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
`
`Q H
`
`Edited by
`J. David Rozzell and Fritz Wagner
`
`With 115 Illustrations and 68 Tables
`
`

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`The Editors:
`Dr. J. David Rozzell, Vice President, Research and Development, Exogene, Monrovia, CA 91016, USA
`Prof. Dr. Fritz Wagner, Institut fiir Biochemie und Biotechnologie der TU Braunschweig, Braun-
`schweig, Germany
`1
`
`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
`especially identified, is not to be taken as a 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 accept any 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).
`,
`p.
`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. II. Wagner, Fritz,
`Dr. rer. nat. .
`TP248.65.A43B56 1992
`660’.63 — dc20
`
`92-26917
`
`’
`
`Die Deutsche Bibliothek — CIP-Einheitsaufnahme
`Biocatalytic production of amino acids and derivatives / J. David
`Rozzell ; Fritz Wagner (ed.). — Munich ; Vlenna ; New York ;
`Barcelona : Hanser, 1992
`
`(Hanser titles in biotechnology)
`ISBN 3-446-15699-2
`NE: Rozzell, J. David [I-Irsg.]
`
`ISBN 3 -446-15699 -2 Carl Hanser Verlag, Munich Vienna New York Barcelona
`ISBN 0 -19 — 520982 - 6 Oxford University Press
`
`All rights reserved. No part of this book may be reproduced or transmitted 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 Kosel, Kempten
`
`
`
`INTRODUC
`
`Amino acids are I
`
`important chemic
`these are optical]:
`are essential for l
`amino acids in i
`
`important phaml
`occur in nature: I
`
`examples found
`secondary metal
`D-phenylglycine
`antibiotics. The
`several hundred!
`
`and L—tryptophal
`drugs, synthesiu
`for biological ted
`it is not surprisil
`pharmaceutical p
`be surprising, I:
`amino acids, bu
`areas.
`
`Worldwide l
`
`annually. For ex
`demand of new
`
`of the high-intell
`than one billion
`
`L-aspaitic acid
`manufactured in
`chelators and s:
`intermediates ii
`
`importance as c
`This book in
`
`production of :
`processes to pr!
`of amino acids
`
`biocatalysts in :
`integrated into
`enzymes in no-
`the cloning and
`and immobilize
`
`production of a
`biotechnology.
`
`
`
`

`

`
`
`

`

`J. D. Rozzell
`306
`[Refs on p 318]M.—
`
`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—workcrs in the
`19605 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. The first
`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—amino acids into their corresponding optically pure enantomeric forms.
`Since that time, enzymes in immobilized form have become increasingly important as
`catalysts for the production of amino acids, as well as numerous other substances.
`By way of definition, immobilized-enzyme biocatalysts consist of the enzyme, in
`varying degrees of purity, 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) of interest may be immobilized. Driving the development of
`this technology is the fact that the immobilization of an enzyme can improve the
`economics of its 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 ease 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 enzyme or cell 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 immobilimtion 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 but rather illustrative of what
`
`

`

`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 technique 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 enzyme is to be used.
`
`13.2.1 Adsorption
`
`l3 Immobilized Enzymes: Techniques & Applications
`
`307
`
`13.2 GENERAL IMMOBILIZATIOI‘} METHODS
`
`Five general techniques have been described for immobilizing enzymes:
`
`affinity for the support relative to other proteinaceous material such that a partial
`
`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, even‘though this immobilization procedure may be straightforward
`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 aqueous solution of the enzyme with the support is all that; is required.
`Occasionally, with judicious selection of the support, the desired enzyme will have an
`
`

`

`
`
`

`

`13 Immobilized Enzymes: Techniques & Applications
`
`309
`
`i" the reaction
`‘ r e bound
`. md attack
`
`‘5
`
`generally
`., within a
`
`‘
`
`protein is detected in the washing solution. These washing procedures are 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
`enzyme activity, it is suspended in 2.5 l 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/hg of preparation, under
`stande 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-
`Dbmethionine 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 considered to be the first
`commercial-scale immobilized—enzyme process.
`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 and silica gel [,5], leucine
`aminopeptidase on hydroxylapatite [6], glutamic-aspartic transaminases on DEAE-
`Sepharose [7], and a-amino-e-caprolactam racemase on DEAE—Sephadex [8]. There
`are numerous reports of the immobilization of other/enzymes 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 found that will 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
`
`in the
`
`‘ drops
`‘ area for
`
`‘ clay,
`titania
`
`=~.~
`
`13.2.2 Covalent Attachment
`
`Covalent attachment of enzymes to surfaces is often employed when leaching of
`enzyme activity 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
`
`

