigenes EM 1 102 n.d. 5.5–6.0 60–65 [54]
Pyrococcus furiosus 110 n.d. 5.5 98 [55]
Thermococcus litoralis 119 n.d. 5.5 98 [55]
Desulfurococcus mucosus n.d. n.d. 5.0 100 [56]
Thermococcus celer n.d. n.d. 5.5 90 [56]
Thermococcus strain TY n.d. n.d. 6.5 100 [56]
Pyrococcus woesei 90 n.d. 6.0 100 [26]
Thermococcus hydrothermalis 110–128 n.d. 5.5 95 [14]
Desulfurococcus mucosus expressed in Bacillus subtilis 66 n.d. 5.0 85 [32]
Neopullulanases
Bacillus stearothermophilus expressed in B. subtilis 62 n.d. 6.0 60–65 [42]
Bacillus stearothermophilus TRS 40 62 n.d. 6.0 60–65 [42]
Bacteroides thetaiotaomicron 95-1 70 n.d. 6.5 37 [57]
Bacillus polymyxa CECT 155 n.d. n.d. 6.0 50 [58]
Isopullulanases
Aspergillus niger ATCC 9642 69–71 n.d. 3.5 40 [36]
Bacillus sp. US 149 200 n.d. 5.0 60 [31]
Alkaline amylopullulanase
Bacillus sp. KSM-1876 120 5.2 10–10.5 50 [59]
Bacillus sp. KSM 1378 210 4.8 9.5 50 [41]
Pullulan hydrolase type III
Thermococcus aggregans expressed in E. coli n.d. n.d. 6.5 95 [37]
a b not
Molecular mass; detected by the researchers.

Table 6: Genetic engineering of pullulanase gene.

Source Expression strain Length (a.a)∗ Vector References

Anaerobranca gottschalkii E. coli BL21 (DE3) 865 pET21a [60]
Bacillus thermoleovorans US105 E. coli DH5α 718 pEBM2 [30]
Bacillus stearothermophilus TS-23 E. coli XL1-Blue MRF 2018 pAP/StuI [61]
Bacillus stearothermophilus strain TRS-128 B. subtilis NA-1 N.A.# pTB522 [62]
Klebsiella pneumonia E. coli K-12 N.A.# pACYC184 [63]
Thermus IM6501 E. coli 718 p6xHis119 [64]
Desulfurococcus mucosus B. subtilis clone JA803 686 JA803 [32]
Fervidobacterium pennivorans Ven5 E. coli FD748 849 pSE420 [29]
Thermococcus hydrothermalis E. coli JM109 (DE) 1339 pAPUΔ2 [65]
Thermotoga maritima E. coli JM83 840 pTPU1 [66]
Caldicellulosiruptor saccharolyticus E. coli Q359 826 pNZ1038 [67]
Bacillus sp. XAL601 II E. coli JM109 2032 pUC18 [68]
∗
a.a: amino acid.
# N.A.: not available.
Enzyme Research 11

the most important industrial application of pullulanase is 5.6. Production of Cyclodextrins (CDs). Pullulanase has also
in the production of high-glucose (30% to 50% glucose; been used to enhance the yield of cyclodextrins by the reac-
30% to 40% maltose) or high-maltose (30% to 50% maltose; tion of CGTase with gelatinized starches and maltodextrin
6% to 10% glucose) syrups [12, 80]. In the saccharification syrups in the presence of cyclodextrins (CDs) complexing
process, pullulanase is normally used in combination with agents [88]. CDs have a wide range of applications in
glucoamylase or β-amylase [13, 20, 80]. complexing materials in foods, pharmaceuticals, plastics,
and agricultural products as emulsifiers, antioxidants, and
stabilizing agents [89].
5.2. Production of High-Maltose Corn Syrup. High-maltose
syrups have mild sweetness, low viscosity in solution, low
hygroscopicity, and good thermal stability. It is well suited 5.7. Others. Pullulanase also finds minor application in
for numerous applications in food processing such as in the the manufacturing of low-calorie beer [79] and in baking
manufacturing of high-quality candy and ice cream [81]. In industry as the antistaling agent to improve texture, volume,
recent years, there has been an increasing interest for pure and flavor of bakery products [12]. It is also possible to use
maltose in the pharmaceutical industry. Maltose may be used pullulanase as a dental plaque control agent [90].
instead of D-glucose for intravenous feeding, where it can
be administered at higher concentrations without elevating 6. Conclusions
blood glucose levels [81]. Pure maltose may also be used
as a starting material for the production of maltitol and Pullulanase capable of hydrolysing α-1,6 linkages of polymer
crystalline maltitol [11]. is widely used in saccharification process for production
of various useful materials such as maltose, amylose, and
5.3. Production of High-Fructose Corn Syrup . High-glucose glucose, by debranching starch with and without α-amylase,
syrup is used as a carbon source in fermentation and feed β-amylase, or glucoamylase, respectively. Clearly, pullulanase
for making high-fructose syrups and crystalline glucose [79, could facilitate a major change in the current strategy for
starch processing and hence, there is a strong demand for
82]. High-fructose syrup is produced by the treatment of
this enzyme around the world with a growing presence in
high-dextrose syrup, especially high-glucose syrup (DE 95–
the industries related to its usage.
96) with immobilized glucose isomerase [12]. High-fructose
corn syrup (HFCS) is a high-quality and clean-tasting caloric
sweetener. HFCS with very high DE value is required in the References
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