Reactive Surfaces Ltd. LLP
`Ex. 1050 (Rozzell Attachment C)
`Reactive Surfaces Ltd. LLP v. Toyota Motor Corp.
`IPR2016-01914
`
`

`

`Biocatalytic Production of
`Amino Acids and Derivatives
`
`anser Publishers, Munich Vienna New York Barcelona
`
`£ H
`
`Edited by
`J. David Rozzell and Fritz Wagner
`
`With 115 Illustrations and 68 Tables
`
`

`

`
`
`The Editors:
`‘
`Dr. J. David Rozzell, Vice President, Research andDevelopment, Exogene, Monrovia, CA 91016, USA
`Prof. Dr. Fritz Wagner, Institut fiir Biochemie und Biotechnologie der TU Braunschweig, Braun-
`schweig, Germany
`*
`
`Distributed in USA and in Canada by
`Oxford University Press
`200 Madison Avenue
`New York, NY 10016
`
`Distributed in all other countries by
`Carl Hanser Verlag
`KolbergerstraBe 22
`D-8000 Miinchen 80
`
`The use of general descriptive names, trademarks,etc., in this publication, even if the former are not
`especiallyidentified,is not to be taken asa sign that such names,as understood by the Trade Marks and
`Merchandise Marks Act, may accordingly be used freely by anyone.
`
`While the advice and information in this book are believed to be true and accurate at the date of going
`to press, neither the authors nor the editors nor the publisher can acceptany legal responsibility for any
`errors or omissions that may be made. The publisher makes no warranty, express or implied, with
`respect to the material contained herein.
`
`Library of Congress Cataloging-in-Publication Data
`Biocatalytic production of amino acids and derivatives /J. David Rozzell, Fritz
`Wagner(editors).
`.
`.
`cm.
`Includes bibliographical references and index.
`ISBN 3-446-15699-2 (Carl Hanser Verlag). ISBN 0-19-520982-6 (Oxford University Press : cloth)
`1. Amino acids — Biotechnology. 2. Amino acids — Synthesis.I. Rozzell, J. David. Il. Wagner, Fritz,
`Dr. rer. nat. .
`TP248.65.A43B56 1992
`660’ .63 — dc20
`Die Deutsche Bibliothek — CIP-Einheitsaufnahme
`Biocatalytic production of aminoacids and derivatives / J. David
`Rozzell ; Fritz Wagner (ed.). — Munich ; Vienna ; New York;
`Barcelona : Hanser, 1992
`(Hansertitles in biotechnology)
`ISBN 3-446-15699-2
`NE: Rozzell, J. David [Hrsg.]
`
`°
`
`:
`
`92-26917
`
`ISBN 3-446-15699-2 Carl Hanser Verlag, Munich Vienna New York Barcelona
`ISBN 0-19-520982-6 Oxford University Press
`
`
`
`INTRODUC
`
`Aminoacids are t
`important chemic
`these are optical}
`are essential for!
`amino acids in fi
`important pharmi
`occur in nature: !
`examples found
`secondary metaby
`D-phenylglycine
`antibiotics. The
`several hundred 1
`and L-tryptophaz
`drugs, synthesize
`for biological tes
`it is not surprisin
`pharmaceutical p
`be surprising, fi
`amino acids, bo
`areas.
`
`Worldwide 1
`annually. For ea
`demand of neazi
`of the high-intes
`than onebillion
`L-aspartic acid
`manufactured in
`chelators and st
`intermediates i
`importance as ¢
`This book im
`production of 2
`processes to pn
`of amino acids
`biocatalysts in
`integrated into
`enzymes in no#
`the cloning and
`and immobilize
`production of:
`biotechnology.
`
`
`
`
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`
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`
`
`Allrights reserved. Nopartof this book may be reproduced ortransmitted in any form or by any means,
`electronic or mechanical, including photocopying or by any information storage and retrieval system,
`without permission from the publisher.
`
`© 1992 Carl Hanser Verlag, Munich Vienna New York Barcelona
`Printed and bound in Germany by Késel, Kempten
`
`
`
`

