关键词: 青霉素酰化酶, 纳米凝胶, 酶修饰, 稳定化

Abstract:

The enhancement of the stability of penicillin acylase at high temperature and in organic solvents is essential to its industrial applications.This research work started with the production of recombinant expressed in E.coliTop10F′/pGEMKTTacPGATag.The crude penicillin acylase was purified using metal chelate chromatography, the penicillin acylase product obtained was of purity 23200 U·L-1.Encapsulation of penicillin acylase into nanogel was accomplished using the aqueous twostep in situ polymerization method, in which the penicillin acylase firstly reacted with N-acryloxysuccinimide so to generate vinyl groups on its surface, and then polymerized in aqueous medium using acrylamide as monomer and ammonium persulfate as initiator.The yield of polymerization reaction was 90%, and the diameter of penicillin acylase nanogel obtained was 12—16 nm. The penicillin acylase nanogel has shown an identical fluorescence emission spectrum compared to its native counterpart.Moreover, the encapsulation into nanogel led to a substantial enhancement of enzyme stability at high temperature and in polar solvents, as shown by the extension of t1/2 by 75fold at 60℃, and 15 and 26fold in the presence of 40%(vol)methanol and ethanol, respectively, compared to its native counterpart.

Key words: 青霉素酰化酶, 纳米凝胶, 酶修饰, 稳定化