Ce

Fig. 2. Approaches to enzyme immobilization, reversible methods.

support is often a primary factor in the overall cost of immobilized catalyst. The reversible immobilization of enzymes is particularly important for immobilizing labile enzymes and for applications in bioanalytical systems (17).

5.1. Adsorption (Noncovalent Interactions)

5.1.1. Nonspecific Adsorption

The simplest immobilization method is nonspecific adsorption, which is mainly based on physical adsorption or ionic binding (39,40). In physical adsorption the enzymes are attached to the matrix through hydrogen bonding, van der Waals forces, or hydrophobic interactions; whereas in ionic bonding the enzymes are bound through salt linkages. The nature of the forces involved in noncovalent immobilization results in a process can be reversed by changing the conditions that influence the strength of the interaction (e.g., pH, ionic strength, temperature, or polarity of the solvent). Immobilization by adsorption is a mild, easy to perform process, and usually preserves the catalytic activity of the enzyme. Such methods are therefore economically attractive, but may suffer from problems such as enzyme leakage from matrix when the interactions are relatively weak.

5.1.2. Ionic Binding

An obvious approach to the reversible immobilization of enzymes is to base the protein-ligand interactions on principles used in chromatography. For example, one of the first applications of chromatographic principles in the reversible immobilization of enzymes was the use of ion-exchangers (4,41,42). The method is simple and reversible but, in general, it is difficult to find conditions under which the enzyme remains both strongly bound and fully active. More recently, the use of immobilized polymeric-ionic ligands has allowed for modulation of proteinmatrix interactions and has thus optimized the properties of the derivative. A number of patents have been filed on the use of polyethyleneimine to bind a rich variety of enzymes and whole cells (43).

However, problems may arise from the use of a highly charged support when the substrates or products themselves are charged; the kinetics are distorted as a result of partition or diffusion phenomena. Therefore, enzyme properties, such as pH optimum or pH stability, may change (44,45). Although this could pose a problem it could also be useful to shift the optimal conditions of a certain enzyme towards more alkaline or acidic conditions, depending on the application (46).

5.1.3. Hydrophobic Adsorption

Another approach is the use of hydrophobic interactions. In this method, it is not the formation of chemical bonds but rather an entropically driven interaction that takes place. Hydrophobic adsorption has been used as a chromatographic principle for more than three decades. It relies on well-known experimental variables such as pH, salt concentration, and temperature (47). The strength of interaction relies on both the hydrophobicity of the adsorbent and the protein. The hydropho-bicity of the adsorbent can be regulated by the degree of substitution of the support and by the size of the hydrophobic ligand molecule. The successful reversible immobilization of P-amylase and amyloglucosidase to hexyl-agarose carriers has been reported (48,49). Several other examples of strong reversible binding to hydrophobic adsorbents have also been reported (50-52).

5.1.4. Affinity Binding

The principle of affinity between complementary biomolecules has been applied to enzyme immobilization. The remarkable selectivity of the interaction is a major benefit of the method. However, the procedure often requires the covalent binding of a costly affinity ligand (e.s., antibody,= or lectin) to the matrix (53).

5.2. Chelation or Metal Binding

Transition metal salts or hydroxides deposited on the surface of organic carriers become bound by coordination with nucleophilic groups on the matrix. Mainly titanium and zirconium salts have been used and the method is known as "metal link immobilization" (15,54,55). The metal salt or hydroxide is precipitated onto the support (e.g., cellulose, chitin, alginic acid, and silica-based carriers) by heating or neutralization. Because of steric factors, it is impossible for the matrix to occupy all coordination positions of the metal; therefore some of the positions remain free to coordinate with groups from the enzymes. The method is quite simple and the immobilized specific activities obtained with enzymes in this way have been relatively high (30-80%) However, the operational stabilities achieved are highly variable and the results are not easily reproducible. The reason for this lack of reproducibility is probably related to the existence of nonuniform adsorption sites and to a significant metal ion leakage from the support. In order to improve the control of the formation of the adsorption sites, chelator ligands can be immobilized on the solid supports by means of stable covalent bonds. The metal ions are then bound by coordination and the stable complexes formed can be used for the retention of proteins. Elution of the bound proteins can be easily achieved by competition with soluble ligands or by decreasing pH. The support is subsequently regenerated by washing with a strong chelator such as ethylene diamine tetraacetic acid (EDTA) when desired. These metal chelated supports were named IMA Immobilized Metal-Ion Affinity (IMA) adsorbents and have been used extensively in protein chromatography (56,57). The approach of using different IMA-gels as supports for enzyme immobilization has been studied using Eschericia coli P-galactosidase as a model (58).

