谷氨酰胺转氨酶在Yarrowia lipolytica中的活性表达  

Expression of active transglutaminase in Yarrowia lipolytica

作　　者：任蕊蕊 刘松[1,2] 李江华[1,2] 堵国成[1,2] 陈坚[1,2] REN Rui-rui;LIU Song;LI Jiang-hua;DU Guo-cheng;CHEN Jian(School of Bioengineering, Wuxi 214122, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)

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

出　　处：《生物学杂志》2019年第1期11-15,共5页Journal of Biology

基　　金：国家自然基金面上项目(31771913);江苏省重点研发计划社会发展项目(BE2016629)

摘　　要：谷氨酰胺转氨酶(EC2. 3. 2. 13,Transglutaminase,TGase)是一种重要的食品酶。基于N-端酶原区对折叠的重要影响,TGase通常以无活性的酶原(pro-TGase)形式在异源宿主中表达。以茂源链霉菌(Streptomyces mobaraensis) pro-TGase为基因来源,重组解脂耶氏酵母(Yarrowia lipolytica po1h)为研究对象,通过在pro-TGase插入宿主Kex2蛋白酶识别位点(策略1)和共表达pro-TGase与谷氨酰胺转氨酶激活金属蛋白酶(TAMEP,策略2),使proTGase在Y. lipolytica中表达后被切除酶原区,而直接转化为活性TGase。摇瓶发酵结果显示,策略1和策略2构建得到的重组菌的TGase活力分别为5. 26 U/m L和6. 77 U/m L。酶学性质研究表明,策略1和策略2得到的重组菌的TGase比酶活、Km及kcat/Km均明显优于S. mobaraensis TGase。基于Y. lipolytica食品安全性,研究结果为TGase的工业化生产提供了新型高产菌种。Transglutaminase (EC2.3.2.13, TGase) is an important food enzyme. Since the N-terminal pro-region of TGase has great effect on its folding, TGase is often expressed as its non-active form (pro-TGase) in heterologous hosts. In this study, Streptomyces mobaraensis pro-TGase was used as the gene source and Yarrowia lipolytica po1h was selected as host. By inserting the Kex2 protease recognition site in pro-TGase (strategy 1) and co-expressing pro-TGase and transglutaminase activating metalloprotease (TAMEP)(strategy 2), pro-TGase was expressed in Y. lipolytica at first, then the pro-region of pro-TGase was excised and the mature TGase was achieved. The results of shake flask fermentation showed that the TGase activities of recombinant strains constructed by strategy 1 and strategy 2 were 5.26 U / mL and 6.77 U / mL, respectively. Moreover, the enzymatic properties were further studied. The results showed that the specific activities, K m and k cat / K m of the recombinant TGase obtained by strategy 1 and strategy 2 were significantly higher than those of S. mobaraensis TGase. Based on the food safety of Y. lipolytica , the results provide two new high-yield strains for the industrial production of TGase.

关 键 词：谷氨酰胺转氨酶 YARROWIA lipolytica 活性表达 蛋白酶识别位点 活化蛋白酶 

分 类 号：Q78[生物学—分子生物学]