Polymer-supported Enzymes: Achievements, Problems and Perspectives Walter Marconi

ASSORENI, 00015 Monterotondo, R o m , Italy

Over the last two decades, intense research activity in the area of enzyme and cell im- mobilisation techniques has fostered the industrial application of several new enzyme- catalysed processes for chemical and pharmaceutical production, as well as for the food industry. Biosensors, based on immobilised enzymes or whole cells, are finding wide- spread applications in the analytical and biomedical fields. The recent remarkable pro- gress attained in the development of recombinant DNA techniques promises to provide any enzyme having properties tailored to meet the specific demands of any user.

Keywords Polymer-supported

enzymes

1. INTRODUCTION

Enzynics are protein catalysts that accelerate the many chemical reactions occurring in living cells and regulate with great efficicncy the metabolic functions of the complete organism. All known enzymes have relatively high molecu- lar weights, exhibit high catalytic activity, show elevated stereochemical and substrate specificities, and catalyse reac- tions under very mild conditions. Despite such striking characteristics, enzymes have several drawbacks like limited stability and therniolability. Moreover, they are water- soluble, a fact that prevents their reuse. In order to circumvent these problems and to develop cat- alysts suited for analytical, biomedical and industrial applic- ations, the immobilisation of enzymes on polymeric sup- ports has been explored. The term ‘immobilized enzyme’ indicates a system or a pre- paration in which the enzyme is confined or localised with- in the semi-permeable micro-cavities of a solid polymeric support or is attached on its surface. There are several ad- vantages in using polymer-supported enzymes. The catalyst can be separated after use and recycled with little or no loss of activity. N o enzyme is left in the reaction mixture, thus minimising by-product formation and facilitating product isolation. Continuous processing is also possible with im- mo bilised enzymes, thus maximising the catalytic efficiency and lowering the costs of process.

2. PREPARATION OF SUPPORTED ENZYMES

The first significant results in preparing polymer-supported enzymes were obtained in the late 1950s by a multidiscip- linary collaboration between biochemists, biophysicists and polymer chemists: Grubhofer, ll2 Manecke 3*4 and Katchal- ~ k i , ~ ~ ~ each with his own expertise, combined their experi- ence in ‘building’ macromolecules in the more exacting task of grafting enzymes onto a well defined polymeric struc- ture. Since then, a multitude of different techniques for enzyme immobilisation have been developed.8 The majority of these methods can be grouped in the following main classes :

(1) Covalent binding to polymeric supports by functional groups not involved in the biological activity of the protein.

Inter- and intramolecular crosslinking by bi- or multi- functional reagents with polymeric supports. Adsorption on natural and synthetic polymers. Entrapment by physical occlusion within microcap- sules and fibres.

The covalent binding approach has been the method most commonly investigated on a laboratory scale. Although this methods - which is tedious, laborious and expensive - leads to significant inactivation of enzymes, the strong chemical bonds formed between the enzymes and the poly- mer supports give a stable conjugate, which is still biologi- cally active and irreversible with respect to microenviron- mental variations (pH, ionic strength, etc.). Two main fac- tors must be considered in coupling enzymes to polymers: (1) the type of functional groups in the protein structure through which the enzymes would be attached to the poly- mer; ( 2 ) the physical and chemical characteristics of the carrier material onto which chemically reactive groups are to be grafted. A variety of functional groups are available in enzymes for the formation of a covalent bond with the support. These include N-terminal amino groups, (€-amino groups of lysine and arginine), C-terminal groups, (0-and y-carboxyl groups of aspartic and glutamic acids), phenol rings of tyrosine, hydroxyl groups of serine and threonine, imidazole of his- tidine and the indole group of tryptophan. The strategy commonly used is to activate the carbonyl, carboxyl, hydroxyl, amino and anhydride groups on the support material, and then to allow these to react with the reactive groups in the protein. The formation of covalent bonds between enzymes and polymer supports is obtained by reactions of acylation, arylation, alkylation and amidation, and by reactions with polymeric aldehydes and diazonium salts. The choice of the reaction conditions should be aimed at causing minimal alterations to the active centre of the enzyme in order to retain maximum enzyme activity after the process of im- mobilisation.

