p obilization
`of Enzymes
`and Cells
`
`Edited by
`Gordon F. Bickerstaff
`
`*HUMANA PRESS
`
`CO2 Solutions Inc.
`Exhibit 2002
`Akermin, Inc. v. CO2 Solutions Inc.
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`

`METHODS IN BIOTECHNOLOGY'"'
`
`METHODS IN BIOTECHNOLOGY"'
`
`John M. Walker, Sews Eagoit
`
`2. Protocols in Sioremediation, edited by David Sheehan, 1997
`1. Immobilization of Enzymes and Cells, edited by Gordon F. Bickerstaff 1997
`
`Immobilization
`of Enzymes and Cells
`
`Edited by
`Gordon F. Bickerstaff
`University of Paisley, Scotland, UK
`
`Humana Press
`
`Totowa, New Jersey
`
`Page 2 of 9
`
`

`

`C 1997 Humana Press Inc.
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`COVET illustration: Fig. I in Chapter 1, "Immob lizalion of Enzymes and Cells! Sallie Pracrical Consider-
`°lions," by Gordon F. Flickerstaff.
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`Library of Congress Cataloging in Publication Data
`
`Main entry under title:
`
`Methods in biotechnology-.
`
`Immobilization of enzymes and cellsled [tad] by Gordon F. Dickerstaff.
`em.—Methods in biotechnology... 11
`p. (cid:9)
`Includes index.
`ISBN 0-89603-386-4 (alit- paper)
`I. Immobilized enzymes-13imeehnology. 2. Immobilized cells—Biotechnology. I. pielcerataff,
`Gordon, F. Il. Series.
`[DNLM: 1. Enzymes, Immobilized. 2. Cells, Immobilized. 3. Biotechnology--methods. Q1_1 135
`1314 19971
`TP248.65.145146 1997
`660'.634--dc20
`IJNLM/DLC
`for Library of Congress (cid:9)
`
`96.29281
`CIP
`
`Preface
`
`immobilization of enzymes, cells, and organelles has expanded greatly.
`in the past 30 years as the advantages of immobilization have been evaluated
`and utilized in analytical, bi otransforrna t ion, and medical applications. A con-
`sequence of this explosion of technology is that there is now a bewildering
`array of permutations for the immobilization of biological material. The pur-
`pose of Immobilization ofEnzymes and Cells is to provide a basic reference
`tool for all academic and industrial research workers seeking to start or expand
`the use of immobilization techniques in their work. The book does not aim to
`provide comprehensive coverage of the vast range of methods available, but
`will serve as a launch pad for potential users of immobilization techniques.
`One reason for the vast expanse of immobilization technology lies in the
`subject material to be immobilized. Biological catalysts (enzymes, organelles,
`and cells) have a high degree of individual variability, and although many
`immobilization techniques have wide applicability, it is impossible for one or
`even a few methods to cater to the great diversity of requirements inherent in
`biological material, This is especially so when the aim is to produce an opti-
`mum system in which the immobilized biocatalyst will function at high levels
`of efficiency, stability, and so on.
`The normal situation faced by research workers is the need to try one or
`more methods of immobilization to reveal the specific requirements dictated by
`the biological catalyst, then adapt the method to these specific circumstances or
`try another method when the first approach places too great a restriction on the
`use or activity of the biocatalyst. This process of discovery can be rewarding, but
`it is also time-consuming, usually frustrating, and the hardest part is making a
`successful start. Immobilization ofEnzymes and Cells has been designed to pro-
`vide a wide range of representative examples of immobilization techniques for
`use by postgraduate, postdoctoral, senior research workers, and technicians
`throughout academia, industry, government, and medical research establishments,
`thereby enabling rapid entry into the world of immobilization.
`All of the chapters, with the exception of Chapter 1, provide detailed
`instructions of the materials and methods employed in the particular immobi-
`lization procedure described. Each author has used the Notes section to pass
`on accumulated experience and valuable "trade secrets" that give greater
`
`Page 3 of 9
`
`