`

`
`
`

`

`J.D. Rozzell
`306
`[Refs. on p 318}TCESOTPPEET
`13.1 INTRODUCTION
`
`has been developed.
`
`Immobilized enzymes have captured the interest of biotechnologists since the 1950s,
`but it was with the work by Katchalski-Katzir and Chibata and co-workers in the
`1960s that research activities in this area began to accelerate, culminating in the First
`Enzyme Engineering Conference in 1971,
`Significant efforts towards improved
`immobilized-enzyme preparations continue today as new uses emerge.
`Thefirst
`industrial application of enzymes in immobilized form was for amino acid production,
`as reported by Chibata and co-workers at Tanabe Seiyaku in Japan in 1969 [1]. This
`group immobilized L-aminoacylase for use in‘a packed-bed reactor in the resolution
`of various DL-aminoacidsinto their corresponding optically pure enantomeric forms.
`Since that time, enzymes in immobilized form have becomeincreasingly importantas
`catalysts for the production of amino acids, as well as humerous other substances.
`By wayof definition, immobilized-enzyme biocatalysts consist of the enzyme,in
`varying degrees ofpurity, attached to or otherwise retained by a support matrix. At
`one extreme,intact dead cells (which are effectively bags of enzymes) may be bound
`to a support for use as a catalyst; at the other extreme, partially purified or purified
`forms of the enzyme(s) ofinterest may be immobilized. Driving the developmentof
`this technology is the fact that the immobilization of an enzyme can improve the
`economicsofits application, improve the quality of the product produced, or both.
`By changing from batch to continuous operation, one can often significantly reduce
`the economics of an enzyme-catalyzed reaction. Other advantages, such as improved
`control of the reaction, leading to better uniformity of the product and greater case of
`product recovery, are also often achieved through the immobilization of an enzyme.
`In assessing the ‘economics of a process using a biological catalyst (enzyme), the
`critical issue is not the cost of the biocatalyst itself but rather the contribution of the
`biocatalyst to the cost of the final product. Biocatalyst costs themselves depend on
`various components, including enzymeorcell production, support matrix, auxiliary
`reagents, and the loss of activity associated with immobilization. However,
`the
`important factors which determine the cost contribution of the biocatalyst are the yield
`of product,
`the volumetric productivity achieved in the process,
`the product
`concentration attained, and the useful
`lifetime of the biocatalyst under operational
`conditions. This chapter will survey immobilization methods, with special attention
`being paid to those which have been found useful in amino-acid production. The
`methods discussed here are not meant to be exhaustive butrather illustrative of what
`
`

`

`13 Immobilized Enzymes: Techniques & Applications
`
`307
`
`13.2 GENERAL IMMOBILIZATION METHODS
`
`Five general techniques have been described for immobilizing enzymes:
`adsorption;
`covalent attachment;
`cross-linking;
`entrapment of an enzyme in a polymeric matrix; and
`encapsulation or confinement of an enzyme in a membrane[2].
`Each will be discussed along with its advantages and disadvantages. Although each
`immobilization techniquc is conceptually distinct, it is important to realize that there
`is often a certain amount of overlap or even combining of techniques in actual
`practice.
`Furthermore,
`the results of over thirty years of work by numerous
`researchers suggest
`that
`there is no one universally applicable immobilization
`technique; rather, a range of methodologies is available and must be evaluated on a
`case by case basis, There is still some art mixed with the science of enzyme
`immobilization. Certain enzymes may be immobilized successfully with one method
`but not with others. Each immobilization method has both advantages and disadvan-
`tages which must be considered within the context of the enzyme to be immobilized
`and the overall process in which the enzymeis to be used.
`
`affinity for the support relative to other proteinaceous material such that a partial
`
`13.2.1 Adsorption
`
`Adsorption is one of the most economical and operationally simple processes by which
`enzymes can be immobilized, and this simplicity is no doubt responsible for its
`attractiveness as an immobilization method. Adsorption was the technique used by
`Chibata and co-workers in their first commercial process involving an immobilized
`enzyme. However, eventhough this immobilization procedure may bestraightforward
`to carry out, the interactions involved in adsorption are complex and not completely
`understood. A good definition of adsorption, given by Messing, is the adhesion of an
`enzyme to the surface of a support which has not been specifically modified for
`covalent attachment [3].
`:
`One of the principal advantages of adsorption is its ease to perform; simply
`contacting an aqueoussolution of the enzyme with the support is all thatis required.
`Occasionally, with judicious selection of the support, the desired enzyme will have an
`
`

