组合策略提高碱性蛋白酶在枯草芽孢杆菌中的表达  

Combined strategy for improving expression of alkaline protease in Bacillus subtilis

作　　者：温树森 吴昌正 侯莎 韦晓群 WEN Shusen;WU Changzheng;HOU Sha;WEI Xiaoqun(College of Food Science,South China Agricultural University,Guangzhou 510642;Foshan Haitian Flavouring and Food Co.,Ltd.,Foshan 528511)

机构地区：[1]华南农业大学食品学院,广州510642 [2]佛山市海天(高明)调味食品有限公司,佛山528511

出　　处：《中国食品添加剂》2025年第2期43-49,共7页China Food Additives

基　　金：国家重点研发计划项目(2023YFD2100200)。

摘　　要：为了促进Shouchella clausii来源的碱性蛋白酶在枯草芽孢杆菌(Bacillus subtilis)WB800n宿主中的高效表达,首先选择3个单启动子替换原始启动子P_(43),其中单启动子P_(amyQ)介导的重组菌经发酵后酶活力最高,为1959 U/mL。其次,通过构建双启动子表达系统发现P_(43)-P_(amyQ)使酶活力进一步提升到2215 U/mL。接着对启动子P_(43)-P_(amyQ)的核心区域(-35和-10区)进行优化,其中Mutant-2的酶活力高达2788 U/mL,是原始菌株酶活力(1746 U/mL)的1.60倍。在此基础上,通过枯草芽孢杆菌内源信号肽替换,筛选得到的带有信号肽SPmpr的重组菌的酶活力进一步提高到4007 U/mL,与信号肽优化前相比提高了43.72%,是出发菌株酶活力的2.30倍。研究结果表明,启动子优化和信号肽筛选的组合策略显著提高了碱性蛋白酶在枯草芽孢杆菌中表达量,为后续研究与应用奠定了基础。Bacillus subtilis WB800n was used as the host strain to heterogeneously express alkaline protease from Shouchella clausii.Firstly,three promoters were selected to replace the original promoter P_(43),among which the recombinant strain mediated by the promoter P_(amyQ) had the highest enzyme activity reaching 1959 U/mL after fermentation.Subsequently,a dual promoter expression system was constructed,P_(43)-P_(amyQ) was found to further increase the enzyme activity to 2215 U/mL;then,the core region(-35 and-10 regions)of the promoter P_(43)-P_(amyQ) was optimized,and the enzyme activity of Mutant-2 strain reached 2788 U/mL,which was 1.60 times higher than that of the original strain(1746 U/mL).On this basis,through the replacement of endogenous signal peptides from Bacillus subtilis,the enzyme activity of the recombinant strain with signal peptide SPmpr was further increased to 4007 U/mL,which was 43.72%higher than that before signal peptide optimization and 2.30 times that of the starting strain. The results showed that the combined strategy of promoter optimization and signal peptide selection significantlyimproved the expression level of alkaline protease in Bacillus subtilis,laying a foundation for future research andapplication.

关 键 词：碱性蛋白酶 枯草芽孢杆菌 异源表达 启动子 信号肽 

分 类 号：TS201[轻工技术与工程—食品科学]