`

`tV
`
`Contributors
`
`MAamiu E B. MEDINA • North Atlantic Area. Eastern Regional Research
`Center, US Department of Agriculture. Philadelphia, PA
`SHown I MOROHA MD • Department of Chemical and Biochemical
`Engineering, Toyama University. Toyama, Japan
`ANDREAS MUSCAT • Institut fir Technologie, Bundesforschungsanstalt fur
`Landwirtschaft (FAL). Braunschweig. Germany
`FLAVIA M. L. PASSOS • Department of Food Science, College of Agriculture
`and Life Sciences, North Carolina State University, Raleigh. NC
`MARION PATERSON • School of Chemistry, University of Birmingham, UK
`GUADALUPE PENZOL • Laboratorio de Technologia Enzymatica, Instituto de
`Catalysis CSIC Campus de Cantoblanco, Madrid, Spain
`GEORGE J. PIAZZA • North Atlantic Area, Eastern Regional Research Center,
`US Department of Agriculture. Philadelphia. PA
`VERONICA RODRIGUEZ • Laboratorio de Technologia Enzymatica, Institute de
`Catdlisis CSIC Campus de Cantoblanco, Madrid, Spain
`CRis-timA M. Rosti.i. • Laboratorio de Technologia Enzymdtica, Campus de
`Cantoblanco, Institute de Catdlisis CSIC Madrid, Spain
`TOSHISUKE SAsnicuita, • Department of Chemical and Biochemical
`Engineering, Toyama University, Toyama, Japan
`Jost V. SINISTERRA • Department of Organic and Pharmaceutical Chemistry,
`Faculty of Pharmacy. University of Complutense. Madrid, Spain
`GLORIA SOLER • Laboratorio de Technologia Enzymcitica, Institute de
`Catdlisis CSIC, Campus de Cantoblanco, Madrid, Spain
`DAVID J. STRIKE • Institute of Microtechnology, University of Neuchatel.
`Switzerland
`HAROLD E, SWAISCrOOD • Department of Food Science, College of Agriculture
`and Life Sciences, North Carolina State University, Raleigh, NC
`PIERRE V1DAL • Department de Genie Chimique, University de Sherbrooke,
`Canada
`GERHARD Von. • Department of Biochemistry, Biomedical Center, Uppsala
`University, Uppsala. Sweden
`KLAUS-DIETER VORLOP • M.Stitrafirr Technologic Bundesforschungsanstalt
`fur Landwirtschaji (FAL), Braunschweig, Germany
`MARIE K. WALSH • Department of Food Science, College of Agriculture and
`Life Sciences, North Carolina State University, Raleigh. NC
`HIROSHI YAMAZAKI • Department of Biology, Carleton University, Ottawa,
`Canada
`QING YANG • Department of Biochemistry, Biomedical Center, Uppsala
`F
`
`1 (cid:9)
`
`
`
`Immobilization of Enzymes and Cells
`
`Some Practical Considerations
`
`Gordon F. Bickerstaff
`
`1. Introduction
`The technology for immobilization of cells and enzymes evolved steadily for
`the first 25 years of its existence (I), but in recent years it has reached a plateau, if
`not a slight decline. However, the expansion of biotechnology, and the expected
`developments that will accrue from advances in genetic technology, has revital-
`ized enthusiasm for immobilization of enzymes and cells (2). Research and devel-
`opment work has provided a bewildering array of support materials and methods
`for immobilization. Much of the expansion may be attributed to developments to
`provide specific improvements fora given application (3), Surprisingly, there have
`been few detailed and comprehensive comparative studies on immobilization
`methods and supports. Therefore, no ideal support material or method of immobi-
`lization has emerged to provide a standard for each type of immobilization. Selec-
`tion of support material and method of immobilization is made by weighing the
`various characteristics and required features of the enzyme/cell application against
`the properties/limitations/characteristics of the combined immobilization/support.
`A number of practical aspects should be considered before embarking on experi-
`mental work to ensure that the final immobilized enzyme and/or cell preparation is
`fit for the planned purpose or application and will operate at optimum effective-
`ness (4-6). This chapter does not aim to provide a review of available methods,
`but does provide some background to assist in choice evaluation for support and
`method of immobilization.
`2. Choice of Support and Principal Method
`In solution, soluble enzyme molecules behave as any other solute in that they
`are readily dispersed in the solution and have complete freedom of movement
`From: memo& ir, Birgechrtoropy, Vot. 1: frnmctiiizarion of Enzymes and COG
`Edited by: G. F. Hickerstaff Humane Press Irm., Toicerra, NJ
`
`Page 4 of 9
`
`