`

`308
`
`J.D. Rozzell
`
`[Refs. on p 318]
`
`rtant advantage of immobilizing the enzyme on a
`a support, or both.
`nonporous, external surface is that diffusional limitations are minimized, However,
`the disadvantages of adsorption on a nonporous material are:
`l.
`a relatively low surface area for bonding maylead to low catalyst activity;
`leaching, already a Problem, may,be worse because the enzyme is more
`Susceptible to physical abrasion and Sloughing off due to shear forces in the
`bulk solution; and
`the enzymeis more exposed to microbial or proteolytic attack.
`3.
`Although immobilization of an
`has one marked disad
`
`Vities are too low.
`
`porous material [1].
`
`speaking, a more stable enzyme system mayresult from immobilization within a
`N reported that, generally
`Typically, Supportparticles usedin adsorption ofenzymewill have diameters in the
`range of 0.5 to 100 micrometers, Smaller particles than this result in Pressure drops
`that are unacceptably
`high;
`larger particles have such a reduced surface area for
`contact with substrate solutio
`
`ly
`
`

`

`13 Immobilized Enzymes: Techniques & Applications
`
`309
`
`generally
`joa within a
`
`in the
`e drops
`© area for
`
`protein is detected in the washing solution. These washing proceduresare carried out
`at room temperature. The resulting precipitate is ready for use in the enzyme-
`catalyzed reaction.
`In order to store the immobilized preparation for a long period or to measure its
`enzymeactivity, it is suspended in 2.5 1 of distilled water and lyophilized. Using this
`procedure, 106 g of immobilized DEAE-Sephadex-aminoacylase is obtained. The
`activity of the preparation is reported to be about 700 »pmol/h-g of preparation, under
`standard assay conditions.
`industrial process for the
`The above method formed the basis for the first
`production of an amino acid by Tanabe Seiyaku Company in Japan. N-Acetyl-
`DL-methionine was passed through the column, where the L-isomer was deacetylated
`stereoselectively. The L-methionine was separated from the unreacted N-acetyl-
`D-methionine, which itself was recovered, racemized and recycled through the process.
`This process was successfully commercialized in 1969 and is consideredto be thefirst
`commercial-scale immobilized-enzymeprocess.
`In this example, the useful lifetime of the immobilized enzyme was approximately
`30 days, and the support could be regenerated each month by adsorbing fresh enzyme
`on the column.
`It was reported that the same batch of support matrix could be used
`for a period of five years, with repetitive regeneration cycles.
`Other enzymes useful for amino acid production which have been immobilized by
`the adsorption method include aspartase on DEAE-cellulose andsilica gel [5], leucine
`aminopeptidase on hydroxylapatite [6], glutamic-aspartic transaminases on DEAE-
`Sepharose [7], and o-amino-€-caprolactam racemase on DEAE-Sephadex[8]. There
`are numerous reports of the immobilization of otherenzymes such as amylase, glucose
`isomerase, glucose oxidase, and proteases on materials such as activated carbon,
`bentonite, and alumina. Given the data base, it is probably fair to surmise that an
`appropriate material may be foundthatwill allow the immobilization by adsorption of
`virtually any enzyme; however,
`the strength of the adsorptive forces binding the
`enzyme to the support will vary considerably from one enzyme to another. Adoption
`of such a catalyst in a process will likely depend on whether the mechanical properties
`and the operational lifetime of the catalyst are sufficient for commercial application.
`
`attachment are also more resistant to attack by proteolysis. The main disadvantage
`
`13.2.2 Covalent Attachment
`
`Covalent attachment of enzymes to surfaces is often employed when leaching of
`enzymeactivity from the support is a concern. This method generally offers the
`advantage of an immobilized enzyme system that is more permanently anchored, and
`may also show greater stability and the ability to withstand a broader spectrum of pH
`conditions,
`ionic strengths, and temperatures. Enzymes immobilized by covalent
`
`

