Reactive Surfaces Ltd. LLP
`Ex. 1049 (Rozzell Attachment B)
`Reactive Surfaces Ltd. LLP v. Toyota Motor Corp.
`IPR2016-01914
`
`

`

`NOLOGYTM
`
`\'
`
`OR
`
`
`
`ado, 2005
`
`Luis Bar/€610, 2005
`I by Jo/m F. T. Spencer
`
`fired by Evgeny N.
`
`’Cz'a Leonor Ragom (Xe
`
`Vil/z'ams and AMI/7017)? A.
`
`Microbiai Enzymgs
`and Biotransformatmns
`
`9, 1999
`
`ucke, 1999
`2000
`
`30ge/‘s
`
`ted by
`
`as J. Men/7, 1999
`
`'z'nically Useful
`arter, 1998
`
`7% 1997
`
`Edited by
`
`josé Luis Barredo
`
`R & D Biology, Antibio’licos S. A,
`Leo’n, Spain
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`© 2005 Humana Press Inc.
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`Cover illustration: Figure 6, from Chapter 2. “Enzyme Biosensors.” by Steven J. Setlord and Jel‘l‘rey D.
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`Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
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`The:
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`overvie
`and of
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`
`Library of Congress Cataloging in Publication Data
`
`Microbial enzymes and biotransformations / edited by Jose Luis Barredo.
`
`p. cm—(Methods in biotechnology)
`
`Includes bibliographical references and index.
`
`ISBN 168829-2536 (alk. paper) E—lSBN 1692598466
`
`1. Microbial enzymes—~Biotechnology-~—Laboratory manuals. 2. Microbial biotechnology——
`
`Laboratory manuals 3. Biotransformation (Metabolism)—Laboratory manuals. l. Barredo Jose Luis.
`
`H. Series.
`TP248.65.E59M54 2004
`
`660.6‘2—dc22
`
`
`-
`
`2004054093
`
`
`
`