`

`2
`
`Bickerstatf
`
`Practical Considerations
`
`3
`
`Table 1
`Fundamental Considerations
`in Selecting a Support and Method of Immobilization
`
`Property
`
`Physical (cid:9)
`
`Chemical (cid:9)
`
`Stability (cid:9)
`
`Resistance (cid:9)
`
`Safety (cid:9)
`
`Economic (cid:9)
`
`Reaction (cid:9)
`
`Points for consideration
`
`Strength, noncompression of particles, available surface area,
`shape/form (beads/sheets/fibers), degree of porosity, pore vol-
`ume, permeability, density, space for increased biomass, flow
`rate, and pressure drop
`Hydrophilicity (water binding by the support), inertness toward
`enzyme/cell, available functional groups for modification, and
`regeneration/reuse of support
`Storage, residual enzyme activity, cell productivity, regeneration of
`enzyme activity, maintenance of cell viability, and mechanical
`stability of support material
`Bacterial/fungal attack, disruption by chemicals, pH, temperature,
`organic solvents, pretenses, and cell defense mechanisms (pro-
`teins/cells)
`Biocompatibility (invokes an immune response), toxicity of com-
`ponent reagents, health and safety for process workers and end-
`product users, specification of immobilized preparation (GRAS
`list requirements for FDA approval) for food, pharmaceutical,
`and medical applications
`Availability and cost of support, chemicals, special equipment,
`reagents, technical skill required, environmental impact, indus-
`trial-scale chemical preparation, feasibility for scale-up, con-
`tinuous processing, effective working life, reuseable support,
`and CRL or zero contamination (enzyme/cell-free product)
`Flow rate, enzyme/cell loading and catalytic productivity, reaction
`kinetics, side reactions, multiple enzyme and/or cell systems,
`batch, CSTR, PBR, FBR, ALR, and so on; diffusion limitations
`on mass transfer of cofactors, substrates, and products
`
`CAL: calculated risk level, CSTR: continuous stirred tank reactor. PBR: packed bed reactor,
`FOR: fluidized bed reactor, ALR: air lift reactor
`
`(I). Enzyme immobilization is a technique specifically designed td greatly
`restrict the freedom of movement of an enzyme. Most cells are naturally immo-
`bilized one way or another, so immobilization provides a physical support for
`cells. The first consideration is to decide on the support material, then the main
`method of immobilization, taking into account the intended use and application.
`Some of the points to consider when making a decision are listed in Table 1, and
`
`Table 2
`Influence of Immobilization Method
`on the BiotransformatIon of Sucrose
`to Isomaltulose by Cells of Envinis rhapontle
`Activity,
`g,/g wet cells%
`
`Free cells
`Immobilization method
`Entrapment in calcium alginate
`Entrapment in poIyacrylamide
`Adsorption to DEAE-cellulose
`Crosslinking with glutaraldehyde
`Entrapment in K-carrageenan
`Entrapment in agar
`Adsorption to bone char
`Reprinted with permission from ref. 21.
`
`0.600
`
`0.325
`0.130
`0.583
`0.153
`0.263
`0.340
`0.010
`
`Half-life,
`h
`
`36
`
`8500
`570
`400
`40
`38
`27
`25
`
`an indication of hoW different methods of immobilization can influence the
`activity and half-life of a cell-based biotransformation is presented in Table 2.
`There are five principal methods for immobilization of enzymes/cells: adsorp-
`tion, covalent binding, entrapment, encapsulation, and crosslinking (see Fig. 1).
`The relative merits of each are discussed briefly below.
`
`2.1. Adsorption
`
`Immobilization by adsorption (see Fig. l) is the simplest method and involves
`reversible surface interactions between enzyme/cell and support material (7,8).
`The forces involved are mostly electrostatic, such as van der Waals forces, ionic
`and hydrogen bonding interactions, although hydrophobic bonding can be sig-
`nificant. These forces are very weak, but sufficiently large in number to enable
`reasonable binding, For example, it is known that yeast cells have a surface
`chemistry that is substantially negatively charged so that use of a positively
`charged support will enable immobilization. Existing surface chemistry between
`the enzyme/cells and support is utilized so no chemical activation/modification
`is required and little damage is normally done to enzymes or cells in this method
`of immobilization. The procedure consists of mixing together the biological
`component(s) and a support with adsorption properties, under suitable condi-
`tions of pH, ionic strength, and so on, for a period of incubation, followed by
`collection of the immobilized material and extensive washing to remove
`nonbound biological components.
`
`Page 5 of 9
`
`(cid:9)
`(cid:9)
`(cid:9)
`