5.3. Formation of Disulfide Bonds

These methods are unique because, even though a stable covalent bond is formed between matrix and enzyme, it can be broken by reaction with a suitable agent such as dithiothreitol (DTT) under mild conditions. Additionally, because the reactivity of the thiol groups can be modulated via pH alteration, the activity yield of the methods involving disulfide bond formation is usually high—provided that an appropriate thiol-reactive adsorbent with high specificity is used (59). Immobilization methods based on this strategy are discussed in Chapter 17.

6. Properties of Immobilized Enzymes

As a consequence of enzyme immobilization, some properties of the enzyme molecule, such as its catalytic activity or thermal stability, become altered with respect to those of its soluble counterpart (11,60). This modification of the properties may be caused either by changes in the intrinsic activity of the immobilized enzyme or by the fact that the interaction between the immobilized enzyme and the substrate takes place in a microenvironment that is different from the bulk solution. The observed changes in the catalytic properties upon immobilization may also result from changes in the three-dimensional conformation of the protein provoked by the binding of the enzyme to the matrix. These effects have been demonstrated and, to a lesser extent, exploited for a limited number of enzyme systems.

Quite often when an enzyme is immobilized its operational stability is improved. The concept of stabilization has thus been an important driving force for immobilizing enzymes. In many cases, the observed operational stabilization is usually the result of loading an excess of enzyme, which in turn makes the process diffusion-controlled. However, true stabilization at the molecular level has also been demonstrated, such as the case of proteins immobilized through multipoint covalent binding (61). Studies carried out by several authors using different methods have demonstrated that there is a correlation between stabilization and the number of covalent bonds to the matrix (62-64). One of the main problems associated with the use of immobilized enzymes is the loss of catalytic activity, especially when the enzymes are acting on macromolecular substrates. Because of the limited access of the substrate to the active site of the enzyme, the activity may be reduced to accessible surface groups of the substrate only. This steric restriction may, in turn, change the characteristic pattern of products derived from the macromolecu-lar substrate (65). There are several strategies to avoid these steric problems such as selection of supports composed by networks of isolated macromolecular chains, careful choice of the enzyme residues involved in the immobilization, and use of hydrophilic and inert spacer arms (66).

7. The Biology of Enzyme Immobilization

Although the science of enzyme immobilization has developed as a consequence of its technical utility, one should recognize that the advantages of having enzymes attached to surfaces have been exploited by living cells for as long as life has existed. An inquiry into the biological role of enzyme immobilization may provide some lessons for biotechnologists and serve as a second point of departure, in addition to the purely chemical one. In fact, there is experimental evidence that the immobilized state might be the most common state for enzymes in their natural environment. The attachment of enzymes to the appropriate surface ensures that they stay at the site where their activity is required. This immobilization enhances the concentration at the proper location and it may also protect the enzyme from being destroyed. Multimolecular assembly depends normally on weak noncovalent forces and hydrophobic interactions, but sometimes on covalent bonds as well (e.g., disulphide bridges) (1,2). All these different forces have been exploited in the development of immobilized enzymes.

