Immobilized enzymes

TIBS-January 1980

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Future Trends Immobilized enzymes Klaus Mosbach In last September's TIBS (1979, N 207) D. Thomas" Future Trends article examined the prospects for enzyme technology. This month Klaus Mosbach looks in detail at one aspect of this rapidly expanding field - - immobilized enzymes - - which he believes have great potential for science, industry and medicine. The last ten years have witnessed a remarkable proliferation of developments in what may be termed 'solid-phase biochemistry'. The common feature of these techniques is the use of solid phase particles, notably agarose, acrylate and glass, as supports for the immobilization of enzymes, cells, nucleotides, inhibitors and other biological molecules. Affinity chromatography, the Merrifield method for the synthesis of polypeptides, and the complementary technique of protein sequencing, are among the most spectacular methodological advances to be based on solid phases. Much research effort has been expended on developing the large variety of polymeric support materials now available, and on the modification of conventional reactions used in.organic chemistry to provide methods for attaching biological molecules to these supports using covalent bonds. The growing understanding among biochemists that a multitude of enzymic reactions within the framework of the cell are carried out on or within membranes and other biological 'solid phases', has paralleled these technological innovations and given impetus to studies of 'solid phase biochemistry'. However, despite the already large number and variety of publications in the field of solid phase biochemistry, its potential is nowhere close to being fully realized. Here the trends for its future development are examined in the oontext of the current state of achievement, with particular emphasis on .immobilized enzymes.

lmmobili,ation methods A large variety of materials'has been employed as supports, mostly beaded solids but also membranes and fibres, and, in a few cases, water-soluble supports such as dextran. Enzymes are usually immobilKlaus Mosbach is head of the Department of Biochemistry at the Institute of Technology~University of Lurid, Sweden.

ized on these supports by one of four ways (Fig. 1): (a) covalent attachment; (b) inclusion within a gel lattice or (micro)-encapsulation within semi: permeable membranes; (c) adsorption, including hydrophobic and affinity-binding; (d) cross-linking to insoluble aggregates. In principle, the same immobilization techniques can or have been applied to microbial cells and organelles such as mitochondria and chloroplasts. For further background, the reader is referred to recent reviews [1-3]. Immobilized enzymes in biochemical

studies It is now widely appreciated that immobilized enzyme systems provide models of conditions in vivo since most enzymes in the living cell are compartmentalized and are often bound to membranes, aggregated in structures, in solid-state assemblies or in gel-like surroundings. Emphasis has been given to assemblies of enzymes which act in metabolic sequences in vivo. For instance, it has been shown that the overall rate of sequentially-acting enzymes, when immobilized near to each other on the same particle, is higher than when the same enzymes act on the same substrates in free solution [4]. From these and simil~ir studies, the importance of the concept of an enzyme's individual microenvironment has been recognized in the understanding of metabolic regulation and enzyme behaviour. As a tool for biochemical studies, immobilization per se is also increasingly utilized, as illustrated by the following examples [1]. Our understanding of the role of monomers (subunits) of oligomeric enzymes has increased from studies of a number of enzymes which, after binding through one subunit, were dissociated,

leaving immobilized single subunits on the support. In the case of the tetrameric enzyme aldolase the immobilized subunit alone appeared to be catalytically active. Conversely,. the immobilized subunits of both lactate and alcohol dehydrogenases were inactive, although the original activity could be reconstituted on solid phase by addition of native monomers. Liver alcohol dehydrogenase, consisting of two monomers, was also reversibly, immobilized through one subunit only, then specifically inhibited with iodoacetate. The preparation obtained after dissociation from the support thus had only one active subunit, permitting studies of 'half-site activity' [5]. More examples could be given to illustrate the applications of immobilization techniques per se, or immobilized enzymes as models, to questions related to fundamental biochemistry. Nevertheless, in my opinion, these approaches are in their infancy and their potential is immense. For these techniques to gain the full confidence of biochemists, however, better characterized immobilized preparations must be obtained in which the intrinsic properties of the enzymes are unchanged by the immobilization procedure. To this end, more sophisticated methods forcharacterization of the immobilized preparations, by chemical or physical means, must be sought. The development of reversible methods for coupling enzymes to supports is an important step in allowing us to check whether the intrinsic properties of the enzyme have remained unaltered through the immobilization.

a b@

Fig. 1. Schematic drawings of the four major types of immobilized enzyme preparations. (a) Covalent binding, (b) entrapment, (c) adsorption, and (d) crosslinking.. O Elsevier/North-Holland Biomedical Press 1980

