Minimal Chemical Modification Minimal Physical Interactions Absence Of Steric Hindrances

Fig. 8. Immobilization of an enzyme without altering its properties.

8. Enzyme Immobilization: Still a Fascinating Challenge

Thousands of protocols for enzyme immobilization have been reported over the past 30 yr. On their surfaces, enzymes have very different structural moieties that are able to interact (i.e., adsorption/covalent attachment) with such activated supports as nucleophilic residues, hydrophobic pockets, carboxylic groups, regions with net-positive charge, and regions with net-negative charge. In a certain way, and some authors maintain this point of view, it can be assumed that the problem of enzyme immobilization has already been solved. However, very simple and very efficient protocols (in terms of activity, stability, selectivity, and absence of inhibitions) for enzyme immobilization have not been fully developed. Therefore, enzyme immobilization must still be considered a fascinating challenge in modern biotechnology—much hard and interdisciplinary work remains. The improvement of protocols for enzyme immobilization should encompass molecular biology, protein chemistry, material science, and chemical engineering, among others. Very simple and efficient protocols for enzyme immobilization would greatly improve the possibilities for a massive implementation of industrial enzymes. A more ambitious and sustainable chemistry could be developed (e.g., the synthesis, under very mild experimental and environmental conditions, of very complex, cost-effective, and useful bioactive molecules).

9. The Immobilization of Enzymes in this Volume

This second edition of Immobilization ofEnzymes and Cells aims to update and complement the first edition. This contributor will show some well-developed protocols for enzyme immobilization in order to encourage the development of new and more efficient immobilization methods. Protocols to improve immobilization and use of immobilized derivatives have been presented according to the following criteria:

1. Very simple, ready for industrial application, protocols for enzyme immobilization.

2. Very efficient protocols that allow for great improvement of the enzyme properties as a consequence of immobilization.

3. Novel protocols for enzyme immobilization that could be very useful in the future.

4. Protocols for characterization of immobilized enzymes that are useful for the improvement of immobilization methods.

5. New reactors for improving the utilization of immobilized derivatives.

6. Protocols for immobilization directed to different uses and applications of industrial enzymes.

References

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2. Robertson, D. E. and Steer, B. A. (2004) Recent progress in biocatalyst discovery and optimization. Curr. Opin. Chem. Biol. 8,141-149

3. van den Burg B. and Eijsink V. G. H. (2002) Selection of mutations for increased protein stability. Curr. Opin. Chem. Biol. 13, 333-337.

4. Fernandez-Lafuente, R, Hernandez-Justiz, O., Mateo, C., et al. (2001) Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicil-lin acylase permits the optimization of ampicillin synthesis under dissociation conditions. Biomacromolecules 2, 95-104.

5. Betancor, L., Hidalgo, A., Fernandez-Lorente, G., Mateo, C., Fernandez-Lafuente, R., and Guisan, J. M. (2003) Preparation of an stable biocatalyst of bovine liver catalase. Biotechnol. Prog. 19, 763-767.

6. Rosell, C. M., Terreni, M., Fernandez-Lafuente, R., and Guisan, J. M.(1998) A criterium for the selection of monophasic solvents for enzymatic synthesis. Enzyme Microb. Technol. 23, 64-69.

7. Guisan, J. M., Alvaro, G., Rosell, C. M., and Fernandez-Lafuente, R. (1994) Industrial design of enzymatic processes catalized by very active immobilised derivatives. Utilisation of diffusinal limitation (gradients of pH) as a profitable tool in enzyme engineering. Biotech. Appl. Biochem. 20, 357-369.

8. Illanes A., and Wilson, L. (2003) Enzyme Reactor Design Under Thermal Inactivation. Crit Rev. Biotechnol, 23, 61-93.

9. Spiess A., Schlothauer, R. C., Hinrichs, J., Scheidat, B., and Kasche, V. (1999) pH gradients in immobilized amidases and their influence on rates and yields of beta- lactam hydrolysis. Biotechnol. Bioeng. 62, 267-277.

10. Bickerstaff G. F., ed. 1997. Immobilization of Enzymes and Cells, Methods is Biotechnology, Humana Press, Totowa, NJ.

11. Katchalski-Katzir, E. (1993) Immobilized enzymes: learning from past successes and failures. TIB 11, 471-478.

12. Fernandez-Lafuente, R. and Guisan, J. M. (1998) Enzyme and protein engineering via immobilization and post-immobilization techniques. In: RecentResearch

Developments in Biotechnology and Bioengineering. (Pandalai, S. G., ed.), Research Signpost, Kerala, India, pp. 299-309.

13. Pessela, B. C. C., Mateo, C., Carrascosa, A. V. et al. (2003) One step purification, covalent immobilization and additional stabilization of a thermophilic poly-his-tagged beta-galactosidase of Thermus sp. strain t2, novel heterofunctional che-late-epoxy supports. Biomacromolecules 4, 107-113.