References

1. Stryer, L. (1995) Biochemistry, Freeman, New York.

2. Creighton, T. E. (1984) Proteins, Freeman, Oxford, UK.

3. Katchalski-Katzir, E. (1993) Immobilized enzymes: Learning from past successes and failures. Trends Biotechnol. 11, 471-478

4. Tosa, T., Mori, T. Fuse, N., and Chibata, I. (1966) Studies on continuous enzyme reactions. I. Screening of carriers for preparation of water-insoluble aminoacylase. Enzymologia. 31, 214-224.

5. Tanaka, A., Tosa, T., and Kobayashi, T. (1993) Industrial Application ofImmobi-lized Biocatalysts, Marcel Dekker, New York, NY.

6. Swaisgood, H. E. (1985) Immobilization of enzymes and some applications in the food industry. In: Enzymes and Immobilized Cells in Biotechnology (Laskin, A. I., ed.), Benjamin Cummings, London, pp. 1-24.

7. Guibault, G. G., Kauffmann, J. M., and Patriarche, G. J. (1991) Immobilized Enzyme Electrodes as Biosensors. In: Protein Immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NT, pp. 209-262.

8. Taylor, R.F. (1991) Immobilized Antibody and Receptor Based Biosensors. In: Protein Immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY, pp. 263-303.

9. Chang, M. S. (1991) Therapeutic Applications of Immobilized Proteins and Cells. In: Protein Immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY, pp. 305-318.

10. Bickerstaff, G. F. (1995) Impact of genetic technology on enzyme technology. Genet. Engineer Biotechnologist 15, 13-30.

11. Hartmeier, W. (1988) ImmobilizedBiocatalysts, Springer-Verlag, Berlin.

12. Trevan, M. (1980) Techniques of Immobilization. In: Immobilized Enzymes. An Introduction and Applications in Biotechnology (Trevan, M., ed.), Wiley, Chichester-New York, pp. 1-9.

13. Brodelius, P. and Mosbach, K. (1987) Immobilization Techniques for Cells/Or-ganelles. In: Methods in Enzymology, volume 135, (Mosbach, K., ed.), Academic Press, London, pp. 173-454.

14. Buchholz, K. and Klein, J. (1987) Characterization of Immobilized Biocatalysts. In: Methods in Enzymology volume 135, (Mosbach, K., ed.), Academic Press, London, pp. 3-30.

15. Cabral, J.M.S. and Kennedy, J. F (1991) Covalent and coordination immobilization of proteins. In: Protein immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY, pp. 73-138.

16. Gemeiner, P. (1992) Materials for enzyme engineering. In: Enzyme Engineering (Gemeiner, P., ed.), Ellis Horwood, New York, NY, pp. 13-119.

17. Gupta, M. and Mattiasson, B. (1992) Unique applications of immobilized proteins in bioanalytical systems. In: Methods of Biochemical Analysis, volume 36, (Suelter, C.H., ed.), Wiley, New York, NY, pp. 1-34.

18. Mattiasson, B. and Kaul, R. (1991) Determination of coupling yields and handling of labile proteins in immobilization technology. In: Protein immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY, pp. 161-179.

19. Scouten, W. H. (1987) A Survey of Enzyme Coupling Techniques. In: Methods in Enzymology, volume 135, (Mosbach, K., ed.), Academic Press, London, pp. 30-65.

20. White, C. A. and Kennedy, J. F. (1980) Popular matrices for enzyme and other immobilizations. Enzyme Microb. Technol. 2, 82-90.

21. Taylor, R. F. (1991) Commercially available supports for protein immobilization. In: Protein immobilization. Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York, NY, pp. 139-160.

22. Lawson, T. G., Regnier, F. E., and Wieth, H. L. (1983) Separation of synthetic oligo-nucleotides on columns of microparticulate silica coated with crosslinked polyethylene-imine. Anal. Biochem. 133, 85-93.

23. Axen, R., Porath, J. and Ernback, S. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214, 1302-1304.

24. Porath, J. and Axen, R. (1976) Immobilization of enzymes to agar, agarose, and Sephadex supports. In: Methods in Enzymology, volume XLIV, (Mosbach, K., ed.), Academic Press, New York, NY, pp. 19-45.