`

`
`
`
`
`J.D. Rozzell310 [Refs. on p 318]cea
`of this type of attachment is its somewhat greater complexity and higher cost to
`prepare. Cost notwithstanding, when covalent attachment results in a significantly
`more stable enzyme system or when the absence of enzyme in the productsolution is
`ofcritical importance,it still may be the method of choice.
`There are three different techniques by which covalentattachment can be effected.
`Thefirst is through exposure of the enzymeto a support which has been preactivated
`to accommodate covalent binding. Operationally, once the activated support has been
`prepared, the immobilization proceeds like adsorption, but the result is an enzyme
`covalently bonded to the support matrix. The second technique involves exposure of
`the enzyme to the support in the presence of an activating or cross-linking reagent.
`Inevitably, some chemical modification or cross-linking of enzyme molecules occurs
`during an immobilization of this type which can lead to a loss in catalytic activity.
`A third possibility—much less commonly used—is to preactivate the enzyme and
`expose it to a support functionalized for covalent binding. The risk of inactivation of
`the enzyme by chemical processes during the preactivation procedure is significantly
`higher, and the procedureis less reproducible. Thus, virtually all practical systems for
`immobilization fall into one of the first two types.
`Frequently, covalent coupling is preferred to other processes in cases where the
`enzyme is multimeric or contains prosthetic groups. There may be a reduced tendency
`to disrupt the complex nature of these enzymessince specific bonds can be formed
`with the functional group to bind the enzyme through multiple points of attachment
`to the support.-
`A list of the amino acid functional groups which are chemically reactive enough
`to participate in covalent binding reactions may be found in Table 13.1.
`Of those functional groupslisted, -NH,, -CO,H, and -SH are involved in most
`immobilization procedures due to their nucleophilicity. The phenolic ring of tyrosine
`is also extremely reactive in diazo-coupling reactions, and its hydroxyl group can be
`an excellent nucleophile at basic pH. The guanidino group of arginine can react with
`aldehydes. Histidine displays a lower nucleophilicity, but can sometimes react with
`supports activated with tosylates, tresylates, or other good leaving groups.
`Functional groups of a wide range of types have been used for the covalent
`attachmentof enzymes. The variety of chemistries available for covalent attachment
`allows the conditions of immobilization to be tailored to each enzyme system. The
`microenvironment of the enzyme may alsobetailored by appropriate modification of
`the support surface; hydrophobic residues or ionically charged groups may be used to §
`alter the support to affect in a desirable way the enzyme-catalyzed reaction of interest
`[9].
`The range of support materials that has been used for covalent attachment, includes
`porous glass, porous ceramics, sand, charcoal, modified cellulose, polymeric resins,
`and metallic oxides. A few examples are described hereto illustrate typical covalent
`
`attachment methodology.
`
`|
`|
`
`