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Practical applications of immobilized

study mesophilic enzyme stabilization employing, for example, the binding of enzymes to the support by multiple points A number of processes on a large scale of attachment; and (c) to develop supports involving immobilized enzymes (known as tailor-made for the process in question. In enzyme technology o r enzyme engineerthe context, however, the potential of free, ing) are already in commercial operation preferentially stabilized enzymes 'immobil[1-3]. These include the hydrolysis of the ized' with ultra-filtration cells should not be side-chain of benzyl penicillin using solidoverlooked. phase penicillin amidase to producethe basic material for semisynthetic penicillins. Other examples of viable commercial pro- Analytical applications Many of the advantages of immobilized duction processes are outlined in the first article in this series (D. Thomas T I B S preparations already mentioned apply equally to analytical work, including their 1979, Sept., N 207-N 209). Despite these isolated successes solid- suitability for continuous monitoring and phase technology has yet to be embraced the possibility of re-using expensive wholeheartedly by industry, contrary to the enzymes [1-3]. In addition, the immobilexpectations Of an over enthusiastic scien- ization technique allows, in principle, the tific community during the early develop- arrangement of a sensor 9(enzyme) in close ment phase of enzyme technology. Why is proximity to a transducer ('measuring this so? One factor may be the practice of 9device'), resulting in a rapid and highly senconcentrating more on the 'engineering' sitive response. This has been realized in problems such as reactor design than on the practice in both the enzyme electrode, e.g. important aspect of enzyme stabilization [6] and the enzyme thermistor, e.g. [1,7]. per se, which is the prerequisite for high Other such 'dual' systems under develop'operational stability'. For solid-phase sys- ment include the 'enzyme mass spectrometems to be cost-effective, they must stand ter', the 'enzyme electrochemical cell', the up to prolonged use. An example of a suc- 'enzyme polarimeter', the 'enzyme speccessful process exhibiting good operational trophotometer', and the 'enzyme stability is the resolution of racemic mix- luminometer'. Some of these devices are tures of amino acids - a system which has presently finding use primarily in clinical been shown to retain 60% of the original analysis. activity after 30 days of operation at 50~ Applications in environmental and pro[1]. It is now realized that, for each particu- cess control are also being investigated. lar application, optimal conditions for Measuring devices containing cells or large-scale use have first: to be found, e n z y m e s - the ultimate 'targets' for before a comparison with the conventional poisonous materials • show great promise process should be made. Other factors that as, for example, environmental 9 contribute to the relatively slow adoption tors. Immobilized acetylcholinesterase of solid phase systems are the additional mounted within an9 cell can cost of the support materials and the reluc- detect a nerve toxin down to one ppm. A tance to abandon tried and trusted pro- microbe thermistor unit has been described cesses. Once systems of acceptable opera- in which living microbial cells are immobiltional stability are developed, however, the ized in proximity to a thermistor that inherent advantages of immobilized measures the total heat of metabolism of enzymes, such as the possibility of repeated these cells. When the unit is immersed in a use (due in part to increased stability), the stream Of water, any pollutants present will absence of contamination of the final pro- be detected by their marked effect on the duct with the 'catalyst', the possibility of metabolic heat pioduction. The advantage applying flow-processes, the reduction of of using intact cells in such a system is that waste, and of labour and energy costs will they are susceptible to many potential polin many instances tip the balance in favour lutants, although their speed of response is of their adoption by industry. less than that of enzymes. Alternatively, Increasing attention is being paid to electrodes can be used to measure changes immobilized intact cells and organelles, in microbial cells immersed in polluted especially those obtained from microbial water. sources, but also from plant and animal Finally, 9I should like to mention briefly cells. In particular, microbial cells immobil- the Use of immobilized enzymes for: the isoized in their 'living and growing' state have lation of inhibitors in affinity chromarecently come much into focus. We can tography [8] and to point to probably the expect that there will be increased efforts: most widespread application of immobil(a) to Utilize both thermophilic and ized enzymes t o d a y - enzyme immunoashalophilic enzymes and cells as with these say [9]. As a technique per se ELISA the risks of contamination are small; (b) to (enzyme-linked immunosorbent assay) has

enzymes

TIBS -January 1980

come close to its ultimate definitiori whereas the full potential of the so-called EMIT method (enzyme-multiplied immunoassay) has yet to be exploited.

