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  • Immobilized enzymes are used in organic syntheses to fully exploit the technical and eco-nomical advantages of biocatalysts based on isolated enzymes. Immobilization enables theseparation of the enzyme catalyst easily from the reaction mixture, and can lower the costs ofenzymes dramatically. This is true for immobilized enzyme preparations that provide a well-balanced overall performance, based on reasonable immobilization yields, low mass transferlimitations, and high operational stability. There are many methods available for immobiliza-tion which span from binding on prefabricated carrier materials to incorporation into in situprepared carriers. Operative binding forces vary between weak multiple adsorptive inter-actions and single attachments through strong covalent binding. Which of the methods is themost appropriate is usually a matter of the desired applications. It is therefore the intention ofthis paper to outline the common immobilization methods and reaction technologies tofacilitate proper applications of immobilized enzymes.

    Keywords: Enzyme immobilization, Mass transfer effects, Operational stability, Immobilizationmethods.

    1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

    2 Why Immobilize Enzymes? . . . . . . . . . . . . . . . . . . . . . 96

    3 Immobilization Methods . . . . . . . . . . . . . . . . . . . . . . . 99

    3.1 Enzyme Functional Groups . . . . . . . . . . . . . . . . . . . . . 1003.1.1 Native Functional Groups . . . . . . . . . . . . . . . . . . . . . . 1003.1.1.1 Amino Acid Side Chains . . . . . . . . . . . . . . . . . . . . . . . 1013.1.1.2 Enzyme-Linked Carbohydrates . . . . . . . . . . . . . . . . . . . 1043.1.2 Synthetic Functional Groups . . . . . . . . . . . . . . . . . . . . . 1043.2 Carrier Materials and Functional Groups . . . . . . . . . . . . . . 1053.2.1 Inorganic Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063.2.2 Organic Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073.2.2.1 Naturally Occurring Organic Carriers . . . . . . . . . . . . . . . . 1073.2.2.2 Synthetic Organic Carriers . . . . . . . . . . . . . . . . . . . . . . 108

    4 Mass Transfer Effects . . . . . . . . . . . . . . . . . . . . . . . . . 112

    4.1 Porous Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . 1134.2 Reaction (Dynamic) and Support-Generated (Static)

    Proton Gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154.3 Temperature Dependence . . . . . . . . . . . . . . . . . . . . . . 118

    Immobilized Enzymes: Methods and Applications

    Wilhelm Tischer 1 Frank Wedekind 2

    1 Boehringer Mannheim GmbH, Nonnenwald 2, D-82372 Penzberg, Germany.E-mail: wilhelm.tischer@roche.com

    2 Boehringer Mannheim GmbH, Nonnenwald 2, D-82372 Penzberg, Germany.E-mail: frank.wedekind@roche.com

    Topics in Current Chemistry, Vol. 200 Springer Verlag Berlin Heidelberg 1999

  • 4.4 Stability Assessment . . . . . . . . . . . . . . . . . . . . . . . . . 1184.5 Other Contributions . . . . . . . . . . . . . . . . . . . . . . . . . 119

    5 Performance of Immobilized Enzymes . . . . . . . . . . . . . . . 119

    5.1 Enzyme Formulation and Activity . . . . . . . . . . . . . . . . . . 1195.2 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

    6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

    7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

    1Introduction

    Man-made usage of binding enzymes onto solid materials goes back to the1950s, when immobilized enzymes, that is enzymes with restricted mobility,were first prepared intentionally [1,2]. Immobilization was achieved by inclu-sion into polymeric matrices or binding onto carrier materials. Considerableeffort was also put into the cross-linking of enzymes, either by cross-linking ofprotein alone or with the addition of inert materials [3].

    In the course of the last decades numerous methods of immobilization on avariety of different materials have been developed. Binding to pre-fabricatedcarrier materials appears to have been the preferred method so far. Recently,cross-linking of enzyme crystals has also been reported to be an interestingalternative [4].

    Immobilized enzymes are currently the object of considerable interest. Thisis due to the expected benefits over soluble enzymes or alternative technologies.The number of applications of immobilized enzymes is increasing steadily [5].Occasionally, however, experimental investigations have produced unexpectedresults such as a significant reduction or even an increase in activity comparedwith soluble enzymes. Thus, cross-linked crystals of subtilisin showed 27 timesless activity in the aqueous hydrolysis of an amino acid ester compared to equalamounts of soluble enzyme [6]. On the other hand, in the application of lipo-protein lipase in the solvent-mediated synthesis of esters there was a 40-foldincrease in activity using immobilized or otherwise modified enzyme prepara-tions as compared to enzyme powder [7].