`

`fs. on p 318]
`tts
`
`
`13 Immobilized Enzymes: Techniques & Applications
`311
`
`gher cost to
`ignifi
`TABLE 13.1 Useful Functional Groups for Covalent Attachmentof Enzymes
`: intly
`to a Support Matrix
`
`# solution is
`
`
`Functional Group
`Corresponding Amino Acid
`be effected. a me
`
`weactivated
`—NH>
`Lysine, N-terminus
`wt has been
`
`am enzyme
`
`
`—COn,H
`Glutamic Acid, Aspartic Acid, C-terminus
`2
`xposure of
`
`_
`Cysteine
`@ reagent.
`SH
`ystein
`les occurs
`
`ic activity.
`
`zyme and
`é \ OH
`Tyrosine
`
`vation of
`NH
`Arginine
`mificantly
`
`~~
`
`4
`NH5
`rstems for
`
`N
`Histidine
`
`CY?
`vhere the
`tendency
`Serine
`
`© formed
`
`tachment
`
`
`
`*
`13.2.2.1 Specific Examples of Covalent Coupling: Binding to Acti-
`€nough
`
`vated Carbohydrate Supports
`in most
`mrosine
`One of the most commonly used procedures for the covalent coupling of enzymes to
`
`
`ict with
`carbohydrate support matrices is based on a preactivation of a support with cyanogen
`
`ct with
`bromide [10, dij. The mechanism ofthis reaction has been studied extensively by
`
`Wilchek and Kohn [12, 13].
`
`The activated carbohydrate will couple generally to an amino group oflysine on
`
`walent
`the protein or the free N-terminusof the protein to yield a covalently bound product.
`Supports which have been preactivated with cyanogen bromide can be prepared in
`“nent
`
`.
`advance andstored for periods of up to one year at freezer temperatures. Preactivated
`-
`kon of
`Supports are also available commercially. Coupling of an enzyme to a CNBr-activated
`sed to
`Support requires no more than exposure of the enzyme to the support in aqueous
`Merest
`solution for a few hours, followed by washing. This method, while extremely popular
`in lab-scale reactions, has not been widely used in large-scale systems due to the
`tudes
`requirements to handle toxic reagents and the poorer mechanical Stability of most
`carbohydrate gels and polymers compared to other support materials. The bond
`Esins,
`ralent
`between the enzyme and the support is also potentially susceptible to hydrolytic
`cleavage.
`
`The
`
`

`

`
`
`[Refs. on p 318]
`J.D. Rozzell
`312
`13.2.2.2 Specific Examples of Covalent Coupling: Carbodiimide
`Coupling
`
`useful for amino acid production [14]. The reaction typically forms a stable amide
`linkage between enzyme-bound carboxy] groups and Support-bound primary amines,
`and the rate of the reaction is relatively rapid. One disadvantage of carbodiimide
`reagentsis their relatively high cost. Chemical modification of the enzymeleading to
`a loss of catalytic activity is another potentially negative side reaction.
`
`
`
`
`13.2.2.3 Specific Examples of Covalent Coupling: Amine-Bearing
`Supports
`Amine-bearing supports are among the most useful and mostutilized supports for the
`covalent attachment of enzymes, and a variety multifunctional reagents has been
`utilized for the covalent attachmentof enzymes to supports bearing pendent amines.
`
`immobilization of proteins, Perhaps the most frequently used technique for the
`covalent attachment to inorganic surfaces is the preactivation and functionalization
`with aminosilane reagents [15, 16, 17].
`This technique involves attaching an
`aminosilane to the inorganic surface,
`leaving the amine available for covalent
`attachment of the enzyme. The most frequently used silane, popularized through the
`developments at Corning Glass Works, is ¥-aminopropyltriethoxysilane [16]. The
`glutaraldehyde, resulting in a support which has been preactivated for enzyme
`attachment. Coupling of enzyme may also be achieved through the use of carbodi-
`imide reagents, in which case it is through enzymic carboxy] groups that attachment ‘
`Another process for immobilizing on amino-functionalized inorganic supports
`involves isocyanate bonding [18]. If the enzymeis attached under alkaline conditions,
`a substituted urea bond is formed between an amine on the protein surface and the
`isocyanate.
`If moderately acidic conditions are employed, then the isocyanate reacts
`
`is achieved.
`
`

