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aInactivations were carried out at pH 8.0 and 50°C (PGA) or 70°C and pH 7.0 (chy-motrypsin).

aInactivations were carried out at pH 8.0 and 50°C (PGA) or 70°C and pH 7.0 (chy-motrypsin).

5. If necessary, verify reference enzyme activity to evaluate the yield and the recovered activity of enzyme during the immobilization process.

6. Amounts and kind of EC-EP or Eupergit supports, ratios, V gel/V suspension used, and reaction times must be established for each enzyme. Avoid magnetic stirring to reduce abrasion of the support, especially for Eupergit supports.

7. If the enzyme activity is decreased because of enzyme inactivation it must be distinguished from a loss in enzyme activity of the supernatant resulting from immobilization process.

8. Multipunctual derivatives can be reached through an extended incubation period of enzyme suspension after the immobilization has concluded. Additional bonds may be formed by keeping the suspension at pH 10.0. The optimum multi-interaction time required for each enzyme must be established for every case. It is necessary to prepare enzyme derivatives with different multi-interaction times and check the thermal stability of each. The optimal time is the shortest period that provides optimal stability and enzyme recovery; this will be a compromise.

References

1. Bickerstaff, G.F. (ed.) (1997) Immobilization of Enzymes and Cells, Methods in Biotechnology, Volume 1. Humana Press, Totowa, NJ.

2. Chibata, I., Tosa, T., and Sato, T. (1986) Biocatalysis: immobilized cells and enzymes. J. Mol. Catal. 37, 1-24.

3. Gupta, M. N. (1991) Thermostabilization of proteins. Biotechnol. Appl. Biochem. 4, 1-11.

4. Hartmeier, W. (1985) Immobilized biocatalysts from simple to complex systems. Trends Biotechnol. 3, 149-153.

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

6. Kennedy, J. F., Melo, E. H. M., and Jumel, K. (1990) Immobilized enzymes and cells. Chem. Eng. Prog. 45, 81-89.

7. Klivanov, A. M. (1983) Immobilized enzymes and cells as practical catalysts. Science 219, 722-727.

8. Rosevear, A. (1984) Immobilized biocatalysts: a critical review. J. Chem. Technol. Biotechnol. 34B, 127-150.

9. Royer, G. P. (1980) Immobilized enzymes as catalysts. Catal. Rev. 22, 29-73.

10. Lasch, J. and Koelsch, R. (1978) Enzyme leakage and multipoint covalent attachment of agarose-bound enzyme preparations. Eur. J. Biochem. 82, 181-186.

11. Kolb, H. J., Renner, R., Hepp, K. D., Weiaa, L., and Wieland, O. (1975) Reevalution of sepharose-insulin as a tool for the study of insulin action. Proc. Natl. Acad. Sci. USA 72, 248-252.

12. Mateo, C., Abian, O., Fernandez-Lafuente, R., and Guisan, J. M. (2000) Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favouring additional multipoint covalent attachment. Enzyme Microb. Technol. 26,509-515.

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

14. Guisan, J. M., Bastida, A., Cuesta, C., Fernandez-Lafuente, R., and Rosell, C. M. (1991) Immobilization-stabilization of chymotrypsin by covalent attachment to aldehyde agarose gels. Biotechnol. Bioeng. 39, 75-84.

15. Mozhaev, V. V., Klibanov, A. M., Goldmacher, V. S., and Berezin, I. V. (1990) Operational stability of copolymerized enzymes at elevated temperatures. Biotechnol. Bioeng. 25, 1937-1945.

16. Kramer, D. M., Lehman, K., Pennewiss, H., and Plainer, H. (1979) Oxirane acrylic adsorption. 26th International IUPAC Symposium on Macromolecules, Mainz, Germany, Sept. 1979.

17. Wheatley, J. B. and Schmidt, D. E. (1993) Salt induced immobilization of proteins on a high-performance liquid chromatographic epoxide affinity support. J. Chromatogr. 644, 11-16.

18. Wheatley, J. B. and Schmidt, D. E. (1999) Salt induced the immobilization of affinity ligands onto epoxide-activated supports. J. Chromatogr. A. 849, 1-12.

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