25. Guisan, J. M. (1988) Agarose-aldehyde gels as supports for immobilization- stabilization of enzymes. Enzyme Microb. Technol. 10, 375-382.

26. Wilchek, M. and Miron, T. (1982) A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters. Anal. Biochem. 126, 433-435.

27. Drobnick, J., Labsky, W., Kudlvasrova, H., Saudek, V., and Svec, F. (1982) The activation of hydroxy groups of carriers with 4-nitrophenyl and N-hydroxy-succinimidyl chloroformates. Biotechnol. Bioeng. 24, 487-493.

28. Parikh, I., March, S., and Cuatrecasas, P. (1974) Topics in the methodology of substitution reactions with agarose. In: Methods in Enzymology, volume XXXIV (Jacoby, W.B. and Wilchek, M. eds.), Academic Press, New York, NY, pp. 77-102.

29. Inman, J. K. and Dintzis, H. M. (1969) The derivatization of cross-linked Polyacrylamide beads. Controlled introduction of functional groups for the preparation of special-purpose, biochemical adsorbents. Biochemistry 8, 4074-4082.

30. Rozprimova, L., Franek, F. and Kubanek, V. (1978) Utilization of powder polyester in making insoluble antigens and pure antibodies. Cesk. Epidemiol. Mikrobiol. Immunol. 27, 335-341.

31. Ngo, T. T., Laidler, K. J., and Yam, C. F. (1979) Kinetics of acetylcholinesterase immobilized on polyethylene tubing. Can. J. Biochemistry 57, 1200-1203.

32. Grubhofer, N. and Schleith L. (1954). Protein coupling with diazotized polyaminostyrene. Hoppe Seylers ZPhysiol. Chem. 297, 108-112.

33. Beitz, J., Schellemberger, A., Lasch, J., and Fischer, J. (1980) Catalytic properties and electrostatic potential of charged immobilized enzyme derivatives. Pyruvate decarboxylase attached to cationic polystyrene beads of different charge densities. Biochim. Biophys Acta 612, 451-454.

34. Hornby, W. E. and Goldstein, L. (1976) Immobilization of enzymes on nylon. In: Methods in Enzymology, volume XXXIV, (Jacoby, W.B. and Wilchek, M., eds.), Academic Press, New York, NY, pp. 118-134.

35. O'Driscoll, K. F. (1976) Techniques of enzyme entrapment in gels. In: Methods in Enzymology, volume XLIV, (Mosbach K., ed.), Academic Press, New York, NY, pp. 169-183.

36. Bernfeld, P. and Wan, J. (1963) Antigens and enzymes made insoluble by entrapping them into lattices of synthetic polymers. Science 142, 678-679.

37. Dinelli, D., Marconi, W., and Morisi, F. (1976) Fiber-entrapped enzymes. In: Methods in Enzymology, volume XLIV, (Mosbach, K., ed.), Academic Press, New York, NY, pp. 227-243.

38. Wadiack, D. T. and Carbonell, R. G. (1975) Kinetic behavior of microencapsulated ß-galactosidase. Biotechnol. Bioeng. 17, 1157-1181.

39. Messing, R. A. (1976) Adsorption and inorganic bridge formations. In: Methods in Enzymology, volume XLIV, (Mosbach, K., ed.), Academic Press, New York, NY, pp. 148-169.

40. Woodward, J. (1985) Immobilized enzymes: adsorption and covalent coupling. In: Immobilized Cells and Enzymes: A Practical Approach, (Woodward, J., ed.), IRL, Oxford, UK, pp. 3-17.

41. Tosa, T., Mori, T., Fuse, N., and Chibata, I. (1967) Studies on continuous enzyme reactions I. Screening of carriers for preparation of water insoluble aminoacylase. Enzymologia 31, 214-224.

