GshF在大肠杆菌中的表达及酶学性质研究

中国生物工程杂志

2013, Vol. 33

Issue (12): 21-28

研究报告

GshF在大肠杆菌中的表达及酶学性质研究

陈永露, 吴绵斌, 林建平, 杨立荣, 岑沛霖

生物质化工教育部重点实验室浙江大学化学工程与生物工程系杭州 310027

Characterization of GshF Expressed in Escherichia coli

CHEN Yong-lu, WU Mian-bin, LIN Jian-ping, YANG Li-rong, CEN Pei-lin

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

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摘要： 谷胱甘肽是参与细胞活动的最普遍的非蛋白巯基化合物，是机体内重要的活性三肽，因其抗氧化作用而在医药、食品、化妆品等行业有着广泛的应用，生物合成是制备谷胱甘肽的主要方法，一直引起广泛关注。近年来发现的双功能酶GshF可同时催化谷胱甘肽生物合成的两步反应，有望提高酶法催化产谷胱甘肽的效率，但该酶的酶学性质尚未得到充分研究。克隆了来自Streptococcus thermophilus的双功能酶基因gshF，并用大肠杆菌表达，研究表明，重组菌于37℃培养4 h后经0.1 mmol/L IPTG诱导，然后在30℃下表达，胞内酶活可达44 U/L。表达产物经破胞、离心、亲和层析等步骤分离纯化后，进一步考察了其相关酶学性质，结果表明，该酶最适pH为8.0，最适温度为37℃，还研究了其最适pH和温度下的稳定性。最终通过重组菌酶法催化合成谷胱甘肽得到的最高产量为2.11g/L。这些对该酶在谷胱甘肽生物合成上的应用有重要的借鉴作用。

关键词： 酶法催化; 谷胱甘肽; GshF; 大肠杆菌; 酶学性质

Abstract: Glutathione, an important active tripeptide, is the most common non-protein thiol compound involved in cell activity. It has a wide range of applications in the pharmaceutical, food, cosmetics and other industries because of its antioxidant effect. And improving glutathione production via biosynthesis is the main method and has always been a focus. The bifunctional enzyme GshF discorvered recently can catalyze the two-step reaction of glutathione biosynthesis simultaneously and is expected to improve the catalytic efficiency. However, the properties of the enzyme have not been sufficiently studied. A bifunctional enzyme gene gshF from Streptococcus thermophilus was cloned and expressed in E. coli. GshF reached its highest activity of 44 U/L when the recombinant was induced with 0.1mmol/L IPTG at 30℃ after being cultivated for 4 hours at 37℃. The related enzymatic properties of GshF were achieved after such purification steps as cell lysis, centrifugation and affinity chromatography. The optimum pH and temperature was 8.0 and 37℃ respectively. The stabilities of GshF under its optimum pH and temperature were also studied. The final maximum yield of glutathione was 2.11g/L by enzymatic synthesis of recombinant bacteria. It is of important reference on the application of the enzyme in the biosynthesis of glutathione.

Key words: Glutathione GshF Escherichia coli Enzymatic properties Enzymatic catalysis

收稿日期: 2013-10-17 出版日期: 2013-12-25

ZTFLH:

Q786

基金资助: 浙江省重点科技创新团队计划资助项目（2011R50002）

通讯作者: 林建平，E-mail：linjp@zju.edu.cn E-mail: linjp@zju.edu.cn

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引用本文:

陈永露, 吴绵斌, 林建平, 杨立荣, 岑沛霖. GshF在大肠杆菌中的表达及酶学性质研究[J]. 中国生物工程杂志, 2013, 33(12): 21-28.

CHEN Yong-lu, WU Mian-bin, LIN Jian-ping, YANG Li-rong, CEN Pei-lin. Characterization of GshF Expressed in Escherichia coli. China Biotechnology, 2013, 33(12): 21-28.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/21

[1] 朱玉强.发酵法生产谷胱甘肽研究进展.发酵科技通讯, 2008, 37(2):22-26. Zhu Y Q. The research development of fermentation production of glutathione. Fermentation Science and Technology, 2008, 37 (2): 22-26.
[2] 王小娟, 卢美欢, 王卫卫, 等. 高产谷胱甘肽菌株选育研究进展.中国酿造, 2011, (5): 5-7. Wang X J, Lu M H, Wang W W, et al. The research development of glutathione yield strain.China Brewing 2011, (5): 5-7.
[3] Sakuda S, Zhou Z Y, Yamada Y. Structure of a novel disulfide of 2-(N-acetylcysteinyl) amido-2-deoxy-alpha-D-glucopyranosyl-myo-inositol produced by Streptomyces sp. Biosci Biotechnology and Biochemistry, 1994, 58: 1347-1348.
[4] 刘振玉.谷胱甘肽的研究与应用.生命的化学, 1995, 15(1):19-21. Liu Z Y. The Research and application of glutathione. Chemistry of Life, 1995, 15 (1): 19-21.
[5] Shelly C L. Regulation of glutathione synthesis. Molecular Aspects of Medicine, 2009, 30(1-2): 42-59.
[6] Masi P L, Veeravalli K, Georgiou G, et al. The many faces of glutathione in bacteria. Antioxidants and Redox Signaling, 2006, 8 (5-6): 753-762.
[7] Liao X Y, Shen W, Chen J, et al. Improved glutathione production by gene expression in Escherichia coli. Letters in Applied Microbiology, 2006, 43(2): 211-214.
[8] Li W, Li Z M, Ye Q. Enzymatic synthesis of glutathione using yeast cells in two-stage. Reaction. Bioprocess and Biosystems Engineering, 2010, 33: 675-682.
[9] Newton G L, Arnold K, Price M S, et al. Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. Journal of Bacteriology, 1996, 178: 1990-1995.
[10] Fahey R C, Brown W C, Adams W B, et al. Occurrence of glutathione in bacteria. Journal of Bacteriology, 1978, 133: 1126-1129.
[11] Janowiak B E, Griffith O W. Glutathione synthesis in Streptococcus agalactiae—One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities. Journal of Biological Chemistry, 2005, 280 (12): 11829-11839.
[12] Gopal S I, Borovok I, Ofer A, et al. A multidomain fusion protein in Listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis. Journal of Bacteriology, 2005, 187 (11): 3839-3847.
[13] Vergauwen B D, Vos D D, Beeumen J V, et al. Characterization of the bifunctional gamma-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida. Journal of Biological Chemistry, 2006, 281 (7): 4380-4394.
[14] Li W, Li Z M, Yang J H, et al. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. Journal of Biotechnology, 2011, 154 (4): 261-268.
[15] 刘云平.三种重要检测用酶的重组表达及酶学特性研究.浙江大学硕士学位论文, 2012.24-25. Liu Y P. Studies on recombinant expression and enzymatical characteristics of three improtant enzymes for analytical applications. Master's Degree Thesis of Zhejiang University, 2012. 24-25.
[16] Nuc P, Nuc K. Recombinant protein production in Escherichia coli. Postepy Biochem, 2006, 52: 44-84.
[17] Tabor S, Richardson C C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proceedings of the National Academy of Sciences of USA, 1985, 82: 1074-1078.
[18] Liedschulte V A, Wachter A, An Z G, et al. Exploiting plants for glutathione (GSH) production: Uncoupling GSH synthesis from cellular controls results in unprecedented GSH accumulation. Plant Biotechnology Journal, 2010, 8 (7): 807-820.

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