青霉素酰化酶纳米凝胶的制备与性质

提高青霉素酰化酶的耐热性和耐有机溶剂性对于其工业应用具有重要的意义.采用E.coli Top10F'/pGEMKT-TacPGA-Tag为表达菌株发酵生产青霉素酰化酶,经金属鳌合层析得到比酶活为23200 U · L-1的青霉素酸化酶样品,将青霉素酰化酶样品与丙烯酰琥珀酰亚胺反应在酶分子表面修饰上丙烯酰基,然后加入丙烯...

Preparation and characterization of penicillin acylase nanogel

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.