`

`and Ferry
`
`
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`initoring to
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`histidine»
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`hyperexw
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`tl, Acad.
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`ymerase
`13—130.
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`(1998)
`isarcina
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`Chem.
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`
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`16E___
`
`\\
`
`Immobilization of Enzymes by Covalent Attachment
`Scott J. Novick and J. David Rozzell
`
`1
`
`l
`
`i
`
`Summary
`Enzymes are finding increasing use for the production of agrochemicals, pharmaceuticals, and
`fine chemicals. They are almost always used in the immobilized form in order to simplify their
`removal from the product stream In addition, immobilization often enhances the stability of the
`methods, properties, and uses of covalently immobilized enzymes
`Key Words: Immobilized enzymes; covalent immobilization.
`
`1. Introduction
`
`i
`
`t
`
`1.1. Historical Perspective
`An immobilized enzyme is generally defined as “the imprisonment of an
`enzyme molecule in a distinct phase that allows exchange with, but is separat—
`ed from, the bulk phase in which substrate effector or inhibitor molecules are
`dispersed and monitored” (1). Immobilized enzyme technology dates back to
`the 1910s to the 1930s, when proteins were physically adsorbed onto surfaces
`such as charcoal, kaolinite, cellulose, and glass beads (2—4). But it was not until
`the 1950s and ’605 with the work of Katchalski~Katzir, and Chibata and co—
`Workers that real advancements were beginning to be made in the development
`and applications of immobilized enzyme materials (5). This early work culmi—
`nated in the First Enzyme Engineering Conference in 1971. The first industrial
`Use of immobilized enzymes was for amino acid production. Chibata and co—
`WOrkers at Tanabe Seiyaku (Japan) in 1969 used an immobilized L—aminoacy—
`1386 in a packed bed reactor to resolve various DL—amino acids into their enan—
`tiOmerically pure forms. Since that time, immobilized enzymes have become
`increasingly important for the production of many important chiral compounds
`(1.6., amines and alcohols) for the pharmaceutical and fine chemical industries.
`
`From: Methods in Bio/eclino/ogy, Vol. [7: Microbial Enzymes and Biol‘ransformarions
`Ediled by: J. L. Barredo © Humana Press Inc, Totowa, NJ
`247
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`Table 1
`
`2
`U)
`
`gation by Spatial fixation of enzyme mole-
`
`ent or adsorptive
`g of the enzyme.
`ciate if all subunits are
`
`
`
`There are
`
`activity is al
`
`possess high
`must come 11'
`
`such as coval
`
`the secondar
`interactions,
`activity. In ac
`nonessential r
`
`supports crea
`biocatalyst (6
`limitations. H
`uct stream an
`
`1.3. Enzyme
`
`In general
`enzymes. It is.
`
`tion system; i
`the enzyme to
`enzyme is to ‘
`ally distinct,
`immobilizatio
`One of the
`
`ing an enzymt
`nonionic inter
`
`polymer ion-e
`and ceramics.
`
`from the suppr
`Cross—linkii
`
`inert protein S
`active enzyme
`tinuous reacto
`used as the CI
`
`result during tl
`Entrapment
`for enzyme im
`monomer and
`
`enzyme. Leach
`of substrate dif
`
`Encapsulatir
`for enzyme in“
`
`Stabilization Effects Immobilization lmparts to Enzymes (8)
`
`1. Prevention of either proteolysis or aggre
`cules to the support.
`. Unfolding of the enzyme is reduced due to multipoint coval
`attachment to the support, and/or intramolecular crosslinkin
`. Multimeric enzymes would have a lower likelihood to disso
`attached to the support.
`4. Denaturing agents (eg, chemical inactivators) can be excluded fro
`m the enzyme
`by t}we support or inactivated by the support before reaching the en
`zyme (e.g.,
`decomposition of hydrogen peroxide, produced during the oxid
`ation of glucose by
`glucose oxidase, catalyzed by activated carbon).
`5. Shifting by a charged support of the local
`pH, thus preventing pH inactivation of
`the enzyme.
`6. Exclusion by the support (e.
`enzyme’s environment
`
`248
`
`Nov/Ck and Rozze/l
`Covalent En
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`The principal advantage of immobilizing en
`
`the reactor. This can greatly improve the economics of a process. For a contin—
`
`uous process, a soluble enzyme would be washed out of the reactor along with
`
`the product stream. A process like this would not be economically feasible if the
`biocatalyst is very expensive (as is often the case) and cannot be reused.
`
`Although an ultrafiltration setup could be used to retain the enzyme, it is often
`
`too costly, both in capital and operation, on a large scale, Also, having a solu-
`
`ble enzyme in the product would not be desirable if the biocatalyst can cause
`the product to undergo side reactions or if there are toxicity effects associated
`
`with the catalyst, as will often be the case if the product is an intravenous drug
`
`(6). Another advantage of immobilizing enzymes is to increase enzyme activi—
`
`
`ty or stability especially under denaturing conditions (7,8). Thermal stability
`can often be improved by many orders of magnitude compared to the soluble
`
`enzyme (9—11). Activity of an enzyme in nonaqueous tnedia can also be signif-
`
`icantly higher than the native enzyme (12—18). Another important advantage iS
`the ability to control the microenvironment of the immobilized enzyme. For
`
`example. by immobilizing an enzyme on an acidic support (such as pOIY
`
`[acrylic acid]), the catalyst can be used at higher pHs, where the substrate may
`
`be more soluble, while the pH of the microenvironment surrounding the
`enzyme could be much closer to the enzyme’s optimum pH. These and other
`
`stabilizing effects of immobilization are listed in Table 1.
`
`
`g., an encapsulation membrane) of proteases from the
`
`7. Increased thermal stability due to mul
`
`tipoint attachment of enzyme to support.
`
`1.2. Reasons for Enzyme Immobilization
`
`
`
`

