Enzyme Immobilization: The Quest for Optimum .Enzyme Immobilization: The Quest for Optimum Performance

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  • DOI: 10.1002/adsc.200700082

    Enzyme Immobilization: The Quest for Optimum Performance

    Roger A. Sheldona,*a Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands

    Fax: (+31)-15-278-1415; e-mail: r.a.sheldon@tudelft.nl

    Received: February 5, 2007

    Abstract: Immobilization is often the key to optimiz-ing the operational performance of an enzyme in in-dustrial processes, particularly for use in non-aque-ous media. Different methods for the immobilizationof enzymes are critically reviewed. The methods aredivided into three main categories, viz. (i) binding toa prefabricated support (carrier), (ii) entrapment inorganic or inorganic polymer matrices, and (iii)cross-linking of enzyme molecules. Emphasis isplaced on relatively recent developments, such as theuse of novel supports, e.g., mesoporous silicas, hydro-gels, and smart polymers, novel entrapment methodsand cross-linked enzyme aggregates (CLEAs).

    1 Introduction2 Types of Immobilization3 Immobilization on Supports: Carrier-Bound En-


    3.1 Synthetic Organic Polymers3.2 Biopolymers3.3 Hydrogels3.4 Inorganic Supports3.5 Smart Polymers4 Entrapment5 Carrier-Free Immobilization by Cross-Linking5.1 Cross-Linked Enzyme Crystals (CLECs)5.2 Cross-Linked Enzyme Aggregates ACHTUNGTRENNUNG(CLEAs=)6 Combi-CLEAs and Catalytic Cascade Processes7 Enzyme-Immobilized Microchannel Reactors for

    Process Intensification8 Conclusions and Prospects

    Keywords: biotransformations; cross-linked enzymeaggregates; entrapment; enzymes; immobilization;support binding

    1 Introduction

    In the drive towards green, sustainable methodologiesfor chemicals manufacture[1] biocatalysis has much tooffer:[2] mild reaction conditions (physiological pHand temperature), a biodegradable catalyst and envi-ronmentally acceptable solvent (usually water), aswell as high activities and chemo-, regio- and stereo-selectivities. Furthermore, the use of enzymes general-ly obviates the need for functional group protectionand/or activation, affording synthetic routes which areshorter, generate less waste and, hence, are both envi-ronmentally and economically more attractive thantraditional organic syntheses.

    Modern developments in biotechnology have pavedthe way for the widespread application of biocatalysisin industrial organic synthesis.[314] Thanks to recombi-nant DNA techniques[15] it is, in principle, possible toproduce most enzymes for a commercially acceptableprice. Advances in protein engineering have made itpossible, using techniques such as site-directed muta-genesis and in vitro evolution via gene shuffling,[1619]

    to manipulate enzymes such that they exhibit the de-sired properties with regard to, inter alia, substrate

    specificity, activity, selectivity, stability and pH opti-mum. Nonetheless, industrial application is oftenhampered by a lack of long-term operational stabilityand difficult recovery and re-use of the enzyme.These drawbacks can often be overcome by immobili-zation of the enzyme.[2025]

    There are several reasons for using an enzyme inan immobilized form. In addition to more convenienthandling of the enzyme, it provides for its facile sepa-ration from the product, thereby minimizing or elimi-nating protein contamination of the product. Immobi-lization also facilitates the efficient recovery and re-use of costly enzymes, in many applications a conditiosine qua non for economic viability, and enables theiruse in continuous, fixed-bed operation. A further ben-efit is often enhanced stability,[26] under both storageand operational conditions, e.g., towards denaturationby heat or organic solvents or by autolysis. Improvedenzyme performance via enhanced stability and re-peated re-use is reflected in higher catalyst productiv-ities (kg product/kg enzyme) which, in turn, deter-mine the enzyme costs per kg product. As a rule ofthumb the enzyme costs should not amount to morethan a few percent of the total production costs. In

    Adv. Synth. Catal. 2007, 349, 1289 1307 D 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 1289


  • the production of 6-aminopenicillanic acid (6-APA)by penicillin G amidase (penicillin amidohydrolase,E.C. hydrolysis of penicillin G, forexample, 600 kg of 6-APA are produced per kg of im-mobilized enzyme and the production of the com-modity, fructose, by glucose isomerase-catalyzed iso-merization of glucose has a productivity of 11,000.[22]

    Indeed, the development of an effective method forits immobilization was crucial for the application ofpenicillin amidase in the industrial synthesis of b-lactam antibiotics.[27]

    Immobilization is generally necessary for optimumperformance in non-aqueous media. In the traditionalmethod of using enzymes as lyophilized (freeze-dried)powders, many of the enzyme molecules are not read-ily accessible to substrate molecules. Furthermore,lyophilization can cause pronounced structural pertur-bations often leading to deactivation. In contrast, dis-persion of the enzyme molecules by immobilizationgenerally provides for a better accessibility and/or anextra stabilization of the enzymes towards denatura-tion by the organic medium.