`

`4 (cid:9)
`
`Ehckerstaft (cid:9)
`
`Practical Considerations (cid:9)
`
`5
`
`ADSORPTION
`
`COVALENT BINDING
`
`ENCAPSULATION
`
`ENTRAPMENT
`
`CROSS-LINKING
`
`Fig, 1. Principal methods of immobilization.
`
`Among the advantages are:
`1. Little or no damage to enzymes/cells.
`2. Simple, cheap, and quick to obtain immobilization.
`3. No chemical changes to support or enzyme/cell.
`4. Reversible to allow regeneration with fresh enzymes/cells.
`
`Disadvantages include:
`1. Leakage of enzymes/cells from the support/contamination of product.
`
`2. Nonspecific binding.
`3. Overloading on the support,
`4. Steric hindrance by the support:,
`
`The most significant disadvantage is leakage of biocatalyst from the sup-
`port. Desorption can occur under many circumstances, and environmental
`changes in pH, temperature, and ionic strength will promote desorption. Some-
`times a cell/enzyme, firmly adsorbed, is readily desorbed during reaction as a
`result of substrate binding, binding of contaminants present in the substrate,
`product production, or other conditions leading to change in protein conforma-
`tion. Physical factors, such as flow rate, bubble agitation, particle—particle abra-
`sion, and scouring effect of particulate materials on vessel walls, can lead to
`desorption. Desorption can be turned to advantage if regeneration of support is
`built into the operational regimen to allow rapid expulsion of exhausted bio-
`catalyst and replacement with fresh biocatalyst.
`Nonspecific binding can become a problem if substrate, product, and/or
`residual contaminants are charged and interact with the support. This can lead
`to diffusion limitations and reaction kinetics problems, with consequent alter-
`ation in parameters V„,,„ and Km (9), Further, binding of protons to the support
`material can result in an altered pH microenvironment around the support with
`consequent shift in pH optimum (1-2 pH units), which may be important for
`enzymes with precise pH optimum requirements (10,11). Unless carefully
`controlled, overloading the support can lead to low cataltyic activity, and the
`absence of a suitable spacer between the enzyme molecule and the support can
`produce problems related to steric hindrance.
`
`2.2. Covalent Binding
`This method of immobilization (see Fig. 1) involves the formation of a
`covalent bond between the enzyme/cell and a support material (8,12.13). The
`bond is normally formed between functional groups present on the surface of
`the support and functional groups belonging to amino acid residues on the sur-
`face of the enzyme. A number of amino acid functional groups are suitable for
`participation in covalent bond formation. Those that are most often involved
`are the amino group (NH2) of lysine or arginine, the carboxyl group (CO2H)
`of aspartic acid or glutamic acid, the hydroxyl group (OH) of serine or threo-
`nine, and the sulfydryl group (SH) of cysteine (14).
`Many varied support materials are available for covalent binding, and the
`extensive range of supports available reflects the fact that no ideal support
`exists. Therefore, the advantages and disadvantages of a support must be taken
`into account when considering possible procedures for a given enzyme immo-
`bilization (15,16). Many factors may influence the selection of a particular
`support, and research work has shown that hydrophilic ity is the most important
`
`Page 6 of 9
`
`