`

`
`
`
`
`249
`
`Covalent Enzyme Immobilization
`
`and Rozze/l
`
`
`
`zyme mole—
`
`ptive
`Lyme.
`.ubunits are
`
`e enzyme
`> (es,
`
`glucose by
`
`vation of
`
`s from the
`
`[pport
`
`catalyst in
`r a contin-v
`
`tlong with
`sible if the
`)e reused.
`
`it is often
`
`.ig a solu—~
`can cause
`
`tssociated
`
`ious drug
`ne activi-
`
`l stability
`e soluble
`
`3e signif—
`antage is
`yme. For
`
`as poly
`rate may
`
`ding the
`.nd other
`
`There are also limitations to immobilizing enzymes. Some inherent catalytic
`activity is almost always lost during the immobilization procedure. Enzymes
`possess highly defined, yet relatively fragile three—dimensional structures that
`must come in contact and interact with the rigid support. These binding forces,
`such as covalent bonds or adsorptive interactions, are often more powerful than
`the secondary forces, such as hydrogen bonding and hydrophobic and ionic
`interactions, which hold proteins in their proper configuration for enzymatic
`activity In addition, no covalent immobilization method is able to bind only the
`nonessential elements of every enzyme (if they even exist) to the support, and all
`supports create asymmetric force fields and change the water activity around the
`biocatalyst (6). In addition, apparent activity can be decreased by mass transfer
`limitations However, the increase in stability and ease of removal from the prod—
`uct stream and reuse often more than make up for any decrease in activity.
`1.3. Enzyme Immobilization Methods
`
`five techniques have been described for immobilization of
`In general
`enzymes. It is important to point out that there is no one universal immobiliza-
`tion system; instead, a range of methodologies must be evaluated depending on
`the enzyme to be immobilized and the overall process in which the immobilized
`enzyme is to be used. Also, most immobilization methods, although conceptu-
`ally distinct, often overlap to a certain extent, and in some cases, multiple
`immobilization methods are employed.
`One of the simplest and most economical immobilization methods is adsorb-
`ing an enzyme onto a support. The enzyme is bound to the support via ionic or
`nonionic interactions. Supports often include carbohydrate—based or synthetic
`polymer ion—exchange resins or uncharged supports such as polymers, glasses,
`and ceramics. The main drawback of this method is the leac
`hing of enzyme
`from the support.
`Cross~linking enz
`yme molecules with themselves, or more often with an
`inert protein such as gelatin or bovine serum albumin, results in an insoluble
`active enzyme preparation that can be readily handled or manipulated in a con—
`tinuous reactor. Glutaraldehyde, adipimate esters, and diisocyanates are often
`used as the cross-linking agent. Significant inactivation of the enzyme may
`result during the cross—linking step and is the major drawback of this method.
`Entrapment of an enzyme within a polymeric matrix is another method used
`for enzyme immobilization. This is often done by mixing the enzyme with a
`monomer and a cross—linker, and polymerizing the monomer around the
`eHZyme. Leaching of the enzyme out of the matrix and mass transfer limitations
`Oi substrate diffusing into the matrix can limit the use of this technique.
`Encapsulating or confining an enzyme within a membrane is another method
`f0r enzyme immobilization. Ultrafiltration membranes or hollow fibers made of
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`
`
`
`250
`
`
`
`NOV/Ck and ROZZe/l
`
`polyethersulfone, cellulose nitrate or acetate, or nylon are often used.
`Th6 pore
`size must be properly chosen to allow substrate and product to enter and exit the
`membrane while still retaining the enzyme. Since the enzyme exists in its sol-
`uble form, activity is usually high. Membrane fouling and reduced flow rates
`are drawback of this technique.
`The fifth immobilization method, covalent attachment of enzym
`port, will be the subject of the rest of this chapter.
`
`BS [0 a SUP-
`
`1.4. Covalent Enzyme Immobilization
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Covalent attachment of enzymes to an insoluble support is an often—used
`method of enzyme immobilization. It is especially useful when leaching of
`enzyme from the support is a concern. The enzyme is usually anchored via mul—
`tiple points and this generally imparts greater thermal, pH, ionic strength, and
`organic solvent stability onto the enzyme since it is more rigid and less suscep-
`tible to denaturation. Covalently immobilized enzymes are also often more
`resistant to degradation by proteolysis.
`There are, however, some drawbacks to covalent enzyme immobilization.
`Typically it is more expensive and complex to covalently immobilize an enzyme
`compared to the other methods due to the higher costs of the support. The sup-
`port often needs to be activated prior to immobilization. The increased stability
`and typically minimal enzyme leaching often more than make up for these short-
`comings.
`Enzymes contain a number of functional groups capable of covalently
`binding to supports. Table 2 lists these groups along with their relative fre-
`quency in a typical protein (19—21). Of the functional groups of enzymes list—
`
`ed, —NH2, —COgH, and —SH are most frequently involved in covalent immo—
`
`bilization. Amines and sulfhydryls are good nucleophiles, while the ability to
`
`activate carboxylates so they are reactive toward nucleophiles makes these
`
`groups important as well. The phenolic ring of tyrosine is also extremely
`
`reactive in diazo—coupling reactions, and its hydroxyl group can be an excel—
`
`lent nucleophile at basic pH. Aldehydes can react with the guanidino group of
`
`arginine and, although histidine displays a lower nucleophilicity, it can react
`
`in some cases with supports activated with tosylates, tresylates, or other good
`
`leaving groups.
`
`The supports to which the enzymes are attached to can vary greatly. They can
`
`be either natural polymers, such as modified cellulose, starch, dextran, agal pOIY‘
`
`saccharides, collagen, and gelatin; or they can be synthetic. such as polystyrene,
`
`polyacrylamide, polyacrylates, hydroxyalkyl methacrylates, and polyamides.
`
`Inorganic supports can also be used, such as porous glass, metal oxides, metals,
`
`sand, charcoal, and porous ceramics. The variety of chemistries available for
`covalent attachment allows the conditions of immobilization to be tailOred t0
`
`
`
`
`Covalent .
`
`Table 2
`Reactive Fl
`in a Typica
`
`Structure of
`reactive gro
`
`“CH2OI
`
`each enzyir
`be tailored l
`eties or ioni
`
`enzyme—cat.
`
`

