豆豉纤溶酶Subtilisin FS33的提取纯化与鉴定  

Preparation and identification of a novel fibrinolytic enzyme FS33 from Bacillus subtilis DC_(33)

作　　者：王成涛[1,2] 籍保平[2] 曹雁平[1] 孙宝国[1] 张慧[1] 

机构地区：[1]北京工商大学化学与环境工程学院,北京100037 [2]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《中国酿造》2009年第2期29-33,共5页China Brewing

基　　金：国家重点基础研究发展计划973项目(2007CB707802)

摘　　要：探讨了凝胶层析法提取纯化新型纤溶酶Subtilisin FS33的方法。芽胞杆菌(Bacillus subtilis)DC33发酵液或固态豆豉抽提液在30%～65%硫酸铵饱和度下分段盐析沉淀,再经DEAE-Sepharose FF、Phenyl Sepharose 6FF、Sephadex G-50连续凝胶层析纯化,得到电泳纯纤溶酶Subtilisin FS33,其最终纯化倍数和回收率分别达到34.6倍和13.0%,酶蛋白比活力达到15495U/mg。该酶在还原和非还原条件下SDS-PAGE电泳分析均呈现单一条带,分子量为30kDa;活性电泳FS33凝胶条带在纤维蛋白平板上也表现出强烈纤溶活性。The method of purifying a novel fibrinolytic enzyme subtilisin FS33 by gel chromography was investigated in this paper. The crude enzyme produced by Bacillus subtilis DC33 was precipitated by 30%-65% saturation of （NH4）2SO4, and the protein collected by centrifugation （10000xg, 15min） was applied to chromatographic procedures including hydrophobic chromatography, anion exchange and gel filtration chromatography. The finally eluted proteins by Sephadex G-50 gel filtration were subjected to SDS-PAGE. Under these conditions, the purified rate and the recovery of final enzyme were 36.6 fold and 13.0%, respectively. The specific activity of the fibrinolytic enzyme was 15495U/mg, and only one band was observed in the purified sample under reducing or non-reducing conditions. The molecular mass was estimated to be approximately 30kDa. The higher fibrinolytic activities of the purified enzyme band were observed on the plasminogen-fxee fibrin plate.

关 键 词：纤溶酶 芽胞杆菌DC33 凝胶层析 电泳 纯化 纤溶活性 

分 类 号：R977.3[医药卫生—药品]