普鲁兰酶可特异性地水解支链淀粉得到直链淀粉，因而在淀粉加工过程中具有重要的应用。本研究从Bacillus naganoensis ATCC 53909基因组中克隆了普鲁兰酶基因pul，并克隆到大肠杆菌-枯草芽孢杆菌穿梭载体pBE中，构建表达载体pBE-pul。在此基础上，将来源于枯草芽孢杆菌、地衣芽孢杆菌以及解淀粉芽孢杆菌中的17个高表达基因的启动子分别克隆到表达载体pBE-pul中，并转化至Bacillus subtilis ATCC6051∆10，成功构建了十七株含有不同启动子介导普鲁兰酶分泌表达的重组菌株。对重组菌株的分泌表达比较发现，启动子P43和PspovG介导的普鲁兰酶活性明显优于其他启动子，其中PspovG介导的普鲁兰酶活性更高。同时，还使用了启动子PspovG介导N端的108个氨基酸缺失的pul324突变体进行分泌表达。通过对17种启动子的比较和两个普鲁兰酶基因的比较，本研究构建的一株重组菌株的普鲁兰酶的表达更为高效，其活性高达389.85 U/mL，后者显著高于现有的相关报道。

Pullulanase can specifically hydrolyze amylopectin to get amylose, which is important in Starch industry applications. In this study, the pullulanase gene pul was cloned from the Bacillus naganoensis ATCC 53909 genome and inserted into the E.coli-Bacillus subtilis shuttle vector pBE to construct the expression vector pBE-pul. Based on these, seventeen strong promoters from Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens were respectively cloned into the expression vector pBE-pul and transformed into Bacillus subtilis ATCC6051∆10. Finally, Seventeen recombinant strains containing different strong promoters were constructed, the secretory expression of pullulanase was achieved and the activity of pullulanase was measured. The results indicated that the activity of pullulanase mediated by promoter P43 and PspovG was significantly higher than that mediated by the other promoters and the activity of pullulanase mediated by promoter PspovG was higher than that mediated by the promoter P43.Meanwhile, the pullulanase gene mutant pul324 which deleted N-terminal 108 amino acids was investigated, which was mediated by promoter PspovG. Through comparing seventeen promoters and two pullulanase genes, a recombinant strain that can efficiently overexpress pullulanase was constructed successfully. The highest pullulanase activity reached 389.85 U/mL, significantly higher than that reported in the literature.

广东省科技计划项目（2016A050503016；2016A010105004）；广东省自然科学基金项目（2017A030313097）；华南理工大学中央高校基本科研业务费资助项目（2015ZP032）；南开大学分子微生物学与技术教育部重点实验室开放课题资助