`

`6 (cid:9)
`
`Bickerstaff
`
`Practical Considerations (cid:9)
`
`7
`
`factor for maintaining enzyme activity in a support environment (17). Conse-
`quently, polysaccharide polymers, which are very hydrophilic, are popular sup-
`port materials for enzyme immobilization. For example, cellulose, dextran
`(Sephadex), starch, and agarose (Sepharose) are used for enzyme immobiliza-
`don. The sugar residues in these polymers contain hydroxyl groups, which are
`ideal functional groups for chemical activation to provide covalent bond for-
`mation. Also, hydroxyl groups form hydrogen bonds with water molecules and
`thereby create an aqueous (hydrophilic) environment in the support. The
`polysaccharide supports are susceptible to microbial/fungal disintegration, and
`organic solvents can cause shrinkage of the gels. The supports are usually used
`in bead form.
`Other popular supports for enzyme immobilization are porous silica and
`porous glass. The microarchitecture of these is shown in Fig. 2. Porous silica
`consists of small spherical particles of silica fused together in such a way as to
`form microcavities and small channels. The support is normally sold in bead
`form, and is very strong and durable. Sintered borosilicate glass may be tem-
`pered to form a system of uniform channels. The diameter of channels depends
`on the tempering conditions. Porous glass is also durable and resistant to
`microbial disintegration or solvent distortion. However, these two supports are
`less hydrophilic than the polysaccharide materials. There are many reaction
`procedures for coupling an enzyme and a support in a covalent bond (18).
`However, most reactions fall into the following categories:
`
`1. Formation of an isourea linkage.
`2. Formation of a diazo linkage.
`3. Formation of a peptide bond.
`4. An alkylation reaction.
`ft is important to choose a method that will not inactivate the enzyme by
`reacting with amino acids at the active site. So, if an enzyme employs a car-
`boxyl group at the active site for participation in catalysis, it is wise to choose
`a reaction that involves amino groups for the covalent bond with the support.
`Basically, two steps are involved in covalent binding of enzymes to support
`materials.
`First, functional groups on the support material are activated by a specific
`reagent, and second, the enzyme is added in a coupling reaction to form a
`covalent bond with the support material. Normally the activation 'reaction
`is designed to make the functional groups on the support strongly electro-
`philic (electron deficient). In the coupling reaction, these groups will react
`with strong nucleophiles (electron donating), such as the amino (NH2) func-
`tional groups of certain amino acids on the surface of the enzyme, to form
`a covalent bond (1).
`
`POLYACRYL AMIDE
`
`POROUS GLASS
`
`AGA ROSE
`
`POROUS SILICA
`
`Fig. 2. Microarchitecture of some support materials used for immobilization of
`enzymes and cells.
`
`Cyanogen bromide (CNBr) is often used to activate the hydroxyl functional
`groups in polysaccharide support materials. In this method, the enzyme and
`support are joined via an isourea linkage. In the case of carbxliimide activa-
`tion, the support material should have a carboxyl (CO2H) functional group,
`and the enzyme and support are joined via a peptide bond. If the support mate-
`rial contains an aromatic amino functional group, it can be diazotized using
`nitrous acid. Subsequent addition of enzyme leads to the formation of a diazo
`linkage between the reactive diazo group on the support and the ring structure
`of an aromatic amino acid, such as tyrosine.
`It is important to recognize that no method of immobilization is restricted to
`a particular type of support material, and that an extremely large number of
`
`Page 7 of 9
`
`

