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

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.