42. Sharp, A.K., Kay, G., and Lilly, M. D. (1969) The kinetics of ß-galactosidase attached to porous cellulose sheets. Biotechnol. Bioeng, 11, 363-380.

43. Bahulekar, R., Ayyangar, N. R., and Ponrathnam, S. (1991) Polyethyleneimine in immobilization of biocatalysts. Enzyme. Microb. Technol, 13, 858-868.

44. Goldstein, L. (1972) Microenvironmental effects on enzyme catalysis. A kinetic study of polyanionic and polycationic derivatives of chymotrypsin. Biochemistry 11, 4072-4084.

45. Goldman, R., Kedem, O., Silman, I., Caplan, S., and Katchalski-Katzir, E. (1968) Papain-collodion membranes. I. Preparation and properties. Biochemistry7, 486-500.

46. Guisan, J.M., Alvaro, G., Rosell, C.M., and Fernandez-Lafuente, R. (1994) Industrial design of enzymic processes catalysed by very active immobilized de-

rivatives: utilization of diffusional limitations (gradients of pH) as a profitable tool in enzyme engineering. Biotechnol .Appl. Biochem. ZG, 357-369.

47. Porath, J. (1987) Salting-out adsorption techniques for protein purification Biopolymers ZG, S193-204.

48. Caldwell, K., Axén, R., Bergwall, M., and Porath, J.(1976) Immobilization of enzymes based on hydrophobic interaction. I. Preparation and properties of a beta-amylase adsorbate. Biotechnol. Bioeng. 18, 1573-1588.

49. Caldwell, K., Axén, R., Bergwall, M., and Porath, J.(1976) Immobilization of enzymes based on hydrophobic interaction. II. Preparation and properties of an amyloglucosidase adsorbate. Biotechnol. Bioeng. 18, 1589-1604.

50. Cashion, P., Lentini, V., Harrison, D., and Javed, A. (1982) Enzyme immobilization on trityl-agarose: Reusability of both matrix and enzyme. Bioechnol. Bioeng. Z4, 1221-1224.

51. Yon, R. (1974) Enzyme purification by hydrophobic chromatography: an alternative approach illustrated in the purification of aspartate transcarbamoylase from wheat germ. Biochem. J. 1S7, 127-130.

52. Dixon, J., Andrews, P., and Butler, L. (1979) Hydrophobic esters of cellulose: properties and applications in biochemical technology. Biotechnol. Bioeng. Zl, 2113-2123.

53. Solomon, B., Hollaander, Z., Koppel, R., and Katchalski-Kazir, E. (1987) Use of monoclonal antibodies for the preparation of highly active immobilized enzymes. In: Methods in Enzymology, volume 135, (Mosbach, K., ed.), Academic Press, London, pp. 160-170.

54. Cabral, J. M. S., Novais, J. M., and Kennedy J. F. (1986) Immobilization studies of whole microbial cells on transition metal activated inorganic supports. Appl. Microbiol. Biotechnol. ZS, 157-162.

55. Kennedy J.F. and Cabral, J.M.S. (1985) Immobilization of biocatalysts by metal-link/chelation processes. In: Immobilized Cells and Enzymes, (Woodward, J., ed.), IRL, Oxford, UK, pp. 19-37.

56. Porath, J. (1992) Immobilized metal ion affinity chromatography. Protein Expr. Purif. S, 263-281.

57. Kâgedal, L. (1998) Immobilized Metal Ion Affinity Chromatography. In: Protein Purification (Janson, J. C. and Rydén, L., eds.), Wiley-VCH, New York, NY, pp. 311-342.

58. Brena, B., Rydén, L., and Porath, J. (1994). Immobilization of ß-galactosidase on metal- chelated- substituted gels. Biotechnol. Appl. Biochem. 19, 217-231.

59. Carlsson, J., Batista-Viera, F., and Rydén, L. (1998) Covalent Chromatography. In: Protein purification: principles, high-resolution methods, and applications, (Janson, J. C. and Rydén, L., eds.), Wiley-VCH, New York, NY, pp. 343-373.