`

`8 (cid:9)
`
`Bickerstaff
`
`Practical Considerations (cid:9)
`
`9
`
`permutations are possible between methods of immobilization and support
`material. This is made possible by chemical modification of normal functional
`groups on a support material to produce a range of derivatives containing dif-
`ferent functional groups. For example, the normal functional group in cellu-
`lose is the hydroxyl group, and chemical modification of this has produced a
`range of cellulose derivatives, such as AE-cellulose (aminoethyl), CM-cellu-
`lose (carboxymethyl), and DEAE-cellulose (diethylarninoethyl). Thus, chemi-
`cal modification increases the range of immobilization methods that can be
`used for a given support material. Derivatization can also be used to modify
`charges on the surface of a support material to improve binding of biocatalyst.
`
`2.3. Entrapment
`
`Immobilization by entrapment (see Fig. 1) differs from adsorption and
`covalent binding in that enzyme molecules are free in solution, but restricted in
`movement by the lattice structure of a gel (1,19). The porosity of the gel lattice
`is controlled to ensure that the structure is tight enough to prevent leakage of
`enzyme or cells, yet at the same time allow free movement of substrate and
`product. Inevitably, the support will act as a barrier to mass transfer, and
`although this can have serious implications for reaction kinetics, it can have
`useful advantages since harmful cells, proteins, and enzymes are prevented
`from interaction with the immobilized biocatalyst (20,21).
`There are several major methods of entrapment:
`
`I. Ionotropic gelation of macromolecules with multivalent cations (e.g., alginate).
`2. Temperature-induced gelation (e.g., agarose, gelatin).
`3. Organic polymerization by chemical/photochemical reaction (e.g., polyacrylamide).
`4. Precipitation from an immiscible solvent (e.g., polystyrene).
`
`Entrapment can be achieved by mixing an enzyme with a polyionic polymer
`material and then crosslinking the polymer with multivalent cations in an
`ion-exchange reaction to form a lattice structure that traps the enzymes/cells
`(ionotropic gelation). Temperature change is a simple method of gelation by
`phase transition using 1-4% solutions of agarose or gelatin. However, the gels
`formed are soft and unstable. A significant development in this area has been
`the introduction of K-carrageenan polymers that can form gels by ionotropic
`gelation and by temperature-induced phase transition, which has introduced a
`greater degree of flexibility in gelation systems for immobilization. '
`Alternatively, it is possible to mix the enzyme with chemical monomers that
`are then polymerized to form a crosslinked polymeric network, trapping the
`enzyme in the interstitial spaces of the lattice. The latter method is more widely
`used, and a number of acrylic monomers are available for the formation of
`hydrophilic copolymers. For example, acrylamide monomer is polymerized to
`
`form polyacrylamide and methylacrylate is polymerized to form polymetha-
`crylate. In addition to the monarffer, a cross linking agent is added during poly-
`merization to form crosslinkagesIlietynen the polymer chains and help to create
`a three-dimensional network lattice. The pore size of the gel and its mechanical
`properties are determined by the relative amounts of monomer and crosslinking
`agent. It is therefore possible to vary these concentrations to influence the lat-,
`tice structure. The formed polymer may be broken up into particles of a desired
`size, or polymerization can be arranged to form beads of defined size. Precipi-
`tation occurs by phase separation rather than by chemical reaction, but does
`bring the cells/enzymes into contact with a water-miscible organic solvent, and
`most cells/enzymes are not tolerant of such solvents. Thus, this method is lim-
`ited to highly stable/previously stabilized enzymes or nonliving cells.
`
`2.4. Encapsulation
`
`Encapsulation (see Fig. 1) of enzymes and or cells can be achieved by envel-
`oping the biological components within various forms of semipermeable mem-
`branes (22-24). It is similar to entrapment in that the enzymes/cells are free in
`solution, but restricted in space. Large proteins or enzymes cannot pass out of
`or into the capsule, but small substrates and products can pass freely across the
`semipermeable membrane. Many materials have been used to construct
`microcapsules varying from 10-100 urn in diameter; for example, nylon and
`cellulose nitrate have proven popular. The problems associated with diffusion
`are more acute and may result in rupture of the membrane if products from a
`reaction accumulate rapidly. A further problem is that the immobilized cell or
`enzyme particle may have a density fairly similar to that of the bulk solution
`with consequent problems in reactor configuration, flow dynamics, and so on.
`It is also possible to use biological cells as capsules, and a notable example of
`this is the use of erythrocytes (red blood cells). The membrane of the erythro-
`cyte is normally only permeable to small molecules. However, when erythro-
`cytes are placed in a hypotonic solution, they swell, stretching the cell
`membrane and substantially increasing the permeability. In this condition,
`erythrocyte proteins diffuse out of the cell and enzymes can diffuse into the
`cell. Returning the swollen erythrocytes to an isotonic solution enables the cell
`membrane to return to its normal state, and the enzymes trapped inside the cell
`do not leak out. A distinct advantage of this method is coimmobilization. Cells
`and/or enzymes may be immobilized in any desired combination to suit par-
`ticular applications.
`
`2.5. CrosslinkIng
`
`This type of immobilization (see Fig. 1) is support-free and involves joining
`the cells (or the enzymes) to each other to form a large, three-dimensional
`
`Page 8 of 9
`
`

`

`10 (cid:9)
`
`Bickerstaff
`
`Practical Considerations (cid:9)
`
`11
`
`complex structure, and can be achieved by chemical or physical methods (25).
`Chemical methods of crosslinking normally involve covalent bond formation
`between the cells by means of a bi- or multifunctional reagent, such as glut-
`araldehyde and toluene diisocyanate. However, the toxicity of such reagents is
`a limiting factor in applying this method to living cells and many enzymes.
`Both albumin and gelatin have been used to provide additional protein mol-
`ecules as spacers to minimize the close proximity problems that can be caused
`by crosslinking a single enzyme.
`Physical crosslinking of cells by flocculation is well known in the biotech-
`nology industry and does lead to high cell densities. Flocculating agents, such
`as polyamines, polyethyleneimine, polystyrene sulfonates, and various phos-
`phates, have been used extensively and are well characterized. Crosslinking is
`rarely used as the only means of immobilization because the absence of
`mechanical properties and poor stability are severe limitations. Crosslinking is
`most often used to enhance other methods of immobilization, normally by
`reducing cell leakage in other systems.
`
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