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

`

`Microbial Enzymes
`and B iotraMnstormat OnS
`
`
`
`
`
`
`
`
`
`
`
`ucke, 1999
`
`2000
`e8ers
`ted by
`
`NOLOG yr
`———eee,
`
`OR
`
`edo, 2005
`Luis Barredo, 2005
`| by John FT. Spencer
`
`hited by Evgenry N.
`
`cia Leonor Ragout de
`
`VilliamsandAnthonyA
`
`5, 1999
`
`us J. Menn, £999
`
`inically Useful
`Iter, 1998
`
`ff, 1997
`
`Edited by
`José Luis Barredo
`K & D Biology, Antibiéticos S. A.,
`
`Leén, Spain
`
`HUMANA PRESS 3K Totowa, NewJersey
`
`

`

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`© 2005 Humana Press Inc.
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`Pref
`
`The:
`
`overvie
`and of
`show ct
`directed
`
`health, r
`toinase/
`nylalani
`salinity
`kinase.
`
`lase, anc
`Micri
`
`experts
`and ind
`biologic
`ogy) be:
`the rang
`I am
`
`agreed tf
`encoura
`
`staff of |
`their eff
`acknow]
`Gonzalc
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`Microbial enzymes and biotransformations / edited by José Luis Barredo.
`p. cm.—(Methods in biotechnology)
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`Includes bibliographical references and index.
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`{. Microbial enzymes-——Biotechnology——-Laboratory manuals. 2. Microbial biotechnology—~
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`Laboratory manuals. 3. Biotransformation (Metabolism)—Laboratory manuals. I. Barredo, José Luis.
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`Il. Series.
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`2004054093
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`

`

`
`
`and Ferry
`
` a
`
`16
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`
`
`immobilization of Enzymes by Covalent Attachment
`Scott J. Novick and J. David Rozzell
`
`roduction of agrochemicals, pharmaceuticals, and
`
`Summary
`Enzymes are finding increasinguse for the
`fine chemicals. They are almost alwa
`removal from the product Stream. In addition,
`methods, properties, and uses of covalently immobilized enzymes.
`Key Words: Immobilized enzymes, covalent immobilization.
`1. Introduction
`1.1. Historical Perspective
`An immobilized enzyme is generally defined as “the imprisonment of an
`enzyme molecule in a distinct phasethat allows exchange with, butis separat-
`ed from, the bulk phase in whichsubstrate effector or inhibitor molecules are
`dispersed and monitored” (J). Immobilized enzyme technology dates back to
`the 1910s to the 1930s, whenproteins were physically adsorbed onto surfaces
`such as charcoal, kaolinite, cellulose, and glass beads (2-4). But it was not until
`the 1950s and ’60s with the work of Katchalski-Katzir, and Chibata and co-
`workers that real advancements were beginning to be madein the development
`and applications of immobilized enzyme materials (5). This early work culmi-
`nated in the First Enzyme Engineering Conference in 1971. Thefirst 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-
`lase in a packed bed reactorto resolve various DL-amino acidsinto their enan-
`tiomerically pure forms. Since that time, immobilized enzymes have become
`increasingly important forthe production ofmany importantchiral compounds
`(.e., amines and alcohols) for the pharmaceutical and fine chemical industries.
`
`;p
`
`From: Methods in Biotechnology, Vol. 17: Microbial Enzymes and Biotransformations
`Edited by: J. L. Barredo © Hurnana Press Inc., Totowa, N}
`247
`
`mitoring to
`
`1 eyewear.
`histidine-
`val for the
`ne is espe-
`t variants,
`
`& catalytic
`is from a
`. The vari-
`rity. It can
`
`a 0.2-um
`
`3-FG02-
`
`3--1414.
`hyperex-
`ise from
`
`-ase/pro-
`tl. Acad.
`
`ymerase
`13-130.
`(1998)
`»~sarcina
`
`ties of
`
`reneous
`Chem.
`
`symatic
`
`- mech-
`kinase
`
`