`

` [Refs. on p 318}
`
`
`
`313
`
` 13 Immobilized Enzymes: Techniques & Applications
`
`
`with a hydroxyl group on the enzyme and a urethane bondis formed. Isothiocyanates
`have also been used successfully [16].
`in providing amino group
`Polyethyleneimine is a common polyamine, useful
`functionality for attachment. It has the advantagethat it is inexpensive and that a wide
`range of supports including those other than inorganic particles may be used.
`Examples are alumina [19], carbon [20], diatomaceous earth [21], and poly(vinyl
`chloride)-silica composites [22, 23].
`A typical procedure involving polyethylenimine activation, which has been used for
`immobilization
`of
`recombinant
`transaminase,
`aspartase,
`and
` aspartate-
`the
`B-decarboxylase in the industrial-scale production of L-aminoacids, has been described
`by Rozzell [24]. The activity retained after immobilization approached 90%, with the
`operational half-lives of the immobilized biocatalysts ranging from 2 to 6 months.
`Recently, Flaschel and co-workers have developed a method for the covalent
`attachment of enzymes involving a mineral or carbon particle coated with chitosan,
`providing a hydrophilic surface of attachment [25]. Chitosan, which is deacetylated
`chitin, contains available amino groups for chemical activation and is easily obtained
`at relatively low cost. Activation with a bifunctional reagent such as glutaraldehyde
`provides a stable immobilized-enzyme preparation. The use of rigid, incompressible
`particles on which the chitosan is deposited allows this catalyst to be used in both
`fixed-bed and fluidized-bed reactors. Yields of activity after immobilization of up to
`90% have been reported.
`.
`
`
`
`
`13.2.2.4 Specific Examples of Covalent Coupling: Oxirane or Epoxy-
`Activated Polymers
`;
`
`Carbodiimide
`
`the formation of an
`|
`gaulic functional groups
`lysis is a competing side
`gation of transaminases
`ly forms a stable amide
`-bound primary amines,
`amtage of carbodiimide
`the enzymeleading to
`: reaction.
`
`Amine-Bearing
`
`silized supports for the
`mal reagents has been
`raring pendent amines.
`for binding. Glutar-
`alk, reacts in complex
`- and produces pendent
`which proteins may
`#8 and diisocyanates,
`1 widely used for the
`sed technique for the
`and functionalization
`avolves attaching an
`vailable for covalent
`spularized throughthe
`toxysilane [16]. The
`mpling agents such as
`wtivated for enzyme
`hh the use of carbodi-
`roups that attachment
`
`Epoxy-activated polymersfall into the catagory of preactivated supports for covalent
`attachment, and they have gained attention as commercially useful support matrices
`for immobilized enzymes [26]. One advantage is the irreversible reaction by which
`“enzymes may be attached to a support through epoxides; as the epoxide opens, in a
`displacementreaction involving a nucleophilic group on the enzyme, a nonhydrolyz-
`able linkage is formed. Another advantage is the ability to activate a wide range of
`different surfaces with epoxides. Yet an additional advantage is that binding through
`epoxides does not appreciably change the charge state of the enzyme onceit is bound.
`For example, the pK, of the secondary amine formed after binding of an enzyme
`through a lysine side chain is not too different from that of the lysine side chain prior
`to coupling.
`An epoxide-activated support has been developed by Rohm Pharma (Darmstadt,
`d inorganic supports
`FRG), and is sold commercially under the trade name of Eupergit. The support ts
`” alkaline conditions,
`based on a methacrylic polymer bead bearing epoxide functionality. Eupergit has been
`otein surface and the
`successfully applied to the immobilization of many enzymes including penicillin
`the isocyanate reacts
`acylase for use in the production of 6-aminopenicillanic acid [27].
`
`
`
`

`

`success in Japan. However, various other materials have also been used. Bauman and
`
`‘method, Novo takes cells which have been
`recovered from the fermentation broth in the form of a Paste and extrudes them in the
`form of spaghetti.
`The Strands are cut
`into uniform pellets and hardened in
`glutaraldehyde. This methodis Particularly useful because it gives particles of a
`controlled size for use in packed-bed reactor systems,
`Although glutaraldehyde is the most popular cross-linking reagent, dimethyl
`adipimidate and diisocyanates have also been used. Diamines may also be used in
`conjunction with carbodiimides, whichactivate carboxyl groups on theproteinsto react
`The enzyme activity of immobilized preparations prepared by cross-linking with
`tee of cross-linking.
`
`tivity of papain was
`A similar effect of glutaraldehyde
`e A was reported by Quiocho and
`Richards [30, 31]. Generally speaking, an increased level of cross-linking will lead
`to a more stable enzyme Preparation, but often at the cost of part of its catalytic
`
`activity.
`
`13.2.4 Entrapmentin a Polymeric Matrix
`Entrapmentis perhaps best viewed as the physical confinement of an enzyme ina
`work or matrix.
`In this technique, an enzyme is typically added to a
`solution of monomer, andthe resulting polymerization entraps the enzyme molecules
`This immobilization technique has been extensively explored since the early work
`of Bernfeld and Wan using polyacrylamide [32]. The most frequently employed
`matrix for entrapment has in fact been polyacrylamide, which has had Significant early
`
`in the matrix.
`
`J.D. Rozzell
`314
`13.2.3 Cross-Linking
`Cross-linking ofenzyme molecules with otherproteins affords an insolublepreparation
`that can be readily handled or manipulated in a continuous reactor. These immobilized
`ions
`are often produced in a particulate form, for use in packedbed reactors,
`
`[Refs. on p 318]
`
`°
`
`with amines.
`
`

