中国传统细菌型豆豉溶栓酶的提取纯化技术研究  

Separation and purification of fibrinolytic enzyme from Chinese traditional bacteria-type Douchi

作　　者：郝征红[1] 张炳文[2] 庞庆芳[3] 

机构地区：[1]山东省农业管理干部学院现代农业技术系,济南250100 [2]济南大学食品科学与营养系,济南250002 [3]山东农业大学食品科学学院,泰安271018

出　　处：《农业工程学报》2008年第3期282-285,共4页Transactions of the Chinese Society of Agricultural Engineering

基　　金：山东省自然科学基金资助项目(Y2007D76)

摘　　要：为深入开发利用中国传统发酵细菌型豆豉溶栓酶,对豆豉溶栓酶分离纯化工艺进行了研究探讨,确认了最佳的纯化条件:首先用饱和度75%的硫酸铵沉淀豆豉溶栓酶;然后利用DEAE-Sepharose FF(Diethylaminoethyl Sepharose Fast Flow,二乙基氨基琼脂糖快速凝胶)阴离子交换柱进行层析,优化的层析条件为用10 mmol/L Tris(Trishydroxymethylaminomen,三羟甲基氨基甲烷)-HCl(pH 8.7)作为上样缓冲液,采用线性梯度洗脱,洗脱液为10 mmol/L Tris-HCl(pH 8.7)和0.6 mol/L NaCl,洗脱流速为1 mL/min;最后经过Sephadex G-75(葡聚糖凝胶G-75)凝胶过滤,得到纯化倍数为11.29倍的豆豉溶栓酶。利用SDS-PAGE(Sodium dodecyl sulphate-polylamide gel electroresis,十二烷基硫酸钠—聚丙烯酰胺凝胶电泳)显示经纯化后的酶无杂蛋白,初步鉴定其分子量接近31 ku。In order to exploit fibrinolysis components of Chinese traditional bacteria-type Douchi, separation and purification of fibrinolytic enzyme were discussed and the best purification method was determined. Firstly, fibrinolytic enzyme was separated by ammonium sulfate with concentration of 75% saturation. Secondly, purification was achieved by Diethylaminoethyl Sepharose Fast Flow（DEAE-Sepharose FF） anion exchange chromatography with 10mmol/L Tris-HC1 （pH 8.7） as sample buffer. The column was eluted stepwise with 0.6 mol/L NaC1 and 10 mmol/L Trishydroxymethylaminomen（Tris-HC1） buffer （pH 8.7） at flow rate of 1 mL/min. Finally, fibrinolytic enzyme was further filtered by Sephadex G-75 chromatography and 11.29 times purified fibrinolytic enzyme was gained. Sodium dodecyl sulphate-polylamide gel electroresis （SDS-PAGE） showed that there was no unpurposcd protein in purified fibrinolytic enzyme and the molecular weight was identified approximately 3 lku preliminarily.

关 键 词：细菌型豆豉 溶栓酶 提取纯化 

分 类 号：TS209[轻工技术与工程—食品科学]