`

`to
`
`Go
`
`tipoint attachment of enzyme to support.
`
`Table 1
`Stabilization Effects Immobilization Imparts to Enzymes (8)
`
`
`. Prevention of either proteolysis or aggregation by spatial fixation of enzyme mole-
`cules to the support.
`. Unfolding of the enzyme is reduced due to multipoint coval
`attachmentto the support, and/or intramolecular crosslinkin
`. Multimeric enzymes would have a lower likelihood to disso
`attached to the support.
`4. Denaturing agents (e.g
`., Chemical inactivators) can be excluded fro
`mthe enzyme
`bythe 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.
`8., an encapsulation membrane) of proteases from the
`enzyme’s environment.
`7. Increased thermal stability due to mul
`
`
`548
`Novick and Rozzel]
`
`Covalent Er,
`
`
`
`
`
`
`
`
`
`
`
`
`
`1.2. Reasons for Enzyme Immobilization
`
`The principal advantage of immobilizing enzymesis to retain the catalyst in
`
`the reactor. This can greatly improve the economics of a process. For a contin-
`
`uous process, a soluble enzyme would be washedoutof the reactor along with
`
`the product stream. A processlike this would not be economically feasible ifthe
`biocatalyst is very expensive (as is often the case) and cannot be reused.
`
`Althoughan 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 enzymein 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 productis 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 media canalso 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 poly
`
`[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 closerto the enzyme’s optimum pH. These and other
`
`stabilizing effects of immobilization are listed in Table 1.
`
`
`ent or adsorptive
`g of the enzyme.
`ciate if all subunits are
`
`There are
`activily 1S al
`possess high
`must come ir
`
`such as cova]
`the secondar
`interactions,
`activity. In ac
`nonessential¢
`
`supports crea
`biocatalyst (6
`limitations. H
`uct stream an
`
`1.3. Enzyme
`
`In general
`enzymes. It is
`tion system; i
`the enzymeto
`enzyme 1s to.
`ally distinct,
`immobilizatio
`One of the
`ing an enzym
`nonionic inter
`polymer ion-e
`and ceramics.
`from the supp:
`Cross-linku
`inert protein s
`active enzyme
`tinuous reacto
`used as the cl
`result duringtl
`Entrapment
`for enzyme im
`monomer and
`enzyme. Leach
`of substrate dif
`Encapsulatir
`
`for enzyme 1m
`
`

