纳豆激酶高效分泌表达系统的构建与最适pH偏酸性改造

血栓导致的心脑血管疾病是当前危害人类健康、导致死亡率最高疾病之一，因此近年来溶栓药物及具有溶栓功能保健食品的研发进展得十分迅速。纳豆激酶具有高效溶血栓作用，是国内外科研热点之一。在前期研究中，我们研究比较了介导纳豆激酶在枯草芽孢杆菌中分泌表达的信号肽。在此基础上，针对目前纳豆激酶开发与应用中的分泌表达量低、酸性环境下稳定性差的问题，本项目拟进行以下两方面的研究：（1）通过启动子、信号肽等调控元件优化与匹配，构建新型分泌表达系统，实现纳豆激酶在枯草芽孢杆菌中高效分泌表达，为工业酶制剂的生产和应用提供理论基础和技术支持；（2）以纳豆激酶的催化机制和晶体结构为基础，结合生物信息学的分析与预测，寻找能够影响酶分子pH作用范围的关键氨基酸位点，通过分子改造来增强纳豆激酶在酸性环境中的稳定性，延长其在机体内的作用时间。

The heart and cerebrovascular disease caused by thrombus is one of the diseases with highest incidence and mortality rates. Researches on thrombolytic drugs and functional food with thrombolytic activity have developed very rapidly in recent decades. Nattokinase (NK) becomes one of the hot research topics due to its high thrombolytic effect. In our previous study, three signal peptides mediated NK expression in Bacillus subtilis were studied. In this project, we aim to enhance the production of recombinant NK and shift the optimum pH of NK towards acid pH, which are the bottleneck for application of NK at present. On one hand, we’ll construct a new system to achieve high-level secretory expression of NK in Bacillus subtilis by optimizing regulatory elements. On the other hand, based on the catalytic mechanism and crystal structure of NK, we are going to find out the key amino acids in NK for shfiting the optimum pH towards acid pH, and induce mutations by side-directed mutagenesis to achieve NK mutants with higher stablility in the acid enviorment. At last but not least, we’ll construct cost-effective engineering strains with high enzyme activity to promote the production and application of NK.