γ-谷氨酰转肽酶在枯草芽孢杆菌中高效表达及发酵培养基优化  

Efficient expression ofγ-glutamyl transpeptidase in Bacillus subtilis and optimization of enzyme-producing culture medium

作　　者：张祖政 毛泽敬 余华顺[2,3,4] 张彦 杨潇[1] 龚大春 郑贤良 ZHANG Zuzheng;MAO Zejing;YU Huashun;ZHANG Yan;YANG Xiao;GONG Dachun;ZHENG Xianliang(College of Biological and Pharmaceutical,China Three Gorges University,Yichang 443002,China;Angel Yeast Co.Ltd.,Yichang 443003,China;Hubei Provincial Key Laboratory of Yeast Function,Yichang 443003,China;National Key Laboratory of Agricultural Microbiology,Yichang 443003,China)

机构地区：[1]三峡大学生物与制药学院,湖北宜昌443002 [2]安琪酵母股份有限公司,湖北宜昌443003 [3]酵母功能湖北省重点实验室,湖北宜昌443003 [4]农业微生物挖掘与利用全国重点实验室,湖北宜昌443003

出　　处：《食品与发酵工业》2024年第24期76-83,共8页Food and Fermentation Industries

基　　金：国家重点研发计划资助(2021YFC2100300);山东省重大科技创新工程项目(2022CXGC020712)。

摘　　要：γ-谷氨酰转肽酶是一种能催化底物谷胺酰胺形成γ-谷氨酰-酶复合体,再被受体底物(如氨基酸、短肽)亲核取代,发生转肽反应形成γ-谷氨酰肽的酶,在食品、医药等领域有重要应用价值。该研究选用枯草芽孢杆菌(Bacillus subtilis)SCK6作为表达宿主,以来源于枯草芽胞杆菌的173种信号肽为基础构建γ-谷氨酰转酶信号肽筛选文库,筛选出最优信号肽SP_(cotC),在此基础上进行启动子筛选,得到最优启动子P_(aprE),最终构建了一株高效分泌表达γ-谷氨酰肽酶的重组菌株BS-gts3,摇瓶发酵胞外酶活力最高为5.04 U/mL。通过单因素试验和响应面试验筛选出最优培养基:乳糖31.73 g/L、FM80250.26 g/L、硫酸镁1.24 g/L、KH_(2)PO_(4)1.25 g/L、K_(2)HPO_(4)0.75 g/L,胞外酶活提高了12%,为5.623 U/mL。并进行5 L发酵罐放大培养,在38 h,胞外酶活力达到最高28.18 U/mL,是已报道的最高水平。该研究对γ-谷氨酰转肽酶的产品开发及应用奠定了基础。γ-Glutamyl transpeptidase is an enzyme that can catalyze glutamine to generate aγ-glutamyl-enzyme complex.Then the complex can be nucleophilically substituted by an acceptor substrate such as an amino acid or a short peptide,resulting in a transpeptidation reaction that forms aγ-glutamyl peptide.This enzyme has significant application value in the fields of food and medicine.In this research,Bacillus subtilis SCK6 was used as the expression host,based on 173 signal peptides of Bacillus subtilis,a signal peptide screening library was constructed,and the optimal signal peptide(SP_(cotC))was identified.Promoter screening was then carried out to obtain the optimal promoter(P_(aprE)).Finally,with the optimal signal peptide and prompter,a recombinant strain BS-gts3 was constructed which could express and secreteγ-glutamyl transpeptidase.The maximum extracellular enzyme activity reached 5.04 U/mL in shake flask fermentation.The optimal medium was further determined through one-way and response surface tests.The optimal composition of the medium was 31.73 g/L of lactose,50.26 g/L of FM802,1.24 g/L of MgSO_(4),1.25 g/L of KH_(2)PO_(4),and 0.75 g/L of K_(2)HPO_(4).The culture was then scaled up in a 5 L fermenter,and the maximum extracellular enzyme activity reached 28.26 U/mL after 38 h fermentation,which was the highest reported level.This study provides a foundation for the development and application ofγ-glutamyl transpeptidase.

关 键 词：枯草芽孢杆菌 Γ-谷氨酰转肽酶 启动子 信号肽 重组表达 

分 类 号：TQ925[轻工技术与工程—发酵工程]