`

`
`
`
`
`ind Rozzel|
`
`
`
`zyme mole-
`
`“ptive
`‘yme.
`ubunits are
`
`© enzyme
`(€.g.,
`glucose by
`
`vation of
`
`3 from the
`
`ipport.
`
`catalyst in
`ta contin-
`uong with
`stble if the
`ye reused.
`
`it is often
`ag a solu-
`can cause
`
`issociated
`ious drug
`ne activi-
`| stability
`e soluble
`je signif-
`‘antage 1s
`yme. For
`as poly
`rate may
`ding the
`nod other
`
`
`
`249
`Covalent Enzyme Immobilization
`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 erizymatic
`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
`In general
`five techniques have been described for immobilization of
`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.
`Oneof the simplest and most economical immobilization methodsis adsorb-
`ing an enzymeonto a support. The enzyme is bound to the support via ionic or
`nonionic interactions. Supports often include carbohydrate-based or synthetic
`polymer ion-exchangeresinsor uncharged supports such as polymers, glasses,
`and ceramics. The main drawback of this method is the leaching of enzyme
`from the support.
`Cross-linking enzyme 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
`Tesult during the cross-linking step andis the major drawback of this method.
`Entrapmentofan 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 atound the
`enzyme. Leaching of the enzyme outof the matrix and masstransfer limitations
`of substrate diffusing into the matrix can limit the use of this technique.
`Encapsulating or confining an enzymewithin a membrane is another method
`for enzyme immobilization. Ultrafiltration membranesor hollowfibers made of
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`
`
`
`Covalent.
`
`
`Novick and Rozzel|
`250
`
`
`polyethersulfone, cellulose nitrate or acetate, or nylon are often used. The pore
`size must be properly chosento allow substrate and productto 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 drawbackof this technique.
`The fifth immobilization method, covalent attachment of enzym€s
`port, will be the subject of therest of this chapter.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`toa sup-
`
`Table 2
`Reactive Fi
`in a Typicaee
`
`Structure of
`reactive gro
`
`—CH,O!
`
`each enzyr
`be tailored t
`eties or ioni
`
`1.4. Covalent Enzyme Immobilization
`Covalent attachment of enzymes to an insoluble Support is an often-useq
`method of enzyme immobilization. It is especially useful when leaching of
`enzyme from the supportis a concern. The enzymeis usually anchored via mul-
`tiple points and this generally imparts greater thermal, pH, ionic strength, and
`organic solventstability onto the enzymesinceit 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-
`quencyin a typical protein (19-21). Of the functional groups of enzymeslist-
`
`ed, -NH2, -CO2H, 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. Aldehydescan react with the guanidino group of
`
`arginine and, althoughhistidine 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 enzymesare attached to can vary greatly. They can
`
`be either natural polymers, such as modified cellulose, starch, dextran, agal poly-
`
`saccharides, collagen, and gelatin; or they can be synthetic, such as polystyrene,
`
`polyacrylamide, polyacrylates, hydroxyalkyl methacrylates, and polyamides.
`
`Inorganic supports can also be used, suchas 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 to
`
`
`enzyme-cat.
`
`

`

`
`
`Covalent Enzyme Immobilization
`
`Table 2
`Reactive Functional Groups in Enzymes and Their Average Occurrence
`
`in a Typical Protein (19-27)
`———___a
`Structure of
`Occurrence
`Reactive group
`reactive group
`in average protein
`
`-NH,
`€-Amino of lysine and N-terminus
`5.9
`
`~CO.H
`
`Carboxylate of glutamic acid,
`aspartic acid, and C-terminus
`
`6.3 (Glu), 5.3 (Asp)
`
`—-SH
`
`Thiol of cysteine
`
`—<_\-on
`
`NH
`
`H 4
`—N=-C
`\
`
`|
`
`‘
`
`NXUN
`
`|
`
`Phenolic of tyrosine
`
`ce
`Guanidino of arginine
`
`Imidazole ofhistidine
`
`1.9
`
`3.2
`
`5.1
`
`2.3
`
`
`251
`
`
`
`
`
`
`
`
`
`
`2zzel]
`
`2 pore
`xit the
`ts sol-
`‘ rates
`
`a sup-
`
`i-used
`ng of
`i mul-
`
`1, and
`
`iscep-
`more
`
`ation.
`
`zyme
`: sup-
`bility
`short-
`
`ently
`2 fre-
`
`3 list-
`nmo-
`
`ity to
`these
`mely
`xcel-
`up of
`react
`good
`
`ycan
`doly-
`rene,
`
`ides.
`stals,
`3, for
`sd to
`
`—S$-sS—
`
`Disulfide ofcystine
`
`Cr
`
`N
`
`Indole oftryptophan
`
`—CH,—-S— Thicether of methionine
`
`—
`
`1.4
`
`2.2
`
`6.8 (Ser), 5.9 (Thr)
`Hydroxyl of serine and threonine
`— CH,OH
`
`
`each enzyme system. This also allows the microenvironment of the enzyme to
`be tailored by appropriate modification ofthe 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
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`oON
`
`oO
`
`N-Enzyme
`
`NH*Enzyme-NH»
`
`C=
`
`oO
`
`O
`
`
`
`Oo
`
` Immobilizedenzyme
`
`ae
`
`C)
`
`Activated
`
`
`
`Immobilizedenzyme
`
`
`
`Activatedsupport
`
`Covalen
`
`epoxide
`ports req
`sections :
`
`L4.70.C
`
`Polyh:
`are amo}
`
`Because
`This is ty
`as S-triaz
`able to c
`lysine or
`hydroxyl
`enzyme |
`Suppo
`for perioc
`available
`requires
`aqueous
`any prote
`This
`widely u:
`mide, is +
`agarose, é
`materials.
`taminatio
`
`
`
`
`
`
`
`support ationontohydroxylcontainingsupportsviaactivationwithcyanogenbromide(top)orS-triazine
`
`SyoNH-EnzymeN
`
`+Enzyme-NH»
`
`a=
`oO
`+
`
`bromide
`
`Striazine
`
`support
`
`+
`
`support
`
`Fig
`
`Cyanogen
`L.Enzymeimmobiliz
`
`
`Hydroxyl-containing
`Hydroxyl-containing
`
`derivatives(bottom). g.
`
`
`enzyme a
`would cat
`
`1.4.2. Cc
`
`Carbox
`acids witt
`port. Thes
`reagent. L
`with carb¢
`tives. The
`is coupled
`or ester lin
`widely us
`propy])-c4!
`bodiimide:
`
`

