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Biocatalysis Research Progress Francesco H. Romano
Digital Instant Download
Author(s): Francesco H. Romano, Andrea Russo
ISBN(s): 9781604566192, 1604566191
Edition: Kindle
File Details: PDF, 4.47 MB
Year: 2008
Language: english
BIOCATALYSIS RESEARCH PROGRESS
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BIOCATALYSIS RESEARCH PROGRESS
FRANCESCO H. ROMANO AND ANDREA RUSSO

EDITORS
Nova Biomedical Books

New York
Copyright © 2008 by Nova Science Publishers, Inc.
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Biocatalysis research progress/Francesco H. Romano and Andrea Russo(editors).

p.;cm.
Includes bibliographical references and index.
ISBN 978-1-61668-287-3 (E-Book)
1.Enzymes—Biotechnology.2.Microbial biotechnology.I.Romano,Francesco H.,1939-II.
Russo,Andrea,1954-
[DNLM:1. Catalysis.2. Enzymes.3.Biochemistry—methods. QU 135 B6147 2008]
TP248.65.E59B568 2008
572’.—dc22 2008012763
Published by Nova Science Publishers, Inc. New York

Contents
Preface vii
Chapter I Nanobiocatalytic Systems: Thin Films of Enzymes 1
Laura Pastorino and Svetlana Erokhina
Chapter II Bioprocesses for the Synthesis of
Nucleosides and Nucleotides 47
Marco Terreni, Daniela Ubiali, Teodora Bavaro,
Davide A. Cecchini, Immacolata Serra
and Massimo Pregnolato
Chapter III Biocatalysis in Environmental Technology 95
D. M. G.Freire, M. L. E. Gutarra,
V. S. Ferreira-Leitão, M. A. Z. Coelho
and M. C. Cammarota
Chapter IV Application of Lipases to Substances with
Pharmacological Importance (Polyunsaturated Fatty Acids) 155
Marie Zarevúcka and Zdeněk Wimmer
Chapter V The Evolution of Directed Evolution 187
Birthe Borup, Lynne Gilson,
Richard Fox and Thomas Daussmann
Chapter VI Biocatalytic Resolution of DL-Pantolactone by
Cross-linked Cells and its Industrial Application 195
Zhi-Hao Sun, Ye Ni, Pu Zheng,
Xin-Fu Guo and Jun Wang
Chapter VII Biocatalytic Potential of Haloalkaliphilic Bacteria 209
Satya P. Singh, Megha K. Purohit,
Jignasha T. Thumar, Sandeep Pandey,
Chirantan M. Raval and Hetal G. Bhimani
vi Contents
Chapter VIII Salt-tolerant Alkaliphilic Actinomycetes

and their Biocatalytic Potential 219
Satya P. Singh and Jignasha T. Thumar
Chapter IX Accelerating Whole-cell Biocatalysis by
Cellular Membrane Engineering 229
Ye Ni and Rachel R. Chen
Chapter X Recent Advances in Enzymatic Synthesis of
Water-Soluble Conducting Polymers 245
Estibalitz Ochoteco, Tomasz Sikora,
David Mecerreyes, Jose A. Pomposo
and Hans Grande
Chapter XI Marine Enzymes for Biocatalysis – Production,
Isolation and Applications 259
K. Muffler, J. Mukherjee and R. Ulber
Chapter XII Immobilized Microbial Cells - Applications
and Mass Transfer Phenomena 281
Venko Beschkov
Chapter XIII Application of Whole-cell Biocatalysis in Chemoenzymatic,
Asymmetric Synthesis of Medically Important Compounds 307
Ewa Żymańczyk-Duda and Paweł Kafarski
Index 345
Preface
Biocatalysis encompasses the use of enzymes or whole cell systems for effecting the
conversion of readily available, inexpensive starting materials to high value products.
Enzymes frequently display exquisite selectivity, particularly chemo-, enantio- and
regioselectivity, making them attractive catalysts for a wide range of chemical
transformations. Enzymes also typically operate under mild conditions of pH and temperature
leading to the formation of products of high purity. As a result of these advantages, enzymes
and whole cells are finding wider application in areas such as the production of intermediates
for pharmaceuticals, fine chemicals, agrichemicals, novel materials, diagnostics, biofuels and
performance chemicals. This new book presents leading-edge research in the field.
Chapter I - The use of enzymes in industry requires their immobilization in order to
reduce the costs and increase the yield of the biocatalytic processes. By reducing the enzyme
mobility, the immobilization affects the enzyme structure and consequently its functionality.
Thus, the immobilized enzymes efficiency is dependent on the peculiarities of the
immobilization technique.
These had been evolved from physical adsorption, membrane entrapment and chemical
surface activation. Although these techniques are at present successfully utilized, the search
for new approaches, which can result in biocatalytic systems with enhanced efficiency, is in
progress. In particular traditional immobilization techniques do not provide effective control
of the immobilization process or of the properties of the biocatalytic system at molecular
level. This lack of control results in the formation of enzyme molecules aggregates leading to
loss in functionality due to mass transfer resistance. This limitation can be overcame by
nanotechnology inspired biocatalytic systems that differ from traditional systems in terms of
preparation, catalytic efficiency and application potential. In particular the “bottom-up”
approach, which consists of building systems molecule-by-molecule, represents a promising
methodology for the precise control of the biocatalytic system structure. Moreover the
molecular dimension of the resulting systems makes economically possible the use of even
highly expensive material, including the most of enzyme molecules. The bottom-up approach
comprises several methods, among them thin film techniques offer the possibility to construct
2 and 3D biocatalytic systems with nm resolution, with predetermined structure and function,
starting from mono/multi layers of enzymes. This can be accomplished by the use of the layer
by layer alternate adsorption of enzymes with oppositely charged polyions (layer-by-layer
viii Francesco H. Romano and Andrea Russo
self assembly technique), or by the Langmuir-Blodgett technique based on the formation of

enzyme-containing monolayers at the air/water interface. Enzyme layers can be deposited
onto solid supports of different shapes or enzymes can be encapsulated into nano- and
microcapsules with layered shell organization for the design of nano-bioreactors. Interest in
this field is rapidly growing and is likely to fuel more exciting developments in the near
future. The aim of this chapter is to review current status of layer-by-layer self assembly
technique and of Langmuir-Blodgett technique for the development of biocatalytic systems,
to give an overview of the techniques for the structural and functional characterization of
enzyme thin films and to give some perspectives of future developments.
Chapter II - Modified nucleosides and nucleotides are routinely used as citotoxic or
antiviral drugs as well as immunosuppressive agents. The therapeutic activity of these
compounds is due to their ability to act as antimetabolites in the RNA and DNA synthesis.
Nucleosides have traditionally been prepared by various chemical methods, involving multi-
step chemical procedures which are plagued by low yields and the formation of undesired by-
products. These drawbacks strongly reduce the performances of the processes used for the
synthesis of unnatural nucleosides and nucleotides, in terms of yields, purity of the final
product, costs and environmental impact.
Enzymatic syntheses have been shown to be an advantageous alternative to chemical
methods due to the high selectivity of the enzymes, the mild reaction conditions and the
overall simplicity of the approach. These bioprocesses can overcome the need of specific
protecting groups often required on the heterocyclic base and/or on the sugar residue in the
glycosylation reaction as well as in the modification of naturally occurring nucleosides.
Consequently, these processes can be very competitive in terms of costs, allowing high
quality products to be obtained in high yields.
Glycosyl-transferring enzymes (e.g. nucleoside phosphorylases, N-
deoxyribosyltransferases) have been used in the synthesis of nucleosides by mediating the
enantioselective transfer of glycosyl residues to acceptor bases. Recent reports involving
glycosyl-transferring enzymes are reviewed herein. In this context, the use of isolated
deaminases to obtain, respectively, cytidine derivatives from uridine, or guanosine
derivatives from adenosine is also considered. Moreover, hydrolases (e.g. lipases, esterases)
will be described in carrying out regio-controlled manipulations on the carbohydrate moiety.
Finally, the enzyme-mediated synthesis of nucleotides through regioselective
phosphorylation of the parent nucleosides catalyzed by kinases and phosphatases are
discussed.
This review will also focus on the importance of the enzyme selection and the design of
the final biocatalyst thereof, particularly by tailor-made immobilization of the protein on
solid support, with the aim to have an active, stable and reusable biocatalyst for preparative
purposes.
Chapter III - New strategies development to solve problems related to waste disposal
may include technologies facing compounds of low biodegradability and/or detoxification of
some residues with the alternative of adding value. There are various chemical and biological
approaches, but biological ones employing enzymes had been showed good results. On the
other hand, environmental use of enzymes depends on costs reduction for biocatalyst
production through the selection of highly-productive strains, development and optimization
Preface ix
of fermentation processes. Production system which employs agroindustrial residues as

culture medium can be an interesting alternative. Microorganisms’growth on solids wastes by
solid state fermentation can add value to these residues by enhancing nutritional quality
and/or producing several enzymes with biotechnological applications, and also can reduce its
pollution potential by degrading toxic compounds. The reduction cost of enzyme production
could make feasible its employment for environmental purposes such as enzymatic
pretreatment of wastewaters with high levels of fats, improving the biological degradation, or
in the treatment of colored and phenolic wastes. The potential of environmental biocatalysis
not only help to solve pollution problems but also simultaneously add value to undesirable
wastes by the generation of biotechnological products or by enhancing its nutritional
proprieties for use as animal feed.
Chapter IV - Polyunsaturated fatty acids and their derivatives are extensively studied
natural compounds with high impact in human medicine. Their sources belong among the
renewable resources, mostly of plant origin.
Lipases are well established biocatalysts for the enantio- and regioselective formation
and hydrolysis of ester bonds in a wide variety of natural and unnatural substrates. Therefore,
they seemed ideally suited also for bioconversion of the plant materials, especially plant oils.
The use of biocatalysts for preparation of partial acylglycerols could provide numerous
advantages as compared to conventional chemical methods such as increased selectivity,
higher product purity and quality, energy conservation and the elimination of application of
toxic catalysts. Two general routes to desired molecules are available, in principle namely
hydrolysis/alcoholysis of triacylglycerols and esterification of glycerol. The reactions can be
provided under conventional conditions or in supercritical fluids. Enzymatic reactions in
supercritical fluids combine the advantages of biocatalysts (substrate specificity under mild
reaction conditions) and supercritical fluids (high mass-transfer rate, easy separation of
reaction products from the solvent, environmental benefits).
Chapter V - The recent increasing usage of biocatalysts as viable alternatives to
conventional chemical reactions is attributable to improved technology in tailoring enzymes
found in nature to the needs of the industry. Arguably the most successful technology in this
field has been directed evolution. Here the authors present the various stages in the evolution
of directed evolution itself. The initial approach employed in directed evolution was
successive rounds of random mutagenesis and screening, from which the top performer was
chosen as parent for the next round of mutagenesis. Although effective, this method is not
very efficient, as all beneficial mutations not found in the top performer are lost in the next
round of evolution. With the advent of DNA recombination methods, mutations found in
lower performing enzymes could be included in the next round. However, there was still
useful diversity left unrecognized and unutilized, since only the top performing enzymes
would be used for recombination. As statistical tools based on sequence-activity relationships
were developed to help identify good mutations for recombination, beneficial diversity from
even poorly performing enzymes could be moved to the next round of evolution. Presently,
the drive to increase knowledge of sequence-activity relationships for enzyme families is
enabling collections of variants to be synthesized that efficiently explore the chemical space
of potential substrates for those enzymes. This will continue to lead to even faster evolution
x Francesco H. Romano and Andrea Russo
cycles, as a more relevant portion of the sequence space to be explored has been designed
into the first library (first set of enzymes to be screened).
Chapter VI - D-Pantoic acid (D-PA) and d-pantolactone (D-PL) are known as important
intermediates for the production of calcium pantothenate, an additive for animal feed. Studies
have showed that several filamentous fungi belonging to the genera Fusarium, Gibberella
and Cylindrocarpon could produce D-PL hydrolases that selectively hydrolyze the D-isomer
of DL-pantolactone to form D-PA,.
In our previous studies, an excellent stereoselective D-lactonohydrolase producing strain
Fusarium moniliforme CGMCC 0536 (SW-902) was obtained from isolation and mutation.
Cells of this strain were successfully applied for the kinetic resolution of DL-pantolactone to
produce D-(−)-isomer (99% e.e.). Furthermore, several immobilization methods were
established to reuse the cells. The rationale for choosing whole cell immobilization is because
it not only eliminates enzyme purification and extraction steps, but also increases enzyme
thermostability, provides higher operational stability, greater resistance to environmental
fluctuations and lower enzyme cost. An important technique is to treat cells with
glutaraldehyde. The cross-linking of enzymes with glutaraldehyde involves the reaction
between this bifunctional reagent and free amine groups of the enzyme. The linkages formed
are irreversible and lead to cross-linked enzymes and cells exhibiting high operational
stability. The wide utilization of glutaraldehyde for whole cells immobilization can be
attributed to the decrease of enzyme leakage by cross-linking among the chemical reagent,
cell wall, and intracellular protein. Therefore, the authors investigated the potential of cross-
linking F. moniliforme CGMCC 0536 with glutaraldehyde for entrapping D-lactonohydrolase
inside the cells and the employment of cross-linked biocatalyst in a stirred reactor.
Their results have showed that cells of F. moniliforme CGMCC 0536 have been
successfully immobilized by cross-linking with glutaraldehyde. The cross-linked cells
exhibited a markedly improved thermal stability and operational stability than free cells.
Kinetic characteristics of immobilized cells were assessed. The Km value of cross-linked
cells (118 mM) was slightly higher than that of free cells (96 mM), while the Vmax value
decreased from 4.18 to 3.87 mM min−1 g−1 wet cells after cross-linking. Furthermore,
glutaraldehyde treatment did not change the stereospecificity, pH, and temperature profile of
the D-pantonohydrolase. The high storage stability reduces fermentation workload.
Significant process engineering advantages were evident for cross-linked cells in repeated
batch operations. The resolution of DL-pantolactone was maintained at a steady level during
110 consecutive batches. The high activity and operational stability of the cross-linked cells
presented in this work have been successfully implemented in the commercial production.
Recently, the authors attempted to operate the resolution continuously by recycling
cross-linked cells in a membrane bioreactor. The data show that glutaraldehyde cross-linking
affords a satisfactory method for preserving the asymmetric hydrolyzing capacity of F.
moniliforme CGMCC 0536. A feasible method with high operational stability for the
production of D-PA catalyzed by cross-linked cells was established in a continuous process
by using membrane bioreactor.
Chapter VII - Haloalkaliphilic organisms have been largely investigated from Soda lakes
around the globe and other habitats for these organisms are rarely explored. During the last
10 years, the authors have been working on the diversity and enzymatic potential of
Preface xi
haloalkaliphilic bacteria from the natural and man made saline habitats along the coastal
Gujarat in Western India. The organisms have displayed varied diversity based on their
cultural and morphological patterns, Gram reaction, biochemical properties, antibiotic
resistance-sensitivity, molecular phylogeny and secretion of extracellular enzymes. The
production of extracellular alkaline proteases and lipases were widely spread among the
isolates, whereas only few secreted amylase. The wide spread distribution of these bacteria
from beyond soda lakes clearly indicated their ecological significance. Besides, the
enzymatic potential would attract several biotechnological applications under alkalinity and
high salt conditions.
While major attention has been focused on molecular phylogeny and diversity, only
limited information is available on their enzymatic potential and enzyme characterization.
Cloning, sequencing and expression of different enzymes from Haloalkaliphilic bacteria and
archaea are limited in literature. Our studies with alkaline proteases from a range of
Haloalkaliphilic isolates revealed that many enzymes were highly resistant to urea
denaturation and displayed catalytic potential under combination of extreme conditions.
However, the ability to catalyse under extreme conditions was salt dependent. The enzymes
displayed unique features for biotechnological applications, besides providing a model
system to study protein folding and stability. It is evident that the secretion and properties of
alkaline proteases would also be very useful in assessment of microbial heterogeneity. The
prospects of metagenomics to explore the novel sequences for biocatalysts from non-
cultivable microbes have emerged as a potential tool in recent years. This would also be
discussed with particular reference to saline habitats. Over all, our findings would be
integrated with the literature and biocatalytic potential of Haloalkaliphilic bacteria and
archaea would be assessed.
Chapter VIII - Extremophiles are distributed over a range of extreme habitats. Among
them, extremophilic actinomycetes have recently attracted greater attention due to their
various natural products and specific mechanism of adapting extreme environments. The
natural and man-made environments may harbor a large population of halophilic and
alkaliphilic actinomycetes. However, they have only recently focused attention of the
researchers. The phylogeny, diversity and biotechnological potential of salt-tolerant
alkaliphilic actinomycetes are still in infancy. It is, therefore, relevant and important to pay
more attention to extreme actinomycetes from unexplored habitats, as a possible way to
discover novel taxa and, consequently, new secondary metabolites. The study would also
enlighten us on their diversity, phylogeny and ecological significance.
During the last decade, there has been a dramatic increase in the need for bioactive
compounds with novel activities. Enzymes, after antibiotics are the most important
biologically derived product having immense potential in catalytic reactions of commercial
interest. Most of the studies related to enzymes have so far focused on halophiles, alkaliphiles
and haloalkaliphiles; however, the enzymatic potential of halo-tolerant alkaliphilic
actinomycete is nearly untouched. From the literature, it is evident that the exploration of the
enzymatic potential of these microbes is just the beginning and till date only few enzymes are
investigated in depth. Salt-tolerant alkaliphilic actinomycetes produce enzymes, such as
alkaline protease, amylase, cellulase and lipase that are functional under extreme conditions.
Consequently, the unique properties of these biocatalysts have potentials in several novel
xii Francesco H. Romano and Andrea Russo
applications in industrial processes. Because of the capacity to survive under nonstandard