`

`13 Immobilized Enzymes: Techniques & Applications
`
`315
`
`: Preparation
`M=amobilized
`
`co-workers reported the immobilization of cholinesterase in polymerized starch [33].
`Vieth and Venkatasubramanian investigated the entrapment of enzymes in collagen
`matrices [34]. Whitesides has published on the use of PAN (polyacrylonitrile) gels
`[35, 36].
`Oneof the major concerns with respect to erftrapped enzymesis that of leaching.
`The enzyme may migrate out of the pore if the pore is too large.
`In many cases, this
`leaching may be overcomeby simply cross-linking the enzyme after entrapment with
`a bifunctional reagent such as glutaraldehyde.
`that of pore diffusion
`The opposite effect is also a concern with entrapment:
`limitations.
`If the substrate is a rather large molecule, such as a protein, it may be
`restricted from entry into the pore and thus be inaccessible to the enzyme.
`Dead cells containing enzymes have been very successfully immobilized by
`entrapment. The size ofthe cells prevents loss of catalyst due to diffusion out of the
`pores of the matrix.
`Fusee described the production of L-aspartic acid using
`polyurethane-immobilized cells containing aspartase [37]. Calton et al. reported the
`production of L-aspartic acid, L-phenylalanine, and L-alanine using cells entrapped in
`a polyazetidine matrix [38]. Chibata and co-workers have commercialized processes
`for L-aspartic acid and L-alanine using cells entrapped in carrageenan gels and
`hardened with hexamethylencdiamine and glutaraldehyde [39]. Calcium alginate gel
`has similarly been used.
`A novel method to overcome the problem of leaching outof activity was developed
`as a part of the polyazctidine method by Calton and co-workers. Cells containing
`enzyme are mixed with a polyazetidine polymer and cured by drying or mild heating.
`The polymer chains contain a reactive N-containing ring which opens under
`nucleophilic attack by cellular material or proteins. The result is believed to be a
`combination of entrapment and covalent attachment,
`rendering a morc stable
`immobilized enzyme preparation in a hydrophilic environment, whichis less prone to
`leaching (40}. Further enhancements to this technique have been developed by Novo
`[41].
`
`tage of this immobilization technique is that only relatively small substrate molecules
`
`13.2.5 Encapsulation or Confinement in a Membrane
`
`Encapsulation is distinguished from entrapment methods by the fact that a solution of
`the enzyme is separated from the bulk solution by a membrane.
`In this approach,
`pioneered by Chang [42, 43], enzymes are encapsulated within membranes that are
`impermeable to the enzymes but permeable to the enzyme substrate. Such semi-
`permeable microcapsules have been used as artificial cells in which the enzymes,
`cofactors, organelles, and other bioactive materials are retained [30]. Encapsulation
`offers the opportunity to immobilize larger quantities of enzyme per unit volume of
`immobilized preparation than any. other procedure [3]. Perhaps the biggest disadvan-
`
`