`

`Cova/ent Enzyme Immobilization
`
`
`
`
`
`
`
`
`
`
`
`
`3 pore
`Kit the
`ts sol~
`' rates
`
`Table 2
`
`Reactive Functional Groups in Enzymes and Their Average Occurrence
`
`in a Typical Protein (19—21)
`—-————~———————_________________
`Occurrence
`
`Structure of
`
`reactive group
`
`Reactive group
`
`in average protein
`
`a sup~
`
`—NH2
`
`-COQH
`
`E-Amino of lysine and N~~terminus
`
`5.9
`
`Carboxylate of glutamic acid,
`aspartic acid, and C—terrninus
`
`6.3 (Glu), 5.3 (Asp)
`
`Thiol of cysteine
`
`Phenolic of tyrosine
`
`Guanidino of arginine
`
`1.9
`
`3.2
`
`5.1
`
`Imidazole of histidine
`
`2.3
`
`Disulfide of cystine
`
`Indole of tryptophan
`
`—CH3—S—
`
`Thioether of methionine
`
`1.4
`
`2.2
`
`~ CHEOH
`
`Hydroxyl of serine and threonine
`
`6.8 (Ser), 5.9 (Thr)
`
`each enzyme system. This also allows the microenvironment of the enzyme to
`be tailored by appropriate modification of the support surface; hydrophobic moi—
`eties or ionically charged groups may be used to alter the support to enhance the
`enzyme—catalyzed reaction of interest. Some supports, such as those containing
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`
` Immobilized
`enzyme
`
`
`
`
`
`
`
`1.Enzymeimmobiliz
`
`Fig.
`
`
`
`/O\
`
`
`
`Immobilizedenzyme
`
`N~—Enzyme
`
`NH+Enzyme-MHZ
`
`
`5
`
`+Enzyme-NH2
`
`Cl
`
`\
`
`WM0
`
`NYN R
`
`
`
`Cl
`
`
`
`Activatedsupport
`
`Striazine
`
`
`
`Hydroxyl-containingsuppon
`
`
`
`Activatedsupport
`
`bromide
`
`support
`
`Cyanogen
`Hydroxyl-containing
`
`
`
`:0: 0
`
`O
`
`E:Z
`
`O+
`
`
`
`
`
`
`
`
`
`
`
`ationontohydi‘oxylcontainingsupportsViaactivationwithcyanogenbromide(top)orS-ti‘iazine
`
`Cova/en
`
`epoxide
`
`ports req
`sections t
`
`7.4.7. C
`
`Polyhj
`are amOi
`
`Because
`
`This is ty
`as S—triaz
`able to o
`
`lysine or
`hydroxyl
`enzyme 1
`
`Suppo
`for periOt
`available
`
`requires
`aqueous
`
`any prote
`This n
`
`widely u:
`mide, is 1
`agarose, 2
`materials,
`taminatio
`
`enzyme a
`would eat
`
`7.4.2. Cc
`
`Carbox
`
`acids witl
`
`port. Thes
`reagent. 1
`with carbt
`tives. The
`
`is coupled
`or ester 11'“
`
`widely “5
`
`
`
`propyl)-Cal
`bodiimide;
`
`
`derivatives(bottom).
`
`
`
`

