902013,Vol.34,No.09
食品科学
※生物工程
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
号214122)
摘要：链莓菌来源的谷氨酰胺转胺酶(microbialtransglutaminase，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。为解决上述间题，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、25C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ODs0m为1.0时测得)。重组型MTG的酶比活力和K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；醇原活化：谷氨酰胺转胺酶：吸水链霉菌
Intein-mediated Activation of Transglutaminase from Streptomyces hygroscopicu
DU Kun', ZHOU Li', DU Guo-cheng', CHEN Jian'2, 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)
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 detemined to be 24 h at 25 C and pH 7.0 in vitro, resulting in an activity of 0.23 U/mL (cell concentration was diluted to ODom at 1.0). The specific activity and Kof 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, cconomic 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
doi:10.7506/spkx1002-6630-201309020
文献标志码：A
谷氨酰胺转胺酶(transglutaminase，TGase，EC 2.3.2.13)是一种能够催化酰基转移反应的酶。它可以催化谷氨酰胺残基的}-羧酰胺基与各种酰基受体发生反应，实现蛋白质分子内、分子间发生交联，从而极大地改变蛋白质的性质"。因此谷氨氮酰胺转胺酶在食品工业有着广泛的应用"，例如重组肉块，增强和改善食品的风味，延长某些食品的保质期等。另外谷氨酰胺转胺酶在生物医
文章编号：1002-6630(2013)09-0090-05
药、组织工程、纺织和皮革工业中也有着广泛的应用前景，例如修饰蛋白类药物以延长其半衰期，用于器官再造技术，增强羊毛类和蚕丝类纺织原料的韧性等。
目前，谷氨酰胺转胺酶的生产方法主要有2种：从动物组织中提取和利用微生物发酵法生产围。由于从动物组织中提取的TGase价格昂贵，限制了其应用，因而开发微生物发酵生产TGase(microbialtranglutaminase
收稿日期：2013-02-25
基金项目：国家自然科学基金项目(31070711)：教育部科学技术研究重大项目(311023)
作者简介：杜坤(1989一)，男，硕士研究生，研究方向为酶学与酶工程。E-mail：dukun2727@163.com
*通信作者：周智敏(1969—)，男，教授，博士，研究方向为酶学与酶工程。E-mail：zhemzhou@jiangnan.edu.cn 万方数据