conditions in non-conventional environments, it is assumed that the properties of
extremophilic enzymes have been optimized for these conditions. Interestingly, our own
studies have also revealed the occurrence of alkaline protease, amylase and cellulase in salt-
tolerant alkaliphilic actinomycetes isolated from vast coastal line and saline habitats along the
Saurashtra Coast Gujarat (Western India). In addition, the results on the alkaline proteases are
suggestive of their unique position in the generation of novel enzymes.
Chapter IX - Whole-cell biocatalysts are preferred in biocatalysis applications involving
cofactors and/or multiple enzymes. However, cell envelope often represents a formidable
permeability barrier, limiting the rate of entry of substrate. As a result, reactions catalyzed by
whole-cells are reportedly orders of magnitude slower than those of by their free enzyme
counterparts. This chapter reviews recent molecular engineering efforts addressing this
critical issue.
Initial studies were carried out with E. coli strains carrying mutations in the outer
membrane structures. The effects of these mutations were investigated by interrogating the
mutant cells with substrates differing substantially in size and hydrophobicity. The reduction
of outer membrane permeability barrier by these mutations led to significant accelerations in
reaction rates of all whole-cell catalyzed reactions investigated. In the case of the
tetrapeptide, a substrate for subtilisin, a single gene mutation in lipopolysaccharide (LPS)
synthesis can render the outer membrane completely permeable to substrate, reaching a
barrier-less condition that maximizes the reaction rate while retaining all the benefits of
whole-cell catalysts. For reaction rates of toluene dioxygenase (TDO)-catalyzed reactions, an
increase of up to 6-fold was observed with lipoprotein mutant for three small, hydrophobic
substrates tested. Mutations in either LPS or lipoprotein are effective for accelerating
reactions with UDP-glucose, a hydrophilic molecule with Mw over 600 Da, resulting in a
striking acceleration (up to 14-fold) of reaction rate. The magnitude of reaction rate
acceleration was found to be dependent upon the substrate concentrations, the enzyme
expression level, and the nature of the mutations. In addition, the mutations were
demonstrated to be far more superior to common permeabilization procedures such as freeze-
thaw and EDTA treatments.
To understand the mechanism of the permeability enhancement in the lipoprotein mutant,
the lpp region was sequenced. The results revealed that Braun’s lipoprotein was absent in the
transposon mutant cells due to multiple stop codons within the 59-bp insertion, suggesting
that the absence, rather than the alteration of Lpp, is responsible for the observed change in
permeability. More importantly, the sequencing result suggests that lpp deletion could
become a general permeabilization method. Subsequent studies were carried out by
generating lpp deletion mutants from strains with different genetic background. It was indeed
shown that lpp deletion generates a useful phenotype, not only effective in enhancing
substrate permeability, but also in reactions limited by product permeability, as demonstrated
in L-carnitine synthesis. Importantly, the deletion has no significant effect on cell growth,
metabolism and recombinant protein expression. Therefore, lpp deletion phenotype could be
applied as a generally applicable method to enhance the outer membrane permeability of
various E. coli strains, and possibly other Gram-negative bacteria with lpp homologs.
Preface xiii
Chapter X - The present chapter introduces the newest synthesis strategies developed by
our research group in order to overcome the main disadvantages derived from enzymatic
biocatalysis as a strategy for producing conducting polymers. First, the enzymatically
catalyzed polymerization of 3,4-ethylenedioxythiophene (EDOT) in the presence of
polystyrenesulfonate (PSS) is achieved showing an electrical conductivity of 2×10-3 S/cm and
an excellent film formation ability as confirmed by atomic force microscopy images. Second,
a simple method for immobilizing horseradish peroxidases (HRP) in the biocatalytic
synthesis of polyaniline (PANI) is presented. This method is based on a biphasic catalytic
system where the enzyme is encapsulated inside the ionic liquide (IL) 1-butyl-3-
methylimidazolium hexafluorophosphate, while other components remain in the aqueous
phase. The enzyme is easily recovered after reaction and reused several times. Finally, a new
bifunctional template (sodium dodecyl diphenyloxide disulphonate, DODD) is proposed in
the synthesis of polyaniline (PANI) as a strategy to improve water solubility as well as
electrical conductivity in the obtained polymer.
Chapter XI - The diversity of marine life offers a variety of novel enzymes which might
have a tremendous potential as biocatalysts for academic research as well as for industrial
processes. Concerning the wide spectrum of ecological habitats, which differs extremely in
temperature, pressure, and salinity, the whole machinery of enzyme expression and stability
of the proteins was adopted to the distinct environmental conditions of the producing micro-
as well as macroorganisms. Therefore the oceans provide an almost untapped reservoir of
biocatalysts showing interesting properties like high salt, pressure, and temperature tolerance.
With respect to the special requirements of industrial processes which are particularly
focused on high mass transfer and high time space yields, respectively, enzymes from marine
origin located at exotically regions can help to fulfill these specific demands. Thus,
applications of such proteins or whole cells as catalysts for manufacturing bulk- and fine
chemicals seems visionary up to know. However, regarding the current trends and
developments in molecular biology as well as genetic engineering it appears more realistic in
the case of long-term view. By means of identifying the origin of the enzymes’ stability one
has the possibility to modulate other so far unstable (terrestrial) enzymes.
This review covers the development and research work done on the processing of
enzymes from marine origin, which can be used as biocatalyst tools for research in academia
and industry.
Chapter XII - Immobilized cells performance has been extensively studied in the last
three decades. The advantages of this type of biocatalysts are in their multiple use,
continuous operation and easy removal from the reaction mixture. Two main types of
immobilization techniques exist: cell entrapment in gels and cell fixation on solid supports.
The drawbacks of cell entrapment are in the diffusion limitations at substrate supply to
and product removal from the gel particles. Such limitations do not exist in cases of cell
fixation on solid supports but other transport phenomena arise, associated with the cell
accumulation and concentration within small area. Both techniques admit changes in the cell
physiology including microbial growth different from that in free culture. That is why
additional impact on the net biocatalyst performance may cause the microbial growth in
immobilized state and the possible cell leakage into the fermentation broth and their
consecutive growth in a free state.
xiv Francesco H. Romano and Andrea Russo
The choice of immobilization method depends on the kinetics of the very microbial
process, on the microbial growth, on the products of reactions, being possible inhibitors or
catalysts, etc.
The purpose of this article is to review and to discuss the advantages and the drawbacks
of these two groups of immobilized biocatalysts from the point of view of the specific
reaction kinetics and the mass transfer limitations. Mathematical modeling is employed to
evaluate the effects of cell growth and detachment from the particles and their contribution in
free and immobilized state. Experimental data are presented to illustrate the discussed effects.
Chapter XIII - Chirality is, in the most cases, the key factor in the safety and efficacy of
many drug products. Usually only one enantiomer is responsible for the desired activity,
whereas its counterpart could be inactive, possess some activity of interest, be an antagonist
of the active enantiomer or have a separate activity that could be either desirable or
undesirable. Only in a few cases specific compositions of a racemate or an enantiomeric pair
demonstrated a synergistic effect. In past decades the pharmacopoeia was dominated by
racemates, but since 1980 number of chiral drugs introduced to market have grown markedly.
Chiral compounds currently account for at least 50% of sales with the annual sales of single-
enantiomer drugs exceeding 150 billions of dollars in 2002. These compounds now represent
one-third of all drug sales worldwide.
Thus, it is not surprising that chiral intermediates and fine chemicals are in high demand,
both from the pharmaceutical and agrochemical industries. During the drug development
process, the question invariably arises of which step and which method to choose for
introducing chirality. Thus, racemates are usually produced by parallel synthesis for drug
candidates - it makes sense to resolve the racemate for the initial animal studies, whereas an
optimized, enantioselective synthesis is employed in the course of production. While
enantiomerically selective organic synthesis is the traditional approach, using enzymes and
enzyme-containing microorganisms to biocatalyze a reaction is becoming increasingly
important. However, only 85% of products obtained by biocatalytic processes are
enantiomerically pure and in 50% of these processes enantiomerically active substrates are
used. This shows that induction of asymmetry is not always the most important feature of
industrial biocatalysis. Quite successfully it can be used to carry out conversions that would
otherwise require difficult or multiple synthetic chemistry steps. In such cases, biocatalysis
can be the preferred route even if chirality is not desired. Moreover, over 25% processes base
on kinetic resolution, reaction which affords the desired products with maximal yield of 50%
and thus seems to be not interesting from industrial point of view. This is because the second
enantiomer is either useful (although in different processes) or is isomerized and processing
back as subtrate.
The microbial biocatalysts demonstrate a wide variation of activities between genera,
within genera and even within species. The range of substrates transformed and the inter- and
intra-species differences in specificity of the individual biocatalysts suggests, that it is
possible to provide multiple catalytic agents, especially when the traditional organic process
fails or is to expensive to perform. Thus, biocatalysis have become an attractive alternative to
conventional catalysts in numerous industrial processes. When compared to chemical
catalysis biocatalysts are exquisitely selective and highly precise due to: their substrate
selectivity, which allowed distinguishing and acting on the subset of compounds within a
Preface xv
larger group of chemically related compounds; their stereoselectivity - the ability to act on a
single enantiomer or diastereoisomer selectively and their regioselectivity – ability to
recognize one location in a molecule and finally because of their selectivity towards defined
functional group in a presence of other equally reactive or more reactive ones. Furthermore,
biocatalysts are able to carry out bioconversions under mild conditions – another benefit of
using them as industrial catalysts.
Biocatalysis in drug reseach and production is usually used in multistep processes in
concert with more traditional production techniques. Thus, biocatalysts (as both isolated
enzyme and whole-cell systems) are increasingly being used to assist in synthetic routes to
complex molecules of industrial interest, in so called chemoenzymatic processes.
Whole-cell biocatalysis is a useful alternative to the use of pure enzymes. Employing
whole – cells is a strategy which allowed overcoming many limitation of the enzymatic
biotransformations and, what is important, is usually cheaper than using purified enzymes.
Additionally, microbial cells used as a catalysts, have their own cofactor regeneration
systems, offer wide range of enzymatic activities towards a number of non-physiological
substrates and moreover, usually there are no side reactions except the expected ones.
Moreover, there is no doubt that, the advantage of microbial biotransformations is the
possibility to induce enzymes of defined, desired activity even if they are not constitutively
presented inside the microbe cells. This enlarge the offer of possible reactions to be carried
out and in fact there exists an enzyme for almost every type of chemical reaction.
In: Biocatalysis Research Progress ISBN: 978-1-60456-619-2
Editors: F. H. Romano, A. Russo © 2008 Nova Science Publishers, Inc.
Chapter I
Nanobiocatalytic Systems:
Thin Films of Enzymes
Laura Pastorino1 and Svetlana Erokhina2

1
Department of Communication, Computer and System Sciences,
University of Genova, Italy
2
Department of Physics, University of Parma, Italy
Abstract
The use of enzymes in industry requires their immobilization in order to reduce the
costs and increase the yield of the biocatalytic processes. By reducing the enzyme
mobility, the immobilization affects the enzyme structure and consequently its
functionality. Thus, the immobilized enzymes efficiency is dependent on the peculiarities
of the immobilization technique.
These had been evolved from physical adsorption, membrane entrapment and
chemical surface activation. Although these techniques are at present successfully
utilized, the search for new approaches, which can result in biocatalytic systems with
enhanced efficiency, is in progress. In particular traditional immobilization techniques do
not provide effective control of the immobilization process or of the properties of the
biocatalytic system at molecular level. This lack of control results in the formation of
enzyme molecules aggregates leading to loss in functionality due to mass transfer
resistance. This limitation can be overcame by nanotechnology inspired biocatalytic
systems that differ from traditional systems in terms of preparation, catalytic efficiency
and application potential. In particular the “bottom-up” approach, which consists of
building systems molecule-by-molecule, represents a promising methodology for the
precise control of the biocatalytic system structure. Moreover the molecular dimension of
the resulting systems makes economically possible the use of even highly expensive
material, including the most of enzyme molecules. The bottom-up approach comprises
several methods, among them thin film techniques offer the possibility to construct 2 and
2 Laura Pastorino and Svetlana Erokhina
3D biocatalytic systems with nm resolution, with predetermined structure and function,