3. TYPES OF POLYMER-SUPPORT

Numerous naturally occurring polysaccharides, such as cellulose, dextrose, starch, agarose and K-carrageenan, and several functionalized chemical derivatives, have been used

222 BRITISH POLYMER JOURNAL, VOLUME 16 DECEMBER 1984

for enzyme i m m ~ b i l i s a t i o n . ~ Polyacrylamides, polyamides, vinyl polymers, copolymers of maleic anhydride and ethy- lene, vinyl polymers, copolymers of maleic anhydride and ethylene, and phenol-formaldehyde resins are the more common synthetic macromolecules tested. In selecting a polymer support, some factors besides chemical reactivity must be considered: mechanical strength, microbial resis- tance, thermal stability, hydrophilicity, permeability, price and availability.

4. CROSS-LINKING OF ENZYME TO SUPPORT

Since all of these factors d o not occur together in any known single support material, the selection of a carrier should be based on a careful evaluation of all the above- mentioned factors. Among a multitude of bifunctional reagents containing both two identical functional groups and groups with different reactivities, glutaraldehyde has emerged as the most widely used crosslinking agent. Glutaraldehyde has also been used as an intermolecular crosslinking reagent producing water-insoluble aggregates of enzymes. The use of bifunctional reagents has been applied success- fully in combination with the adsorption of enzymes onto a polymeric material. In this case, desorption phenomena, due t o the weakness of the binding forces (electrostatic, hydrophobic interactions, hydrogen bonds, etc.) involved in adsoprtion, are avoided by crosslinking of the enzyme t o the adsorption support.

5. ENTRAPMENT WITHIN MEMBRANES

The physical entrapment of enzymes inside microcapsules, membranes, spun fibres,1° hollow fibres and polymeric gels can maintain the enzymes in a micro-environment similar to that in vivo. This approach has been developed mostly for the preparation of immobilised multi-enzyme systems, or- ganelles, microbial cells and other living systerns."J2 Among the various methods of entrapment utilising both natural and synthetic polymers, resin photocrosslinkable prepolymers and urethane prepolymers have recently been d e ~ e l o p e d . ' ~

6. LARGE-SCALE USE OF JMMOBILISED ENZYMES

The immoblisation methods described above have been accepted as useful techniques not only in the laboratory but also on an industrial scale.I4 They have promoted the industrial application of several new enzyme catalysed pro- cesses. One of the oldest immobilised enzyme processes is the resolution of optical isomers of amino acids by the deacetylation of N-acetylated L-aminoacids by L-amino a c y l a ~ e . ~ ~ The hydrolysis of penicillin G by penicillin acylase l6 t o produce 6-aminopenicillanic acid (6-APA), the hydrolysis of lactose in milk and cheese whey by B- galactosidase l 7 t o yield galactose and glucose, and the production of malic acid from fumarate by immobilised fumarase were developed soon after.18 By far the largest application of immobilised enzymes is the isomerisation of glucose to glucose/fructose syrups using glucose isomerase. Currently about 2.5 x lo6 tons/ year of isomerised sugars are produced requiring about 1000 tons of glucose isomerase.

7. ANALYTICAL, BIOMEDICAL AND ELECTRONIC USES OF IMMOBILISED ENZYME

Analytical and biomedical 19q20 applications are other areas where immobilised enzymes play an important role. Over the last decade, interactions between the area of biology and electronics, more specifically between enzymology and transducer technology, have led t o the development of various biosensors.21,22 The common feature of these devices is the close proximity of the immobilised enzyme t o a suitable transducing ele- ment designed t o convert the chemical signal produced by the enzymatic reaction into an electrical response. The transducer may take the form of a semiconductor elec- trode, an ion-selective elecerode, a thermistor, a piezoelec- tric crystal, a transistor or an optoelectronic device. These devices, applied in the diagnostic, biomedical and en- vironmental fields, combine the selectivity and sensitivity of enzymatic methods of analysis with the speed and sim- plicity of the electrochemical device.

8. GENETIC MODIFICATION OF ENZYMES

The remarkable progress achieved in recent years in the de- velopment of recombinant DNA techniques promises to provide virtually any desired enzyme at relatively low prices and on a large scale. A new phase of enzymology has begun, as molecular genetics has found the tools to build man-made enzymes having properties which may be target- ed, enhanced or modulated in response to the specific de- mands of industrial catalysis. The list of changeable proper- ties include: substrate and cofactor specificity and affinity, reaction stereoselectivity, stability t o non-aqueous solvents, heat, oxidising media, in vivo stability, proteolysis suscep- tibility and catalytic efficiency. The ability t o build these unnatural catalysts is achieved by coupling the enzymatic methods of genetic engineering with the chemical synthesis of oligonucleotides. Both these techniques allow the assembly and replication of altered genes, and hence pro- teins of altered aminoacid sequence, offering the opportun- ity t o study the relationship between protein structure and enzyme function and a c t i v i t ~ . ~ ~ ? ~ ~

