植物乳杆菌环二腺苷酸合成酶的克隆表达与活性分析  

Cloning, expression and characterization of c-di-AMP-synthesizing enzyme from Lactobacillus plantarum

作　　者：杜斌 刘喜朋[2] 韦曾传 汪肖 刘正红 林栋 DU Bin;LIU Xipeng;WEI Zengchuan;WANG Xiao;LIU Zhenghong;LIN Dong(College of Food and Pharmacy Engineering,Guiyang University,Guiyang,Guizhou 550005,China;School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]贵阳学院食品与制药工程学院,贵州贵阳550005 [2]上海交通大学生命科学技术学院,上海200240

出　　处：《微生物学通报》2021年第4期1130-1139,共10页Microbiology China

基　　金：贵州省科学技术基金(黔科合基础[2017]1003);国家自然科学基金(U1832161);贵阳市财政支持贵阳学院学科建设与研究生教育项目(SY-2020);贵州省大学生创新创业训练计划项目(20190265139016);贵州省教育厅青年科技人才成长项目(黔教合KY字[2017]241);贵州省高等学校工程研究中心建设项目:(黔教合KY字[2019]051)。

摘　　要：【背景】环二腺苷酸(Cyclic Diadenosine Monophosphate,c-di-AMP)是一种主要存在于革兰氏阳性菌中的重要的第二信使分子,其参与细菌的生长、生存、抗逆性等多种生理活动,但目前关于乳酸菌中c-di-AMP的研究甚少。【目的】从植物乳杆菌(Lactobacillus plantarum)中克隆得到c-di-AMP合成酶基因,在大肠杆菌中进行可溶性表达并研究其体外活性。【方法】使用高效液相色谱以及质谱分析对植物乳杆菌-YRA7细胞内容物中的c-di-AMP进行检测;以植物乳杆菌-YRA7基因组DNA为模板,克隆c-di-AMP合成酶基因(lpDacA),构建重组表达载体pET-28a-lpDacA并在大肠杆菌BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化后进行体外活性研究。【结果】在植物乳杆菌中检测到c-di-AMP分子;成功构建了c-di-AMP合成酶基因的重组表达质粒,该重组蛋白在大肠杆菌中得到可溶性表达;体外活性分析显示,该重组蛋白可以催化ATP生成c-di-AMP,其活性依赖于二价阳离子的存在,在Mg2+存在以及碱性环境下活性较强;RHR是合成酶活性的关键基序,是环二腺苷酸合成酶与ATP的结合位点。【结论】植物乳杆菌c-di-AMP合成酶的克隆表达及活性分析为进一步研究c-di-AMP在植物乳杆菌中的作用奠定了基础。[Background] Cyclic diadenosine monophosphate(c-di-AMP) is an important secondary messenger molecule produced primarily within Gram-positive bacteria and is involved in cell growth, survival, stress resistance and many other aspects of bacterial physiology;however, there are few studies on c-di-AMP in lactic acid bacteria currently. [Objective] The gene of c-di-AMP-synthesizing enzyme from Lactobacillus plantarum was cloned and efficiently heterologous expressed in Escherichia coli, and its biochemical activity in vitro was studied. [Methods] c-di-AMP in L. plantarum-YRA7 was detected by HLPC and ESI-MS using the cell extract. Then the c-di-AMP-synthesizing enzyme gene(lpDacA) was cloned from the genomic DNA of L. plantarum-YRA7 and the recombinant plasmid pET-28a-lpDacA was constructed. The recombination protein was successfully expressed in E. coli BL21(DE3), and purified by Ni-NTA affinity chromatography. Then enzymatic characteristics in vitro was studied. [Results] c-di-AMP was detected in L. plantarum-YRA7. The recombinant expression plasmid pET-28 a-lpDacA was constructed and the purified recombinant protein was obtained. The biochemical activity study showed that the purified recombinant protein converted ATP into c-di-AMP in vitro. Its c-di-AMP-synthesizing activity was dependent on divalent metal ions and exhibited higher activity in the presence of Mg2+ at a basic pH. We also found that RHR motif is essential for the c-di-AMP-synthesizing activity and is the ATP binding site of lpDacA. [Conclusion] Cloning, expression and characterization of c-di-AMP-synthesizing enzyme from L. plantarum will laid a solid foundation for future exploration on the physiological role of c-di-AMP in L. plantarum.

关 键 词：环二腺苷酸 植物乳杆菌 原核表达 

分 类 号：TS201.3[轻工技术与工程—食品科学] Q78[轻工技术与工程—食品科学与工程]