starting from mono/multi layers of enzymes. This can be accomplished by the use of the
layer by layer alternate adsorption of enzymes with oppositely charged polyions (layer-
by-layer self assembly technique), or by the Langmuir-Blodgett technique based on the
formation of enzyme-containing monolayers at the air/water interface. Enzyme layers can
be deposited onto solid supports of different shapes or enzymes can be encapsulated into
nano- and microcapsules with layered shell organization for the design of nano-
bioreactors. Interest in this field is rapidly growing and is likely to fuel more exciting
developments in the near future. The aim of this chapter is to review current status of
layer-by-layer self assembly technique and of Langmuir-Blodgett technique for the
development of biocatalytic systems, to give an overview of the techniques for the
structural and functional characterization of enzyme thin films and to give some
perspectives of future developments.
Introduction
Enzymes molecules are catalysts and they speed up the chemical reactions at the basis of
the metabolism of all living organisms with extremely high selectivity and efficiency.
Moreover, they are not part of the final product of the catalysed reaction, and they operate at
mild conditions, such as room temperature and physiological pH [Hartmeier, 1988]. In this
respect enzymes can be considered remarkable catalysts and their catalytic properties
represent the environmentally friendly solution to industrial problems. From the application
point of view, it is very important that many enzymes are commercially available, and
numerous industrial applications have been described. Enzymes have been used for more
than 50 years in the detergent, textile, food and feed industries [van Beilen, 2002]. Moreover,
biocatalysis has an important role in the industrial synthesis of bulk chemicals [Koeller,
2001], pharmaceutical [Pollard, 2006] and agrochemical intermediates [Aleu, 2006].
Biocatalysis also holds considerable promises in environmental fields [Alcade, 2006], such as
enzymatic bioremediation [Whiteley, 2006; Wu, 2008], enzyme-based biofuel cells [Kim,
2006a] and biodiesel production [Rashid, 2008; Hernández-Martín, 2008]. These are only
few examples of the growing impact of biocatalysis on different industrial sectors.
However, the potentiality of enzymes in industrial applications is not exhaustively
exploited, because of some bottlenecks such as the high cost of the most of enzymes, the low
activity and/or stability under working conditions and low reaction yields [Coward-Kelly,
2006]. Recent advances in tools and techniques for biocatalysis research and development
should help to overcome these limitations [Bommarus, 2006; Coward-Kelly, 2006; van
Beilen, 2002]. Advances in metabolic and protein engineering, high-throughput screening,
nanotechnology and other technologies will increase the impact of biocatalysis in industry
[Rubin-Pitel, 2006; Coward-Kelly, 2006] and new industrial applications are expected to be
realized [Schmid, 2001; Schoemaker, 2003].
Different steps can be individuated in the development of a biocatalytic process. They
span from the identification of the target reaction, to the selection of the more suitable
biocatalyst, to its characterization and modification, in order to arrive finally to its application
Nanobiocatalytic Systems: Thin Films of Enzymes 3
[Schmid, 2001]. The economic feasibility of a biocatalytic process depends on several factors
involved in these steps and differs for each process under study since requirements vary
enormously depending on the biocatalyst itself. One critical point to be taken into account is
represented by the cost of the biocatalyst itself, as many enzymes are expensive. To minimize
the influence of this cost on the whole process, recombinant DNA technologies [Petersen,
1999; Sheldon, 2004] can be used to enable the production of enzymes with competitive
prices on the one hand and immobilization techniques make possible the reuse of the same
biocatalyst in different production cycles on the other hand [Hartmaier, 1988; Schmid, 2001;
van Beilen, 2002]. As relates to immobilization techniques, they represent a powerful tool not
only to reduce the cost related to the enzyme itself but also to simplify the design of the
production plant, as the reaction product can be easily recovered without enzyme
contamination, to simplify the process control and finally to improve some enzyme properties
such as operational stability [Katchalsky-Katzir, 1993; Mateo, 2007].
Depending on the process requirements (enzyme characteristics, substrate, reaction type
and reactor configuration) specific immobilized enzymes should be designed. Moreover,
several possible restrictions, such as activity losses and diffusional limitations, should be
taken into account in the design of immobilized enzymes [Cao, 2005]. For these reasons
different immobilization strategies have been set up so far. They can be divided into five
main groups based on the kind of interactions between the enzyme molecules and the solid
support, and specifically they are: adsorption, micro-encapsulation, entrapment, cross-linking
and covalent bonding [Hartmaier, 1988; Chibata, 1986, Katchalski-Katzir, 1993].
Even if many strategies are already available and have also found some applications at
the industrial scale, the search for innovative and more efficient approaches for the
immobilization of enzymes is still under progress [Cao, 2005]. One of the main limitation of
traditional approaches is the lack of the effective control of the immobilization process at the
molecular level. This lack of control results mainly in the formation of aggregates of enzyme
molecules, which have partly lost their active conformation, in which the active sites could be
not exposed to the substrate molecules and where the catalytic activity is lowered also by
mass transfer resistance. It is then evident how the development of immobilization techniques
able to control the positioning and the orientation of the enzyme molecules is required in
order to control finally the properties of the realized biocatalytic medium. In that sense,
nanotechnology appears to have a pivotal role in the development of the next generation of
biocatalysts.
Nanotechnology has found applications in almost all fields of research from aerospace to
electronics, biology, medicine and biotechnology. Recently in the field of biotechnology a
new area has appeared, which is nanoscale enzymatic biocatalysis [Wu, 2004]. The area of
enzymatic nano-biocatalysis comprises both the use of nanostructured materials as hosts for
enzyme immobilization [Kim, 2006b; Wang 2006], via approaches including adsorption,
covalent attachment and so on, and the fabrication of biocatalytic systems molecule-by-
molecule with a predetermined structure and, therefore, function [Huie, 2003; Schäffer,
2007]. This last approach to nanofabrication is the so called “bottom-up” one and is based on
the use of chemical or physical forces at the nanoscale level to assemble molecules, acting as
building blocks, into complex structures with a predetermined architecture and function
[Shimomura, 2001; Seeman, 2005; Zhou, 2005]. Extensive research has been performed on
the bottom-up approach in the last years and a number of bottom-up methods have been
developed also due to the appearance of techniques allowing a direct characterization at the
molecular scale.
Among the bottom-up nanofabrication methods, thin film techniques represent a
powerful tool for the assembling of molecules into highly ordered architectures [Ulman,
1991; Seeman, 2005]. In particular thin film techniques are well suited for the manipulation
of enzymes which display the capability to form highly packed assemblies and to generate 2D
and 3D structures. The properties of the developed structures can be tailored by incorporating
appropriate molecules and functional groups. There are different mechanisms by which thin
films can be accomplished, two of them are the Langmuir-Blodgett technique (LB) and the
layer-by-layer self assembly (LbL) technique. The LB technique, which was developed at the
beginning of the twentieth century [Blodgett, 1935; Blodgett, 1937], is based on the
formation of a closely packed monolayer at the air/water interface and on it subsequent
transfer onto the surface of a solid support. This technique was mainly developed for the
deposition of amphiphilic molecules. However, extensive work has been carried out on the
formation of thin films of biological interest including enzyme molecules for biocatalytic
purposes. The LbL technique, a more recently developed technique, is based on the alternate
assembly of oppositely charged polyelectrolytes for the deposition of complex multilayered
nanostructures [Iler, 1966; Decher, 1997]. This process is a very simple one and can be used
for the deposition of multilayered films onto supports of any shape with the precise control of
the structure and of the function. Enzyme molecules can be assembled using this technique
for the fabrication of biocatalytic media or can be entrapped into nanocapsules for the
fabrication of nanobioreactors. Variations of film composition and sequence of layer
alternation influence the properties of the biocatalytic film. Moreover sequential catalytic
reactions can be realized in multi-enzymes films. Enzymes immobilized in thin films have
shown to posses unique properties in terms of stability and functionality. Therefore, these
new types of biocatalytic systems would have a great impact on the industrial application of
enzymes.
The main aim of this chapter is to overview some of the more advanced and promising
nanotechnological methods for the immobilization of enzymes and specifically the
fabrication of organized thin film architectures by the LB technique and the LbL self
assembly technique. The current status and applicability of these methods in the biocatalysis
field, as well as prospective applications and developments are discussed.
The Langmuir-Blodgett Technique
LB Introduction
Langmuir-Blodgett (LB) technique is one of the most powerful tools for the realization
of functional organic structures with molecular resolution in one direction. In particular, it
was widely used for realization of layers with biological molecules. Lipids and lipid-like
molecules are traditional objects of the applicability of the method. Proteins were also
deposited using LB technique. However, in the case of proteins, the method not always can
be applied directly for the deposition of protein-containing layers and, therefore, must be
modified according to the nature of each particular protein. Strictly speaking, monolayers of
only membrane proteins can be directly formed at the air/water interface. For all other
proteins, including enzymes, special tools and approaches must be developed in order to form
functionally active molecules. Reviews on the approaches allowing to overcome the
difficulties can be found in [Erokhin, 2000; Erokhin, 2002].
Enzymes are proteins, whose function is to perform the activity of the extremely
effective biological catalysers. Therefore, mutual orientation of the active zones in the
enzyme molecule, responsible for the electron transfer from and to biological molecules
(substrates), involved into the reaction, is very important for their functional activity. This
feature is crucial considering the applicability of LB method for the enzyme layers
realization. Essential step of the LB technique is spreading and compression of the layer at
the air/water interface. This step is very important for all the protein monolayers in general,
and even more critical for enzymes in particular. Exposition to the action of the surface
tension of the pure water surface (72 mN/m) can result in the complete denaturation or at
least partial modification of tertiary and/or secondary structure of the enzyme. In fact, The
structure of the enzyme globule is stabilized by the molecular interactions within
hydrophobic nucleus of the protein, that is comparable with the surface tension. These
modifications of the protein structure can result in the irreversible decrease of the enzymatic
activity.
However, LB technique is still widely used for the working with enzyme molecules. It is
worth to mention that the very first work on the application of the LB technique for enzyme
layers formation was carried out in 1938 by I. Langmuir and V. Schaefer [Langmuir, 1938].
This work is well-known as it describe the first application of the horizontal deposition
method (Langmuir-Schaefer technique). However, we would like to recall the fact (rarely
mentioned now), that the work was performed on pepsin and urease monolayers.
20
Number of articles / year
18
16
14
12
10
8
6
4
2
0
1938 1948 1958 1968 1978 1988 1998 2008
Year
Figure 1. Number of articles on enzyme-containing Langmuir and LB films per year (the dependence was
averaged for 5 years).
Literature analysis allows to monitor the genesis of the activity in the field of the
application of LB technique to study properties of enzymes and/or deposition of enzyme-
containing layers. Averaged dependence of the number of articles concerning the enzyme-
containing Langmuir and LB layers on the year of publication is presented in Figure 1.
Averaging was performed for 5 years in order to smooth occasional scattering and to reveal
existing tendencies. Let us analyse the figure.
First publication of I. Langmuir and V. Schaefer had not attract the attention of
researched. Only one other article was published during almost 3 decades. However, at the
end of the sixties we can see an increasing activities in the field coming to the maximum at
the end of seventies. These activities can be connected to the fundamental investigations of
the action of enzymes on the model membranes. In fact, Langmuir monolayer at the air/water
interface can be consider as a model of the half of the biological membrane. Its composition
can be varied by the variation of spreading solution using natural mixtures of lipids and/or
mono- or multi-component solutions of different amphiphilic molecules. The state of the
model membrane can be also varied by changing its surface pressure, modelling, therefore,
liquid-expanded, liquid-condensed or solid regions of the biological membrane. Enzyme
molecules were placed into the subphase under the monolayer and its activity was analysed
by all available methods (in the early stages of the research, restricted number of techniques
was available; the most of works were performed by the analysis of the monolayer area and
surface potential variations). As it is practically always in the scientific activity, after the
peak we can observe some decrease of the activity coming to the steady state situation. LB
technique was found to be rather useful for the investigation and understanding of interaction
of enzyme molecules with model membranes and such kind of works are still performed by
different research groups.
At the end of eighties we can observe a significant increase of the activity in the field of
enzyme-containing LB films coming to the maximum in the beginning of nineties. This
period coincides perfectly with wide spreading of the ideas of molecular and bio-electronics,
as well as the beginning of the numerous works on the biosensors construction. This stage of
researches are mainly dedicated to the incorporation of enzymes in thin layers and their
transfer onto the surface of adequate transducer, capable to register the enzymatic reaction
and, therefore, the presence of the substrate molecules in the solution under investigation. As
in the previous case, some decrease of the activity was observed in the second part of nineties
and we can see currently rather steady state situation, indicating continuous interest of
research groups to the application of LB method for the construction of enzyme-containing
functional molecular structures.
The part of the work, dedicated to the Langmuir and LB films techniques for study the
activity and construction of enzyme-containing layers, will be organized in the following
manner. First, we will briefly present the basic principle of the LB technique in its classic
form. Then, we will illustrate the applicability of the technique for the investigation of the
interaction of enzymes with model membranes considering some examples, described in
literature. Third, we will describe several special modifications of LB technique, allowing
deposition of enzyme layers with preserved activity. It will be illustrated by some special
cases of different enzymes. We will also present some examples of biosensors, using enzyme-
containing LB layers as active sensitive elements. Finally, we will present a list of enzymes,
ever used for LB studies, in order to provide reference points for further researches.
Basic Principles of Langmuir-Blodgett Technique
Monolayers at the Air/Water Interface
Salts of fatty acids are “classic” objects of the LB technique [Blodgett, 1937; Gaines,
1956; Overbeck, 1993; Peterson, 1992]. General structure of fatty acid molecules is:
CH 3 (CH2 ) n COOH
Fatty acids, which form stable monolayers are stearic (n=16) one, arachidic (n=18) one,
and behenic (n=20) one.
Being place at the air/water interface, these molecules arrange themselves in such a way,
that its hydrophilic part (COOH) penetrates water due to its electrostatic interactions with
water molecules, which can be considered as electric dipoles or, more frequently, as electric
charge, as head-group can be in a dissociated form. Hydrophobic part (aliphatic chain) faces
itself to air, because it can not penetrate water due to entropy reasons. Therefore, if rather few
molecules of such type were placed to the water surface, they form 2-dimensional molecular
system at the air/water interface.
60
Surface pressure (mN/m)
50
40
30
20
10
0
0 10 20 30 40 50 60 70 80
Area per molecule A (A²)
Figure 2. π - A isotherm of stearic acid monolayer at the air-water interface.
Let us consider what will happen when the layer is compressed with some kind of
barrier. We will consider surface pressure as a parameter describing the monolayer state.
Surface pressure is determined as:
π = σ H O − σ ml
2
where σH 0 is the surface tension of the pure water and σml is the surface tension of the
2
monolayer covered water surface. In other words, surface pressure can be considered as the
decrease of the water surface tension due to the presence of the monolayer on it.
Compression isotherm of the stearic acid monolayer is presented in Figure 2. This
important characteristics represents the dependence of the surface pressure upon the area per
one molecule, obtained at constant temperature. Usually, this dependence is called π-A
isotherm. The measurements of the isotherm are practically always performed for studying
the behaviour and phase transitions of the monolayers at the air/water interface.
Let us consider the isotherm. Initially, the compression does not result in the surface
pressure variations. Molecules at the air/water interface are rather fare from each other and do
not interact. In some cases they also form domains, containing several molecules. This state
of the monolayer is refereed as “two-dimension gas”. Further compression results in the
increase of the surface pressure. Molecules come closer one to the other and begin to interact.
This state of the monolayer is referred as “two-dimensional liquid”. For some compounds it is
possible to distinguish also liquid-expanded and liquid-condensed phases. Continuation of
the compression results in the appearance of “two-dimensional solid state” phase,
characterized by the sharp increase in the surface pressure even for small decrease in the area
per molecule. Dense packing of molecules in the monolayer is reached in this case. Further
compression results in the collapse of the monolayer. 2-D structure does not exist anymore.
Not controllable multilayers are formed at the water surface.
Two instruments are usually considered for surface pressure measurements, namely,
Langmuir balance [Langmuir,1917] and Wilhelmy balance [Wilhelmy,1863]. Langmuir
balance measuring principle is the following. Barrier, separating the clean water surface from
that covered with the monolayer, is the sensitive element. Even if this balance measures
directly the surface pressure, it is not so frequently used. Mainly it is used when precise
measurements of the monolayer conditions at the air/water interface are studied, and it is
practically not used when the monolayer is supposed to be transferred onto solid substrates.
There are several reasons for its restricted applications. First of all, the utilization of the
Langmuir balance supposes that the compression of the monolayer is from one direction only.
This fact can result in the gradient of the monolayer density, what is absolutely undesirable in
particular cases. Second, the measurement of the surface pressure is performed in the other
point with respect to that, where the deposition takes place. This feature can result in the
weak control of the monolayer state during its transfer onto solid substrates. Third, there is
rather large area of the monolayer, which cannot be used. The first and the second drawbacks
are very critical when working with rigid monolayers. It was shown that it is possible to
observe a gradient of the monolayer density (and so the surface pressure), if the compression
is anisotropic one. The third drawback is very important when working with expansive
substances, such as biomolecules (including enzymes), as a significant part of the layer must
be wasted.
Wilhelmy balance had found more applications, even if it does not provide the direct
measurement of the surface pressure. However, it allows to avoid all 3 drawbacks of the
Langmuir balance mentioned above.
The sensitive element of the Wilhelmy balance is a plate (very often it is made from
paper). The measurement principle is illustrated in the Figure 3.
The forces acting to the plate are:
mg -weight of the plate;

