Abstract
Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support’s possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an “intermolecular” covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme-support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.
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References
Bickerstaff G.F. (ed.) (1997) Immobilization of Enzymes and Cells, Methods in Biotechnology, Volume 1. Humana Press, Totowa, NJ.
Chibata I., Tosa T., and Sato T. (1986) Biocatalysis: immobilized cells and enzymes. J. Mol. Catal. 37, 1–24.
Gupta M. N. (1991) Thermostabilization of proteins. Biotechnol. Appl. Biochem. 4, 1–11.
Hartmeier W. (1985) Immobilized biocatalysts from simple to complex systems. Trends Biotechnol. 3, 149–153.
Katchalski-Katzir E. (1993) Immobilized enzymes-learning from past successes and failures. Trends Biotechnol. 11, 471–478.
Kennedy J. F., Melo E. H. M., and Jumel K. (1990) Immobilized enzymes and cells. Chem. Eng. Prog. 45, 81–89.
Klivanov A. M. (1983) Immobilized enzymes and cells as practical catalysts. Science 219, 722–727.
Rosevear A. (1984) Immobilized biocatalysts: a critical review. J. Chem. Technol. Biotechnol. 34B, 127–150.
Royer G. P. (1980) Immobilized enzymes as catalysts. Catal. Rev. 22, 29–73.
Lasch J. and Koelsch R. (1978) Enzyme leakage and multipoint covalent attachment of agarose-bound enzyme preparations. Eur. J. Biochem. 82, 181–186.
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.
Mateo C., Abian O., Fernandez-Lafuente R., and Guisán 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.
Guisán J. M. (1988) Aldehyde gels as activated support for immobilization-stabilization of enzymes. Enzyme Microb. Technol. 10, 375–382.
Guisán 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.
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.
Kramer D. M., Lehman K., Pennewiss H., and Plainer H. (1979) Oxirane acrylic adsorption. 26th International IUPAC Symposium on Macromolecules, Mainz, Germany, Sept. 1979.
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.
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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Mateo, C. et al. (2006). Immobilization-Stabilization of Enzymes by Multipoint Covalent Attachment on Supports Activated With Epoxy Groups. In: Guisan, J.M. (eds) Immobilization of Enzymes and Cells. Methods in Biotechnology™, vol 22. Humana Press. https://doi.org/10.1007/978-1-59745-053-9_4
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DOI: https://doi.org/10.1007/978-1-59745-053-9_4
Publisher Name: Humana Press
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