`

` Covalent Enzyme Immobilization
`
`7.4. 7. Covalent Attachment Onto Polyhydroxy/ Supports
`Polyhydroxyl supports, such
`as porous glass, and especially polysaccharides
`are among the most commonl
`y used matrices for enzyme immobilization.
`Because hydroxyl groups are
`poor leaving groups they must first be activated.
`This is typically done with cyanogen bromide (22). Other activating agents such
`as S-triazine derivatives have also been used. Once the support is activated it is
`able to covalently couple to an enzyme usually through the e—amino group of
`lysine or through the amino terminus. The mechanism of derivatization poly—
`hydroxyl supports with the above two derivatizing agents and the subsequent
`enzyme immobilization is shown in Fig. 1.
`Supports that have been preactivated with cyanogen bromide can be stored
`for periods of up to 1 yr at freezer temperatures. Preactivated supports are also
`available commercially. Once the support is activated, coupling of the enzyme
`requires no more than exposing the enzyme to the activated support in an
`aqueous solution for a few hours, followed by extensive washing to remove
`any protein that is not covalently bound.
`This method is extremely popular in the lab scale; however, it has not been
`3 Widely used in large—scale applications. The activating
`‘ mide,
`is extremely toxic
`
`tamination and degradation are a concern. Finally,
`the bond between the
`enzyme and the support is potentially susceptible to hydrolytic cleavage, which
`would cause leaching of the enzyme from the support over time.
`7.4.2. Covalent Attachment onto Carboxylic Acid—Bearing Supports
`Carboxylic acid—containing su
`pports, such as copolymers of (meth)acrylic
`acids with (meth)acrylic esters h
`ave also been used as an immobilization sup—
`port. These must also be activ
`ated and this is usually done with a cai‘bodiimide
`reagent. Under slightly acidic conditions (pH 4.75—50) carbodiimides react
`With carboxylic acid groups to give the highly reactive O—acylisourea deriva-
`tives. The supports are then washed to remove excess reagent and the enzyme
`iS Coupled to the activated support at neutral pH to give stable amide, thioester,
`0r ester linkages, depending on the residue reacting with the support. The most
`Widely used water—soluble carbodiimides are l—ethyl~3-(3~dimethylamino
`PrOpyl)—carbodiimide (EDC) and l—cyclohexyl—3~(2—morpholino—ethyl)—car—
`bOdiimide (CMC), both of which are available commercially. The reaction
`
`.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`
`
`Fig.2.Activationofacarboxylicacidcontainingsupportwithacarbodiimidefollowedbyenzymecoupling.
`
`
`
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`Fig.3.Activationofanamine—bearingsupportwithglutaraldehydefollowedbyenzymecoupling.
`
`Covalent En.
`
`scheme for 0
`
`enzyme coup
`
`7.4.3. Coval.
`
`Amine-beg
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`ports for COVa
`ic or inorganj
`nique for intr
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`attachment (2
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`groups on the
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`A simplified e)
`be reduced to ]
`increase the st:
`
`Crump and <
`acid transamin
`
`that was activa
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`uaJ activity we
`bound to the 81
`
`approximately
`for immobilize
`and immobilize
`
`Enzymes cal
`Via the enzyme
`iInide or simila
`
`enzyme inactiv:
`
`

