Enzyme Immobilization: the Quest for Optimum Performance
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REVIEWS 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: [email protected] Received: February 5, 2007 Abstract: Immobilization is often the key to optimiz- 3.1 Synthetic Organic Polymers ing the operational performance of an enzyme in in- 3.2 Biopolymers dustrial processes, particularly for use in non-aque- 3.3 Hydrogels ous media. Different methods for the immobilization 3.4 Inorganic Supports of enzymes are critically reviewed. The methods are 3.5 Smart Polymers divided into three main categories, viz. (i) binding to 4 Entrapment a prefabricated support (carrier), (ii) entrapment in 5 Carrier-Free Immobilization by Cross-Linking organic or inorganic polymer matrices, and (iii) 5.1 Cross-Linked Enzyme Crystals (CLECs) cross-linking of enzyme molecules. Emphasis is 5.2 Cross-Linked Enzyme Aggregates(CLEAsACHTUNGRE ) placed on relatively recent developments, such as the 6 Combi-CLEAs and Catalytic Cascade Processes use of novel supports, e.g., mesoporous silicas, hydro- 7 Enzyme-Immobilized Microchannel Reactors for gels, and smart polymers, novel entrapment methods Process Intensification and cross-linked enzyme aggregates (CLEAs). 8 Conclusions and Prospects 1 Introduction 2 Types of Immobilization Keywords: biotransformations; cross-linked enzyme 3Immobilization on Supports: Carrier-Bound En- aggregates; entrapment; enzymes; immobilization; zymes support binding 1 Introduction specificity, activity, selectivity, stability and pH opti- mum. Nonetheless, industrial application is often In the drive towards green, sustainable methodologies hampered by a lack of long-term operational stability for chemicals manufacture[1] biocatalysis has much to and difficult recovery and re-use of the enzyme. offer:[2] mild reaction conditions (physiological pH These drawbacks can often be overcome by immobili- and temperature), a biodegradable catalyst and envi- zation of the enzyme.[20–25] ronmentally acceptable solvent (usually water), as There are several reasons for using an enzyme in well as high activities and chemo-, regio- and stereo- an immobilized form. In addition to more convenient selectivities. Furthermore, the use of enzymes general- handling of the enzyme, it provides for its facile sepa- ly obviates the need for functional group protection ration from the product, thereby minimizing or elimi- and/or activation, affording synthetic routes which are nating protein contamination of the product. Immobi- shorter, generate less waste and, hence, are both envi- lization also facilitates the efficient recovery and re- ronmentally and economically more attractive than use of costly enzymes, in many applications a conditio traditional organic syntheses. sine qua non for economic viability, and enables their Modern developments in biotechnology have paved use in continuous, fixed-bed operation. A further ben- the way for the widespread application of biocatalysis efit is often enhanced stability,[26] under both storage in industrial organic synthesis.[3–14] Thanks to recombi- and operational conditions, e.g., towards denaturation nant DNA techniques[15] it is, in principle, possible to by heat or organic solvents or by autolysis. Improved produce most enzymes for a commercially acceptable enzyme performance via enhanced stability and re- price. Advances in protein engineering have made it peated re-use is reflected in higher catalyst productiv- possible, using techniques such as site-directed muta- ities (kg product/kg enzyme) which, in turn, deter- genesis and in vitro evolution via gene shuffling,[16–19] mine the enzyme costs per kg product. As a rule of to manipulate enzymes such that they exhibit the de- thumb the enzyme costs should not amount to more sired properties with regard to, inter alia, substrate than a few percent of the total production costs. In Adv. Synth. Catal. 2007, 349, 1289 – 1307 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1289 REVIEWS Roger A. Sheldon In this review we shall focus on recent develop- Roger Sheldon (1942) ments in novel immobilization techniques, such as the is Professor of Bioca- use of novel supports, e.g., smart polymers, novel en- talysis and Organic trapment methods, and the recent development of Chemistry at Delft cross-linked enzyme aggregates (CLEAs), in the University of Technol- quest for optimum performance in biotransforma- ogy (Netherlands). He tions. Another approach to facilitating the recovery received a PhD in or- and re-use of enzymes is to perform the reactions, ganic chemistry from with the free enzyme, in a membrane bioreactor con- the University of Lei- sisting of an ultrafiltration membrane which retains cester (UK) in 1967. the enzyme on the basis of its size but allows sub- This was followed by strates and products to pass through. This combines post-doctoral studies ease of recovery and recycling with the high activity with Prof. Jay Kochi in of the free enzyme. The methodology is applied com- the USA. From 1969 to 1980 he was with Shell Re- mercially, for example, by Degussa in the commercial search in Amsterdam and from 1980 to 1990 he was synthesis of enantiomerically pure amino acids using R&D Director of DSM Andeno. His primary re- hydrolases or dehydrogenases.