14. Abian, O., Grazú, V., Hermoso, J., et al. (2004) Stabilization of Penicillin G Acy-lase from Escherichia coli: site directed mutagenesis of the protein surface to increase multipoint covalent attachment. Appl. Envir. Microb. 70, 1249-1251.

15. López-Gallego, F., Montes, T., Fuentes, M., et al. (2005) Chemical increase of the amount of reactive groups on enzyme surface to improve its stabilization via multipoint covalent attachment. J. Biotechnol. 116, 1-10.

16. Betancor, L, Fuentes, M., Dellamora-Ortiz, G., et al. (2005) Dextran aldehyde coating of glucose oxidase immobilized on magnetic nano-particles prevents in-activation by gas bubbles. J. Mol. Catal., B Enzym. 32, 97-101.

17. Guisán, J. M. (1988) Aldehyde gels as activated support for immobilization-stabilization of enzymes. Enzyme Microb. Technol. 10, 375-382.

18. Mateo, C., Abian, O., Bernedo, M., et al.(2005) Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb. Technol. In press.

19. Mateo, C., Abian, O., Fernández-Lorente, G., Predoche, J., Fernández-Lafuente, R., and Guisán, J. M. (2002) Sepabeads: a novel epoxy-support for stabilization of industrial enzymes via very intense multipoint covalent attachment. Biotechnol. Progr. 18, 629-634.

20. Mateo, C., Torres, R., Fernández-Lorente, G., et al. (2003) Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method. Biomacromolecules 4, 772-777.

21. Poltorak, O. M., Chukhary, E. S., and Torshin, I. Y. (1998) Dissociative thermal inactivation, stability and activity of oligomeric enzymes. Biochemistry(Moscow) 63,360-369.

22. Fernández-Lafuente, R., Rodríguez. V., Mateo, C., et al. (1999) Strategies for the stabilization of multimeric enzymes via immobilization and post-immobilization techniques. J. Mol. Catal, B Enzym. 7, 181-189.

23. Wilson, L., Betancor, L. Fernández-Lorente, G., et al. (2004) Crosslinked aggregates of multimeric enzymes: a simple and efficient methodology to stabilize their quaternary structure.Biomacromolecules 5, 814-817.

24. Bastida, A., Sabuquillo, P., Armisen, P., Fernández-Lafuente, R., Huguet, J., and Guisán, J. M. (1998) A single step purification, immobilization and hyperactivation of lipases via interfacial adsorption on strongly hydrophobic supports. Biotechnol. Bioeng. 58, 486-493.

25. Fernández-Lafuente, R., Armisen, P., Sabuquillo, P., Fernández-Lorente, G., and Guisán, J. M. (1998) Immobilization of lipases by selective adsorption on hydrophobic supports. Chem. Phys. Lipids. 93, 185-197.

26. Palomo, J. M., Muñoz, G., Fernández-Lorente, G., Mateo, C., Fernández-Lafuente, R., and Guisán, J. M. (2002) Interfacial adsorption of lipases on very hydrophobic support (octadecyl Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases. J. Mol. Catal, B Enzym... 19-20C, 279-286.

27. Fernandez-Lafuente, R., Rosell, C. M., Alvaro, G., and Guisan, J. M. (1992) Additional stabilisation of penicillin G acylase by controlled chemical modification of immobilised/stabilised derivatives. Enzyme Microb. Technol. 14, 489-495.

28. Fernandez-Lafuente, R., Rosell, C. M., Guisan, J. M., Caanan-Haden, L., and Rodes, L. (1999) Facile synthesis of artificial enzyme nano-environments via solid-phase chemistry of immobilized derivatives dramatic stabilization of penicillin acylase versus organic solvents. Enzyme Microb. Technol. 24, 96-103.

29. Abian O, Wilson L., Mateo, C., et al. (2002) Preparation of artificial hyper-hydro-philic micro-environments (polymeric salts) surrounding immobilized enzyme molecules. New enzyme derivatives to be used in any reaction medium. J. Mol. Cat B Enzymatic. 19-20C, 295-303.

30. Wilson, L., Illanes, A., Abian, O., Pessela, B. C. C., Fernandez-Lafuente, R., and Guisan, J. M. (2004) Co-aggregation of penicillin g acylase and polyionic polymers: a simple methodology to prepare enzyme biocatalysts stable in organic media. Biomacromolecules 5, 852-857.

31. Penzol, G., Armisen, P., Fernandez-Lafuente, R., Rodes, L., and Guisan, J. M. (1998) Use of dextrans as long, inert and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules. Biotechnol. Bioeng. 60, 518-523.