`

`
`J.D. Rozzell316 {Refs. on p 318]aES.onp518)
`
`
`can be utilized with the intact membranes. Depending on the type of membrane used,
`
`encapsulation can also be a relatively expensive way to immobilize enzymes due to
`
`the high cost of membranes.
`
`Chang has described the encapsulation of enzymes in a variety of membranes[44].
`Two of the membranesthat he used most successfully were cellulose nitrate and nylon.
`In another example,the Snamprogetti Companyin Italy has entrapped aminoacylase
`
`
`and hydantoinase, for the production of amino acids in hollow fibers of cellulose
`acetate [45]. The half-lives were greater than one month under operating conditions.
`
`Other enzymes have been similarly immobilized for producing a range of different
`
`products, showing wide applicability of this general method.
`Another membrane system which has shown promising results in biocatalytic
`applications has been developed by Sepracor
`[46, 47].
`This system has been
`particularly effectivein facilitating contact between an aqueousphase and a non-water-
`miscible phase. The enzymeis entrapped against the membranesandheld in place by
`a slight positive pressure. As long as the stability of the enzymeis high enough, this
`System can operate continuously with little down time.
`In addition, old, deactivated
`enzyme can be flushed out and new enzyme loaded while the membrane cartridge is
`in place.
`
`
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`
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`13.3 CONCLUSIONS
`
`Perhapsthe most important conclusion one can draw from the pastthirty years of work
`on enzyme immobilization is that, due to the range of methods that has been
`developed,
`it can be asserted with a high degree of confidence that a successful
`immobilization method can be developed for virtually any enzyme. That does not
`mean, however, that one method will be successful for almost all enzymes.
`In fact,
`the method of choice will likely vary from one case to another. All five methods
`surveyed and described here have been found useful in the past in certain instances,
`Even more importantly, a numberofthese methods have been proven successful at the
`commercial scale, thus giving the researcher several options to choose from which
`have withstood the “test of practicality” at the manufacturing scale.
`Products manufactured today using immobilized enzymes include high-fructose corn
`syrup, 6-aminopenicillanic acid, certain fatty acids and derivatives, L-malic acid, and
`isomaltulose. Specifically, a numberof amino acidsare also made using immobilized
`enzyme catalysts, including L-aspartic acid from fumaric acid and ammonia, L-alanine
`from L-aspartic acid, D-4-hydroxyphenylglycine from the corresponding hydantoin,
`L-omithine and L-citrulline from L-arginine, L-tryptophan from indole and L-serine.
`Depending on the economics, technology has been developed for other amino acids
`*
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`

`CHARACTERIZATION
`
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`
`13 Immobilized Enzymes: Techniques & Applications
`
`317
`
`NATIVE ENZYME
`
`CLONING OF GENE
`
`EXPRESSION IN
`SUITABLE HOST
`
`
`
`
`as well,
`including L-lysine, L-phenylalanine, L-tyrosine, L-serine, L-4-phenyl-
`2-aminobutanoic acid, L-norvaline, and L-DOPA. Genetic engineering has played a
`
`
`key role in a numberofcases in making enzymes available in quantity at costs that
`
`
`previously could not be achieved.
`
`
`Future applications will
`likely have an increasing dependence on genetic
`
`
`engineering as a tool to enhance enzyme-catalyzed processes by allowing improve-
`
`
`ments in the properties of the enzymes: modified substrate specificity, improved
`stability, and more suitable temperature or pH optima. Figure 13.1 illustrates in flow
`
`
`chart form how this may be done.
`
`
`
`
`
`
`been
`
`a-water-
`
`
`place by
`
`agh, this
`
`
`ctivated
`
`
`
`rade is
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`
`
`
`SELECTION OF NEW
`MODIFIED ENZYMES
`
`of work
`
`Desired
`Sub-optimal .
`Bi
`ikas been
`
`PropertiesProperties
`
`
`
`
`
` does not
`
`
`SITE-DIRECTED
`PRODUCTION AND
`
`
`Tn fact,
`MUTAGENESIS
`APPLICATION
`
`methods
`
`Figure 13.1. .Using Genetic
`Engineering to Improve the
`COMMERCIALIZ ATION
`Catalytic Properties of.
`
`
`
`Enzymes.
`On) which
`
`
`
`
`
`
`
`Thefield of immobilized biocatalysts is rich with technology awaiting exploitation.
`
`acid, and
`Furthermore, biocatalysis is not standing still. The use of enzymes in non-aqueous
`obilized
`
`solutions is rapidly expanding the scope of applications for biological catalysts.
`L-alaninc
`
`
`Continuing advances in genetic engineering, as mentioned above, and also in reactor
`
`
`Sydantoin,
`de