Immobilized enzymes in organic synthesis 9A recent use of immobilized enzymes is iri synthetic organic chemistry, an area closely related to enzyme technology. Obviously inreacti0ns where enzymes can be employed, 9conversions with minimal side reactions are likely to occur. The greatest attraction 9 of enzymes to the organicchemist lies in their ability to discriminate between optical enantiomers. Apart from their current use in the resolution of racemic mixtures of amino acids and in steroid transformations (such as specific 11 /3:hydroxylation of Reichstein compound S to cortisol) more applications of this kind are likely to come, both for largescale processes and for the smaller-scale preparation of fine chemicals. It is likely that the capacity of proteolytic enzymes for stereospecific hydrolysis of esters or of alcohol dehydrogenase for stereospecific reduction-oxidation reactions will soon be exploited employing the inherent advantages of immobilized enzymes. In some cases it may even be a prerequisite to use immobilized enzymes in particular when a reaction has to be carded out in organic solvents, so that hydrolytic reactions may be reversed or the solubility of the reactants may be increased. Examples of the former kind are the synthesis of peptide polymers from dipeptides using immobilized proteolytic enzymes in high concentrations of organic solvents .and the synthesis of esters using oe-chymotrypsin. Free enzymes would be inactivated or precipitated under such conditions. These examples, to my mind, presage future developments in this area.

Therapeutic applications Immobilized enzymes have 10ng been thought to have potential for enzyme replacement therapy, and as substitutes for at least partial function of organs [10,11].By administering enzymes in their immobilized,, entrapped state, the normally observed immunological reactions which normally lead to anaphylactic shock or loss of activity, may be avoided. Further, the degradation of the enzymes administered by proteolytie enzymes is likely to :be reduced considerably..Some examples of applications of immobilized enzymes in therapy include the use of liposomes for embedding various enzymes, microencapsulation of catalase in nylon beads, and the use of red blood cells as hosts for asparaginase [10,11].

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TIBS -January 1980 These are isolated successes in an extremely complex area in which only limited progress has been made so far, despite much effort. Researchers, however, will undoubtedly persevere in the therapeutic use of enzymes for the removal of toxic substances and the replacement of 'missing' enzymes in the many known enzyme deficiency diseases. Two approaches suggest themselves: either the direct injection of immobilized enzyme preparations into the blood stream, or their placement in extracorporal shunts. Much work is required before direct injection will be feasible, particularly because ideally these preparations should be both biocompatible and biodegradable. For the use of enzymes in flow systems in extracorporal shunts, their immobilization is a prerequisite. A promising example of the latter approach is the removal of bilirubin by immobilized albumin packed in an extracorporal shunt system. " Clearly much more effort must be devoted to.obtaining immobilized enzyme preparations with prolonged lifetimes. The reported stabilization of enzymes 97 vivo throughcovalent attachment of carbohydrates is of special interest. We also need to develop self-sufficient, 'closed' immobilized enzyme-coenzyme systems, which do not perturb the medium, as a number of clinically interesting systems depend on the participation of coenzymes. The use of immobilized enzymes for therapy is certain to increase gradually. As an artificial organ for the relief of uremic disorders, urease has been entrapped with an ion exchange resin or, alternatively, it can be entrapped with glutamate dehydrogenase and activated carbon; the entrapment is usually within microcapsules and these a r e placed in an extracorporal shunt. The semipermeable membranes of the capsules allow urea to enter. The productsare carbon dioxide, which is exhaled, and toxic ammonium ions, which are either taken up by the ion exchange resin or transformed by the second enzyme t o glutamate, which is adsorbed On to carbon. Other tentative steps towards building artificial biological organs include studies with immobilized enzymes forextracorporal det0xification in cases of liver failure, and the simulation of lung function by immobilization of carbonic anhydrase and catalase on silicone to give a system capable of exchanging oxygen and carbon dioxide. Implantable glucose electrodes have been specially designed to allow controlled administration of insulin in a simulation of pancreatic function. Finally, mention should be made of the potential of immobilized drugs or enzymes tailor-made

for drug-targeting [ 11 ]. Among the various approaches under discussion, magnetic microspheres as carriers may also have a role [12].

general thinking employed in the numerous immobilization techniques we are familiar with. Conclusion