`

` Covalent Enzyme Immobilization
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`253
`epoxide groups can be used directly for enzyme binding. However, most sup-
`ports require preactivation before enzymesare able to bind to it. The following
`sections describe some typical covalentattachment methodologies.
`1.4.1. Covalent Attachment Onto Polyhydroxy! Supports
`Polyhydroxyl Supports, such as porous glass, and especially polysaccharides
`aré arnong the most commonly used matrices for enzyme immobilization.
`Because hydroxy! groups are poor leaving groups they mustfirst be activated.
`This is typically done with cyanogen bromide (22). Other activating agents such
`as S-triazine derivatives have also been used. Once the Supportis activatedit is
`able to covalently couple to an enzyme usually through the ¢-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 periodsof up to 1 yr at freezer temperatures. Preactivated Supports are also
`avatlable commercially. Once the Supportis 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
`- widely used in large-scale applications. The activating agent, cyanogen bro-
`~ mide,
`is extremely toxic, and most carbohydrate Supports, such as cellulose,
`agarose, and dextran, have poor mechanical stability compared to other support
`materials. Also, since the supports are natural polysaccharides, microbial con-
`tamination and degradation are a concern. Finally,
`the bond between the
`enzyme and the supportis potentially susceptible to hydrolytic cleavage, which
`would cause leaching ofthe enzyme from the support overtime.
`1.4.2. Covalent Attachment onto Carboxylic Acid-Bearing Supports
`Carboxylic acid-containing Supports, such as copolymers of (meth)acrylic
`acids with (meth)acrylic esters have also been used as an immobilization sup-
`Port. These mustalso be activated and this is usually done with a carbodiimide
`feagent. Under slightly acidic conditions (pH 4.75-5.0) carbodiimides react
`With carboxylic acid groups to give the highlyreactive O-acylisourea deriva-
`tives. The Supports are then washed to remove excess reagent and the enzyme
`ls coupled to the activated Supportat neutral pH to give stable amide, thioester,
`OF ester linkages, depending on the residue reacting with the Support. The most
`Widely used water-soluble carbodiimides are !-ethyl-3-(3-dimethylamino
`Propyl)-carbodiimide (EDC) and !-cyclohexy!-3-(2-morpholino-ethy!)-car-
`Odiimide (CMC), both of which are available commercially. The reaction
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`
`
`
`
`
`
`
`
`
`
`
` Fig.2.Activationofacarboxylicacidcontainingsupportwithacarbodiimidefollowedbyenzymecoupling.
`
`
`Enzyme-NH>
`
`
`|
`z<
`|
`
`
`
`=x
`
`—
`
`+ o
`
`w
`x=
`Zz
`
`Oo
`
`oO
`
`+Ht
`
`(@-Z=O=2-a
`
` 4
`
`a
`r=
`2©
`EaN
`w
`
`cW
`
`=
`
`Oo
`
`by-product
`
`Substitutedurea
`
`Immobilizedenzyme
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`Fig.3.Activationofanamine-bearingsupportwithglutaraldehydefollowedbyenzymecoupling.
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`Covalent En,
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`scheme for 9
`enzyme Coup,
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`1.4.3. Covah
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`A simplified e
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`Crump and <
`acid transamin
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`ual activity we
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`for immobilize
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`Enzymescal
`via the enzyme
`imide or simila
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`