FA - Archimedic force;
Fs - surface tension induced force.
The last force (FS) is just the product of the surface tension and the plate perimeter. As
the weight of the plate is constant, and the Wilhelmy balances now are equipped by systems,
maintaining the plate immersion depth into the water at the same level, providing, therefore,
the Archimedic force constant, it is possible to attribute the zero value to the clean water
surface, and the differences from this value will be directly the surface pressure.
The construction of the balance allows to perform measurements in the point exactly
corresponding to the deposition point with respect to the barrier position. It provides the
precise control of the surface pressure value, maintained by the feedback system. The other
advantage of the Wilhelmy balance is the possibility of compressing the monolayer from both
sides, performing better homogeneity of the monolayer.
Figure 3. Illustration of the working principle of the Wilhelmy balance.
Figure 4. Typical dependences of the surface pressure (a) and surface potential (b) on the barrier coordinate
for the monolayer at the air/water interface.
As it was mentioned above, an important aspect of the Wilhelmy balance utilization is

connected to the necessity of the maintenance constant of the plate position with respect to
the water surface level, in order to avoid the variations in the Archimedic force. Usually, it is
reached by special construction of the balance. Wilhelmy plate is connected to the magnet,
which is inserted into the solenoid. Variations in the surface pressure displace the magnet
position, and electronics provides the current in solenoid, which brings back the initial
magnet position. The position is usually controlled by optical methods. The value of the
current is proportional to the surface pressure value. Mentioned advantages allow to state that
the most of measurements of the surface pressure are currently performed with Wilhelmy
balance.
The other parameter, which can be controlled when working with monolayers at the
air/water interface, is the surface potential. The surface potential appearance results from the
orientation of molecular charges and dipoles during the compression of the monolayer.
Three different parts of the monolayer are usually considered for the surface potential
interpretation [Oliveira, 1992]. The first one is due to the orientation of the C-H bonds in the
hydrocarbon chains of the amphiphilic molecules during the monolayer formation. The
second one is connected to the regular arrangement of the polar head-groups.
And the third one is due to the dipol orientation of water molecules in the area just under
the monolayer. The relative input of each regions can be different and it is due to the nature
of the molecules forming the monolayer.
Typical dependence of the surface potential of the monolayer upon the barrier coordinate
is presented in Figure 4 [Yasutake, 1996] (the dependence of the surface pressure is presented
in the same figure for the comparison). It is interesting to note, that there are differences in
the behavior of the surface potential with respect to that of the surface pressure. The variation
of the surface potential begins much before than the surface pressure begins to increase
significantly. Such behavior is due to the fact, that molecules begin to aggregate, forming
dimers, trimers and small domains, at the initial stage of the monolayer formation. Being
aggregated, molecules tend to orient themselves in energetically adequate position, giving
rise to the variation in the surface potential. It happens when there is practically no increase
of the surface pressure. In the latter stage of the monolayer compression the variation of the
surface potential is mainly due to only the increase of the monolayer density.
Kelvin probe is the tool, which is usually used for the surface potential measurements
[Surplice, 1970; Yasutake, 1996; Di Natale, 1999]. The instrument is equipped with a
vibrating electrode, placed near the water surface. The reference electrode is inserted into the
water subphase. Vibrating electrode and water surface form a capacitor. Vibration of the
electrode provides the modulation of the capacity, resulting, therefore, in the appearance of
the alternating current proportional to the value of the surface potential.
The value of the surface potential of the monolayer can be both positive and negative and
the sign of the surface potential is determined by the nature of the molecules in the
monolayer.
Monolayer Transfer onto Solid Substrates
The floating monolayer can be transferred onto the surface of solid supports. Two main
techniques are usually considered for the monolayer deposition, namely, Langmuir-Blodgett
(or vertical lift) one [Blodgett, 1934] and Langmuir-Schaefer (or horizontal lift) one
[Langmuir, 1938].
The scheme of the Langmuir-Blodgett deposition is illustrated in Figure 5. Specially
prepared substrate with hydrophobic surface is passing vertically through the monolayer. The
monolayer is transferred onto the substrate surface during this passing. The important point of
such deposition is connected to the necessity of having the monolayer in the electrically
neutral state. If some charges in the monolayer molecule head-groups will be uncompensated,
the deposition will not be performed – electrostatic interaction of these charges with water
molecules will be higher with respect to the hydrophobic interactions of their chains with the
hydrophobized substrate surface. It will make impossible the monolayer transfer to the solid
substrates.
Let us consider again the monolayer of fatty acids in order to demonstrate the necessity
of the head group neutrality. If the monolayer is formed at the surface of distilled water (pH
is about 6.0) it cannot be transferred onto solid substrate. Its head group is dissociated and
contains negative charge (COO-). There are two ways to provide the possibility of deposition.
In the first case one must use LS technique (horizontal lift) and to provide the conditions
when all head groups of the fatty acid molecules are in the protonated (not dissociated) form
[Erokhin, 2000]. It requires the decrease of the pH of the subphase. In fact, the deposition
begins to take place when the pH value is less then 4.0 when there is practically no
dissociation.. However, the monolayer of pure fatty acids is very rigid and its transfer results
usually in defective LB films at solid substrates. Therefore, usually fatty acid salt monolayers
are deposited instead of pure fatty acids [Agarwal, 1973; Alekseev, 1987; Amador, 1998]. In
this case, bivalent metal ions are added into the water subphase. Normally, their
concentration is of the order of magnitude of 10-4 M. The metal ions attach themselves
electrostatically to the dissociated fatty acid head groups, providing their electric neutrality.
At the air/water interface, these ions are in a dynamic equilibrium with the fatty acid groups
in the monolayer. In the deposited layer, the bivalent metal ion coordinates 4 oxygen atoms in
two fatty acid molecules in adjescent monolayers [Erokhin, 1989]. This coordination is
illustrated schematically in the Figure 6. Metal atom is in the center of the tetraeder formed
by four oxygen atoms. Such coordination implies that the metal ions are bound to the fatty
acid molecules in the adjacent layers and their attachment, very likely, takes place when the
substrate pass through the meniscus during the upward motion.
The other method of the monolayer transfer from the air/water interface onto solid
substrates is illustrated in Figure 7. The method is called Langmuir-Schaefer (LS) technique
(horizontal lift). It was developed in 1938 by I. Langmuir and V. Schaefer for the deposition
of protein layers [Langmuir, 1938]. Specially prepared substrate touches horizontally the
monolayer, and the layer transfer itself onto its surface. The method is often used for the
deposition of rigid and protein monolayers. In both cases the application of Langmuir-
Blodgett method is not desirable as it results in the deposition of defective films.
Figure 5. Scheme of the LB deposition: downward (a) and upward (b) motion of the solid support through
the monolayer at the air/water interface.
Figure 6. Tetrahedral coordination of bivalent metal ions (central circle) by head-groups of adjacent
monolayers.
In the case of the application of LS method to rigid monolayers special cares must be
performed. The monolayer at the air/water interface must be divided into parts after reaching
the desired surface pressure. It must be done with a special grid with windows, corresponding
to the solid support sizes [Aktsipetrov, 1995]. The main reason of the utilization of the grid is
the following one. If the monolayer is rigid, the removal of some its part will result in the
formation of empty regions in the monolayer. As the layer is rigid, these empty zones will be
maintained for a very long time. Repeating of the deposition will result in the formation of
many defects in the monolayer, and the resulting transferred layer will be absolutely not
homogeneous. The use of the grid provides also the guaranty, that only one monolayer is
transferred during one touch.
In the case of proteins (mainly, membrane proteins) the monolayer is soft [Erokhin,
2002]. Therefore, the problems, mentioned above, do not exist in this case and the use of the
grid can be avoided. In fact, the monolayer structure in the case of protein layers is
practically amorphous, what is easy to reveal by Brewster angle microscopy – a powerful tool
for the monolayer domain structure visualization. Therefore, the removal of some monolayer
regions can be rapidly compensated by the feedback system without the loss of the monolayer
homogeneity. However, there is the other problem when applying the LS technique for the
protein monolayer transfer. The situation on the solid support after the touching of the
monolayer is schematically shown in the Figure 8. Regular closely packed monolayer is at

the surface of the solid support. Some amount of water, transferred together with the
monolayer, forms a drop at the substrate [Erokhin, 2000]. Some protein molecules can form
not regular layer at the top of this drop. If the sample will be dried in a usual way, these
molecules will form not homogeneous layer in not controllable way. Therefore, these
additional molecules must be removed before the sample drying. The effective way to realize
it is to use rather strong jet of inert gas, such as nitrogen or argon. It removes the water drop
together with randomly distributed protein molecules on its top, leaving only regular layer,
faced to the substrate surface.
Figure 7. Scheme of the LS deposition.
Figure 8. Scheme of the water and protein molecules distribution on the substrate surface after horizontal
touching of the protein monolayer.
According to the transfer procedure, deposited films are usually divided into 3 types,
schematically shown in the Figure 9, namely, X-, Y-, and Z-types [Roberts, 1990]. As it is
clear from the Figure 9, the Y-type is a centrosymmetric one, while X- and Z-types are polar
ones and differ one from the other by only the orientation of the head-groups and
hydrocarbon chains with respect to the substrate surface. Such division appeared due to the
fact, that in some cases there is no monolayer transfer during upward or downward motion of
the substrate in the case of LB deposition. In the case of the LS deposition, moreover, the
layers seem to be always transferred in a polar manner. However, the X- and Z-types are
practically never realized in practice [Lvov, 1986]. Even if some nonlinear properties, such as
pyroelectricity [Blinov, 1984], realizable only in polar structures, were observed, and the
structures were considered as polar ones, detailed investigations revealed that the films are of
Y-type with only not equal density of add and even layers [Roberts, 1989]. Therefore,
thermodynamically stable structures, that in a case of LB films are bilayers, must be realized.
Moreover, in the case of LS deposition of fatty acid films, it was shown that the last 3
transferred monolayers are involved into the structural reorganization during the meniscus
passing, in order to realize thermodynamically stable Y-type packing [Kato, 1987]. This
reorganization provides the orientation of the hydrocarbon chains to the air in the last
deposited monolayer.
Figure 9. Types of the structures of LB films.
Figure 10. Fromherz trough. Circular trough is separated into sections (a). Monolayer formation and
interactions with enzyme take place in different sections. The monolayer can be transferred from one section
to the other by simultaneous motion of both barriers in the same direction due to the deformation of water
subphase meniscus during passing the section walls.
Generally, packing of amphiphiles in LB films is determined by the head-to-head and

tail-to-tail packing. The tilting angle of the hydrophobic chains with respect to the film plane
can be different, depending on the size of the head-group, but it must provide the close
packing of the chains. However, several examples of the other packing with interdigitation of
the hydrocarbon chains of adjescent layers were observed [Erokhin, 1989].
Significant increase of the interest to LB method appeared after the works published by
the H. Kuhn group [Kuhn, 1970; 1972; 1979]. They had shown the possibility to realize
molecular architecture with this method. Complicated structures with alternation of layers of
different molecules were realized. Such systems were used for studying the energy transfer
processes. Layers of donors and acceptors were separated by spacer layers of different
thickness.
Lb Films of Enzymes: Fromherz Trough
Application of the LB technique for the deposition of enzyme films meets the same
difficulties as for the most of other proteins [Erokhin, 2002]. These difficulties are resulted
from the organization of such objects. Practically all proteins (except membrane ones),
including enzymes, have the most of charged groups at their surface, while hydrophobic
groups are in their center maintaining the globular tertiary structure. Therefore, it is difficult
to suppose that such molecule will remain in the native form at the air/water interface.
Observed surface activity of protein molecules in the most of cases is mainly due to the
complete or partial denaturation of the protein molecule. Hydrophobic interactions,
maintaining the protein globule structure, are of the same order of magnitude as the action of
the surface tension (72 mN/m for pure water). Thus, arriving to the air/water interface,
protein molecules (including enzymes) must rearrange their structure. Hydrophobic groups
will be oriented towards air, while charged groups will remain in water. Native protein
globular structure is lost. Especially for enzyme molecules, such reorganization of the protein
globule is very critical for the functioning. In fact, enzyme can be considered as an extremely
effective catalyser and mutual orientation of specific groups, responsible for the improved
electron transfer between involved reagents (substrates), is a key parameter. Even partial
denaturation will result in the complete lost of the enzymatic activity.
Above considerations have demonstrated the necessity of the modification of the
traditional LB technique if we want to deposit enzyme-containing layers. Such modifications
were developed basing mainly on the formation of complex layers, including lipid (or other
surfactant) monolayers with attached enzyme layer. In this case the lipid layer is formed at
the air/water interface. Its presence decrease the surface tension for the value of the reached
surface pressure. Thus, enzyme molecules will be much less affected by the external forces
when arriving to the air/water interface. However, in order to provide a stable complex
between lipid monolayer and enzyme molecules, we need to involve some forces of
interactions. The most frequently used interactions have the electrostatic nature [Peschke,
1987; Pachence, 1991; Edmiston, 1998]. Similarly to the case of LbL deposition, one need to
determine the isoelectric point of the enzyme that is planed to be deposited. All the further
manipulations will be performed at the pH, when the enzyme is charged and to find lipid
molecule, capable to form stable monolayers and having an opposite charge in the head-
group at the chosen pH value. Thus, the procedure must be the following one. Lipid solution
must be spread at the air/water interface and the monolayer must be compressed till the target
surface pressure. After equilibrating, enzyme solution must be injected under the monolayer.
It is important to inject the enzyme solution after the monolayer is already formed, otherwise
enzyme molecules can be denatured during the contact with the pure water surface.
Incubation time can vary from some minutes till some hours depending on the particular
enzyme and lipid molecules as well as on the composition and pH of the subphase. The
attachment of the enzyme molecules can be revealed by the variation of the surface pressure
(if the feedback is switched off) or surface area, covered by the monolayer (if the feedback is
switched on) as well as by measuring of the monolayer surface potential. After the
incubation, such layer can be transferred onto solid supports using generally Langmuir-
Shaefer technique (horizontal lift).
A special device was designed for such purposes. It was developed by Fromherz and was
called Fromherz trough [Fromherz, 1971; Fromherz, 1975]. The scheme of the device is
shown in Figure 10.
Usually, it has a circular geometry and is divided in several sections as it is shown in the
figure 10a. Compression is provided by two barriers that are radii of the circular trough and
move towards each other. Each section must have its own surface pressure sensor. Let us
consider briefly the operation principles of this device. The formation of the lipid monolayer
takes place in the section 1. The subphase can contain some additions (usually, salts), suitable
to the formation of the stable layer. When the target pressure is reached and the monolayer is
equilibrated, it must be transferred to the section 2 with the composition and pH facilitating
the detachment of ions, attached during the previous stage, suitable for the stable monolayer
formation but not useful for the interactions with the enzyme molecules. We can call this
operation “two-dimensional washing”. The transfer of the monolayer from one section to the
other is performed by the simultaneous motion of both barriers in the same direction. Then,
the monolayer must be transferred to the third section, already containing enzyme molecules
in the subphase volume. Adsorption of enzyme molecules onto the lipid head-groups takes
place in this section. Composition and pH of the subphase must provide the most effective
interactions. When the enzyme layer is formed, the complex layer will be transferred to the
section 4 where the subphase is suitable to the layer transfer to the solid supports.
In principle, it is possible to use not only electrostatic interactions, but some specific
recognition [Uzgiris, 1987; Morgan, 1992]. In this case, the lipid molecules must have some
groups providing high affinity to the other groups, already present or attached to the enzyme
molecule surface. Of course, this approach is more complicated and expensive, as it demands
synthesis stages. However, it can provide a desirable orientation of the enzyme molecules
towards the analysed solution.
Special Modifications of LB Technique: Sliding Plate Method
One other technical decision, suggested by V. Troitsky, allows to deposit enzyme-

containing layers [Troitsky, 1996]. The method combines the LB deposition and self-
assembling. Important feature of the method is a special construction of the sample-holder.

