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。

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 temperature were 8.0 and 50 ℃, respectively. And the recombinant nattokinase was stable at pH of 5.0~11.0 and temperature of less than 60 ℃. This study could be useful for genetic reconstruction of nattokinase and improvement of expression level of nattokinase in B. subtilis.

新世纪优秀人才项目（NCET-10-0461）；教育部重大项目（311023）