`

`[Refs on p 318]
`J. D. Rozzell
`310
`________________—-————————-
`
`'
`"
`
`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 product solution is
`of critical importance, it still may be the method of choice.
`There are three different techniques by which covalent attachment can be effected.
`The first is through exposure of the enzyme to 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 procedure is 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 enzymes since 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 groups listed, -NH2, -C02H, 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
`attachment of 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 also be tailored 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 here to illustrate typical covalent
`
`attachment methodology.
`
`

`

`
`
`\—
`fl. on p 318]
`
`—CO H
`2
`
`13 Immobilized Enzymes: Techniques & Applications
`311
`
`g-
`_ COSI [0
`TABLE 13.1 Useful Functional Groups for Covalent Attachment of Enzymes
`'
`ngmficantly
`
`to a Support Matrix
`_
`t solution is
`
`
`Functional Group
`Corresponding Amino Acid
`be effected.
`,
`
`lmctivated
`—NH2
`Lysine, N—terminus
`It has been
`an enzyme
`xposure of
`g reagent.
`the occurs
`it: activity
`zyme and
`livation of
`:nificantly
`stems for
`
`Glutomic Acid, Aspartic Acid, C—terminus
`C ste ne
`y
`I
`.
`Tyrosrne
`
`Arginine
`
`where the
`lurdency
`bchment
`
`! enou h
`g
`in most
`22:12:
`n 'th
`at with
`w‘
`al
`CW ent
`:hment
`
`Histidine
`
`Serine
`
`'
`
`.
`.
`.
`.
`.
`13.2.2.1 Specrfic Examples of Covalent Coupling: Binding to Acti—
`vated Carbohydrate Supports
`One of the most commonly used procedures for the covalent coupling of enzymes to
`carbohydrate support matrices is based on a preactivation of a support with cyanogen
`bromide [10, .11]. The mechanism of this reaction has been studied extensively by
`Wilehek and Kohn [12, 13].
`The activated carbohydrate will couple generally to an amino group of lysine on
`'
`the protein or the free N-terminus of the protein to yield a covalently bound product.
`Supports which have been preaetivated with cyanogen bromide can be prepared in
`advance and stored for periods of up to one year at freezer temperatures. Preactivated
`supports are also available commercially. Coupling of an enzyme to a CNBr-activated
`support requires no more than exposure of the enzyme to the support in aqueous
`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
`requirements to handle toxic reagents and the poorer mechanical stability of most
`carbohydrate gels and polymers compared to other support materials. The bond
`between the enzyme and the support is also potentially susceptible to hydrolytic
`cleavage.
`‘
`
`

`

`
`
`[Refs on p 318]
`I. D. Rozzell
`312
`13.2.2.2 Specific Examples of Covalent Coupling: Carbodiimide
`Coupling
`
`through the formation of an
`Carbodiimide reagents activate carboxyl groups
`O-acylisourea intermediate, which reacts rapidly with nucleophilic functional groups
`
`
`
`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,
`l6, 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 y—aminopropyluiethoxysilane [16]. The
`
`Another process for immobilizing on amino-functionalized inorganic supports
`involves isocyanate bonding [18). If the enzyme is 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 isocyanatc reacts
`
`

