一种碱性γ-谷氨酰转肽酶在Bacillus subtilis 168中的分泌表达与酶学性质分析  

Homologous Cloning and Characterization of Bacillus subtilis 168 γ-Glutamyltranspeptidase

作　　者：Bernard Gitura Kimani 杨套伟[1,2] 饶志明[1,2] 张显[1,2] 徐美娟[1,2] 和斐[1,2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2017年第2期149-155,共7页Journal of Food Science and Biotechnology

基　　金：国家973计划项目(2012CB725202);国家863计划项目(2015AA021004);国家自然科学基金项目(31400082);教育部重点项目(113033A);江苏高校优势学科建设工程资助项目

摘　　要：将γ-谷氨酰转肽酶(GGT)在Bacillus subtilis 168同源过量表达,发现该酶能够自动分泌到细胞外,且重组菌酶活比原始菌提高了3 568倍。通过SDS-PAGE分析发酵液上清液发现,该γ-谷氨酰转肽酶是由一个相对分子质量为42 000的大亚基和一个22 000的小亚基组成。通过酶学性质分析发现,该γ-谷氨酰转肽酶是一个耐碱性的酶,最适反应pH为10;同时该酶具有良好的耐热性,最适反应温度达到50℃;另外,加入5.0 mmol/L的Mg^(2+)、Ca^(2+)、K^+、La^(3+)、Li^+和NH_4^+对GGT转肽活力具有明显的促进作用,其中添加的NH_4^+对GGT活力促进作用最为显著,GGT活力提高45%以上,而Cu^(2+)和Zn^(2+)则对其具有抑制作用。Gammaglutamyltranspeptidase gene from Bacillus subtilis 168 was cloned and overexpressed with p MA5 vector in Bacillus subtilis 168 for overproduction of GGT. GGT was secreted in the extracellular space. The overexpressed enzyme was 3568 fold higher in activity than that from the parent strain and exhibited a specific transpeptidase activity of 49.8 U/mg. The molecular mass of two sub-units was estimated to be 42 000 and 22 000 respectively,by SDS-PAGE.The overexpressed GGT was a very versatile enzyme in alkaline p H. Its optimal temperature was 50℃ showing a moderately high thermostability. The effect of ions on purified GGT was also examined. Results showed that NH_4~＋ had the highest activation effect on GGT（45%）,other ions such as Ca^（2＋）、K~＋、Mg^（2＋）、Li~＋ and La^（3＋） had a significant activation effect on the enzyme,whereas Cu^（2＋）and Zn^（4＋） recorded the highest deactivation on GGT activity.

关 键 词：Γ-谷氨酰转肽酶 分泌表达 酶学特征 Bacillus SUBTILIS 168 

分 类 号：Q55[生物学—生物化学]