蟹源副溶血弧菌胞外蛋白酶的纯化与特性分析

Purification and characterization of extracellular proteases of Vibrio parahemolyticus from Chinese mitten crab Eriocheir sinensis

采用硫酸铵盐析、Sephadex G-100凝胶层析和DEAE—Cellulose离子交换柱层析等方法，从中华绒螯蟹Eriocheir sinensis弧菌病病原菌——副溶血弧菌Vibrio parahemolyticus的胞外产物中分离纯化出两种具有致病作用的胞外蛋白酶。相对分子质量为39600的蛋白酶是一种金属蛋白酶，EDTA可抑制其活性，金属离子Cu^2＋、Mg^2＋、Fe^2＋对该酶有抑制作用，而Ca^2＋对其则有一定程度的激活作用；该酶在50—60℃下热稳定性最好，最适pH为9。而相对分子质量为20000的蛋白酶属于丝氨酸蛋白酶，EDTA对其酶活力几乎没有影响，但苯甲基磺酰氟（PMSF）可抑制其酶活性；该酶的最适温度为50℃，最适pH为8。

Two kinds of extracellular proteases were purified from the extracellular products of the Vibrio parahemolyticus isolated from Chinese mitten crabs, Eriocheir sinensis by ammonium sulfate, Sephadex G - 100 and DEAE - Cellulose. The two kinds of purified extracellular proteases had molecular weights of 39 600 and 20 000 by SDS - PAGE, one being stable and showing high proteolytic activity at water temperature of 50 -60℃ and pH 9.0, and the other 50 ℃ and pH 8.0. The 39 600 protease activity was inhibited by EDTA and some metal ions （ Cu^2＋ , Mg^2＋ ,Fe^2＋ ）. On the contrary, Ca^2＋ activated the enzyme to some extent, indicating that it was a melalloprotease. The enzymatic activity of the 20 000 protease as a serine protease was inhibited by PMSF. The two purified protea- ses were found to be toxic to Vero cells in vitro and in cells of hepatopancreas in the crab.