重组枯草芽孢杆菌α-淀粉酶基因工程菌构建与表达  

Construction and Expression of Gene Engineering Bacterium of α-amylase in Recombinant Bacillus subtilis

作　　者：谢光蓉[1] 乔代蓉[1] 曹毅[1] 

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,四川成都610064

出　　处：《食品与发酵科技》2012年第3期13-17,共5页Food and Fermentation Science & Technology

基　　金：四川省科技厅项目支持(2008GZ0245;2011HH0038)

摘　　要：根据GenBank枯草芽孢杆菌α-淀粉酶基因序列设计引物,以枯草芽孢杆菌基因组为模板,PCR克隆α-淀粉酶基因(amy),将α-淀粉酶基因插入穿梭表达载体pP43C,构建重组质粒pP43Camy。随后将重组质粒转化八种蛋白酶缺陷的宿主枯草芽孢杆菌WB800,经筛选获得重组枯草芽孢杆菌α-淀粉酶基因工程菌WB800/pP43Camy1026,工程菌摇瓶发酵酶活力达960U。性质研究表明,重组α-淀粉酶的最适作用温度为70℃,最适反应pH为6.0,具有良好的应用潜力。According to a-amylase gene sequence from Bacillus subtilis published in GenBank, primers were designed. Gene sequence （amy） was obtained by PCR method. The cloned sequence was inserted into the E.coli-BaciUus subtilis shuttle expression vector pP43C to construct recombinant plasimd： pP43Camy. Then, recombinant plasmids was transferred into eight proteases deficient host WB800, and the engineering strains with high expression level： Bacillus subtilis WB800/pPd3Camy was obtained by screening. The enzyme activity of Engineering strain reached 960U by flask fermentation. Optimum temperature and optimum pH of the recombinant a-amylase were respectively 70 ℃ and 6.0. It has a good potential for application.

关 键 词：Α-淀粉酶 枯草芽孢杆菌 基因克隆 基因表达 

分 类 号：TQ925.1[轻工技术与工程—发酵工程]