Adsorption of the enzyme molecules can be performed on the lipid monolayer head-groups
not only at the air/water interface, but also at solid supports. However, the substrate with
deposited lipid layer must be transferred to the reaction medium (enzyme solution) without
its exposition to air, otherwise not controllable reorientation of lipid molecules can take
place. In fact, as it was demonstrated, last 3 monolayers of fatty acids reorient themselves by
flip-flop transitions during the deposition by Langmuir-Shaefer technique (horizontal lift).
Second important remark is connected to the necessity to avoid the contact of the enzyme
molecules with air, what can result in the degradation of their properties. The developed
construction of the sample holder allows to overcome these difficulties.
The scheme of the sample-holder (sliding plate sample-holder) and principles of its
functioning are shown in Figure 11. An essential part of the sample-holder is the presence of
the sliding plate in the vicinity of the sample surface (about 1 mm). The plate is realized from
hydrophilic material. It can be in two positions. In the “open” position the sample surface is
exposed to the environmental medium. In the “close” position, water can be kept between the
sample and the sliding plate by capillary forces. Figure 11 illustrates the deposition procedure
realized by the described device [Pastorino, 2
002]. Sample with hydrophobic surface is attached to the sample-holder with sliding
plate in “open” position. It pass through the already formed lipid monolayer at the air/water
interface and the monolayer is transferred onto its surface with head-groups oriented to the
water volume. In the bottom position, the sliding plate is transferred into the “close” state
(Figure 11a). Thus, during the upward motion nothing will be transferred onto the sample
surface and the water between the sample and the sliding plate will prevent the molecules in
the monolayer from reorientation (Figure 11b). Then, the sample is transferred to the volume
containing enzyme solution. The sliding plate is transferred to the “open” state and the
adsorption of the enzymes takes place (Figure 11c). Similarly to the previous case, the
enzyme-lipid interactions can have an electrostatic origin or can be based on high affinity of
specific molecular groups. After the enzyme layer formation, the sliding plate is transferred to
the “close” state and the sample is passed to the Langmuir trough. Still maintaining the plate
in the “closed” state, the sample must go through the lipid monolayer till the bottom position.
In this position, the sliding plate is transferred into the “open” state and the sample passes
through the lipid monolayer during upward motion. Transferred lipid layer provide the
protection of the enzyme molecules from the exposition to the air (Figure 11d). Figure 11e
demonstrates the motion of the sliding plate sample-holder between different sections of the
equipment. The effectiveness of the method was initially demonstrated for the layers
containing cytochrome P450 [Troitsky, 1996]. Then, using electrostatic interactions the
method was successfully applied for the deposition of enzyme-containing films, in particular,
films containing penicillin G acylase [Pastorino, 2002]. It is to mention, that the temporal
stability of enzymes in such protected layers was shown to be significantly higher with
respect to the enzymes in solution and in films prepared by traditional immobilization
techniques.
Figure 11. Deposition method using sliding plate sample-holder. Figures 11a-11d represent different stages
of the enzyme-containing structure formation, while Figure 11e shows the motion of the sample-holder
between different sections of the deposition equipment.
Langmuir Monolayers for Study of Enzyme-Membrane Interactions
Monolayer at the air/water interface can be considered as a half of a model biological

membrane. Its composition can be varied allowing to model different situations. Moreover,
variation of the surface pressure can allow to identify different membrane regions that are
more affected to the enzyme activity actions.
Different measurements can be performed during such investigations. Practically in all
cases when the interaction of enzymes with Langmuir monolayers is considered,
measurements of the variation of the surface pressure and/or monolayer area are carried out.
Usually, the monolayer of a desirable composition (containing natural or synthetic lipids) is
spread at the air/water interface and compressed till the target pressure. Then enzyme solution
must be added to the water subphase. Measurements can be performed when the feedback,
maintaining the fixed surface pressure, is switched on or off. In the case of the switched on
feedback, one will observe the variation of the monolayer area during the interaction. Two
distinct situations of the enzyme effect on the monolayer can occur. They are schematically
illustrated by the Figure 12.
Initial organization of the lipid monolayer at the air/water interface is shown in Figure
12a. In the case of the increase of the monolayer area (Figure 12b), one can conclude that the
enzyme is attached to the monolayer and, in some cases, can even partially penetrate into it.
Instead, it is possible to observe also the decrease of the monolayer area. It can mean that the
enzyme action on the model membrane result in the formation of local collapses and/or lipid
micelles, that then leave the air/water interface and penetrate the aqueous subphase. This
situation is illustrated by the Figure 12c. Of course, more precise interpretation of what really
happened will demand additional investigations with other, more powerful techniques. Some
of such useful technique will be mentioned below. However, even the information on the
monolayer area variation can be very useful and can allow even quantitative estimation of the
process. In the case of the switched off feedback, the monolayer area is fixed and one can
register the surface pressure variation during the enzyme-model membrane interactions. In
principle, the information obtained during such investigations is comparable with that in the
case of switched on feedback. However, the difference is that in the case of “feedback on”
situation the state of monolayer is maintained constant during the interactions, while in the
“feedback off” case the variation of the surface pressure can result in the phase transitions of
the monolayers state (for example, from liquid expanded to liquid condensed). Therefore, the
interactions of enzyme molecules with the monolayer can be varied from the beginning till
the end of the process.
The other possibility to study these interactions is to measure the surface potential
variations. As it was mentioned above, the measurements of the surface potential is usually
performed with the Kelving probe and can provide the information of the charges and dipoles
distribution across the monolayer. Therefore, in some cases the variation of the surface
potential can indicate the attachment of the enzyme to the monolayer (as it contains its own
charges and dipoles) as well as reorganization and reorientation of the molecules in the
monolayer. In this last case the conformational changes of the lipid head-groups will be more
evident as their polar nature result in a significant contribution to the total value of the
surface potential.
Considering the morphology of the monolayer and its variation during the interactions
with enzyme molecules in the subphase, two types of microscopies are widely utilized.
Fluorescence microscopy [Lösche, 1984; Chi, 1987; Schwartz, 1993] is based on the
addition of the fluorescently labelled probes to the system under investigation. The system in
this case must be illuminated with the light of the wavelength, corresponding to the
fluorescence excitation. Imaging must be performed after filtering of the exciting light. These
dye molecules can be added to the spreading solution of lipids. During the monolayer
compression, the dye molecules will not be able to penetrate regions with lipid molecules
close packing, visualizing therefore the domain structure of the model membrane. The
domain sizes and shape can be varied during the interaction with enzymes and these
morphology variations can be easily visualized even in a real time. The other possibility is to
work with a monolayer without dye addition, but to use fluorescently labelled enzymes. In
this case it will be possible to determine and to localize the interaction of the enzymes with
the monolayer registering the appearance of the fluorescence.
Figure 12. Lipid monolayer at the air/water interface (a). The interaction with enzyme molecules in the water
subphase can result in the protein adsorption at the lipid head-groups and even in their partial penetration
into the monolayer and, therefore, in the increase of the monolayer area at the given value of the surface
pressure, maintained by the feedback system (b). In other cases, interactions with enzymes can result in the
local monolayer collapses and partial transfer of lipid molecules into the volume of the water subphase in the
form of micelles (c).
Second type of the microscopy, widely used for the monolayer investigations, is called
Brewster angle microscopy (BAM) [Overbeck, 1994; Tsao, 1995; Angelova, 1995]. It is
based on the fact that polarized light, incident to the interface at the Brewster angle, is not
reflected. Thus, BAM instrument is equipped by goniometric system with attached laser and
CCD device, allowing to vary angle of incidence and, accordingly, reflectance. Usually, the
incidence angle corresponds to the Brewster angle for the air/water interface. Thus, the pure
water surface will be seen as the dark field in the acquired image. The presence of the
monolayer will vary Brewster conditions for both water/monolayer and monolayer/air
interfaces resulting, therefore, in the appearance of bright areas in the image, corresponding
to the position of monolayer domains. It is clear that this technique can be also used for the
study of interaction of enzymes with model membrane, as it will allow to visualize the
variation of the domain sizes and shape during such interactions. In addition, BAM has one
significant advantage with respect to the fluorescence microscopy. In the case of BAM we do
not need to add any dye or other molecules into the system under investigation. In other
words, the system in the case of BAM is much less affected by the artefacts added for the
visualization possibility in the case of fluorescence microscopy.
Other technique that is used for the monolayer study and can be useful for the
investigation of the interaction of enzymes with model membranes is ellipsometry [Feachem,
1934; den Engelsen, 1974; Ducharme, 1985; Kim, 1990]. The variation of the polarization
state of light reflected from the interface can give the information about the variation of
thickness and refractive index of the thin layer at the interface.
However, it seems even more effective to use X-ray reflectivity measurements for these
reasons [Helm, 1987; Kjaer, 1988; Majewski, 1998; Kaganer, 1999]. Modelling of the
experimental curves can give the information not only about the thickness of the layer, but
even to reconstruct the electron density profile in the direction normal to the monolayer
plane. In fact, in the case of the monolayer interaction with DNA it has allowed to register
not only the fact of the DNA adsorption, but also the state of DNA in complexes with
different lipids [Erokhina, 2007; Cristofolini, 2007]. In the case of interactions with enzymes,
such measurements can help in more precise localization of the enzymes in model
membranes during the interaction processes. Effective utilization of the X-ray reflectivity
measurements demand the use of synchrotron radiation as it provides high signal-to-noise
ratio, necessary for the reliable model construction. As the most serious drawback of the
method, we can mention that it is rather time consuming and does not allow real time
monitoring of the process. However, current works in the field, especially based on the
analysis of the diffuse scattering allow to forecast that in the nearest future these limitation
will be successfully overcame.
Of course, as more techniques are available for the characterization, as better and
precisely the interaction process will be described.
Just to illustrate the previous considerations, let us consider some specific example of the
application of the monolayer technique for the investigation of the interactions of enzymes
with model membranes. Historically, these examples will correspond to the first peak of the
activity, shown in Figure 1, when the effectiveness of such studies was well demonstrated.
The most of the current studies are based on the same approaches and assumptions, as it was
30 years ago, with the only difference that much more powerful characterization techniques
are available.
Investigation of the hydrolysis of the lecithin monolayer by Crotalus adamanteus α-
phospholipase A2 had allowed to demonstrate that the active dimeric form of the enzyme is
in equilibrium with inactive subunit. The study had also demonstrated that the calcium ions
presence is required for the surface reaction. Thus, authors have demonstrated the
applicability of the monolayer technique for studying lipid-enzyme interactions [Lagocki,
1970].
In the other study similar system, containing lecithin monolayer at the air/water interface
and phospholipase A in the subphase, was investigated in order to identify the relative
position and orientation of the hydrophilic groups of the various membrane constituents
[Colacicco, 1971]. These work has two essential features. First, it was explicitly shown that
the measurement of the surface potential can provide a very important information about the
interaction of lipid molecules in the monolayer with enzymes in the water subphase. Second,
it was demonstrated that the kinetics of the interaction processes depends on the initial
surface pressure of the monolayer and, therefore, on the state of the model membrane.
Enzyme-Containing LB Films as Sensitive Layers of Biosensors
We have attributed the second increase of the activities in the field of enzyme-containing
LB films (Figure 1) to the possibility of using these layers for molecular and biomolecular
electronics and as sensitive elements of enzymatic biosensors. Several specific features of the
deposition method have attracted the increased attention as practically no other method can
provide similar possibilities. First of all, in the case of the LB technique we can form a film,
containing only one molecular layer of the enzyme. This feature can be very important for the
industrial applications when we have to work with expensive enzymes. Second, protection of
the enzyme sublayer by its incorporation into the lipid layers can, as we have discussed
above, increase significantly its temporal stability, preventing the contact with undesirable
surrounding containing polutants. Third, the LB method allows to realize complex structures
where enzyme layers can be included into the structures, containing also layers of conducting
molecules that can be considered as mediators, facilitating electron transfer between the
enzymes, substrates and products. In the most of cases, conducting polymers, such as
polyaniline, polythiophene, etc., are used for these purposes.
Let us consider particular examples of the utilization of the enzyme-containing LB films
as the sensitive layer of biosensors. Glucose oxidase (GOD) is probably the mostly used
enzyme as its practical usefulness is obvious [Okawa, 1989; Rosilio, 1997; Anicet, 1998].
The working principle of the biosensors for the glucose detection is based on the following
reaction:
GOD
GLUCOSE GLUCONIC ACID + H2O2
Thus, sensitive GOD layer must be deposited onto the working electrode. The sensor
chamber must contain also reference electrode, that is usually realized from Ag-AgCl
material. It must also contain counter electrode, usually realized from Pt, that must provide
electrical carriers necessary for the maintaining the potential difference between reference
and working electrodes and not to be involved directly to the electrochemical reactions.
Therefore, the current in the measuring circuit will be proportional to the concentration of the
hydrogen peroxide and, therefore, to the concentration of the glucose in the solution under
the investigation.
We must also consider several possibilities that can improve the performance of the
sensitive enzymatic layer and, therefore, the performance of entire biosensor.
First of all, the GOD layer is deposited always together with the surfactant monolayer.
For some particular reasons, easiness of the glucose access, for example, the presence of this
surfactant layer can be undesirable. It was shown the possibility to remove such layer after
the active layer formation practically without the damage of the enzyme properties in the
active sensitive layer. In particular, when the GOD layer was transferred together with
behenic acid monolayer, the removal of the last was possible by isopropanol treatment of the
complex layer. It is to note, that such selective removal of one compound of the formed
complex layers is rather well-known and was used for the removal of fatty acid molecules
from the layers leaving only fatty acid salt molecules at the solid support surface. This
process was called skeletonization process. It is very interesting itself, as it allows to form the
layer with desirable porosity. In fact, the ratio of the fatty acid – fatty acid salt molecules in
the layer can be easy controlled by the pH variation. After the layer deposition, this ratio is
preserved in the film. Organic solvent treatment will remove only soluble counterpart (acid)
maintaining, however, the overall structure of the film. Therefore, desirable fraction of
cavities will be realized. Other example of the selective removal of one compound from the
complex layer is connected to the formation of aggregated inorganic nanoparticulate layers
starting from LB organic precursors. As it was shown, the method allows to remove organic
molecules selectively from the layer, leaving on the support surface only ultrathin inorganic
films [Facci, 1994; Erokhina, 2002]. The accuracy of the film thickness realization in this
case can reach the value of about 0.5 nm what is comparable with the most developed,
complicated and expansive techniques, available now, such as molecular beam epitaxy.
As it was mentioned above, the method allows also to deposit complex layers, where
GOD can be attached to conducting polymer sublayers instead of traditional lipids or
surfactants. Such complex layers were shown to exhibit the improved properties when they
are used as sensitive layers.
Finally, let us present an overview of the enzymes involved into investigations using
Langmuir and Langmuir-Blodgett films.
About 200 works were published where different lipases were involved into the study.
The fact does not seem strange if we consider the membrane-active properties of these
enzymes. Here we present references to only some representative works with these enzymes
(Lagocki, 1970; Colacicco, 1971; Shen, 1975; Bianco, 1989; Davies, 1991; Ivanova, 1993;
Mirsky, 1994; Grandbois, 1999; Estrela-Lopis, 2001; Jensen, 2002; Nielsen, 2002; Pastorino,
2002; Wang, 2005).
Glucose oxidase is the other enzyme widely studied applying Langmuir technique. About
50 articles were published on this object (Okahata, 1989; Sun, 1991; Lee, 1993; Zaitsev,
1993; Rinuy, 1999; Zhang, 2000; Zayats, 2002; Watanabe, 2005; Lee, 2007). Rather low cost
and obvious usefulness of this enzyme for sensor applications are main reasons for such
activities.
Other relatively widely studied enzymes are peroxidases (Chasovnikova, 1992; Razumas,
1996; Berzina, 1998; Tang, 2002; Morandat, 2004), urease (Langmuir, 1938; Gidalevitz,
1999; Hou, 2002; Singhal, 2002) and acetylcholinesterase (Choi, 1997; Dziri, 1999; Choi,
2001; Wang, 2006).
Other enzymes to whome Langmuir technique was applied are: ATPase (Babakov, 1979;
Prokop, 1995; Wu, 2001), luciferase (Marron-Brignone, 1996; Marron-Brignone, 2000;
Palomba, 2006), cytochrome c oxidase (Tredgold, 1979; Salamon, 1993; Cullison, 1994),
glutamate dehydrogenase (Hourdou, 1995; Girard-Egrot, 1997), hydrogenase (Nakamura,
1998; Noda, 1998; Qian, 2002), catalase (Nitsch, 1990; Maksymiw, 1991), cholesterol
oxidase (Slotte, 1992; Slotte, 1993), choline oxidase (Girard-Egrot, 1997; Girard-Egrot,
1998), cholinesterase (Danilov, 1975; Godoy, 2005), monoamine oxidase (Barmin, 1994),
tyrosine hydroxylas (Eremenko, 1991), protease (Gole, 2000; Vinod, 2007), alkaline
phosphatase (Petrigliano, 1996; Giocondi, 2007), alcohol dehydrogenase (Pal, 1994), alpha-
chymotripsin (Anzai, 1989), anhydrolase (Mello, 2001, Mello, 2003), cutinase (Ivanova,
2003), DNA polymerase (Mizushina, 2000; Matsuno, 2001), RNA polymerase (Rajdev,
2007), DNA gyrase (Lebeau, 1990; Lebeau, 1992), glutathione S-transferase (Paddeu, 1996),
penicillin G acylase (Pastorino, 2002), sphingomyelinase (Harte, 2005; Rao, 2005), xylanase
(George, 2002), endonuclease (Matsuo, 2005), glycosyltransferase (Nagahori, 2003),
hexokinase (Castro, 2007), human erythrocyte catalase (Harris, 1995), malate dehydrogenase
(Peters, 1975), nuclease (Inbar, 1976), phytase (Caseli, 2006), saccharase (Sobotka, 1941),
transglutaminase (Faergemand, 1997), trypsin (Fromhertz, 1975), xanthine oxidase
(Kristensen, 1998).
The Electrostatic Layer by

