青霉素酰化酶研究——Ⅰ.抑制作用,纯化和性质

Study on Penicillin Acylase Ⅰ. Inhibition By Organic Compounds, Purification and Characteristics

丙酮、乙醇和苯丙氨酸是大肠杆菌青霉素酰化酶的竞争性抑制剂,分别抑制模拟底物3-苯乙酰胺基6-硝基苯甲酸水解的活力。青霉素酰化酶粗酶液经硫酸铵盐析,pH5沉淀,SE-Sephadex C50层析,苯丙氨酸-Sepharose 4B疏水层析和DEAE-Sephadex A25层析可得纯酶,聚丙烯酰胺凝胶梯度电泳呈现一条带,比活为27.5u/mg蛋白。纯化总活力回收率为28.1%。青霉素酰化酶分子量约为90000,有两个亚基,分子量分别为70000和20000,其最适温度为40℃,最适pH为8.0,催化青霉素G水解的米氏常数为2.94mmol/L。

The inhibition of penicillin acylase by organic compounds has been studied and its purification and characteristics are reported. Ethanol, acetone and phenylalanine are the competitive inhibitors of the penicillin acylase activity.E. coli 5852 penicillin acylase has been purified by ammonium sulfate salting out, pH 5 precipitation, SE-Sephadex C 50 chromatography, hydrophobic chromatography and DEAE-Sephadex A 25 successively. The specific activity of the purified enzyme is increased from 2.1 to 27.5 u/mg protein. The isolated enzyme is proved by polyacrylamide gradient gel electrophoresis to be homogeneous with molecular weight about 90000. The resuh of SDS gel electrophoresis shows that the enzyme consists of two subunits with molecular weight around 70000 and 20000. The optimum pH and temperature are found to be 8 and 40℃ respectively. The K_m of the enzyme for penicillin G is 2.94 mmol/L.