纳豆激酶基因工程菌的构建及酶活力分析  

Construction of Genetically Engineered Strain for Nattokinase Production and Enzyme Activity Analysis

作　　者：崔青[1] 钱炳俊[1] 姚晓敏[1] 季顺利 鲁飞凤 吴静[1] 张建华[1] CUI Qing;QIAN Bingjun;YAO Xiaomin;JI Shunli;LU Feifeng;WU Jing;ZHANG Jianhua(Bor S. Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China)

机构地区：[1]上海交通大学农业与生物学院陆伯勋食品安全研究中心,上海200240

出　　处：《食品科学》2017年第14期1-8,共8页Food Science

基　　金：国家自然科学基金面上项目(31171737)

摘　　要：纳豆激酶由纳豆芽孢杆菌(Bacillus subtilis natto)的aprN基因编码,在体内外具有很强的溶解纤维蛋白活性。利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增B.subtilis natto的aprN基因,并依据B.subtilis的密码子偏好性优化了起始30个氨基酸的密码子,构建了重组表达质粒pHT01-aprN。经限制性酶酶切、PCR扩增和测序验证了其编码的正确性。通过电击法将含有强启动子的pHT01-aprN导入B.subtilis,利用氯霉素抗性筛选获得B.s 168/pHT01-aprN工程菌。经IPTG诱导表达,摇瓶发酵培养最高酶活力为(289.00±3.42)U/mL,是野生菌的3.9倍,酶活力表达稳定性良好。Nattokinase(NK),encoded by the aprN gene of Bacillus subtilis natto,has strong fibrinolytic activity both invitro and in vivo.In this research,the aprN gene from B.subtilis was cloned and the codons which encode the first30aminoacids were optimized on the basis of the codon preference of B.subtilis.Recombinant vector pHT01-aprN was constructed.Through restriction enzyme digestion analysis,PCR amplification and sequencing,the subcloned gene was confirmedto be aprN.The pHT01-aprN was transformed into B.subtilis168by electroporation,and the engineered bacterium(B.s168/pHT01-aprN)was isolated on LB plates containing chloramphenicol.NK expression was induced by IPTG,and thehighest enzyme activity in shaking flask culture was up to(289.00±3.42)U/mL with good stability,which was3.9times ashigh as that of wild-type B.subtilis natto.

关 键 词：纳豆激酶 aprN基因 枯草芽孢杆菌 基因工程菌 酶活力 

分 类 号：Q786[生物学—分子生物学]