`

` [Refs on p 318]
`
` 13 Immobilized Enzymes: Techniques & Applications
`
`313
`
`
`
`with a hydroxyl group on the enzyme and a urethane bond is formed.
`lsothiocyanates
`have also been used successfully [16].
`in providing amino group
`Polyethyleneiminc is a common polyamine, useful
`
`
`functionality for attachment. It has the advantage that 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
`
`
`
`the
`immobilization
`of
`recombinant
`transaminase,
`aspartase,
`and
`aspartate-
`B-decarboxylase in the industrial-scale production of L~amino acids, 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.
`.
`
`
`
`
`
`Carbodiimide
`
`the formation of an
`I
`wrilic functional groups
`W5 is a competing side
`Mon of transaminases
`'1 forms a stable amide
`-bound primary amines,
`““386 of carbodiimide
`f the enzyme leading to
`traction.
`
`Amine—B earing
`
`nilized supports for the
`ml reagents has been
`firing pendent amines.
`for binding. Glutar—
`Ilk, reacts in complex
`:md produces pendent
`h which proteins may
`:3 and diisocyanates.
`I widely used for the
`wd technique for the
`and functionalization
`'nvolves attaching an
`vailable for covalent
`Ipularized through the
`mysilane [16]. The
`upling agents such as
`ctivated for enzyme
`:1: the use of carbodi-
`mups that attachment
`
`d inorganic supports
`aalkalinc conditions,
`stein surface and the
`the isocyanate reacts
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`13.2.2.4 Specific Examples of Covalent Coupling: Oxirane or Epoxy-
`Activated Polymers
`’
`
`Epoxy-activated polymers fall 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
`displacement reaction 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 once it is bound.
`For example, the pKa 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,
`FRG), and is sold commercially under the trade name of Eupergit. The support is
`based on a methacrylic polymer bead bearing epoxide functionality. Eupergit has been
`successfully applied to the immobilization of many enzymes including penicillin
`acylase for use in the production of 6-aminopenicillanic acid [27].
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`J. D. Rozzell
`314
`13.2.3 Cross-Linking
`
`[Refs on p 318]
`
`us other materials have also been used. Bauman and
`
`in the matrix.
`This immobilization technique
`of Bemfeld and Wan using poly
`matrix for entrapment has in fact been polyacrylamide, which has had significant early
`'
`
`,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 method is particularly useful because it gives particles of a
`controlled size for use in packed-bed reactor systems.
`
`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
`
`

`

`13 Immobilized Enzymes: Techniques & Applications
`
`315
`
`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].
`One of the major concerns with respect to entrapped enzymes is that of leaching.
`The enzyme may migrate out of the pore if the pore is too large.
`In many cases, this
`leaching may be overcome by 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 of the 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 a]. 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 czuragcenan gels and
`hardened with hexamethylenediamine and glutaraldehyde [39]. Calcium alginate gel
`has similarly been used.
`A novel method to overcome the problem of leaching out'of activity was developed
`as a part of the polyazetidine 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 more stable
`immobilized enzyme preparation in a hydrophilic environment, which is 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. Rozzell
`316
`[Refs on p 318]\
`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 membranes that he used most successfully were cellulose nitrate and nylon.
`In another example, the Snamprogetti Company in 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 effective in facilitating contact between an aqueous phase and a non-water-
`miscible phase. The enzyme is entrapped against the membranes and held in place by
`a slight positive pressure. As long as the stability of the enzyme is 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.
`
`13.3 CONCLUSIONS
`
`
`
`
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`
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`
`
`Perhaps the most important conclusion one can draw from the past thirty 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 number of these 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 number of amino acids are also made using immobilized
`enzyme catalysts, including L—aspartic acid from fuman'c 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
`4"
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`

`

`13 Immobilized Enzymes: Techniques & Applications
`
`317
`
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`
`
`including L—lysine, L-phcnylalanine, L-tyrosine, L-sen'ne. L-4-phenyl-
`as well,
`2-aminobutanoic acid. L—norvaline, an'd L—DOPA. Genetic engineering has played a
`key role in a number of cases 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.
`
`
`
`
`
`
`
`
`
`
`
`of work
`Desrred
`Sub—optimal
`.
`
`‘ has been
`Propevtve
`Properties
`
`
`
`successful
`
`does not
`
`
`
`SITE- DIRECTED
`PRODUCTION AND
`
`In fact,
`MUTAGENESIS
`APPLICATION
`
`
`
`
`
`Figure 13.]. Using Genetic
`Engineering to Improve the
`COMMERCIALIZATION
`Catalytic Properties of_
`
`
`Enzymes.
`
`I'll which
`
`
`
`
`
`
`The field of immobilized biocatalysts is rich with technology awaiting exploitation.
`acid, and
`
`
`
`I iilizcd
`Furthermore, biocatalysis is not standing still. The use of enzymes in non—aqueous
`
`L—alanine
`solutions is rapidly expanding'the scope of applications for biological catalysts.
`
`
`Continuing advances in genetic engineering, as mentioned above, and also in reactor
`iydanloin,
`
`
`L-scrine.
`design, should help to accelerate the realization of the potential benefits of this
`
`technology.
`.
`| acids
`
`
`NATIVE ENZYME
`
`CLONING OF GENE
`
`EXPRESSION IN
`SUITABLE HOST
`
`CHARACTERIZATION
`
`SELECTION OF NEW
`MODIFIED ENZYMES
`
`
`
`
`
`
`
`

