内含肽介导谷氨酰胺转胺酶酶原的活化

doi:10.7506/spkx1002-6630-201309020

食品科学 ›› 2013, Vol. 34 ›› Issue (9): 90-94.doi: 10.7506/spkx1002-6630-201309020

内含肽介导谷氨酰胺转胺酶酶原的活化

杜坤，周丽，堵国成，陈坚，周哲敏

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.江南大学食品科学与技术国家重点实验室，江苏无锡 214122

收稿日期:2013-02-25 修回日期:2013-04-14 出版日期:2013-05-15 发布日期:2013-05-07
通讯作者: 周哲敏 E-mail:zhmzhou@jiangnan.edu.cn; zhmzhou@yahoo.com
基金资助:
教育部科学技术研究重大项目;国家自然科学基金项目

Intein-mediated Activation of Transglutaminase from Streptomyces hygroscopicus

DU Kun，ZHOU Li，DU Guo-cheng，CHEN Jian，ZHOU Zhe-min

1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China； 2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

Received:2013-02-25 Revised:2013-04-14 Online:2013-05-15 Published:2013-05-07

摘要/Abstract

摘要： 链霉菌来源的谷氨酰胺转胺酶(microbial transglutaminase，MTG)以酶原(pro-MTG)的形式表达，需要蛋白酶将其活化。蛋白酶有降解MTG及其底物的可能，且增加MTG的分离纯化成本。为解决上述问题，将一种pH值依赖型的内含肽(intein)插入pro区和MTG之间，用以介导二者之间的断裂，实现MTG的pH值控制活化。结果表明：重组蛋白(pro-Intein-MTG)在大肠杆菌中实现了可溶高表达。在pH7.0、25℃的体外环境下，重组蛋白经24h即可完全转化为MTG，且最高酶活力为0.23U/mL(菌体浓度稀释至OD600nm为1.0时测得)。重组型MTG的酶比活力和Km值分别为14.3U/mg、69.4mmol/L，与野生型MTG相近；且圆二色谱显示二者具有相似的二级结构，说明内含肽的插入对于MTG的功能和结构无显著影响。

关键词: 蛋白质剪接技术, 内含肽, 酶原活化, 谷氨酰胺转胺酶, 吸水链霉菌

Abstract: Microbial transglutaminase (MTG) from Streptomyces is secreted as an inactive form of pro-MTG, which can be activated by protease. However, protease has the potential for the hydrolysis of MTG as well as its substrates and can improve product separation costs. In this study, a pH-dependent mini-intein was inserted between the pro-region and MTG to mediate the cleavage of both parts by controlling pH. The recombinant protein (pro-Intein-MTG) was successfully overexpressed in Escherichia coli. The time required for converting recombinant protein to MTG was determined to be 24 h at 25 ℃ and pH 7.0 in vitro, resulting in an activity of 0.23 U/mL (cell concentration was diluted to OD600nm at 1.0). The specific activity and Km of the recombinant MTG was determined to be 14.3 U/mg and 69.4 mmol/L, respectively, which was consistent with that of wild type MTG from Streptomyces hygroscopicus. In addition, circular dichroism analysis showed that the recombinant and wild type MTG had similar secondary structure. These results indicated that the insertion of intein between the pro-region and MTG did not significantly affect the structure and function of MTG. This method is simple, economic and effective in the activation of pro-enzyme and has a great potential for industrial application.

Key words: protein splicing, intein, pro-enzyme activation, transglutaminase, Streptomyces hygroscopicus

中图分类号:

Q816

杜坤，周丽，堵国成，陈坚，周哲敏. 内含肽介导谷氨酰胺转胺酶酶原的活化[J]. 食品科学, 2013, 34(9): 90-94.

DU Kun，ZHOU Li，DU Guo-cheng，CHEN Jian，ZHOU Zhe-min. Intein-mediated Activation of Transglutaminase from Streptomyces hygroscopicus[J]. FOOD SCIENCE, 2013, 34(9): 90-94.

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内含肽介导谷氨酰胺转胺酶酶原的活化

Intein-mediated Activation of Transglutaminase from Streptomyces hygroscopicus

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