`

` Cova/enl‘ Enzyme Immobilization
`
`
`255
`scheme for of activating a carboxylic acid—containing support and subsequent
`enzyme coupling is shown in Fig. 2.
`
`
`
`
`
`Amine-bearing supports are among the most used and the most useful Sup~
`ports for covalent enzyme immobilization. These supports can either be organ—
`ic or inorganic supports bearing amine functionality. The most frequent tech—
`nique for introducing amine groups on inorganic supports is via aminosilane
`attachment (23—25). For example, 3—aminopropyltriethoxysilane can be cou—
`pled to porous glass to give pendent amine groups (26). This silane has been
`developed through the pioneering work at Corning Glass Works (23).
`Another common amine-bearing support is polyethyleneimine—coated par-
`
`imately 25% primary amines, 25% tertiary amines, and 50% secondary
`amines. This polymer can be coated onto various supports including alumina
`;
`(27), carbon (28), diatomaceous earth (29), and polyvinyl chloride—silica
`; composites (30,31).
`
`I}
`
`‘
`-1
`
`: carbonyl functionalities through which enzymes may attach. Enzyme attach—
`ment is accomplished simply by mixing the enzyme with the activated support.
`"‘ .A simplified example of this is shown in Fig. 3. The acid-labile Schiff bases can
`be reduced to more stable secondary amine bonds with sodium borohydride to
`increase the stability of the enzyme—support linkage.
`Crump and coworkers (32) have described the immobilization of an L-amino
`acid transaminase onto a polyethyleneimine coated PVC—silica support matrix
`that was activated with glutaraldehyde. Very high binding efficiency and resid—
`Ual activity were obtained. After washing, 93% of the enzyme offered was
`bOLmd to the support (total loading was about 10%) and the enzyme retained
`approximately 89% of the soluble activity. Both these values are unusually high
`fOT immobilized enzymes, but not necessarily atypical for this type of support
`i and immobilization chemistry.
`. Enzymes can also be covalently bonded directly to amine—bearing supports
`l
`
`' Y1?! the enzymes carboxyl groups. These must first be activated with a carbodi—
`
`lmide or similar reagent prior to immobilization. The activation step can cause
`
`enZYme inactivation and thus this method is not used as often.
`
`
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`Nov/Ck and Rozze/l
`
`Covalent El
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`Enzyme—NH2
`
`OH
`|
`CH—‘CH2 ~NH—Enzyme
`
`Eupergit
`
`immobilized enzyme
`
`Fig. 4. Enzyme immobilization to Eupergit via free amino groups.
`
`Diisocyanates have also been used as a coupling agent between amine—bear-
`ing supports and enzymes (33). If alkaline conditions are used a substituted urea
`bond is formed between an amine on the enzyme and the isocyanate. If moder—
`ately acidic conditions are employed, the isocyanate will react with a hydroxyl
`group on the enzyme and form a urethane bond. Isothiocyanates have also been
`used successfully (23).
`Another amine—bearing support, developed by Leuta and coworkers (34), is
`mineral or carbon particles coated with chitosan. Chitosan is deacylated chitin,
`a polymer of glucosamine, and contains an available amino group for chemical
`activation and enzyme binding using methods similar to those described for the
`other amine—bearing supports.
`
`7.4.4. CovalentAttachment to Reactive Polymer Supports
`
`Due to the preactivated nature of epoxy—containing supports, these materials
`have gained considerable attention as commercially useful support matricies for
`enzyme immobilization. A commercial epoxy—containing support is available
`from Rohm Pharma Polymers (Piscataway, NJ) under the trade name Eupergit.
`The material is a crosslinked copolymer of methacrylamide and oxirane contain-
`ing monomers and consists of spherical beads of about 200 um in diameter.
`Eupergit is available in two varieties, Eupergit C and Eupergit C 250 L, with their
`differences being their oxirane content and pore size. Eupergit C has average pore
`radius of 10 nm and an oxirane content of 600 limOl/g, while Eupergit C 250 L
`has a pore size and oxirane content of 100 nm and 300 umol/g, respectively (35)-
`Eupergit C 250 L is targeted for the immobilization of large molecular weight
`enzymes.(>l 00 kDa). Immobilization of enzymes to Eupergit is relatively simple.
`The enzyme solution is brought in contact with the Eupergit beads either quies-
`cently or with slight mixing (magnetic stirbars should be avoided to prevent frac—
`tionation of the beads) for 2496 h. This can be done either at room temperature,
`or if the enzyme is unstable, 4°C will also work, Various pHs can be used for the
`binding. Under neutral and alkaline conditions the amino groups on the enzyme
`are principally responsible for binding to the support (Fig. 4). Under acidic and
`neutral conditions sulfhydryl and carboxyl groups take part
`in binding.
`
`Immobilizat
`
`Typically, it
`is Optimum
`
`type, immot
`buffer or net
`
`enzyme imnr
`support, the
`range, from I
`do not effect
`After the
`
`epoxy group
`
`will slowly l
`
`pounds that
`by making it
`the stability
`threitol, Tris
`
`glycine), am
`have been us
`
`altered deper
`There hav
`immobilizati
`the immobili
`addition to E
`for the covali
`
`Polyacrole
`enzyme imm
`them into a;
`
`polyaldehyde
`vated support
`been attachec
`
`and the enzyrs
`through then
`poly(lysine),
`attached t0 3
`
`groups using!
`acrolein SUPP‘
`carbodiimide]
`ers has Showr1
`
`weight subsm
`acrolein beads
`fied polyacr01€
`
`
`
`
`
`

