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Immobilized Enzymes: Methods and ApplicationsWilhelm Tischer 1 Frank Wedekind 21 2

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

Immobilized enzymes are used in organic syntheses to fully exploit the technical and economical advantages of biocatalysts based on isolated enzymes. Immobilization enables the separation of the enzyme catalyst easily from the reaction mixture, and can lower the costs of enzymes dramatically. This is true for immobilized enzyme preparations that provide a wellbalanced overall performance, based on reasonable immobilization yields, low mass transfer limitations, and high operational stability. There are many methods available for immobilization which span from binding on prefabricated carrier materials to incorporation into in situ prepared carriers. Operative binding forces vary between weak multiple adsorptive interactions and single attachments through strong covalent binding. Which of the methods is the most appropriate is usually a matter of the desired applications. It is therefore the intention of this paper to outline the common immobilization methods and reaction technologies to facilitate proper applications of immobilized enzymes.Keywords: Enzyme immobilization, Mass transfer effects, Operational stability, Immobilization

methods.

1 2 3 3.1 3.1.1 3.1.1.1 3.1.1.2 3.1.2 3.2 3.2.1 3.2.2 3.2.2.1 3.2.2.2 4 4.1 4.2 4.3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Why Immobilize Enzymes? . . . . . . . . . . . . . . . . . . . . . 96

Immobilization Methods . . . . . . . . . . . . . . . . . . . . . . . 99 Enzyme Functional Groups . . . . . . . Native Functional Groups . . . . . . . . Amino Acid Side Chains . . . . . . . . . Enzyme-Linked Carbohydrates . . . . . Synthetic Functional Groups . . . . . . . Carrier Materials and Functional Groups Inorganic Carriers . . . . . . . . . . . . . Organic Carriers . . . . . . . . . . . . . Naturally Occurring Organic Carriers . . Synthetic Organic Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 100 101 104 104 105 106 107 107 108

Mass Transfer Effects . . . . . . . . . . . . . . . . . . . . . . . . . 112 Porous Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Reaction (Dynamic) and Support-Generated (Static) Proton Gradients . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Temperature Dependence . . . . . . . . . . . . . . . . . . . . . . 118Topics in Current Chemistry, Vol. 200 Springer Verlag Berlin Heidelberg 1999

96 4.4 4.5 5 5.1 5.2 6 7 Stability Assessment Other Contributions

W. Tischer F. Wedekind

. . . . . . . . . . . . . . . . . . . . . . . . . 118 . . . . . . . . . . . . . . . . . . . . . . . . . 119

Performance of Immobilized Enzymes . . . . . . . . . . . . . . . 119 Enzyme Formulation and Activity . . . . . . . . . . . . . . . . . . 119 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

1 IntroductionMan-made usage of binding enzymes onto solid materials goes back to the 1950s, when immobilized enzymes, that is enzymes with restricted mobility, were first prepared intentionally [1,2]. Immobilization was achieved by inclusion into polymeric matrices or binding onto carrier materials. Considerable effort was also put into the cross-linking of enzymes, either by cross-linking of protein alone or with the addition of inert materials [3]. In the course of the last decades numerous methods of immobilization on a variety of different materials have been developed. Binding to pre-fabricated carrier materials appears to have been the preferred method so far. Recently, cross-linking of enzyme crystals has also been reported to be an interesting alternative [4]. Immobilized enzymes are currently the object of considerable interest. This is 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 unexpected results such as a significant reduction or even an increase in activity compared with soluble enzymes. Thus, cross-linked crystals of subtilisin showed 27 times less activity in the aqueous hydrolysis of an amino acid ester compared to equal amounts of soluble enzyme [6]. On the other hand, in the application of lipoprotein lipase in the solvent-mediated synthesis of esters there was a 40-fold increase in activity using immobilized or otherwise modified enzyme preparations as compared to enzyme powder [7]. This is why it is mandatory to have some basic knowledge of the essential contributions of the chemical forces of binding and of the physicochemical interactions during an enzyme reaction which generally is a matter of heterogeneous catalysis.

2 Why 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

Immobilized Enzymes: Methods and Applications

97

EnzymeBiochemical properties Reaction type & kinetics

Carrier

Chemical characteristics Mechanical properties

Immobilization method yield [%]

Mass transfer effects efficiency [h]

Operational stability cycles [#]

Performance productivity [units/kg product] Enzyme consumption [kg product/unit]

Fig. 1. Characteristics of immobilized enzymes

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 applications and supports a reliable and efficient reaction technology. On the other hand, reuse of enzymes provides cost advantages which are often an essential prerequisite for establishing an enzyme-catalyzed process in the first place. The properties of immobilized enzyme preparations are governed by the properties of both the enzyme and the carrier material. The specific interaction between the latter provides an immobilized enzyme with distinct chemical, biochemical, mechanical and kinetic properties (Fig. 1). Of the numerous parameters [810] which have to be taken into account, the most important are outlined in Table 1. As far as manufacturing costs are concerned the yield of immobilized enzyme activity is mostly determined by the immobilization method and the amount of soluble enzyme used. Under process conditions, the resulting activity may be further reduced by mass transfer effects. More precisely, the yield of enzyme activity after immobilization depends not only on losses caused by the binding procedure but may be further reduced as a result of diminished availability of enzyme 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 compensate for such drawbacks, resulting in an overall benefit. Altogether, these interactions are a measure of productivity or of enzyme consumption, for example, expressed as enzyme units per kg of product. If we replace enzyme units by enzyme costs we obtain 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 necessary to consider the individual manufacturing steps and their contribution to

98Table 1. Selected characteristic parameters of immobilized enzymes

W. Tischer F. Wedekind

Enzyme

Carrier

Immobilized enzyme

Biochemical properties molecular mass, prosthetic groups, functional groups on proteinsurface, purity (inactivating/protective function of impurities) Enzyme kinetic parameters specific activity, pH-, temperature profiles, kinetic parameters for activity and inhibition, enzyme stability against pH, temperature, solvents, contaminants, impurities Chemical characteristics chemical basis and composition, functional groups, swelling behavior, accessible volume of matrix and pore size, chemical stability of carrier Mechanical properties mean wet particle diameter, single particle compression behavior, flow resistance (for fixed bed application), sedimentation velocity (for fluidized bed), abrasion (for stirred tanks) Immobilization method bound protein, yield of active enzyme, intrinsic kinetic parameters (properties free of mass transfer effects) Mass transfer effects consisting of partitioning (different concentrations of solutes inside and outside the catalyst particles), external and internal (porous) diffusion; this gives the effectiveness in relation to free enzyme determined under appropriate reaction conditions, Stability operational stability (expressed as activity decay under working conditions), storage stability Performance productivity (amount of formed product per unit or mass of enzyme) enzyme consumption (e.g. units kg1 product, until half-life)

the overall costs. Firstly, these comprise the costs for biomass from plant and animal sources, or from microbial fermentations. In the latter case, the costs are determined mainly by the fermentation scale and the expression rate of the enzymes. Secondly, downstreaming is needed to achieve the required purity but is accompanied by loss in activity. The use o

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