[32] However, since it in- search interests are in the application of catalytic volves the free enzyme it falls outside the scope of methodologies – homogeneous, heterogeneous and this review. enzymatic – in organic synthesis, particularly in rela- tion to fine chemicals production. He developed the concepts of E factors and atom utilization for assess- 2 Types of Immobilization ing the environmental impact of chemical processes. Basically, three traditional methods of enzyme immo- bilization can be distinguished, binding to a support (carrier), entrapment (encapsulation) and cross-link- the production of 6-aminopenicillanic acid (6-APA) ing. by penicillin G amidase (penicillin amidohydrolase, (i) Support binding can be physical (such as hydro- E.C. 3.5.1.11)-catalyzed hydrolysis of penicillin G, for phobic and van der Waals interactions), ionic, or co- example, 600 kg of 6-APA are produced per kg of im- valent in nature. However, physical bonding is gener- mobilized enzyme and the production of the com- ally too weak to keep the enzyme fixed to the carrier modity, fructose, by glucose isomerase-catalyzed iso- under industrial conditions of high reactant and prod- merization of glucose has a productivity of 11,000.[22] uct concentrations and high ionic strength. Ionic bind- Indeed, the development of an effective method for ing is generally stronger and covalent binding of the its immobilization was crucial for the application of enzyme to the support even more so, which has the penicillin amidase in the industrial synthesis of b- advantage that the enzyme cannot be leached from lactam antibiotics.[27] the surface. However, this also has a disadvantage: if Immobilization is generally necessary for optimum the enzyme is irreversibly deactivated both the performance in non-aqueous media. In the traditional enzyme and the (often costly) support are rendered method of using enzymes as lyophilized (freeze-dried) unusable. The support can be a synthetic resin, a bio- powders, many of the enzyme molecules are not read- polymer or an inorganic polymer such as (mesopo- ily accessible to substrate molecules. Furthermore, rous) silica or a zeolite. lyophilization can cause pronounced structural pertur- (ii) Entrapment via inclusion of an enzyme in a po- bations often leading to deactivation. In contrast, dis- lymer network (gel lattice) such as an organic poly- persion of the enzyme molecules by immobilization mer or a silica sol-gel, or a membrane device such as generally provides for a better accessibility and/or an a hollow fiber or a microcapsule. The physical re- extra stabilization of the enzymes towards denatura- straints generally are too weak, however, to prevent tion by the organic medium. enzyme leakage entirely. Hence, additional covalent Another benefit of immobilization is that it enables attachment is often required. The difference between the use of enzymes in multienzyme and chemoenzy- entrapment and support binding is often not clear. matic cascade processes (see later).[28–31] A major For the purpose of this review we define support problem encountered in the design of catalytic cas- binding as the binding of an enzyme to a prefabricated cade processes is the incompatibility of the different support (carrier) irrespective of whether the enzyme catalysts and a possible solution is compartmentaliza- is situated on the external or internal surface. Entrap- tion (i.e., immobilization) of the different catalysts ment requires the synthesis of the polymeric network thus circumventing their mutual interaction which in the presence of the enzyme. For example, when an could result in inhibition and or deactivation. enzyme is immobilized in a prefabricated mesoporous 1290 asc.wiley-vch.de 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2007, 349, 1289 – 1307 Enzyme Immobilization: The Quest for Optimum Performance REVIEWS silica the enzyme may be situated largely in the meso- average particle size of 170 mm and a pore diameter pores but this would not be entrapment. On the other of 25 nm.[38] It is highly hydrophilic and stable, both hand when the enzyme is present during the synthesis chemically and mechanically, over a pH range from 0 of a silica sol-gel the enzyme is entrapped. to 14, and does not swell or shrink even upon drastic (iii)ACHTUNGRE Cross-linking of enzyme aggregates or crystals, pH changes in this range. It binds proteins via reac- using a bifunctional reagent, to prepare carrierless tion of its oxirane moieties, at neutral or alkaline pH, macroparticles. with the free amino groups of the enzyme to form co- The use of a carrier inevitably leads to dilution of valent bonds which have long-term stability within a activity, owing to the introduction of a large portion pH range of pH 1 to 12 (see Figure 1).