60. Trevan, M. (1980) Effect of Immobilization on Enzyme Activity. In: Immobilized Enzymes. An Introduction and Applications in Biotechnology (Trevan, M. ed.), Wiley, Chichester-New York, pp. 11-56.

61. Blanco, R. M., Calvete J. J., and Guisán, J. M. (1989) Immobilization-stabilization of enzymes. Variables that control the intensity of the trypsin (amine)-agar-ose-(aldehyde) -multipoint attachment. Enzyme Microb. Technol. 11, 353-359.

62. Koch-Schmidt, A. and Mosbach, K. (1977) Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal sta bility of nicotinamide adenine dinucleotide dependent dehydrogenases. Biochemistry 16,2101-2105.

63. Koch-Schmidt, A. and Mosbach, K. (1977). Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 2. Specific activity and thermal stability of enzymes bound weakly and strongly to Sepharose CL 4B. Biochemistry 16, 2105-2109.

64. Gabel, D., Steinberg, I., and Katchalski-Kazir, E. (1971) Changes in conformation of insolubilized trypsin and chymotrypsin, followed by fluorescence. Biochemistry 10, 4661-4669.

65. Boundy, J., Smiley,.K.L., Swanson, C.L., and Hofreiter, B.T. (1976) Exoenzymic activity of alpha-amylase immobilized on a phenol-formaldehyde resin Carbohydr. Res. 48, 239-244.

66. Guisan, J. M., Penzol, G., Armisen, P., et al. (1997) Immobilization of enzymes acting on macromolecular substrates.. In: Immobilization of Enzymes and Cells, (Bickerstaff, G. F., ed.), Humana Press, Totowa, NJ, pp. 261-275.

Cross-Linked Enzyme Aggregates

Roger A. Sheldon, Rob Schoevaart, and Luuk M. van Langen

Summary

The economic viability of biocatalytic conversions is often dependent on finding an effective method for immobilization of the enzyme involved. This provides for its improved operational stability and facile recovery and re-use. Cross-linked enzyme aggregates (CLEAs®) constitute an effective methodology for enzyme immobilization with broad scope. The technique is exquisitely simple, involving precipitation from aqueous buffer and subsequent cross-linking of the resulting physical aggregates of enzyme molecules, and amenable to rapid optimization. The resulting CLEAs are stable, recyclable biocatalysts exhibiting high activity retention, in some cases higher than that of the free enzyme they were derived from. The enzyme does not need to be of high purity since the methodology essentially combines purification and immobilization into a single operation. The technique can also be applied to the preparation of combi-CLEAs containing two or more enzymes. For example, an oxynitrilase/nitrilase CLEA for the one-pot synthesis of (S) mandelic acid from benzaldehyde in high yield and enantioselectivity.

Key Words: Immobilized enzymes; cross-linked enzyme aggregates; CLEAs®; hyperactivation; lipase CLEAs; combi CLEAs; biocatalysis.

1. Introduction

Considerable effort has been devoted (1,2) to developing effective immobilization techniques to increase the operational stability of enzymes and to facilitate their recovery and recycling, thereby reducing the contribution of the enzyme to the cost of the final product. In some cases, immobilizing the enzyme can also lead to enhanced selectivity (3). Indeed, an effective method for enzyme immobilization is often essential for making a biotransformation commercially viable.

Immobilization methods can conveniently be divided into three types: binding to a support (carrier), encapsulation (e.g., in a polymeric gel), or via cross-linking techniques (4). The first method is the most common. It can involve physical adsorption on organic polymers like polypropylene or inorganic oxides, such as

From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ

silica. A disadvantage of physical adsorption is that the enzyme is readily leached from the surface in an aqueous medium.