`

`
`
`' Rozze/l
`
`Cova lent Enzyme Immobilization
`
`257
`
`nzyme
`
`ne--bear-
`ited urea
`f moder—
`
`1ydroxyl
`lso been
`
`(34), is
`d chitin,
`hemical
`i for the
`
`Jaterials
`icies for
`available
`
`iupergit.
`contain—
`iameter.
`ith their
`
`tge pore
`3 250 L
`
`:1y(35).
`weight
`simple.
`r quies—
`:nt frac—
`erature,
`
`l for the L
`
`enzyme
`die and
`
`finding
`
`Immobilization to Eupergit does not change the charged state of the enzyme.
`Typically, it is best to bind the enzyme to the support at the pH at which activity
`is Optimum for the enzyme. The various parameters mentioned above—mixing
`type, immobilization time, temperature, pH, and also ionic strength (0.5—1 M
`buffer or neutral salt is often optimal)——can be varied to optimize the amount of
`enzyme immobilized and the residual activity. Once the enzyme is bound to the
`support, the binding is stable over the long term and it is stable over a wide pH
`range, from 1.0 to 12.0. Also, because Eupergit is electrically neutral, pH changes
`do not effect the swelling of the gel.
`After the enzyme has been bound, typically only about 1% of the available
`epoxy group are involved in enzyme immobilization. The remaining groups
`will slowly hydrolyze into diols or they can be quenched with a variety of com-
`pounds that can effect the microenvironment around the immobilized enzyme
`by making it more hydrophilic, hydrophobic, or charged. This in turn can effect
`the stability or activity of the bound enzyme. Bovine serum albumin, dithio—
`threitol, Tris—buffer, mercaptoethanol, various amino acids (i.e.,
`lysine or
`glycine), and ethanolamine are among some of the quenching reagents that
`have been used, and in many cases activity of the immobilized enzyme can be
`altered depending on the quenching reagent.
`There have been two extensive reviews recently published concerning the
`immobilization of enzymes to Eupergit (35,36). In these reviews, the details of
`the immobilization of nearly two dozen different enzymes are presented. In
`addition to Eupergit, other epoxy—containing polymers have been investigated
`for the covalent attachment of enzymes (37—42).
`Polyacrolein beads is another useful reactive—polymer carrier for covalent
`enzyme immobilization. Margel (43) synthesized such beads and encapsulated
`them into agarose prior to enzyme binding. Because these supports are
`polyaldehydes, enzymes are bound in a similar way as with glutaraldehyde acti—
`vated supports. Various oligomers such as poly(lysine) and poly(glycine) have
`been attached to the polyacrolein beads to act as spacers between the particles
`and the enzyme. In both cases the poly(amino acids) are attached to the support
`through their terminal amino groups, or e—amino groups in the case of
`p01y(lysine), via Schiff bases (which can then be reduced). The enzyme is
`attached to the poly(lysine)--derivatized polyacrolein via the lysine e-amino
`groups using glutaraldehyde as a linker. For the poly(glycine)-derivatized poly—
`acrolein support, the terminal carboxyl group is activated with a water—soluble
`Catbodiimide followed by enzyme binding. In some cases the use of these spac-
`EFS has shown a significant increase in activity, especially for large—molecular—
`Weight substrates. Covalent enzyme immobilization to paramagnetic poly—
`2lcrolein beads has also been investigated (44). Binding of enzymes to unmodi—
`fied polyacrolein is shown in Fig. 5.
`
`
`
`
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`

