β-淀粉酶（β-amylase，AmyM）是一种重要的工业用酶，广泛应用在啤酒酿造、制糖等行业中。本研究利用Bacillus megatherium DSM 319来源的β-淀粉酶基因在枯草芽孢杆菌（Bacillus subtilis ATCC 6051△10）中分泌表达。首先，构建不同单启动子和双启动子介导β-淀粉酶表达的重组菌株，在摇瓶培养条件下，由P43-P43介导的双启动子重组菌株在发酵培养27 h时，分泌β-淀粉酶酶活力最高达2950.76 U/mL；随后研究碳代谢阻遏效应（Carbon Catabolite Repression，CCR）对重组β-淀粉酶表达的影响，发现对分解代谢控制蛋白（Catabolite control protein A，CcpA）在DNA上顺式调控元件的碱基位点进行定点突变，可以有效缓解碳阻遏效应，β-淀粉酶酶活提高到4663.03 U/mL；最后，将重组β-淀粉酶应用在糖化反应过程中，结果表明麦芽糖转化率可以达到57.62%。综上，本研究成功构建出β-淀粉酶在枯草芽孢杆菌中的高效表达体系，为工业化生产β-淀粉酶提供了理论数据支撑。

β-amylase (AmyM) is an important industrial enzyme, which is mainly used in beer brewing and sugar industry. In this study, AmyM was first amplified from Bacillus megaterium DSM 319, and its expression in B. subtilis ATCC 6051△10 was analyzed. First, recombinant strains were successfully obtained using different single and dual promoters, and they were then cultured in shaking flasks. AmyM synthesized under the control of the P43-P43 dual promoter showed the maximum enzymatic activity (approximately 2950.76 U/mL) after 27 h culture. Subsequently, the impact of carbon catabolite repression on the expression of recombinant AmyM was examined. Results showed that site-directed mutagenesis of bases at sites controlled by catabolite control protein A could effectively alleviate carbon repression, increasing the enzymatic activity of AmyM up to 4663.03 U/mL. Finally, recombinant AmyM was used for glycation, where a maximum maltose conversion rate of 57.62% was achieved. In summary, this study provided a high-efficiency expression scheme for AmyM in B. subtilis and supported the production of recombinant AmyM for industrial applications.

广东省重点领域研发计划项目（2020B020226007）；广州市科技计划项目（202002030207）