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` Covalent Enzyme Immobilization
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`ring supports
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`255
`scheme for of activating a carboxylic acid-containing Support and subsequent
`enzyme Coupling is shownin Fig, 2.
`1.4.3. Covalent Attachment Onto Amine-Bearing Supports
`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 of inorganic supports bearing amine functionality. The most frequent tech-
`nique for introducing amine Soups 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-
`ticles. Polyethyleneimineis a common polyamine derived from the polymer-
`ization ofethyleneimineto give highly branched polymers containing approx-
`imately 25% primary amines, 25% tertiary amines, and 50% secondary
`amines. This polymercan be coated onto various Supports including alumina
`(27), carbon (28), diatomaceous earth (29), and polyvinyl chloride-silica
`_ composites (30,31).
`The coupling ofan enzyme to amine-bearing supports can be done in a num-
`ber ofways. The most common way is through the use ofdifunctional reagents,
`such as diimidate esters, diisocyanates, and dialdehydes. Glutaraldehyde is
`- often used,as it is one of the least expensive difunctional reagents available in
`bulk. This reagentreacts in a complex fashion to form Schiff bases with amine
`S gfoups on the support and produces pendent aldehydes and «,B-unsaturated
`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 Schiffbases can
`_ be reduced to more stable secondary amine bonds with sodium borohydride to
`increase the stability of the enzyme-supportlinkage.
`_
`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
`bound to the Support (total loading was about 10%) and the enzyme retained
`‘Pproximately 89% ofthe soluble activity. Both thes
`for immobilized enzymes, but not necessarily
`e values are unusually high
`atypical for this type of support
`_ and immobilization chemistry.
`Enzymes can also be covalently bonded directly to amine-bea
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`_ Mla the enzyme’s carboxyl groups. These mustfirst be activated with a carbodi-
`~Mide orsimilar reagent prior to immobilization. The activation step can cause
`_ zymeinactivation and thus this method is not used as often.
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` Novick and Rozzel]
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`fied polyacrolé
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`Enzyme-NH»
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`Eupergit
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`Immobilized enzyme
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`Fig. 4. Enzyme immobilization to Eupergit via free amino groups.
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`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 hydroxy]
`group on the enzyme and forma 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 deacylatedchitin,
`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.
`1.4.4, Covalent Attachment 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 Réhm Pharma Polymers (Piscataway, NJ) under the trade name Eupergit.
`The material is a crosslinked copolymerof methacrylamide and oxirane contain-
`ing monomers and consists of spherical beads of about 200 lim in diameter.
`Eupergit is available in twovarieties, Eupergit C and Eupergit C 250 L, with their
`differencesbeing their oxirane content and pore size. Eupergit C has average pore
`radius of 10 nmand an oxirane content of 600 umol/g, while Eupergit C 250 L
`has a pore size and oxirane content of 100 nm and 300 tmol/g, respectively (35).
`Eupergit C 250 L is targeted for the immobilization of large molecular weight
`enzymes (>100 kDa). Immobilization of enzymes to Eupergit is relatively simple.
`The enzymesolution is brought in contact with the Eupergit beads either quies-
`cently or with slight mixing (magnetic stirbars should be avoidedto prevent frac-
`tionation of the beads) for 24—96 h. This can be done either at room temperature,
`orif the enzymeis unstable, 4°C will also work. Various pHscan be used for the
`binding. Underneutral and alkaline conditions the amino groups on the enzyme
`are principally responsible for binding to the support (Fig. 4). Underacidic and
`neutral conditions sulfhydryl and carboxyl groups take part
`in binding.
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`Covalent F)
`
`Immobilizat
`Typically, it
`is Oopumum
`type, immot
`buffer or nev
`enzyme imur
`support, the
`range, from |
`do not effect
`After the
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`epoxy grout
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`poundsthat
`by makingit
`the stability
`threitol, Tris
`glycine), anc
`have been us
`altered depex
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`immobilizati
`the immobili
`addition to E
`for the covale
`Polyacrole
`enzyme imm
`them into a:
`polyaldehyde
`vated support
`been attachec
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`through theit
`poly(lysine),
`attached to t
`groups using {
`acrolein supp
`carbodiimide!
`ers has shown
`weight subst
`acrolein beads
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`