Layer Self-assembly Technique
General Principles
The LbL technique was firstly introduced by Iler in 1966 for the alternate assembly of
oppositely charged layers of colloidal particles, such as silica and alumina [Iler, 1966]. In the
1990’s Decher and co-workers reported the fabrication of multicomposite films of charged
materials trough LbL adsorption from aqueous solutions [Decher, 1991; Knoll, 1996; Decher,
1997]. Since then, extensive work has been carried out on the application of this technique to
the fabrication of multilayered ultrathin films incorporating a broad range of charged
macromolecules including synthetic polyions, biopolymers, viruses, ceramics and
nanoparticles [Lvov, 1995; Ariga, 1997; Caruso, 1997; Lvov, 2000; Salditt, 2002].
Figure 13. Scheme of the LbL deposition procedure, based on the successive adsorption of polyanion and
polycation layers on the solid supports.
The general principle of the LbL technique is very simple and, as already mentioned, is
based on the alternate adsorption of oppositely charged species on the surface of a charged
solid support. Figure 13 schematically describes the assembly procedure.
In the figure a negatively charged support is dipped into a diluted aqueous

solution/dispersion of a cationic specie for a fixed period of time, which has to be
experimentally determined in order to reverse the charge of the support surface. As a result, a
thin layer of the cationic specie is formed on the support, thereby generating a positively
charged surface. The positively charged support is then rinsed in water, in order to remove
the unbound material, and dipped into a diluted aqueous solution/dispersion of an anionic
specie for a given period of time. The rinsing step is repeated and the support, which is now
negatively charged can be used again for the assembly of a successive positively charged
layer. By repeating the described steps, a stable and uniform multilayered structure can be
deposited with a desired thickness ranging from few nanometers to 10 μm, with precision
better than 1 nm and with a predetermined structure and function. The driving force of the
assembly process is the electrostatic one, but other interactions can be utilized such as
covalent bonding [Schultz, 2005; Bai 2006; Lu, 2007] and biomolecular recognition [Sano,
1996; Cassier, 1998; Dai, 2007;]. The physico-chemical properties of the multilayer
architectures can be modified by varying the deposition conditions, such as pH and ionic
strength of the solutions [Voigt, 2003; Schonhoff, 2003]. Finally different charged species
can be utilized in the same assembly for the fabrication of complex structures.
What makes this technique so promising is that it does not require the use of complex
equipments, as the assembly in the laboratory is carried out in beakers, the use of harmful
regents, as the assembly is carried out from aqueous solution and finally the assembly is not
limited to flat surfaces but it can be carried out onto 3D supports of any shape.
The above considerations marks clearly the LbL technique as a powerful tool also for
processes to be conducted at the industrial scale considering that the simplicity of the
technique implies that no complex industrial plant would be required, that this technique can
be regarded as environmentally clear and that its versatility guarantees applications in a wide
range of technological fields.
LbL Films of Enzymes: Structure and Properties
Assembly Procedure on Planar Supports
Multilayers containing biomolecules, including enzymes have been fabricated by means

of the LbL technique and their functionality has been characterized [Lvov, 2000; Lvov, 2002;
Lvov, 2003]. The first publication on enzyme LbL multilayer formation was published by
Lvov et al in 1995 [Lvov, 1995] and assembly of cytochrome, myoglobin, glucose oxidase
and peroxidase in alternation with oppositely charged linear polyelectrolytes and nanoclay
was described. Proteins in the LbL multilayer preserved their conformation and enzymatic
activity. Regular multilayers of different proteins were also prepared forming by this protein
supperlattices.
The starting point in the set up of an assembly protocol for the use of each particular
protein, is represented by the determination of its isoelectric point. The working pH of all the
solutions has to be set apart from this value so that the protein molecules are sufficiently
positively or negatively charged. It is interesting to note how the same protein can be utilized
either as a positively or negatively charged specie [Liang, 2005]. When a decision has been
made on the charge to be adopted for the protein molecules, the polyions to be used in the
assembly process can be defined and thus the architecture of the multilayered structure. For a
successful assembly of proteins, it is important to alternate the protein layers with linear or
branched polyions as it has been demonstrated that flexible polyions penetrate the protein
layer acting as an “electrostatic glue” [Lvov, 2000].
The following steps can be individuated in the fabrication of a protein containing
multilayer on the surface of a planar support:
(1) prepare the protein and polyions solutions at a concentration between 0.1 and 1
mg/ml and between 1 and 2 mg/ml respectively at the established pH (1 or 2 units below or
above the isoelectric point of the protein under consideration). When working with expensive
proteins it is important to use as less material as possible in a single experiment, in this case
the protein solution concentration can be kept at lower values and the adsorption time
consequently has to be longer; (2) prior to start the protein multilayer fabrication, take your
planar support having a charged surface (e.g. silicon, quartz, glass slides) and deposit on it
minimum three polyion layers in order to provide an uniform surface with a well defined
charge. It has been demonstrated that precursor films are necessary to provide liner mass
increase for the following layers [Lvov, 1995]. Let’s consider as an example the case of a
negatively charged support. During the first step it is dipped into a water solution of a
polycation, such as poly(dimethyldiallylammonium chloride) (PDDA), at a concentration of 2
mg/ml for 10 min and then it is rinsed for 1 minute using a buffer solution at the same pH of
the solutions used in the assembly in order to keep the polyions and protein ionised. The
same procedure is then repeated for a polyanion solution, such as poly(styrenesulfonate)
(PSS). Repeat the alternate adsorption of PDDA and PSS in order to deposit a two bilayer
precursor. For the subsequent protein adsorption, the precursor will have PDDA or PSS as
outmost layer depending on the charge of the protein. (3) the modified support bearing the
right charge for protein adsorption is now dipped in the protein solution for a time between
10-60 min depending on the protein concentration [Caruso, 1997; Brynda, 2005; Lu, 2007].
As proteins generally are not stable at ambient temperature, it is recommendable to carry out
the protein adsorption step at 4 °C. After rising the sample, (4) it is dipped in the solution of
the oppositely charged polyion for further deposition. The steps can be repeated for the
deposition of the desired multilayered structure. The final structure is then stored in buffer
solution preferably at 4 °C. This is a general procedure which applies to the fabrication of
protein containing multilayers and which has to be experimentally tailored to the specific
molecules and biomolecules under the use.
Structure Characterization of LbL Films on Planar Supports
The progressive build-up of the multilayered films onto a planar support can be
monitored using different well-established techniques such as UV-Vis spectroscopy, quartz
crystal microbalance (QCM) and surface plasmon resonance (SPR).
The easiest way to characterize the deposition process is represented by the employment
of UV-Vis spectroscopy. In this case, UV-Vis absorption spectra of the film deposited onto a
quartz slide are recorded after each layer or bilayer deposition. The multilayer growth is then
monitored by the enhancement of the absorbance peak intensities of the deposited species.
Typically proteins display an absorbance peak around 280 nm, a linear increase of the
adsorbance at this wavelength, plotted against the number of deposited layers, would confirm
a proper and uniform deposition.
Another useful technique, which does not require expensive and complicated
instruments, is QCM.
QCM is based on the properties of piezoelectric quartz crystals to vary frequency of their
resonant oscillations according to the state of the resonator surface. The
deposition/adsorption of molecules onto the surface of the quartz determines a decrease in the
oscillating frequency. A direct relation between the frequency shift and the deposited mass
can be obtained using the Sauerbrey equation [Sauerbrey, 1959; Facci, 1993]:
Δf = - Δm x C
where
C = 2f02 / A(ρqμq)½
Δf = measured frequency shift,
f0 = resonant frequency of the fundamental mode of the crystal,
Δm = mass change per unit area (g/cm2),
A = piezo-electrically active area,
ρq = density of quartz
μq = shear modulus of quartz
The Sauerbrey equation can also be expressed as:
ΔL = -Δf / Cρ
where
ρ = density of the deposited material.
The density of the protein / polyion film has been found to be approximately 1.3 g/cm3
[Lvov, 1995]. QCM is a very sensitive tool to detect changes in mass and thus a helpful
method to sense adsorption processes both at solid/gas or solid/liquid interfaces.
For measurements at solid/gas interface, the assembly process is carried out by dipping
the quartz crystal in the adsorption solution for the given amount of time then, after the rising
step, the quartz crystal is dried in a nitrogen stream and the resonance frequency shift is
measured. For measurements at the solid/liquid interface, only one electrode is usually used.
In this case the quartz crystal is mounted in a flow chamber where one electrode is placed in a
permanent contact with the adsorption solutions while the electrode on the other side is
maintained in the air. In the case of the necessity of the utilization of both oscillator
electrodes, each of them must be placed in separate reaction chambers, avoiding possible
Exploring the Variety of Random
Documents with Different Content
and Loli, and Kohenemonemo Loli, ame ka laua wahine o
the wife of the two men. Kohenemonemo.
When Lonoikamakahiki was I ko Lonoikamakahiki wa

quite young, when he was just opiopio, oiai ua hoomaka ae
about beginning to reason for kona noonoo ana, ia manawa
himself, he looked up one day nana ae la o Lonoikamakahiki, e
and saw the various implements kau ana na mea lealea a kona
used by his father in the different makuakane he nui maloko o ka
games, which were hanging up hale alii; a ike ae la oia e kau
in the palace; when he saw the ana na ihe-pahee, nana loihi ae
long spear used in the game of la oia, a liuliu, alaila, ninau aku la
pahee 3 he looked at it for a long oia i kona mau kahu: “Heaha
time and then asked his keia mau mea loloa e kau nei
retainers: “What are those long iluna o ka hale?” I aku la na
things hanging up there on the kahu: “He ihe-pahee.” Ninau hou
side of the house?” The retainers aku la o Lonoikamakahiki:
replied: “They are pahee “Heaha kana waiwai?” Alaila hai
spears.” Lonoikamakahiki again aku la na kahu: “Elua mau
asked them: “What are they kanaka e manao ana e lealea pili
used for?” The retainers then waiwai, alaila hele laua a ma ke
told him: “When two men wish to kahua pahee; a i ka hiki ana
wager certain articles of value, malaila, alaila, olelo ka pili a
they would proceed to the pahee holo, ina he mau waiwai ka pili, a
grounds and upon arriving at the i ole, he mau aina paha; a ina
place they would decide first as aole i pili ia ma ia mau waiwai,
to the wager, whether it be alaila, o ka pili no i na iwi ka pili,
articles of value or pieces of alaila pahee, ina he
land. If they do not wager these umikumamalima ka ai (ka helu).
things, then they would put up A ina ua hiki e aku kekahi i ka ai
other things, such as their eo (i ka helu pau) a emi mai
bones, meaning their lives. After paha kona hoa pahee, alaila o
the bets are agreed on, they ke eo ae la no ia, ina paha o ka
would then proceed to play the waiwai ke kumu pili, alaila o ke
game of pahee. If the points to eo ae la no ia o ka waiwai; ina
be scored in order to win the ua nui ka waiwai o ka pili ana,
game be made fifteen, then the ekolu, eha hale e piha i ka
one who first obtains this number waiwai. Aka ina o na kino o laua
of points would win and the one ka pili, alaila, o ka make no o
with the lesser points would lose; kekahi o laua ka hope. Aole nae
then the winner takes the articles he pili nui ia oia mea; aia no a ku
wagered, or whatever had been ka hoopaapaa mawaena o na
placed as wagers. Sometimes aoao elua, e hoole ana kekahi a
the articles of value would be so me kekahi i na akamai o laua,
great that it would take three and alaila pili kino ia; a oia la, pela
four houses to hold them all. But iho la ka waiwai o ia mea.”
if the things wagered be their
bones, then death of course
would be meted out to the loser.
Wagering for bones was not
made very often, only when the
parties entered into the merits of
their skill by long and spirited
arguments, each claiming to be
superior to the other. That is the
use of those long things you
see.”
When Lonoikamakahiki heard A lohe o Lonoikamakahiki i keia

this explanation he replied: mea, olelo ae la oia: “Aole ana
“Those things are worthless and waiwai; aka, he waiwai no,
have very little use; the great hookahi no hewa, o ka pili ana i
objection I have against them is na iwi ke hiki mai i ka manawa e
that they are used by men for the hoopaapaa ai na mea pahee i ko
purpose of making wagers, even laua mau ike, nolaila ka waiwai
to the extent of their bones, on ole oia mea.” I aku la na kahu:
the result of their skill after “Oia iho la no ka waiwai oia mea
heated arguments. That is the (pahee) i malamaia ai e kou
reason they are [258]worthless.” makuakane.” [259]
The retainers then said: “That is
what the pahee spears are used
for and the reason why they are
being kept by your father.”
Lonoikamakahiki again looked Ia manawa, nana hou ae la oia,

up and saw a round, flat stone a iki i ka olohu (ulu maika) ninau
and again asked: “What is that hou ae la oia: “Heaha kela
thing?” The retainers replied: “It mea?” Hai aku la no na kahu:
is called an olohu.” 4 “He olohu.” Ninau aku o
Lonoikamakahiki again asked: Lonoikamakahiki: “Heaha hoi ka
“What is it used for?” Then the waiwai oia mea?” Alaila hai aku
retainers told him that it was la no na kahu e like me ka olelo
used in the same way and for ana no ka ihe pahee. I hou aku o
the same purpose as the pahee Lonoikamakahiki: “E kiola, aole
spears. At this Lonoikamakahiki ana waiwai.”
replied: “Throw it away; it is also
worthless.”
Again Lonoikamakahiki looked, Nana hou ae la no ua o

and when he saw the sugar- Lonoikamakahiki, a ike i ka pua
cane top, used as an arrow, he kea, ninau hou aku la no i na
asked of his retainers: “What is kahu: “Heaha hoi kela?” Hai aku
that?” The retainers replied: “It is la na kahu: “He pua kea.” Ninau
an arrow made from the sugar- hou aku no ua o
cane top.” Lonoikamakahiki Lonoikamakahiki: “A pehea hoi
again asked: “And what is it used kana hana?” Hai aku la no na
for?” The retainers replied: “It is kahu: “He mea lealea no ia; ina
also used in games. If two or elua a ekolu paha mau mea e
three fellows wish to play the kea pua ana, a ina i lele ka
game with the arrows 5 they go to kekahi a oi loa mamua o ka
the playground and see who kekahi mau mea alaila, o ka eo
could glide his arrow on the ae la no ia. A ina he pili ma ka
ground the farthest. The one waiwai, ua like no ka waiwai me
who can send it the farthest ko ka pahee ana, ke nui no hoi
wins. If articles of value have ka pili ana.” I aku la o
been placed as wagers the Lonoikamakahiki: “Aole ana
winner takes them. It is used in waiwai, e pono ke haihai a kiola
the same way and for the same aku.”
purpose as the pahee spears,
and large wagers have been lost
and won on the game.”
Lonoikamakahiki then replied: “It,
too, is worthless; you had better
break it up and throw it away.”
Again Lonoikamakahiki looked Nana hou ae la no ua o