`

`
`
`Covalent
`
`E Z
`” We
`
`‘NH
`
`2
`
`CHIN—Enzyme
`
`
`NaBH
`4»
`
`
`
`‘
`Polyacrolein
`support
`
`hnmoszedenzyme
`(oxidized form)
`
`
`Nov/Ck and [30229]]
`
`
`
`
`
`
`
`
`
`
`
`—-CH2—NH—Enzyme
`
`Innnobmzedenz me
`(reduced formy)
`
`7.5.2. D.
`
`Enzyme
`
`It is oi
`
`support 2
`when opl
`ual enzyi
`
`the partic
`In the
`
`determin
`bilizatior
`done on
`
`enzyme
`solutions
`In the
`
`determin
`method,
`
`is used (A
`solution.
`same ma
`
`of peptic
`purple—o
`the amor
`
`as protei
`Coon
`amounti
`
`to the e1
`removec
`
`by addii
`this solu
`
`support
`In an
`
`protein
`brown v
`
`nm'). WI
`cles, s01
`and the
`
`correlat
`BSA or
`
`that is i
`
`Also, if
`
`in g of t
`
`Fig. 5. Enzyme immobilization to unmodified polyacrolein via free amino groups,
`followed by reduction of the Schiff base with sodium borohydride.
`
`
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`
`
`1.5. Assaying the Properties of Immobilized Enzymes
`
`There are three important properties of immobilized enzymes that are often
`evaluated: activity, enzyme loading, and stability. Prior to the measurement of
`these properties, the immobilized enzyme materials should be washed exten—
`sively to remove any unbound enzyme that may be entrapped in the pores of the
`particles or loosely bound through noncovalent interactions.
`
`7.5. 7, Activity Assay
`
`There are two basic methods to measure activity—~batch and continuous. In the
`batch method the immobilized enzyme is added to a flask or vial and the substrate
`solution is then added to initiate the reaction. At various time points, an aliquot of
`the mixture is removed and filtered (this is most easily done through a syringe fil-
`ter) to remove any of the immobilized enzyme particles and to quench the reac—
`tion. This aliquot can then be analyzed using the appropriate analytical method,
`such as liquid chromatography, gas chromatography or spectrophototometry. If
`product continues to be produced in this aliquot after filtration, it is a good indi—
`cation that there may be significant leaching of soluble enzyme off the support.
`This can occur if the support is not washed extensively enough after immobiliza-
`tion or if the binding is labile under the assay conditions. To get more accurate
`activity measurements the supports should be rewashed.
`There are two basic methods for performing a continuous activity assay. In
`the packed—bed plug—flow tubular reactor (PFTR) method,
`the immobilized
`enzyme is packed into a column and substrate is pumped though the column
`and the substrate and/or product concentration is measured in the effluent. In
`the continuous stirred tank reactor, the solution and the immobilized enzyme
`are well mixed so there are minimal concentration gradients. To prevent the loss
`of immobilized enzyme out of the exit, a filter is added at the effluent or a tub6
`is added at the exit that is long enough such that at the given flow rat