`

`318
`
`1. D. Rozzell
`
`13 .4 REFERENCES
`
`Chibata, 1., Tosa, T., Takamatsu, S., Methods Enzymol. 136, 472 (1987).
`
`”999993“?
`Nb—At—At—‘p—At—‘i—Ip—tHy—tb—d53.0.“.“99‘29959599
`
`21.
`22.
`
`MNPS”
`
`Chibata, I. Immobilized Enzymes; John \Viley & Sons, New York, 1978.
`Mosbach, K., Methods Enzymol. 44, v-vi (1974).
`Messing, R. A., Methods Enzymol. 44, 148 (1974).
`Chibata, T. et (11., Methods Enzymol. 44, 746 (1974).
`Tosa, T., Soto, T., Mori, T., Matuo, Y., Chibata, 1., Biotechol. Bioeng. 15, 69 (1973).
`Schwabe, C., Biochemistry 8, 795 (1969).
`Rozzell, 1. D., unpublished results.
`Fukumura, T., Jpn. Pat. 74-15795 (1974).
`Goldstein, L., Biochemistry 11, 4072-4084 (1972).
`Axen, R., Porath, 1., Ernback, S., Nature (London) 214, 1302 (1967).
`Porath, 1., Axen, R., Ernback, S., Nature (London) 215, 1491 (1967).
`Kohn, 1., Wilchek, M., Enzyme and Microbial Technology 4, 161-163 (1982).
`Kohn, 1., Wilchek, M., Appl. Biochem and Biotechnology 9, 285-305 (1984).
`Rozzell, 1. D., Methods Enzymol. 136, 479 (1987).
`Messing, R. A., Weetall, H. H., U.S. Pat. 3,519,538 (1970).
`Weetall, H. 11., Science 166, 615 (1969).
`Weetall, H. H., Filbert, A. M., Methods Enzymol. 34, 59 (1974).
`Messing, R. A., Yaverbaum, S., U.S. Pat. 4,071,409 (1978).
`Rohrbach, R. P., U.S. Pat. 4,525,456 (1985).
`Lantero, O. 1., U.S. Pat. 4,438,196 (1984).
`Chiang, 1. P., Lantero, O. 1., U.S. Pat. 4,713,333 (1987).
`Goldberg, B. S., U.S. Pat. 4,102,746 (1978).
`Goldberg, B. S., U.S. Pat. 4,169,014 (1978).
`Crump, S. P., Meier, 1. S., Rozzell, 1. D. In Biocatalysis; Abramowicz, D. A., Ed.; Van
`Nostrand Reinhold, New York, 1990; pp 115—133.
`Leuba, 1.-L., Renker, A., Flaschel, E., U.S. Pat. 4,918,016 (1990).
`Bigwood, M. P., Naples, 1. 0., U.S. Pat. 4,582,860 (1986).
`Bihari, V., Buchholz, K., Biotech. Letters 6, 571-576 (1984).
`Paulsen, P., Enzyme and Microbial Technology 3, 271 (1981).
`Silman, I. H., Albu-Weissenberg, M., Katchalski, E., Biopolymers 4, 441 (1966).
`Quiocho, F. A., Richards, F. M., Proc. Natl. Acad. Sci. USA 52, 833 (1964).
`Quiocho, F. A., Richards, F. M., Biochemistry 5, 4062 (1986).
`Bemfield, P., Wan, 1., Science 142, 678 (1963).
`Bauman, E. U., Goodson, L. U., Guilbault, G. G., Kramer, D. N., Anal. Chem. 37, 1378
`(1965).
`Vieth, W. R., Venkatsubramanian,K., Methods Enzymol. 44, 243 (1976).
`Pollack, A., Blumerfeld, H., Baughlin, R. L., Whitesides, G. M., J. Am. Chem. Soc. 102,
`6324-6336 (1980).
`Crans, D. C., Kazlavskas, R. 1., Hirschbein, B. L., Wong, C.-H., Abril, 0., Whitsides, G.
`M., Methods Enzymol. 136, 263 (1987).
`Fusee, M. C., Methods Enzymol. 136, 463 (1987).
`Calton, G. 1., Wdod, L. L., Campbell, M. L., Methods Enzymol. 136, 497 (1987).
`
`

`

`13 Imm