A distinct disadvantage of carrier-bound enzymes, whether they involve binding to or encapsulation in a carrier, is the dilution of catalytic activity resulting from the introduction of a large proportion of noncatalytic mass, generally ranging from 90 to >99% of the total mass. This inevitably leads to lower volumetric and space-time yields and lower catalyst productivities. Moreover, attempts to achieve high enzyme loadings usually lead to loss of activity. The third type of immobilization, via cross-linking of enzyme molecules with a bifunctional cross-linking agent, most commonly glutaraldehyde, does not suffer from this disadvantage. Because the molecular weight (MW) of the cross-linking agent is negligible compared with that of the enzyme the resulting biocatalyst essentially comprises 100% active enzyme.

1.1. Cross-Linking of Enzymes

The technique of protein cross-linking via the reaction of glutaraldehyde with reactive NH2 groups on the protein surface was initially developed in the 1960s (5). However, this method of producing cross-linked enzymes (CLEs) had several drawbacks, such as low activity retention, poor reproducibility, low mechanical stability, and difficulties in handling the gelatinous CLEs.

Mechanical stability and ease of handling could be improved by cross-linking the enzyme in a gel matrix or on a carrier but this led to the disadvantageous dilution of activity just mentioned. Consequently, in the late 1960s, emphasis switched to carrier-bound enzymes, which became the most widely used method for enzyme immobilization for industrial use.

The cross-linking of a crystalline enzyme by glutaraldehyde was first described by Quiocho and Richards in 1964 (6). Their main objective was to stabilize enzyme crystals for X-ray diffraction studies, but they also showed that catalytic activity was retained. The use of cross-linked enzyme crystals (CLECs) as industrial biocatalysts was pioneered by scientists at Vertex Pharmaceuticals (7) and subsequently commercialized by Altus Biologics (8). The initial studies were performed with CLECs of thermolysin, of interest in the manufacture of aspartame, but the method was subsequently shown to be applicable to a broad range of enzymes (9-11).

CLECs proved significantly more stable to denaturation by heat, organic solvents, and proteolysis than the corresponding soluble enzyme or lyophilized (freeze-dried) powder. CLECs have been formulated as robust, highly active immobilized enzymes of controllable size, varying from 1 to 100 ^m, for use in industrial biotransformations. Their operational stability and ease of recycling, coupled with their high catalyst and volumetric productivities, render them ideally suited for industrial application.

An inherent disadvantage of CLECs is the need to crystallize the enzyme, often a laborious procedure requiring enzyme of high purity. Hence, we reasoned that comparable results could possibly be achieved by simply precipitating the enzyme from aqueous solution, using standard techniques, and cross-linking the resulting physical aggregates of enzyme molecules. This indeed proved to be the case and led to the development of a new family of cross-linked enzymes, which we have called cross-linked enzyme aggregates (CLEA).

1.2. The CLEA Technology

It is well-known (12) that the addition of salts, organic solvents, or nonionic polymers to aqueous solutions of proteins leads to their precipitation as physical aggregates of protein molecules without perturbation of their tertiary structure, that is without denaturation. Indeed, aggregation induced by addition of ammonium sulfate, poly(ethyleneglycol), and some organic solvents is a commonly used method of protein purification.

These solid aggregates are held together by noncovalent bonding and readily collapse and redissolve when dispersed in water. We surmised that cross-linking of these physical aggregates would produce CLEAs in which the pre-organized superstructure of the aggregates, hence, their catalytic activity, would be maintained.

Initial experiments were performed (13) with penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11), an industrially important enzyme used in the synthesis of semi-synthetic penicillin and cephalosporin antibiotics (14). The free enzyme has limited thermal stability and a low tolerance to organic solvents, making it an ideal candidate for stabilization as a CLEA. Indeed, penicillin G acylase CLEAs, prepared by precipitation with, for example, ammonium sulfate or tert-butanol, proved to be effective catalysts for the synthesis of ampicillin (15). They exhibited activities comparable with those of CLECs of the same enzyme, with substantially less competing hydrolysis of the side chain. Analogous to the corresponding CLECs, the penicillin G acylase CLEAs also maintained their activity in organic solvents.

