介导纳豆激酶分泌表达信号肽比较.pdfVIP

现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.5 介导纳豆激酶分泌表达的信号肽比较 何孝天，刘中美，崔文璟，周哲敏 （江南大学生物工程学院，工业生物技术教育部重点实验室，江苏无锡 214122） 摘要：以纳豆芽孢杆菌（Bacillus natto ）的基因组为模板，PCR 分别扩增编码信号肽、前导肽、成熟肽的前纳豆激酶原基因序列 （pre-pro-nk ），编码前导肽、成熟肽的纳豆激酶原基因（pro-nk ），构建了含有三种不同信号肽的纳豆激酶重组质粒 pMA0911- wapA-pro-nk 、pMA0911-yncM-pro-nk 、pMA0911-pre-pro-nk (m)。通过 3 种信号肽介导的纳豆激酶的分泌表达、酶活性质分析等实验 结果表明，wapA 信号肽介导的纳豆激酶的分泌效率以及纤溶酶活性最高。对 wapA 信号肽介导的纳豆激酶表达产物进行了纯化，纯 化倍数及回收率分别为 2.15 和 21.5%。酶学性质分析结果表明重组纳豆激酶的最适温度、最适pH 分别为 50 ℃和pH 8.0 ，纳豆激酶 在温度低于 60 ℃及 pH 5.0~11.0 比较稳定。本研究为纳豆激酶的基因工程改造以及进一步提高纳豆激酶在枯草芽孢杆菌中的表达量奠 定了一定的基础。 关键词：纳豆激酶；信号肽；表达；酶学性质；纳豆芽孢杆菌 文章篇号：1673-9078(2014)5-62-68 Comparison of Signal Peptides for Nattokinase Secretory Expression HE Xiao-tian, LIU Zhong-mei, CUI Wen-jing, ZHOU Zhe-min (The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China) Abstract: The gene pre-pro-nk (encoding signal peptide, propeptide and mature peptide) and pro-nk (encoding propeptide and mature peptide) were amplified from Bacillus natto chromosome by PCR. Three recombinant vectors (pMA0911-wapA-pro-nk, pMA0911- yncM-pro-nk , pMA0911-pre-pro-nk (m)) with different signal peptides were constructed. Overexpression of nattokinase with those different signal peptides were achieved in B. subtilis expression system. The recombinant nattokinase with wapA signal peptide was expressed at the highest level, and exhibited the strongest fibrinolytic activity. The recombinant nattokinase was purified, and the purification factor and activity recovery of the nattokinase were 2.15 and 21.5%, respectively. The enzymatic property was analyzed. The optimal pH and temper