青霉素酰化酶纳米凝胶的制备与性质

Preparation and characterization of penicillin acylase nanogel

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.coliTop10F′/pGEMKT-TacPGA-Tag为表达菌株发酵生产青霉素酰化酶,经金属螯合层析得到比酶活为23200 U.L-1的青霉素酰化酶样品,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,然后加入丙烯酰胺单体,通过聚合反应得到凝胶直径在12-16 nm的青霉素酰化酶纳米凝胶,聚合反应的酶活收率为90%。与青霉素酰化酶样品相比,青霉素酰化酶纳米凝胶具有相同的荧光发射光谱,其在60℃时的酶活半衰期是自由酶的75倍;在30℃,40%（体积）的甲醇、乙醇中的酶活半衰期分别是自由酶的15倍、26倍。

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.