本研究通过串联启动子方式实现纳豆激酶在枯草芽孢杆菌WB800中的高效分泌表达。通过对几种现有报道的强启动子的比较并对其进行串联操作，确定生产纳豆激酶的最优启动子及纳豆激酶的最高产量。本研究首先在枯草芽孢杆菌WB800中成功构建五种含不同强启动子的重组质粒pSG101（PHpaII），pSG102（PBcaprE），pSG103（PluxS），pSG104（PgsiB）和pSG105（PyxiE），实现纳豆激酶分泌表达，并对其纤溶活性进行测定。结果表明，启动子PHpaII介导的纳豆激酶纤溶活性（110.80 FU/mL）明显优于其他四种启动子。通过对启动子PHpaII进行多次串联，成功构建质粒pSG106（PHpaII-PHpaII），pSG107（PHpaII-PHpaII-PHpaII）和pSG108（PHpaII-PHpaII-PHpaII-PHpaII）。数据显示，菌株Bacillus subtilis WB800/pSG107（PHpaII-PHpaII-PHpaII）纳豆激酶产量最高为213.30 FU/mL，相比单个启动子PHpaII，提高了92.51%。通过对五种强启动子的比较以及对其进行串联操作，成功实现纳豆激酶在枯草芽孢杆菌WB800的高效表达，纤溶活性最高为213.30 FU/mL，与现有相关报道相比有明显优势。

In Bacillus subtilis WB800, the efficient secretory expression of nattokinase by tandem promoters was investigated. Several previously reported strong promoters were compared and arranged in tandem repeat sequence, and the optimal promoter for nattokinase and its maximum yield were determined. In this study, five recombinant strains harboring five different strong promoters were successfully constructed in Bacillus subtilis WB800, including pSG101 (PHpaII), pSG102 (PBcaprE), pSG103 (PluxS), pSG104 (PgsiB), and pSG105 (PyxiE), the secretory expression of nattokinase was achieved and the fibrinolytic activity was measured. The results indicated that the fibrinolytic activity of nattokinase mediated by promoter PHpaII (110.80 FU/mL) was higher than that mediated by the other four promoters. Subsequently, three plasmids were built by arranging the promoter PHpaII in multi-tandem sequences, including pSG106 (PHpaII-PHpaII), pSG107 (PHpaII-PHpaII-PHpaII), and pSG108 (PHpaII-PHpaII-PHpaII-PHpaII). The data showed that the highest yield of nattokinase (213.30 FU/mL) was achieved under promotion mediated by PHpaII-PHpaII-PHpaII, which increased the production of nattokinase by 92.51% compared with that achieved by mediation by the single promoter PHpaII. Through comparing five strong promoters and arranging them in tandem repeat sequences, the efficient expression of nattokinase was successively achieved in B. subtilis WB800. The highest fibrinolytic activity reached 213.30 FU/mL, significantly higher than that reported in the literature.

国家自然科学基金资助项目（31400078）；国家高技术研究发展计划（863计划）（2014AA021304）；江苏省产学研联合创新资金-前瞻性联合研究项目（BY2014023-21）