尖镰孢青霉素V酰化酶的纯化及性质

PURIFICATION AND PROPERTIES OF PENICILLIN V ACYLASE FROM FUSARIUM OXYSPORUM

通过硫酸铵沉淀、硅藻土吸附、DEAD-纤维素离子交换层析和Sephadex G-200凝胶过滤,由尖镰孢(Fusarium oxysporum)FP941培养滤液中得到了聚丙烯酰胺凝胶电泳均一的青霉素V酰化酶。酶作用最适pH为7.0,最适温度为50℃。酶在pH6.0—8.0和42℃以下稳定。酶作用青霉素V的米氏常数Km为4.65×10^(-3)mol/L;苯氧乙酸是酶的竞争性抑制剂,抑制常数Ki为23.87×10^(-3)mol/L;6-氨基青霉烷酸是酶的非竞争性抑制剂,抑制常数Ki为30.01×10^(-3)mol/L。某些金属离子对酶有抑制作用,Fe^(2+)最强,其次是Hg^(2+)和Cu^(2+)。用SDS凝胶电泳测定酶亚基分子量为77600;用分子筛测定自然酶分子量为148000。

Penicillin V acylase from the crude culture filtrate of Fusarium oxysporum FP 941 was purified with a single band on non-denaturing electrophoresis by means of (NH4)2SO4 fractional precipitation, adsorption on, and elution from, celite, ion-exchange column chromatography with DEAE-cellulose, and Sephadex G-200 gel filtration chromatography. The optimum pH and temperature for enzyme action were 7.0 and 50℃ respectively. The purified enzyme was stable below 42℃ and pH 6.0- 8.0. The Michaelis constant ( Km) of this enzyme for penicillin V was found to be 4.65 x 10-3 mol/L. Phenoxyacetic acid was a competitive inhibitor of penicillin V acylase with an inhibitory constant (Ki) of 23.87 × 10-3 mol / L, whereas 6-aminopenicillanic acid was noncompetitive in nature with a Ki of 30.01 × 10-3 mol/L. The enzyme activity was inhibited by Hg2+, Cu2+ and strongly by Fe2+. The molecular weight of the subunit was 77 600, which was estimated by SDS-PAGE. The molecular weight of the whole enzyme was 148 000, determined by gel filtration chromatography.