纳豆激酶的原核表达、纯化及其特性分析（英文）  

Prokaryotic expression,purification and characterization of nattokinase

作　　者：艾海新[1,2,3] 张力[1,2] 张新刚[1] 陈思瑶[1] 胡桓[1,3] 魏洪运[1,4] 蒙新睿 王天琦[5] 赵健[1] 刘宏生[1,2,3] 

机构地区：[1]辽宁大学生命科学院,辽宁沈阳110036 [2]辽宁省生物大分子计算模拟与信息处理工程技术研究中心,辽宁沈阳110036 [3]辽宁省药物分子模拟与设计工程实验室,辽宁沈阳110036 [4]沈阳神州天盛生物科技有限公司,辽宁沈阳110036 [5]辽宁大学药学院,辽宁沈阳110036

出　　处：《微生物学杂志》2017年第1期20-27,共8页Journal of Microbiology

基　　金：support by Nature Science Foundation(2013225086);Science of Public Research Foundation(2014001015) from the Scientific and Technological Department of Liaoning Province and Innovation Team Project(LT2015011);General Research Project Foundation(L2014001) from the Education Department of Liaoning Province;Large-scale Equipment Shared Services Project(F15165400);Applied Basic Research Project(F16205151) from the Science and Technology Bureau of Shenyang~~

摘　　要：纳豆激酶(Nattokinase)作为一种新型溶解血栓的纤维蛋白溶解酶,有着广阔的市场前景和巨大的产业化潜力。本研究从枯草芽胞杆菌(Bacillus subtilis natto)发酵液中提取、纯化纳豆激酶,依次通过硫酸铵盐析、凝胶过滤层析和疏水层析方法纯化纳豆激酶,纯化倍数为5.2,纯化率为46.3%。纯化后的纳豆激酶经SDS-PAGE电泳显示相对分子量约为28 kD a,纤维蛋白平板法显示活性为4 580 IU/mg。但纳豆激酶在枯草芽胞杆菌发酵液中的含量较低,因此在原核表达载体大肠埃希菌(Escherichia coli)BL21(DE3)中克隆并高效表达了纳豆激酶基因,其纳豆激酶产量显著提高,但酶活相对降低。Nattokinase is a fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy.In this study,nattokinase was purified from fermentation broth of a Bacillus subtilis strain by ammonium sulfate saltingout,gel filtration chromatography,and hydrophobic interaction chromatography with a purification fold of 5.2 and at a yield of 46.3%.The purified enzyme has molecular mass of 28 kD a and fibrinolytic activity of 4 580 U/mg.Since the concentration of nattokinase on fermentation broth was quite low,we cloned nattokinase gene from B.subtilis and expressed it in E.coli BL21（DE3）.Nattokinase was actively expressed in the recombinant strain.The yield of nattokinase was increased significantly,but the activity of the protein produced by recombinant strain was low.

关 键 词：纳豆激酶 纯化 原核表达 

分 类 号：Q939.9-3[生物学—微生物学]