节杆菌肌酐水解酶的纯化及特性研究

Purification and Characteristics of Creatininase from Arthrobacter sp.

研究了产自于一株节杆菌的肌酐水解酶。该肌酐水解酶经热处理、硫酸铵分级沉淀、DEAE Cellulose离子交换、疏水层析后 ,酶提纯了 14 5倍 ,比活力达 2 0 9u mg。SDS PAGE测定显示该酶亚基质量为 33 7kD。对酶特性的研究表明 :酶在pH6 0～ 9 0、6 0℃以下稳定 ,对肌酐的Km值为 2 1 14mmol L。Ag+ 、Hg2 + 和邻菲罗啉能使酶完全丧失活性 ,Co2 + 、Mn2 + 对酶活性有明显促进作用。

A creatininase produced from a Arthrobacter sp. was purified 145-fold by a series of steps including heat treatment, ammonium sulfate precipitation, D EAE-Cellulose ion-exchange and hydrophobic chromatography. The specific activi ty of the pure enzyme was 209u/mg. The subunit molecular mass of creatininase was estimated to be 33 700D by SDS-PAGE. The creatininase was stable in the pH rang e between 6.0～9.0 and below 60℃. Its Km value for creatinine was estimate d to be 21.14mmol/L. The enzyme was markedly inactivated by incubation with 1mmol/L of Hg 2+, Ag 2+, Li+, Cu 2+ and 20mmol/L of 1,11-Phananthroli ne respectively. Activation was observed when the enzyme was incubated with 1mmo l/L of Co 2+ and Mn 2+.