We then examined the effect of various parameters, such as the precipitant and the addition of additives such as surfactants and crown ethers, on the activities of CLEAs prepared from seven commercially available lipases (16). The activation of lipases by additives, such as surfactants, crown ethers and amines, is well documented and is generally attributed to the lipase being induced to adopt a more active conformation (17). We reasoned, therefore, that cross-linking of enzyme aggregates, resulting from precipitation in the presence of such an additive, would "lock" the enzyme in this more favorable conformation. Moreover, because the additive is not covalently bonded to the enzyme, the additive can subsequently be washed from the CLEA with, for example, an appropriate organic solvent.

Using this procedure we succeeded in preparing a variety of lipase CLEAs exhibiting levels of activity even higher than the corresponding free enzyme, that is, up to three times the hydrolytic activity and up to ten times the activity of the free enzyme in organic solvents (18). In addition, we also demonstrated that the experimental procedure for CLEA preparation could be further simplified by combining precipitation, in the presence or absence of additives, with cross-linking into a single operation.

Hence, the potential of the CLEA technology for preparing immobilized enzymes with high catalyst and volumetric activities, in some cases with activities significantly exceeding those of the native enzymes they were derived from, was firmly established (3). The method is exquisitely simple and can, in principle, be performed with relatively impure samples of enzymes.

Indeed, the methodology essentially combines purification of the enzyme (via precipitation) and immobilization into one step. It must be noted, however, that if

Table 1

Activity Recovery of a Variety of CLEAs

Enzyme Activity recovery (%)

Penicillin acylase from E. coli 53

Penicillin acylase from A. faecalis 58

Lipase A from C. antarctica 263

Lipase B from C. antarctica 177

Lipase from Thermomyces lanuginosa 327

Lipase from A. niger 116

Hydroxynitrile lyase from Prunus amygdalus 72

Hydroxynitrile lyase from Manihot esculenta 110

Nitrilase from Pseudomonas fluorescens 50

Pyruvate decarboxylase from Zymomonas mobilis 90

Galactose oxidase from Dactylium dendroides 95

ß-Galactosidase from A. oryzae 100

an impure sample containing a mixture of enzymes is used, this can lead to a CLEA containing more than one enzyme. We have used this to our advantage in the deliberate preparation of combi-CLEAs, containing two or more enzymes, for use in multistep, biocatalytic cascade processes, for example an oxynitrilase and a nitrilase.

1.3. Scope of CLEA Technology

Subsequent studies were aimed at demonstrating the applicability of the CLEA technology to the effective immobilization of a broad range of enzymes, including cofactor-dependent oxidoreductases and lyases, and the influence of the many parameters (temperature, pH, concentration, stirring rate, precipitant, and cross-linking agent) on the properties of the resulting CLEA. The relative simplicity of the operation ideally lends itself to high-throughput methodologies.

We have shown that, by a suitable optimization of the procedure, which may differ from one enzyme to another, the CLEA methodology is applicable to essentially any enzyme. We have prepared active and stable CLEAs from, in addition to penicillin G acylase and lipases, esterases, trypsin, oxynitrilases, nitrilases, galactosidase, an alcohol dehydrogenase, formate dehydrogenase, glucose oxidase, galactose oxidase, catalase, laccase, phytase, and pyruvate decarboxylase. Table 1 gives examples of the activity retention observed with various enzymes.

Activity yields exceeding that of the native enzyme were first observed with lipases, but were subsequently also observed with other enzymes. Table 2 gives some examples of activity recoveries obtained using a variety of precipitants with four different enzymes.

Various cross-linking agents are known and can be used. Although glutaralde-hyde remains cheap and versatile, some enzymes are inactivated by this reagent. Most likely, active site residues are modified. To prevent the cross-linker from entering the active site or to prevent extreme conformational changes, we devel-

Table 2

Example of Typical Precipitants and Activity Recovery After Precipitation

Table 2

Example of Typical Precipitants and Activity Recovery After Precipitation

Precipitant

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