9 .FUTURE DEVELOPMENTS

The perspective of obtaining known or novel enzymes or hybrid enzymes carrying functions and properties from two or more natural protein catalysts, predicted by computa- tional methods, has led to a strong revival of interest in im- mobilised enzymes.25 Many potential enzymatic processes, too expensive t o com- pete with conventional homogeous or heterogeneous cataly- sis in the pharmaceutical and chemical areas, promise to be- come competitive in the future. The chief target for the ex- ploitation o f these emerging biocatalysts could be the ac- quisition of a variety o f chemical transformations involving not only degradation reactions and modifications of chemi- cal compounds but also complex sequential chemical re- actions for organic synthesis.26

References

1 Grubhofer, N. & Schleith, L., Naturwissenschaften. 1953,40, 508.

BRITISH POLYMER JOURNAL, VOLUME 16 DECEMBER 1984 223

2

3

4 5

6

7

8

9

10

11

12

13

Grubhofer, N. & Schleith, L., Hoppe-Seyler’s Z. Phjviol. Chem., 1954,297,108. Manecke, G . & Gillert, K.E., Naturwissenschaften. 1955, 42,212. Manecke, G. & Singer, S., Makromol. Chem., 1960, 37, 119. Bar-Eli, A. & Katchalski, E., Nature (London), 1960, 188, 856. Katchalski, E., in Polyamino Acids Polypeptides, Proteins, Ed. Stahman, M A . , 1962, p283, Madison: University of Wisconsin Press. Methods in Enzymology, Vol. 44, 1976, Ed. Mosback, K., New York: Academic Press. Chibata, I . , in ‘Immobilized Enzymes’, 1978, New York: John Wiley &Sons. Messing, R.A., in jidvances in Biochemical Engineering’, Vol. 10, 1978, p51, Berlin: Springer-Verlag. Marconi, W. & Morisi, F.. in ‘Applied Biochemistry and Bio- engineering’, Vol. 2, 1979, p209, New York: Academic Press. Mosbach, K., Birnbaum, S., Hardy, K., Davies J . & Bulow, L., Nature, London, 1983, 302,543. Nilsson, K., Sheirer, W., Merten, O.W., Ostberg, L., Liehl, E., Katinger, W.D. & Mosback, K., Nature, (London), 1983, 302,629. Fukui, S. & Tanake, A., in ‘Advances in Biochemical Engin- eeringlBiotechnoIogy, Vol. 29, 1984, 1, Berlin: Springer- Verlag.

14 15

16

17

18

19

20

21

22

23

24 25 26

Klibanov, A.M.,Science, 1983, 219, 722. Chibata, I., in ‘Food Process Engineering’, Vol. 2, p l , 1979, London: Applied Science Publishers Ltd. Marconi, W., Bartolii F., Cecere, F., Galli, G . & Morisi, F., Agric. Riol. Chem., 1975, 39, 1, 277. Marconi, W., Bartoli, F . & Morisi, I:., in Enzyme Engineer- ing, Vol. 5. 1980, 269, New York: Plenum Press. Chibata, I., Tosa, T. & Takata, I., Trends in Biotechnol., 1983, 1(I) , 9 . Chang, T.M.S., Biomedical Applications of immobilized Enzymes and Proteins, Vol. 2, 1977, New York: Plenum Press. Callegaro, F. & Deriti, I:., J. Intern. o fAr t i f : Organs, 1983, 6 , 107. Susuki, S. & Karube, I., in Applied Biochemistry and Bio- engineering, Vol. 3, 1981, p.145, London: Academic Press. Guibaul t , G.C., in Enzyme Engineering, vo1.6, 1982, p395, New York. Plenum Press. Winter, G., F‘ersht, A.R., Wilkinson, A.J., Zoller, M. & Smith M., Nature, (London), 1982,299, 756. Ulmer, K., Science, 1983, 219,666. Drexler, KB. , Proc. Natl. Acad. Sci. USA, 1981, 78, 5275. Oyama, K. & Kihara, K., Chemtech, 1984, 2, 100.

224 BRITISH POLYMER JOURNAL, VOLUME 16 DECEMBER 1984