`

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`
`'Rozzell
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`Covalent Enzyme Immobilization
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`257
`
`nzyme
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`ne-bear-
`ited urea
`f moder-
`1ydroxyl
`Iso been
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`(34), is
`d chitin,
`hemical
`1 for the
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`aaterials
`icies for
`wailable
`wupergit.
`contain-
`iameter.
`ith their
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`ige pore
`> 250 L
`ly (35).
`weight
`simple.
`r quies-
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`erature,
`| for the |
`
`enzyme
`dic and
`nding.
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`Immobilization to Eupergit does not change the charged state of the enzyme.
`Typically, it is best to bind the enzymeto 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 enzymeis bound to the
`support, the binding is stable over the long term andit is stable over a wide pH
`range, from 1.0 to 12.0. Also, because Eupergitis electrically neutral, pH changes
`do noteffect the swelling ofthe gel.
`After the enzyme has been bound, typically only about 1% ofthe 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 makingit 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 (.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 €poxy-containing polymers have been Investigated
`for the covalentattachment 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 glutaraldehydeacti-
`vated supports. Various oligomers such as poly(lysine) and poly(glycine) have
`been attached to the polyacrolein beadsto act as spacers between the particles
`and the enzyme.In both cases the poly(aminoacids) are attached to the support
`through their terminal amino groups, Or
`€-amino
`groups in the case of
`Poly(lysine), via Schiff bases (which can then be reduced). The enzymeis
`attached to the poly(lysine)-derivatized polyacrolein via the lysine ¢-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
`carbodiimide followed by enzyme binding. In some cases the use of these spac-
`ers has showna significant increase in activity, especially for large-molecular-
`Weight substrates. Covalent enzyme immobilization to paramagnetic poly-
`acrolein beads has also been investigated (44). Binding of enzymes to unmodi-
`fied polyacrolein is shown in Fig. 5.
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` Covalent
`
`Novick and Rozzel|
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`
`—CH» -NH-Enzyme
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`Enzyme-NH2
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`NaBH,
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`CH=N-Enzyme
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`Immobilized enzyme
`Immobilized enzyme
`.
`(reduced form)
`(oxidized form)
`Peer
`Fig. 5. Enzyme immobilization to unmodified polyacrolein via free amino groups,
`followed by reductionof the Schiff base with sodium borohydride.
`
`1.5. Assaying the Properties of Immobilized Enzymes
`There are three important properties of immobilized enzymesthat 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 enzymethat may be entrapped in the poresof the
`particles or loosely bound through noncovalent interactions.
`
`1.5.2. Di
`Enzyme
`
`It is of
`
`support <
`when opt
`ual enzyl
`the partic
`in the
`
`determin
`bilizatior
`done on
`
`enzyme
`solutions
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`
`determin
`method,
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`solution.
`same ma
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`1.5.1. Activity Assay
`There are two basic methods to measure activity—batch and continuous. In the
`batch method the immobilized enzymeis addedto a flask or vial and the substrate
`solution is then addedtoinitiate the reaction. At various timepoints, an aliquot of
`the mixture is removed andfiltered (this is most easily done through a syringefil-
`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