食品科学 ›› 2017, Vol. 38 ›› Issue (14): 1-8.doi: 10.7506/spkx1002-6630-201714001

• 生物工程 •    下一篇

纳豆激酶基因工程菌的构建及酶活力分析

崔青,钱炳俊,姚晓敏,季顺利,鲁飞凤,吴静,张建华   

  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)
  • 出版日期:2017-07-25 发布日期:2017-09-06
  • 基金资助:
    国家自然科学基金面上项目(31171737)

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

CUI Qing, QIAN Bingjun, YAO Xiaomin, JI Shunli, LU Feifeng, WU Jing, ZHANG Jianhua   

  1. (Bor S. Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China)
  • Online:2017-07-25 Published:2017-09-06

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。

关键词: 纳豆激酶, aprN基因, 枯草芽孢杆菌, 基因工程菌, 酶活力

Abstract: Nattokinase (NK), encoded by the aprN gene of Bacillus subtilis natto, has strong fibrinolytic activity both in vitro and in vivo. In this research, the aprN gene from B. subtilis was cloned and the codons which encode the first 30 amino acids 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 confirmed to be aprN. The pHT01-aprN was transformed into B. subtilis 168 by electroporation, and the engineered bacterium (B.s 168/pHT01-aprN) was isolated on LB plates containing chloramphenicol. NK expression was induced by IPTG, and the highest enzyme activity in shaking flask culture was up to (289.00 ± 3.42) U/mL with good stability, which was 3.9 times as high as that of wild-type B. subtilis natto.

Key words: nattokinase, aprN gene, Bacillus subtilis, enzyme activity, engineered bacteria

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