Solid phases in 'synthetic biochemistry' The organic chemists, originally preoccupied with Nature's own products, eventually turned their attention to 'independent' synthetic research. Genetic engineers have done so recently, and t h e time has come for us enzymologists to take, based on solid-phase techniques, a similar lateral step to modify enzymes, Nature's own supercatalysts. One way would be to alter the amino acid sequence of enzymes using the Merrifield or other techniques. But what I have particularly in mind is the possibility of preparing, on solid-phase systems, new metabolic sequences not normally found in an organism, or replacing parts of an enzyme molecule by a support, or of changing drastically the properties of an enzyme either by binding to a support or by employing the immobilization technique as such. Tentative steps in this direction have already been taken. An example is the binding of phosphorylase b to a support already substituted with the enzyme's effector, AMP, in such a manner that no more free AMP is required for activity [13]. Alternatively, by using cr0ss-linking agents, conformational 'freezing' in the presence of an effector was achieved with dCMP aminohydrolase [14]. This gives an enzyme preparation !n the activated form. Or, again, by freezing in the presence of a substrate, as for asparate transcarbamoylase, an enzyme with higher substrate affinity can be obtained [15]. Related to this is the immobilization of enzymes to supports in the presence of substrates or inhibitors which leads to enzymes with altered properties [16]. Attempts to change the conformation of immobilized enzymes by direct deformation of the support in what has been coined 'mechanochemistry of enzymes' is further example of such 'synthetic' biochemistry [1]. The covalent immobilization of NAD directly on to alcohol dehydrogenase in such a fashion that NAD can be recycled is an example of modifying the normally freely-diffusing NAD coenzyme into a kind of prosthetic group [17]. Covalently binding a synthetic ravin to the active site of the hydrolytic enzyme, papain, changes it into an oxidation-reduction catalyst [18] another example of molecular engineering. Although in some of the above examples solid phases have not been used, the examples are based on the same chemistry and

The use of immobilized enzymes for pure and applied studies is still in its infancy. By the turn of the century, it is likely that solid-phase technology will be used in many processes and analytical applications. Medicine will have found applications to improve the quality of life for patients suffering from what are now intractable diseases. Finally, it is my firm conviction that the merging of 'solid phase techniques' as applied to the immobilization of enzymes, with a general understanding of enzyme bchaviour, will allow us to prepare artificial biocatalysts that, in many aspects, will simulate Nature's own enzymes. References 1 'Immobilized Enzymes' in: Methods bz Enzymology (Mosbach, K., ed.), Vol. 44, Academic Press, New York, 1976 2 Enzyme Engineering (Pye, E. K. and Wingard, L. B. Jr, Pye, E. K. and Weetall, H. H., Brown, G. B. and Manecke, G. and Wingard, L. B. Jr, eds), Vols 2-4, Plenum Press, New York, 1974 and 1978 3 Applied Biochemistry and Bioengineering, Vol. i (Wingard, L. B. Jr, Katchalski-Katzir, E. and G01dstein, L., eds), Academic Press, New York, 1976 4 Mosbach, K. and Mattiasson, B. in Current Topics in Cellular Regulation (Horecker, B. L. and Stadtman, E. R. eds), Vol. 14, 1978, pp. 187-241 5 Andersson, L. and Mosbach, K. (1979) Eur. J. Biochem. 94, 565-571 6 Guilbault, G. G. (1976) tlandbook of Enzymatic Methods of Analysis, Dekker, New York (and references cited therein including Clark, Hicks and Updike) 7 Danielsson, B., Mattiasson, B. and Mosbach, K. (1979), Pureand Applied Chemistry, Vol. 51, No. 7, 1443-1455 8 '.Affinity Techniques' in: Methods in Enzymology, Vol. 34 (Jacoby, W. B. and Wilchek, M., eds), Academic Press, New York, 1974 9 Wisdom, B. (1976) Clin. Chem. 22, 1243-1255 10 BiomedicqlApplications of lmmobilized Enzymes and Proteins (Chang, T. M. S., ed.), Vols 1 and 2, Plenum Press, New York, 1977 11 Gregoriadis, G. (1977) Nature (London) 265, 407--411 12 Mosbach, K. and Schroeder, U. (1979) FEBS Lett. 102, 112-116 13 Mosbach, K. and Gestrelius, S. (1974) FEBS Lett. 42, 200-204 14 Nucci, R., Raia, C. A., Vaccaro, C., Sepe, :5., Scarano, E. and Rossi, M. (1978) J. MoL Biol. 124, 133-145 15 Enns, C. A. and Chan, W. W. -C. (1978) J. BioL Chem. 253, 2511-2513 16 Royer, G. P., Jkeda, S. and Lee, T. (1977b) J. Biol. Chem. 252, 8775-8777 17 M~nsson, M. O., Larsson, P. -O. and Mosbach, K. (1979) FEBS Lett. 98.309-313 18 Levine, tt. L. and Kaiser, E.T. (1978)JACS 100, 7670-7677