up, and when he saw a wooden Lonoikamakahiki, a ike ae la i ka
club he asked: “And what is that laau palau, ninau ae la: “Heaha
thing?” The retainers replied: “It hoi kela?” I aku na kahu: “He
is a wooden club.” 6 laau palau?” Ninau hou aku la o
Lonoikamakahiki again asked: Lonoikamakahiki: “Heaha kana
“And what is its purpose?” The hana?” Hai aku la na kahu: “He
retainers replied: “It is an mea pepehi aku i na kanaka, ina
implement of war and used to kill paha he hoouka kaua mai ko
people with. If a battle is being kekahi aoao, a hoouka aku ko
fought with one side opposing kekahi aoao, alaila o ka laau
the other then the war club palau ka mea e luku aku ai. A
comes in use as an implement of ina e kaua aku me ua laau palau
war. When this club is used in nei, alaila, he kanaha kanaka e
war it can kill as many as forty make i ka laau palau hookahi, a
people, and sometimes it will kill ina no he nui aku, oia no.” I aku
more people than that.” la ua o Lonoikamakahiki: “Aole
Lonoikamakahiki then said: “That no ana waiwai, hookahi ana
thing is also without value. Its waiwai, he ulu imu.”
only use would be for a stick to
turn over the stones in an umu.” 7
Again Lonoikamakahiki looked Nana hou ae la no ua o

up and saw a bundle of war Lonoikamakahiki nei, e kau ana
spears; 8 he then asked: “What ka ihe kaua, ninau ae la: “Heaha
are those things?” The retainers kela?” I aku na kahu: “He mea
replied: “They are also used to luku kanaka no, he mea luku aku
kill people with. In times of war i na kanaka ke kaua mai, ina i
when men are fighting each kahi e, e hou aku ai, ku aku la no
other these spears are used at ke kanaka. A ina he akamai mai
close quarters by thrusting, and kekahi aoao i ka pale ana o ka
at long range by throwing, at the ihe, alaila, aole e ku.” I aku o
enemy. These spears in the Lonoikamakahiki: “Ae, he mea
hands of strong men can be waiwai ia, aka, o ka mea akamai
thrown for some distance. If the i ka alo ihe ke kanaka waiwai;
person on the other side is of nolaila, he hana waiwai ia a kuu
great skill he could ward off one makuakane, nolaila, ina eia no
or more spears at a time, and in kuu piko ke waiho nei, alaila, e
that way avoid being hit.” nikii pu i kuu piko me ka pua ihe
Lonoikamakahiki then said: “Yes, a kuu makuakane.”
those things are of some value;
but the person who can skilfully
ward them off is of more
importance. These things of my
father’s are of some value;
therefore, if my navel string is
still in your keeping, then tie it
together with my father’s bundle
of war spears.” [260]
Lonoikamakahiki again looked, Nana hou ae la no ua o
and seeing the strings of a sling Lonoikamakahiki, a o ke kau a
hanging he asked: “What is the ke kaula maa, ninau ae la:
use of those strings hanging “Heaha ka waiwai o kela mau
from the wall?” The retainers kaula e lewalewa mai nei?” Hai
replied: “They belong to the aku la na kahu: “He maa.” Ninau
sling.” 9 Lonoikamakahiki again hou aku o Lonoikamakahiki:
asked: “What is it used for?” The “Heaha hoi kona waiwai?” Hai
retainers replied: “A stone is aku la na kahu: “E hookomo i ka
placed in the opening in the pohaku ma ka puka o ka maa,
middle of the sling, then the ends alaila, e pelu mai ina piko elua o
of the strings are brought ua maa la, a e hoopaa ae i ka
together and held in the palm of piko o na kaula o ua maa la i ka
the sling hand; then swing the poho o ka lima e makaukau
sling around the head and when [261]ana i ka hana; alaila, e wili
you think it time to let go, one of ae, a kowali ae, a e like me kona
the ends of the string is released manawa i manao ai e hoolele
which allows the stone to fly out aku i ka pohaku, alaila, e haalele
at the same time. Sometimes the loa aku i kekahi piko o ka maa,
stone would fly over forty alaila e lele aku ka pohaku, he
fathoms, and if a person is struck kanaha a oi aku na anana e lele
with it the force would kill the ai, a ina i pa aku i ka pohaku,
person. It is, however, used as make loa kekahi kanaka. I
an implement of war.” hanaia no nae no ke kaua.” I aku
Lonoikamakahiki then said: “That la o Lonoikamakahiki: “Alua mea
makes two things of value waiwai a kuu makuakane; nakii
belonging to my father. Tie it up pu ia aku me ka pua ihe.” Pela
with the bundle of spears.” kona ninau ana i na mea lealea
Lonoikamakahiki in this manner a pau a kona makuakane; ua
inquired into the use of all the hooleia ka waiwai o na mea
things kept by his father. He apau, a elua wale no mau mea a
denied the usefulness of Lonoikamakahiki i mahalo.
everything but two, which two
things he had the greatest desire
to reserve for his own use.
Sometime after this, I kekahi manawa ae, hele aku la

Lonoikamakahiki again visited no o Lonoikamakahiki a ka hale i
the house where the different waiho ai na mea lealea, nana ae
implements of war and games la, e kau ana no na mea ana i
were kept, and again looked and olelo ai i na kahu, e haihai a
saw the things he had ordered to kiola; nolaila, hoi aku la oia a
be broken and destroyed still kona mau kahu, olelo aku la:
hanging in their respective “Kai noa, ua kiola olua i na mea
places, so he returned and a’u i olelo aku ai ia olua?” I aku
asked of his retainers: “I thought la kona mau kahu: “Aole e hiki ia
you two had destroyed those maua ke kiola i na mea a ko
things that I told you to.” His two makuakane; make mai paha
retainers answered him saying: maua, no ka mea, o ka laau
“We cannot destroy the things palau a ko makuakane, he laau
belonging to your father, for he hai kanaka ia.” A no ka paakiki
would consider it a matter loa o Lonoikamakahiki, nolaila,
sufficient to cause our death, hele aku la na kahu, a hai aku la
because the war club is one of ia Keawenuiaumi, i keia mau
the things highly valued by your hana a kana keiki.
father, for it has been used in his
great battles, and it has been the
means of killing many of his
enemies.” Lonoikamakahiki
becoming very stubborn in the
matter, the retainers therefore
went to Keawenuiaumi and
reported to him the wish of his
son.
When Keawenuiaumi heard this Ia manawa, lohe ae la o

report he was greatly surprised Keawenuiaumi, alaila, haohao
because of the strange wish iho la oia i keia hana kupanaha a
expressed by his son. He kana keiki; nolaila, hele aku la
therefore sought out oia e ninau maopopo ia
Lonoikamakahiki with the Lonoikamakahiki i ke kumu o ko
intention of asking him why he ke keiki manao ana pela. Nolaila
wished to have these things i ka hiki ana aku o
destroyed. When Keawenuiaumi Keawenuiaumi i kahi i hanai ia ai
came to the place where the boy e na kahu, aia nae ua o
was being cared for by the Lonoikamakahiki i ka
retainers, he found that hooholowaa me kekahi mau
Lonoikamakahiki was out canoe kahu ona. A hoi mai la ua o
sailing with some of his other Lonoikamakahiki, e noho aku
retainers. When ana o Keawenuiaumi, hele mai
Lonoikamakahiki returned la ke keiki a noho iho la i luna o
Keawenuiaumi was waiting for na uha o ka makuakane; alaila, i
him; the boy then went up to the mea e maopopo ai ia
father and sat on his lap. 10 In Keawenuiaumi ko
order to have the matter Lonoikamakahiki manao, nolaila,
understood by his son properly lawe ae la kona makuakane iaia
Keawenuiaumi took i kahi i waiho ai na mea lealea. A
Lonoikamakahiki to the house hiki aku la laua, me na kahu pu
where the different implements ma ka hale i waiho ai na mea
of war and games were kept, lealea, ninau aku la o
and there the father asked the Keawenuiaumi: “Heaha kou
son: “What do you think of these manao no neia mau mea (na
things?” meaning the mea lealea ame na mea kaua) e
implements of war and games kau nei?” I ae la ke keiki
hanging on the wall. The son (Lonoikamakahiki): “Aole he
replied: “These things are of no waiwai iki o keia mau mea, ua
value or use. I have told those olelo aku wau ia laua ’la (Hauna
two (Hauna and Loli) to destroy ame Loli) e kiola keia mau mea
them all, but to keep the bundle a pau, a o ka ihe kaua ame ka
of spears and the sling, for they maa na mea waiwai.” I aku la o
are of value.” Keawenuiaumi Keawenuiaumi: “Aole pela ko’u
then said to the boy: “That is not manao, aia no a hiki i kou noho
what I think about those things. aimoku ana, alaila, nau no e
When the time comes for you to kiola, ke ike aku la oe, he mea
assume the care of the whole waiwai ole kela.”
island, then you will be in a
position to do as you like; you
can then throw these things
away if you see no use in
retaining them.”
After this incident Keawenuiaumi Ma ia hope mai, nalu wale iho la

for some time thought over the no o Keawenuiaumi i ka hope o
future of the boy and wondered keia keiki ke nui ae. I iho a ka
what would become of him after makuakane: “Ane kipi wale aku
he had grown up. The father said no koe o keia keiki ma kona
[262]to himself: “It looks as though noho ai aina ana, a heaha la ka
the boy will some day go hana a keia alii ke kanaka
contrary to all the laws that have makua aku.” [263]
heretofore governed the
apportioning of lands, and I
wonder what this chief will do
after he has grown up.”
Sometime after this I kekahi manawa ma ia hope

Lonoikamakahiki entered the mai, komo ae la ua o
temple with his retainers and Lonoikamakahiki i loko o ka
there saw the images standing heiau me kona mau kahu, a ike
up in one of the corners, when aku la i na kii e ku mai ana ma
he asked of his retainers: “Who kuono o ka heiau, ninau aku la i
are those persons standing there na kahu: “Owai kela mau kanaka
within the wall?” His parents and e ku mai la i loko o ka pa?” I aku
retainers replied: “They are not la na makua a me na kahu:
persons; they are the gods of our “Aole ia he kanaka, he akua ia o
parents, your grandparents.” ko makou mau makua, na
When Lonoikamakahiki heard kupuna hoi ou.” A lohe o
that the images were gods he Lonoikamakahiki he akua ia mau
was sore afraid 11 and held on to kii, alaila puliki ikaika aku la i na
his parents with all his strength, makua, no ka mea, ua makau o
for he had been told by his Lonoikamakahiki, a no ka mea
playmates that ghosts were hoi, ua lohe mua oia i ka
things to be avoided and feared, hoomakaukauia e na hoa paani
and he thought the images were ona, a nolaila oia i puliki paa ai i
the ghosts. Because na makua, no kona manao o pau
Lonoikamakahiki held on to his mai i ke akua, no ka mea, ua
parents they said to him: “You oleloia e kona mau hoa kamalii:
must not be afraid; what you see “E lono-e! A-pa-u. A pau i ke
are not ghosts; they are the gods akua lapu.” A no ko
who own this place.” Lonoikamakahiki puliki ana aku, i
Lonoikamakahiki then asked of aku la na makua: “Mai makau
his parents: “What are they good oe, aole ia he akua lapu, he
for?” The parents made reply: akua ia nona keia wahi.” I aku o
“The reason why they are kept is Lonoikamakahiki i na makua:
this: If in case of battle one is “Heaha kana waiwai?” I aku la
taken captive or defeated, they na makua: “Eia kona mea i
offer a prayer to the gods, and malama ia ai; ina he kaua a pio
then the gods will direct the paha, alaila, hoomanamana aku
person to safety. If, on the other i ke akua, alaila, na ua akua la e
hand, a canoe is capsized out in alakai i kahi e pakele ai. A ina he
mid-ocean, prayers are offered waa kahuli ma ka moana, pule
to the gods and those in the no i ke akua, ola no; ina he kau
canoe will be saved. If a season wi, a pule no i ke akua, alaila ea
of famine should come, prayers mai no ka ai. Oia ka waiwai o ke
are offered to the gods and the akua i malama ia ai.” I aku o
food would again appear out of Lonoikamakahiki ia
the earth. These are some of the Keawenuiaumi: “Akolu wale no
benefits why a god should be au mea waiwai i malama ai; o
kept.” Lonoikamakahiki then said keia mau mea au ka’u e
to his father, Keawenuiaumi: malama.”
“That makes three things in your
keeping that are of value. I will
take care of these things.”
Sometime after Lonoikamakahiki Mahope mai o ko

had outgrown his childhood days Lonoikamakahiki mau la opiopio,
and had almost attained ma ka hookanaka makua iki ana
manhood, he began to learn the ae, ao ae la oia i ka alo ihe a me
art of dodging and throwing the ka oo ihe ana, a ao ae la no hoi
spear; he also learned how to oia i ke kui ame ka mokomoko, a
box and wrestle. These things akamai ae la oia ma ia mau
were in time mastered by him. hana. A i ka manawa i akamai
When he became proficient in ai, alaila, hailona aku la na kumu
these arts of defense and of war, nana i ao i kela mau hana ma ka
the teachers who had charge of ai lolo ana. A i ka ai lolo ana, ua
his training in these matters then ku kana mau hana a pau i ka
held the last customary pono ma ke akamai. A o ka
ceremonies, as a sign of hailona o ke kui ma ka lolo ana,
foretelling how he would act in oia ka lolo i ino. Nolaila olelo aku
life. The signs were favorable in la ke kumu kui: “Aole oe e pono
all the different arts with one ke ao i ke kui, no ka mea, ua ku
exception, that of boxing, which, kau lolo i ka pono ole, a nolaila,
not being favorable in this one e pono ke haalele.” Nolaila,
thing, he was advised to haalele iho la o Lonoikamakahiki
eliminate this one art from the list i ke ao ana i ke kui. Aka, ma ka
of those he was to participate in. mokomoko, oia ka oihana i oi
In other words, he was forbidden aku ko Lonoikamakahiki ike ame
from ever going into any boxing ke akamai maoli.
contest. Because of this
Lonoikamakahiki relinquished his
claims as a boxer. It was in the
art of wrestling, however, that
Lonoikamakahiki proved himself
to be the most proficient.
CHAPTER II. MOKUNA II.
How Lonoikamakahiki Ko Lonoikamakahiki Imi