    This is why it is mandatory to have some basic knowledge of the essentialcontributions of the chemical forces of binding and of the physicochemicalinteractions during an enzyme reaction which generally is a matter of heteroge-neous catalysis.

    2Why Immobilize Enzymes?

    There are several reasons for the preparation and use of immobilized enzymes.In addition to a more convenient handling of enzyme preparations, the two

    96 W. Tischer F. Wedekind

  • main targeted benefits are (1) easy separation of the enzyme from the product,and (2) reuse of the enzyme.

    Easy separation of the enzyme from the product simplifies enzyme applica-tions and supports a reliable and efficient reaction technology. On the otherhand, reuse of enzymes provides cost advantages which are often an essentialprerequisite for establishing an enzyme-catalyzed process in the first place.

    The properties of immobilized enzyme preparations are governed by theproperties of both the enzyme and the carrier material. The specific interactionbetween the latter provides an immobilized enzyme with distinct chemical, bio-chemical, mechanical and kinetic properties (Fig. 1).

    Of the numerous parameters [810] which have to be taken into account, themost important are outlined in Table 1.

    As far as manufacturing costs are concerned the yield of immobilized enzymeactivity is mostly determined by the immobilization method and the amount ofsoluble enzyme used. Under process conditions, the resulting activity may befurther reduced by mass transfer effects. More precisely, the yield of enzymeactivity after immobilization depends not only on losses caused by the bindingprocedure but may be further reduced as a result of diminished availability ofenzyme molecules within pores or from slowly diffusing substrate molecules.Such limitations, summarized as mass transfer effects, lead to lowered efficiency.On the other hand, improved stability under working conditions may compensatefor such drawbacks, resulting in an overall benefit. Altogether, these interactionsare a measure of productivity or of enzyme consumption, for example, expressed asenzyme units per kg of product. If we replace enzyme units by enzyme costs weobtain the essential product related costs, for example, in US$ per kg of product.

    In order to estimate the cost advantages of immobilized enzymes, it is neces-sary to consider the individual manufacturing steps and their contribution to

    Immobilized Enzymes: Methods and Applications 97

    Fig. 1. Characteristics of immobilized enzymes

    Immobilization methodyield [%]

    Performanceproductivity [units/kg product]

    Enzyme consumption [kg product/unit]

    Operational stabilitycycles [#]

    EnzymeCarrier

    Mass transfer effectsefficiency [h]

    Biochemical properties Chemical characteristics

    Reaction type & kinetics Mechanical properties

  • the overall costs. Firstly, these comprise the costs for biomass from plant andanimal sources, or from microbial fermentations. In the latter case, the costs aredetermined mainly by the fermentation scale and the expression rate of theenzymes. Secondly, downstreaming is needed to achieve the required purity butis accompanied by loss in activity. The use of larger fermentation scales may benecessary in order to compensate for loss in activity and also for some increasein cost (Fig. 2). Thirdly, costs for the immobilization procedure further increasethe manufacturing expense. Thus, aside from the potential advantage of easierremoval of the enzyme from the product formed, immobilized enzymes so farprovide no cost benefit. However, cost savings will be achieved by multiple reuseof an immobilized enzyme. On the other hand, prolonged use also meansdownscaling of the unit operation and thus increased costs for manufacture ofthe enzyme itself which must also be taken into account. In conclusion, onlymultiple use will lead to dramatic cost reduction. In practice, this can be moni-tored by consideration of the amount of enzyme required per kg of formedproduct.

    98 W. Tischer F. Wedekind

    Table 1. Selected characteristic parameters of immobilized enzymes

    Enzyme Biochemical propertiesmolecular mass, prosthetic groups, functional groups on protein-surface, purity (inactivating/protective function of impurities)Enzyme kinetic parametersspecific activity, pH-, temperature profiles, kinetic parameters foractivity and inhibition, enzyme stability against pH, temperature,solvents, contaminants, impurities

    Carrier Chemical characteristicschemical basis and composition, functional groups, swellingbehavior, accessible volume of matrix and pore size, chemical stability of carrierMechanical propertiesmean wet particle

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