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谷氨酰胺转氨酶在Yarrowia lipolytica中的活性表达

  

  1. 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
  • 出版日期:2019-02-18 发布日期:2019-02-18
  • 通讯作者: 刘松,副教授,主要从事酶工程技术等研究,E-mail: liusong@jiangnan.edu.cn;李江华,教授,主要从事酶技术、发酵过程优化与控制等研究,E-mail: lijianghua@jiangnan.edu.cn
  • 作者简介:任蕊蕊,硕士研究生,主要研究方向发酵工程与酶工程,E-mail: renruiruijiangnan@163.com
  • 基金资助:
    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)

Expression of active transglutaminase in Yarrowia lipolytica

  1. 1. School of Bioengineering; 2. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
  • Online:2019-02-18 Published:2019-02-18

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pro-TGase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/mL和6.77 U/mL。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。

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

Abstract: 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, Km and kcat/Km 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.

Key words: transglutaminase, Yarrowia lipolytica, activity expression, protease recognition site, activating protease

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