Searched Into the Ana i na Hana oi o ka
Most Useful Things. Waiwai.
When Lonoikamakahiki became I ko Lonoikamakahiki wa i

older and more matured in hoonaauao loa ae ai, makemake
thought he expressed a desire to ae la oia e ike maopopo i na
know the things that would be of hana oi o ka waiwai, a nolaila,
the most use to him, especially hoao pakahi aku la oia i na hana
in the games, so he tried each lealea, ame na hana kaua a
one of them, as well as the kona makuakane, na hana hoi
different arts of warfare indulged ana i olelo ai i kona mau kahu,
in by his father, the things that he mau hana waiwai ole. [265]
were told him by his retainers as
the things most desired. [264]
After Lonoikamakahiki had tried A i ko Lonoikamakahiki hoao

these different things he was ana, maopopo iho la no he
convinced that they were of no waiwai ole ia mau mea, a e like
use, as he had said. The thrust hoi me kana olelo mua, o ka alo
and dodging spear, the sling, ihe ame ka oo ihe, ka maa ame
and the care of the god, ka malama i ke akua na hana
however, were of value. He waiwai. Aka, no ko
therefore made a visit around the Lonoikamakahiki makemake nui
island of Hawaii accompanied by e ike i ka hana i oi aku o ka
his parents and retainers. waiwai, nolaila, kaahele ae la ia
ma ka mokupuni o Hawaii, oia
ame kona mau makua ame na
kahu pu.
Hauna and his younger brother O Hauna nae ame kona kaikaina
Loli, the personal attendants or me Loli, na kahu hoi o ua o
retainers of Lonoikamakahiki, Lonoikamakahiki, he mau kaula
were prophets; they were men laua, he mau kanaka haipule
who paid attention strictly to the hoi, a ua oleloia he mau kanaka
laws of the gods, and it was said mana laua, a he hiki ia laua ke
that they were men who hana i na hana mana he nui ma
possessed supernatural powers, ka inoa o ko Keawenuiaumi
and that they were able to akua, ame ko laua akua hoi.
perform many miracles in the
name of the god of
Keawenuiaumi, and also in the
name of their own god.
In this circuit of the island made Ma keia kaapuni ana o

by Lonoikamakahiki and his Lonoikamakahiki me kona mau
parents, upon their arrival at Hilo makua, a hiki ma Hilo, a noho
they made their abode at iho la ma Kanokapa, kahi e pili
Kanokapa, a place adjoining the pu ana me ka nuku o ka muliwai
mouth of the Wailuku river, o Wailuku. E noho ana o
where lived a man by the name Kawaamaukele malaila, he
of Kawaamaukele, a great priest kahuna kakaolelo nui, ua
and counselor. He was a very elemakule oia, a poohina no hoi.
old man, his head was wholly Aka, he mea haohao nae ia ia
gray. Lonoikamakahiki i kona ike ana
aku i kela elemakule, no ka mea,
o kela kanaka ke kanaka ano e i
hiki mai i ke alo o
Keawenuiaumi, a ua loloa hoi
kona lauoho a hiki i lalo i ka
puhaka, e like mau me ke ano o
na kahuna nui.
When Lonoikamakahiki saw the A ike aku la o Lonoikamakahiki i

old man he was greatly ua elemakule nei, oiai e noho pu
surprised, because this man was ana oia me kona mau kahu.
the only man that differed from Ninau malu aku la: “He akua
the rest of the men that came in anei kela elemakule lauoho
the presence of Keawenuiaumi; loloa?” I aku la na kahu: “Aole he
his hair was so long that it akua, he kanaka no, he
reached below his waist, a thing kakaolelo nae, he kahuna nui oia
common with the high priests, ma na oihana kahuna apau.”
however. When Ninau hou aku la ua o
Lonoikamakahiki, who was Lonoikamakahiki: “Heaha ka
sitting with his attendants, had waiwai a ia elemakule?” I aku na
looked at the old man for some kahu: “O ke kanaka ike i ke
time he asked: “Is that old man kakaolelo, he kanaka nui ia imua
with the long hair a god?” The o ke alo alii; he kanaka akamai i
attendants replied: “He is not a ka olelo, ma kana olelo e olelo
god; he is a human being, but ai, malaila ke alii e hoolohe ai;
not of the ordinary kind; he is a nana e ike ka pomaikai o ka aina
counselor. He is also the high ame ke kanaka, he hiki i kela
priest, higher than all the others.” kanaka ke iki mai i ke kanaka
Again Lonoikamakahiki asked: waiwai ame ka waiwai ole, ke alii
“What is the old man good for?” waiwai ame ka waiwai ole.”
The attendants replied: “The
man who is a counselor is a very
great man in the court of the
king; he must be a man who is
skilful in language, and whatever
advice he gives the king, the
king will take heed. He can
predict the coming of prosperity
to the land and the people. That
man can tell whether a common
person will become rich or poor,
or the chief who will become
wealthy or not.”
When Lonoikamakahiki heard A lohe o Lonoikamakahiki i keia

these remarks from one of his olelo a ke kahu, he mea puiwa
retainers he was greatly loa ia nona, no kona lohe ana i
impressed that such a thing ka olelo, he hiki ke ike i ke alii
could be possible, that is, that waiwai, ame ka waiwai ole, a
the man could tell whether a nolaila, olelo aku la oia i kona
chief will become rich or poor. mau kahu, me ka i aku: “A, e ike
He therefore asked of his mai no auanei kela elemakule la
attendants: “And will that old ia’u?” I aku na kahuna: “Ae, aole
man be able to recognize me?” oe e nalo, a me kau hana
The attendants: “Yes, he will not mahope aku.” I hou aku la o
overlook you 12 and also your Lonoikamakahiki i na kahu: “He
doings in the future.” kanaka kapu anei kela, aole e
Lonoikamakahiki again asked kamailio ia aku e kamalii? O na
them: “Is there any restriction kanaka makua wale no anei?” I
placed on that man, that is, aku la na kahu: “Nau e kamailio
something that will prevent kela elemakule, i malamaia hoi
young people from addressing na kakaolelo ame na kahuna no
him? And are the grown up oukou no na ’lii.”
people the only ones that are
allowed to speak to him?” The
attendants replied: “You are
indeed privileged to address that
old man. Counselors and priests
are retained and cared for to be
used by the chiefs.”
Because of this Lonoikamakahiki A no keia mea hoouna aku la o

sent one of his attendants to go Lonoikamakahiki i kekahi kahu
and bring the aged counselor, ona e kii i ke kakaolelo ia
Kawaamaukele. When he came Kawaamaukele. A hiki mai la i
in the presence of mua o Keawenuiaumi me
Keawenuiaumi and Lonoikamakahiki, i aku la o
Lonoikamakahiki, Lonoikamakahiki: “I kiiia aku nei
Lonoikamakahiki spoke up oe no ko’u lohe ana he
saying: “You have been elemakule akamai oe i ka ike
requested to come here because mai i ke alii waiwai ame ka
I have been told that you are an waiwai ole; nolaila, e nana mai
old man who is learned in the oe ia’u, malia paha he alii ilihune
things of the future and can tell wau ma keia manawa aku, a e
whether a chief will become rich hai mai oe i ka’u mau hana
or poor; therefore I want you to [267]ma keia hope aku.” I mai o
make an examination of me and Kawaamaukele: “He alii waiwai
tell me what I am to be in the no oe i kekahi manawa, aia a
future.” [266]Kawaamaukele then hike aku i kou wa kanaka
replied: “You are going to be a makua, alaila, ilihune oe aole ou
wealthy chief at times, but when kanaka, aka, he alii koa oe.” I
you reach maturity then you will hou aku la o Lonoikamakahiki: “I
become poor, in that you will be aha ka’u hana e hana ai i waiwai
without followers; but you are ai? A ina ua ike oe i ka hana
going to be a brave chief.” waiwai no’u, alaila, e ao no
Lonoikamakahiki then again kaua.” Noho ke kahuna a liuliu
asked him: “What profession me ke kali ana i kona manawa e
shall I take up in order that I may olelo mai ai ia Lonoikamakahiki,
become wealthy? If you know alaila, olelo aku la: “O ka hana e
what I can take up that will be kaulana ai oe a puni na moku, o
profitable as a profession, then ke kakaolelo, ame ka hoopapa;
we will take it up and you instruct ina e akamai oe ma na hana
me in its detail.” The priest hoopapa, alaila, waiwai oe.” Ma
paused for a while, thinking of ka olelo a ke kahuna kakaolelo,
what Lonoikamakahiki had hoolohe aku la no o
asked, and then replied: “The Lonoikamakahiki.
professions that will make you
famous all over the islands are
that of a counselor and
hoopapa. 13 If you can be an
expert in this profession of
hoopapa, then you will become
wealthy.” Lonoikamakahiki took
to heart every word spoken by
the high priest.
Sometime after this the Mahope iho oia manawa, ao ae

profession of hoopapa was taken la oia i ka oihana hoopapa ma
up by Lonoikamakahiki and he ka aoao kakaolelo, a naauao oia
was educated into the different ma ia hana, a oia ka oihana i
things of the profession kaulana nui ai o
pertaining to that portion relating Lonoikamakahiki a puni na
to language, and after he had moku, o ke kolu no hoi ia o ka
mastered it he in later years did Lonoikamakahiki mau hana
become famous all over the akamai a hiki i kona make ana; a
islands. This made the third thing nui loa ka pilikia o kekahi poe alii
that Lonoikamakahiki became iaia.
proficient in up to the time of his
death, and he caused no end of
trouble for certain chiefs.
After completing the study of Mahope mai o kona ao ana i ka

hoopapa in Hilo he returned with oihana hoopapa ma Hilo, hoi aku
his parents to Napoopoo, where la oia me kona mau makua a
they took up their residence and noho ma Napoopoo, a hoomaka
he immediately practiced his aku la oia i ka hoopapa me na
profession on his playmates, and hoa paani ona, a lilo iho la ka
in this manner he made practical hana hoopapa i mea makemake
use of it. In this way the nui na Lonoikamakahiki a pau ka
profession of hoopapa became a la, a pela aku. Aka, o
favorite thing with him, making Lonoikamakahiki, ua
use of it day after day. After a hoolawehala wale aku oia i kona
time, however, Lonoikamakahiki mau hoa paani, i mea e hoopapa
began to ensnare his playmates ai, he mea e hoao ai i kana
by getting into argument with oihana hoopapa. O na puulu
them in order to test his kamalii a pau o Kealakekua, ua
profession of wrangling. All the hoopapa mau ia e
crowds of children in Kealakekua Lonoikamakahiki, aka nae, aole i
were taken up by ike o Lonoikamakahiki i kona
Lonoikamakahiki and defeated. akamai ma ia hana hope ana i
In thus making practical tests of ao ai, aka, o ka mea nana i ao
his vocation Lonoikamakahiki, aku, ua ike aku oia i ke akamai
although making great headway, ma ka hoopapa ana.
was at the same time unaware of
his advance in his profession;
but the person who had charge
of his education was well aware
of his skill in argument.
When Lonoikamakahiki grew to Ma ko Lonoikamakahiki mau la

the age of maturity he took unto hookanaka makua, lawe ae la
himself his cousin Kaikilani to be oia i kona kaikuahine ia Kaikilani
his wife. During the early part of i wahine nana. Mai ia manawa
their married life they lived in mai, he pono wale no ko laua
peace and happiness, and noho aua, aole i loaa ia laua ka
nothing occurred between them mea ino ma ko laua noho pu
to cause any dissatisfaction. ana. Iloko o ko laua manawa i
During all the time that they lived noho ai, aole i loaa keiki laua a
as man and wife they did not hiki i ko laua make ana. Aka, o
have issue; but Kaikilani had Kaikilani ka mea i hanau na keiki
three children with ekolu me kekahi mea e ae me
Kanaloakuaana, an uncle of Kanaloakuaana, he makuakane
Kaikilani’s. When no no ua o Kaikilani. Ia ike ana o
Kanaloakuaana took Kaikilani to Kanaloakuaana me Kaikilani,
be his wife their issue was loaa o Kalanioumi ame
Kalanioumi and Kealiiokalani, Kealiiokalani, he mau
who were girls, and Keakealani, kaikamahine laua, a o
a boy. Keakealani, ke keikikane.
Before Keawenuiaumi died he Mamua o ko Keawenuiaumi

requested Lonoikamakahiki to make ana, kauoha ae la oia ia
take the head of the government, Lonoikamakahiki e noho ma ka
but Lonoikamakahiki did not noho alii, aka, aole pela ko
think it proper to do so. What Lonoikamakahiki manao. O ko
Lonoikamakahiki told his father Lonoikamakahiki manao i olelo
was, that he did not wish to take aku ai i kona makuakane, aole
charge of the affairs of state at ona makemake e ku koke i ka
that time, but to defer the time moku, aia a makaukau oia ma
until he was able to master the na mea kaua, a ailolo hoi, alaila,
arts of warfare, when he could ku i ka moku. A nolaila, hooili ae
become expert therein; then he la o Keawenuiaumi i ka aina a
would take charge. Because of puni o Hawaii no Kaikilani. A
this, Keawenuiaumi left the make aku la o Keawenuiaumi,
whole island of Hawaii in the ku ae la o Kaikilani i ka moku,
care of Kaikilani. 14 After the oia ka wahine alii i ai i ka moku.
death of Keawenuiaumi, [269]
Kaikilani took charge of the
government. She was the first
chiefess who became the ruler of
the land. [268]
After Kaikilani had assumed the Ma ia hope mai o ko Kaikilani ai

care of the government, moku ana, kaapuni ae la o
Lonoikamakahiki made a circuit Lonoikamakahiki ia Hawaii a
of the island of Hawaii making puni, e hoiki ana i kona ike ma
public competitions in all the na mea ana i ao ai o ka oihana
different arts of warfare kaua, a lanakila ae ia oia ma ia
mastered by him, in which he mau hana. Aka, kui aku la keia
was always victorious. Word of mau hana a Lonoikamakahiki a
these accomplishments of lohe o Kanaloakuaana; a i ka hoi
Lonoikamakahiki was in time ana aku o Lonoikamakahiki mai
carried to the hearing of kana huakai kaapuni aku,
Kanaloakuaana. When hoomaka ae la oia i ka
Lonoikamakahiki arrived home mokomoko me Kanaloakuaana,
after making this circuit, he no ka mea, he akamai oia i na
competed in boxing against oihana kaua a pau. Aka, i mea e
Kanaloakuaana, for he, too, was ike ai o Kanaloakuaana i ke
skilful in all the arts of warfare. akamai o Lonoikamakahiki,
Kanaloakuaana did not demand nolaila, hoao hou laua i ka
this competition for any other mokomoko. Alaila, hoao aku la
purpose than to test for himself no o Kanaloakuaana ma ka oo
how proficient Lonoikamakahiki ihe; i aku o Lonoikamakahiki:
was, therefore they tried at “Aole wau i ao i ka oo ihe, aka, o
boxing and Kanaloakuaana ka alo ihe ka’u mea i ao.” A hoao
found that he was skilful. laua i ka alo ihe, ia
Kanaloakuaana then took up Kanaloakuaana nae ka ihe, o ka
spear throwing as the next thing. alo ka Lonoikamakahiki; ia hoao

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FRANCESCO H. ROMANO AND ANDREA RUSSO

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NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Biocatalysis research progress/Francesco H. Romano and Andrea Russo(editors).

Published by Nova Science Publishers, Inc. New York

Chapter VIII Salt-tolerant Alkaliphilic Actinomycetes

self assembly technique), or by the Langmuir-Blodgett technique based on the formation of

of fermentation processes. Production system which employs agroindustrial residues as

applications in industrial processes. Because of the capacity to survive under nonstandard

Laura Pastorino1 and Svetlana Erokhina2

3D biocatalytic systems with nm resolution, with predetermined structure and function,

The Langmuir-Blodgett Technique

Basic Principles of Langmuir-Blodgett Technique

Monolayers at the Air/Water Interface

Figure 2. π - A isotherm of stearic acid monolayer at the air-water interface.

The forces acting to the plate are:

mg -weight of the plate;

Figure 3. Illustration of the working principle of the Wilhelmy balance.

As it was mentioned above, an important aspect of the Wilhelmy balance utilization is

Monolayer Transfer onto Solid Substrates

monolayer is schematically shown in the Figure 8. Regular closely packed monolayer is at

Figure 7. Scheme of the LS deposition.

Figure 9. Types of the structures of LB films.

Generally, packing of amphiphiles in LB films is determined by the head-to-head and

Lb Films of Enzymes: Fromherz Trough

Special Modifications of LB Technique: Sliding Plate Method

One other technical decision, suggested by V. Troitsky, allows to deposit enzyme-

assembling. Important feature of the method is a special construction of the sample-holder.

Langmuir Monolayers for Study of Enzyme-Membrane Interactions

Monolayer at the air/water interface can be considered as a half of a model biological

Enzyme-Containing LB Films as Sensitive Layers of Biosensors

The Electrostatic Layer by

In the figure a negatively charged support is dipped into a diluted aqueous

LbL Films of Enzymes: Structure and Properties

Assembly Procedure on Planar Supports

Multilayers containing biomolecules, including enzymes have been fabricated by means

Structure Characterization of LbL Films on Planar Supports

The Sauerbrey equation can also be expressed as:

ρ = density of the deposited material.

When Lonoikamakahiki was I ko Lonoikamakahiki wa

When Lonoikamakahiki heard A lohe o Lonoikamakahiki i keia

Lonoikamakahiki again looked Ia manawa, nana hou ae la oia,

Again Lonoikamakahiki looked, Nana hou ae la no ua o

Again Lonoikamakahiki looked Nana hou ae la no ua o

Again Lonoikamakahiki looked Nana hou ae la no ua o

Sometime after this, I kekahi manawa ae, hele aku la

When Keawenuiaumi heard this Ia manawa, lohe ae la o

After this incident Keawenuiaumi Ma ia hope mai, nalu wale iho la