双启动子促进亮氨酸脱氢酶在Bacillus subtilis中表达及发酵研究  

To Promote the Expression of Leucine Dehydrogenase in Bacillus subtilis via Dual-Promoter and Fermentation Research

作　　者：张玲[1] 王男 金吕华 林荣 杨海麟[1] ZHANG Ling;WANG Nan;JIN Lv-hua;LIN Rong;YANG Hai-lin(Key laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室

出　　处：《中国生物工程杂志》2018年第12期21-31,共11页China Biotechnology

基　　金：江苏省产学研(BY2016022-40);国家轻工技术与工程一流学科自主课题(2018-23)资助项目

摘　　要：为了促进亮氨酸脱氢酶在Bacillussubtilis中高效表达,采取在质粒pMA5自带的启动子PHpaII之后分别添加诱导型和组成型启动子,考察双启动子对酶表达的影响。选取2种诱导型启动子(Pgrac、Pgl-M1)和4种组成型启动子(P43、Plaps、PHpaII、PamyQ)进行构建表达,其中组成型启动子PamyQ与PHpaII构成的双启动子效果最好,有效地将胞外活性提高到31.24U/ml,是单启动子PHpaII的3.4倍。在以上最优双启动子的基础上分别融合Sec途径和Tat途径的4种信号肽,但信号肽与双启动子共同作用并没有获得更高的酶活性。选用酶活最高的双启动子突变菌株Bacillussubtilis168/pW6(PHpaII-PamyQ)进行7.5L发酵罐补料发酵产酶研究,LeuDH酶活达到217.96U/ml,是摇瓶水平的6.97倍,对工业上产亮氨酸脱氢酶有一定参考价值。In order to promote the efficient expression of leucine dehydrogenase in Bacillus subtilis,the inducible and constitutive promoters were inserted downstream of PHpaII,which is the native promoter of plasmid pMA5,to investigate the effect on LeuDH by dual-promoter.Two inducible promoters (Pgrac,Pglv-M1) and four constitutive promoters (P43,Plaps,PHpaII,PamyQ) were selected for expression of LeuDH.The dual promoter was consisted in the constitutive promoter PamyQ and PHpaII had the best performance,reaching 31.24U/ml,which was 3.4 times higher than that of the single promoter PHpaII.Four signal peptides of Sec pathway and Tat pathway were fused to behind the best dual-promoter PHpaII-PamyQ,respectively.However,this combination did not give higher enzyme activity.The strain B.subtlis 168/pW6 with the best activity of producing leucine dehydrogenase were analyzed in 7.5L fermenter.The enzyme activity reached 217.96U/ml,which was 6.97 times higher than the shake flask level.The results for industrial production of leucine dehydrogenase have a certain reference value.

关 键 词：亮氨酸脱氢酶 枯草芽孢杆菌 启动子 信号肽 发酵产酶 

分 类 号：Q78[生物学—分子生物学]