    Another benefit of immobilization is that it enablesthe use of enzymes in multienzyme and chemoenzy-matic cascade processes (see later).[2831] A majorproblem encountered in the design of catalytic cas-cade processes is the incompatibility of the differentcatalysts and a possible solution is compartmentaliza-tion (i.e., immobilization) of the different catalyststhus circumventing their mutual interaction whichcould result in inhibition and or deactivation.

    In this review we shall focus on recent develop-ments in novel immobilization techniques, such as theuse of novel supports, e.g., smart polymers, novel en-trapment methods, and the recent development ofcross-linked enzyme aggregates (CLEAs), in thequest for optimum performance in biotransforma-tions. Another approach to facilitating the recoveryand re-use of enzymes is to perform the reactions,with the free enzyme, in a membrane bioreactor con-sisting of an ultrafiltration membrane which retainsthe enzyme on the basis of its size but allows sub-strates and products to pass through. This combinesease of recovery and recycling with the high activityof the free enzyme. The methodology is applied com-mercially, for example, by Degussa in the commercialsynthesis of enantiomerically pure amino acids usinghydrolases or dehydrogenases.[32] However, since it in-volves the free enzyme it falls outside the scope ofthis review.

    2 Types of Immobilization

    Basically, three traditional methods of enzyme immo-bilization can be distinguished, binding to a support(carrier), entrapment (encapsulation) and cross-link-ing.

    (i) Support binding can be physical (such as hydro-phobic and van der Waals interactions), ionic, or co-valent in nature. However, physical bonding is gener-ally too weak to keep the enzyme fixed to the carrierunder industrial conditions of high reactant and prod-uct concentrations and high ionic strength. Ionic bind-ing is generally stronger and covalent binding of theenzyme to the support even more so, which has theadvantage that the enzyme cannot be leached fromthe surface. However, this also has a disadvantage: ifthe enzyme is irreversibly deactivated both theenzyme and the (often costly) support are renderedunusable. The support can be a synthetic resin, a bio-polymer or an inorganic polymer such as (mesopo-rous) silica or a zeolite.

    (ii) Entrapment via inclusion of an enzyme in a po-lymer network (gel lattice) such as an organic poly-mer or a silica sol-gel, or a membrane device such asa hollow fiber or a microcapsule. The physical re-straints generally are too weak, however, to preventenzyme leakage entirely. Hence, additional covalentattachment is often required. The difference betweenentrapment and support binding is often not clear.For the purpose of this review we define supportbinding as the binding of an enzyme to a prefabricatedsupport (carrier) irrespective of whether the enzymeis situated on the external or internal surface. Entrap-ment requires the synthesis of the polymeric networkin the presence of the enzyme. For example, when anenzyme is immobilized in a prefabricated mesoporous

    Roger Sheldon (1942)is Professor of Bioca-talysis and OrganicChemistry at DelftUniversity of Technol-ogy (Netherlands). Hereceived a PhD in or-ganic chemistry fromthe University of Lei-cester (UK) in 1967.This was followed bypost-doctoral studieswith Prof. Jay Kochi inthe USA. From 1969 to 1980 he was with Shell Re-search in Amsterdam and from 1980 to 1990 he wasR&D Director of DSM Andeno. His primary re-search interests are in the application of catalyticmethodologies homogeneous, heterogeneous andenzymatic in organic synthesis, particularly in rela-tion to fine chemicals production. He developed theconcepts of E factors and atom utilization for assess-ing the environmental impact of chemical processes.

    1290 asc.wiley-vch.de D 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim Adv. Synth. Catal. 2007, 349, 1289 1307

    REVIEWS Roger A. Sheldon


  • silica the enzyme may be situated largely in the meso-pores but this would not be entrapment. On the otherhand when the enzyme is present during the synthesisof a silica sol-gel the enzyme is entrapped.

    ACHTUNGTRENNUNG(iii) Cross-linking of enzyme aggregates or crystals,using a bifunctional reagent, to prepare carrierlessmacroparticles.

    The use of a carrier inevitably leads to Kdilution ofactivityL, owing to the introduction of a large portionof non-catalytic ballast, ranging from 90% to >99%,which results in lower space-time yields and produc-tivities.[22] Moreover, immobilization of an enzyme ona carrier often le