Multi-Point Covalent Immobilization of Enzymes on Supports Activated with Epoxy Groups: Stabilization of Industrial Enzymes

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.