海洋贝类肠道弧菌21Z1碱性蛋白酶的分离纯化及酶学性质

Purification and Characterization of Alkakine Protease from Marine Shelfish Vibrio sp. 21Z1

由威海海域贝类肠道中分离出一株产胞外碱性蛋白酶的细菌,经16S rDNA分析鉴定为弧菌21Z1。该菌发酵液上清经60%饱和度的硫酸铵溶液沉淀、透析、Sephadex-G100分子筛层析等步骤,获得电泳纯的21Z1碱性蛋白酶,聚丙烯酰胺凝胶电泳(SDS-PAGE)显示其分子量约为31 kDa。酶学性质测定结果显示:该酶最适反应温度为40℃,在20~40℃稳定;最适反应pH为8.5,在pH=8.5~11.0间稳定;Na^+、K^+、Ca^(2+)、Mn^(2+)对该酶具有激活作用,Fe^(2+)、Fe^(2+)、Zn^(2+)及Cu^(2+)则不同程度地抑制其活性;该酶的耐盐性检测结果显示,在Na^+浓度为4 mol/L时,残余酶活在62%以上,表明21Z1碱性蛋白酶具有较强的耐盐性。

A strain was isolated from intestinal of shellfish in W eihai waters which could produce extracellular alkaine protease,and was identified as Vibrio sp.21Z1by16S rD N A analysis.The purified alkaine enzyme21Z1was obtained through ammonium sulfate precipitation,dialysed and Sephadex-GlOO column chromatography.Its molecular weight was determined as about31kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAG E)analysis.The results show that the optimal temperature of this enzyme is40T!,and it is stable at20?40T!.The optimal pH of this enzyme is8.5,and it is stable at pH ranging from8.5?11.0.N a+,K+,Ca2+,and M n2+have effect on its enzyme activity while Fe2+,Fe3+,Zn2+and Cu2+have various degrees of inhibition effect upon this enzyme.The salt tolerance test of enzyme21Z1exhibits the residual protease activity remains more than62%at4m o l/L concentration of N a+,so alkaine protease21Z1has good salt resistance.