32. Turkova, J. (1999) Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function. J. Chromatography B 722, 11-31.

33. Fuentes, M., Mateo, C., Guisan, J. M., and Fernandez-Lafuente, R. (2005) Preparation of inert magnetic nano-particles for the directed immobilization of antibodies. Biosen. Bioelec. 20, 1380-1387.

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35. Fuentes, M., Mateo, C., Garcia, L., Tercero, J. C., Guisan, J. M., and Fernandez-Lafuente, R. (2004) The directed covalent immobilization of aminated dna probes on aminated plates. Biomacromolecules 5, 883-888.

36. Pessela, B. C. C, Torres, R., Fuentes, M., Mateo, C., Fernandez-Lafuente, R., and Guisan, J. M. (2004) Immobilization of rennet from Mucor miehei via its sugar chain—its use in milk coagulation. Biomacromolecules 5, 2029-2033.

37. Fuentes, M., Mateo, C., Rodriguez, A., et al. (2005) Detecting minimal traces of dna by using dna covalently attached to superparamagnetic nanoparticles and PCR-ELISA in one step. Biosen. Bioelec. In press.

Immobilization of Enzymes

A Literature Survey

Beatriz M. Brena and Francisco Batista-Viera

Summary

The term "immobilized enzymes" refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously.". Besides the application in industrial processes, the immobilization techniques are the basis for making a number of biotechnological products with applications in diagnostics, bioaffinity chromatography, and biosensors. Initially, only immobilized single enzymes were used, but the 1970s saw the development of more complex systems—including two-enzyme reactions with co-factor regeneration and living cells. The major components of an immobilized enzyme system are the enzyme, the matrix, and the mode of attachment. The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable cova-lent bonds. The covalent reactions commonly employed give rise to binding through amide, ether, thio-ether, or carbamate bonds. As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered. These effects have been demonstrated and exploited. The concept of stabilization has been an important driving force for immobilizing enzymes. True stabilization at the molecular level has been demonstrated (e.g., proteins immobilized through multipoint covalent binding).

Key Words: Bioaffinity chromatography; biosensors; enzyme stabilization; immobilization methods; immobilized enzymes.

1. Background

Enzymes are biological catalysts that promote the transformation of chemical species in living systems. These molecules, consisting of thousands of atoms in precise arrangements, are able to catalyze the multitude of different chemical

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

reactions occurring in biological cells. Their role in biological processes and in health and disease has been extensively investigated. They have also been a key component in many ancient human activities, especially food processing, well before their nature or function was known (1).

Enzymes have the ability to catalyze reactions under very mild conditions with a very high degree of substrate specificity, thus decreasing the formation of by-products. Among the reactions catalyzed are a number of very complex chemical transformations between biological macromolecules, which are not accessible to ordinary methods of organic chemistry. This makes them very interesting for biotechnological use. At the beginning of the 20th century, enzymes were shown to be responsible for fermentation processes and their structure and chemical composition started to come under scrutiny (2). The resulting knowledge led to widespread technological use of biological catalysts in a variety of other fields such as the textile, pharmaceutical, and chemical industries. However, most enzymes are relatively unstable, their costs of isolation are still high, and it is technically very difficult to recover the active enzyme, when used in solution, from the reaction mixture after use.

Enzymes can catalyze reactions in different states: as individual molecules in solution, in aggregates with other entities, and as attached to surfaces. The attached—or "immobilized"—state has been of particular interest to those wishing to exploit enzymes for technical purposes. The term "immobilized enzymes" refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously" (3). The introduction of immobilized catalysts has, in some cases, greatly improved both the technical performance of the industrial processes and their economy (Table 1).

The first industrial use of immobilized enzymes was reported in 1967 by Chibata and co-workers, who developed the immobilization of Aspergillus oryzae aminoacylase for the resolution of synthetic racemic d-l amino acids (4). Other major applications of immobilized enzymes are the industrial production of sugars, amino acids, and pharmaceuticals (5) (Table 2). In some industrial processes, whole microbial cells containing the desired enzyme are immobilized and used as catalysts (6).

Aside from the application in industrial processes, the immobilization techniques are the basis for making a number of biotechnological products with applications in diagnostics, bioaffinity chromatography, and biosensors (7,8). Therapeutic applications are also foreseen, such as the use of enzymes in extracorporeal shunts (9).

In the past three or four decades, immobilization technology has developed rapidly and has increasingly become a matter of rational design; but there is still the need for further development (10). Extension of the use of immobilized enzymes to other practical processes will require new methodologies and a better understanding of current techniques.

Table 1

Technological Properties of Immobilized Enzyme Systems (3)

Technological Properties of Immobilized Enzyme Systems (3)

Table 1

Advantages

Disadvantages

Catalyst reuse

Loss or reduction in activity

Easier reactor operation

Diffusional limitation

Easier product separation

Additional cost

Wider choice of reactor

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