大肠杆菌茶氨酸合成酶基因γ-GGT的生物信息学分析
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摘要
γ-谷氨酰转肽酶(γ-GGT)是催化L-茶氨酸合成的关键酶之一,对其编码基因的解析有助于进行茶氨酸的合成调控和异源表达生产研究,本论文利用生物信息学方法,对大肠杆菌(Escherichia coli)γ-GGT基因的结构和特性进行分析。结果显示,E.coliγ-GGT基因编码580个氨基酸,主要氨基酸为甘氨酸和亮氨酸,γ-GGT酶蛋白等电点为5.38,分子量为61.7 kDa,属于酸性不稳定亲水性蛋白,具有信号肽序列和保守功能结构域,在细胞质周质中发挥作用,其二级结构元件主要是α-螺旋和无规卷曲,在微生物中进化较为保守。研究结果为进一步研究γ-GGT基因功能及其应用奠定了基础。
γ-Glutamyltranspeptidase(γ-GGT) is one of the key enzymes catalyzing the synthesis of L-theanine. Analyzing its coding gene is great significance for the production of theanine. In this study, a bioinformatics approach was used to analyse the structure and function of genes in Escherichia coli γ-GGT. The results showed that the γ-GGT gene of E.coli encoded 580 amino acids. The main amino acids were Gly and Leu. The isoelectric point of γ-GGT enzyme protein was 5.38 with a molecular weight of 61.7 kDa. It was an unstable hydrophilic protein and had a signal peptide sequence and a conserved functional domain and played a role in the periplasm. The secondary structural elements were mainly Helix and Coil, which played a conservative role in the evolution.
γ-Glutamyltranspeptidase(γ-GGT) is one of the key enzymes catalyzing the synthesis of L-theanine. Analyzing its coding gene is great significance for the production of theanine. In this study, a bioinformatics approach was used to analyse the structure and function of genes in Escherichia coli γ-GGT. The results showed that the γ-GGT gene of E.coli encoded 580 amino acids. The main amino acids were Gly and Leu. The isoelectric point of γ-GGT enzyme protein was 5.38 with a molecular weight of 61.7 kDa. It was an unstable hydrophilic protein and had a signal peptide sequence and a conserved functional domain and played a role in the periplasm. The secondary structural elements were mainly Helix and Coil, which played a conservative role in the evolution.
引文
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[3] 吕毅,郭雯飞,倪捷儿,等. 茶氨酸的生理作用及合成[J].茶叶科学,2003,23(1):1-5.
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[19] 郭建华. 蔗糖浓度对渗透休克法提取细胞周质重组蛋白的影响[J].安徽农业科学,2017,45(4):147-148.
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[21] Suzuki H,Kumagai H. Autocatalytic Processing of γ-Glutamyltranspeptidase[J].Journal of Biological Chemistry,2002,277(45):43536-43543.
[22] Ishiye M, Yamashita M, Niwa M. Molecular cloning of the γ-glutamyltranspeptidase gene from a Pseudomonas strain [J].Biotechnol ogy Progree,1993,9 (3):323-331.
[23] Suzuki H,Kumagai H. Gamma-glutamyltranspeptidase from Escherichia coli K-12[J].Seikagaku the Journal of Japanese Biochemical Society,1989,61(6):491-496.
[24] Chevalier C,Thiberge J M,Ferrero R L,et al. Essential role of Helicobacter pylori γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice[J].Molecular Microbiology,2010,31(5):1359-1372.
[25] Xu K,Strauch MA. Identification,sequence,and expression of the gene encoding gamma-glutamyltranspeptidase in Bacillus subtilis[J].Journal of Bacteriology,1996,178(14):4319-4322.
[26] Lin LL,Chou PR,Hua YW,et al.Overexpression,one-step purification,and biochemical characterization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis[J].Applied Microbiology and Biotechnology,2006,73 (1):103-112.
[27] JeanF. Tomb,Owen White,Anthony R. Kerlavage,et al. The complete genome sequence of the gastric pathogen Helicobacter pylori[J].Nature,1997,388(6642):539.
[2] Sakato Y. The chemical constituents of tea :III. A new amide theanine[J]. Nippon Nogeikagaku Kaishi,1949(23):262-267.
[3] 吕毅,郭雯飞,倪捷儿,等. 茶氨酸的生理作用及合成[J].茶叶科学,2003,23(1):1-5.
[4] 王丽鸳,王贤波,成浩,等. 基因工程菌生物合成茶氨酸条件研究[J].茶叶科学,2007,27(2):111-116.
[5] Yokogoshi H,Kobayashi M. Hypotensive effect of gamma-glutamylmethylamide in spontaneously hypertensive rats[J].Life Sciences,1998,62(12):1065-1068.
[6] Kamath AB,Wang L,Das H,et al. Antigens in tea-beverage prime human Vγ2Vδ2 T cells in vitro and in vivo for memory and nonmemory antibacterial cytokine responses[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(10):6009-6014.
[7] Yokogoshi H,Mochizuki M,Saitoh K. Theanine-induced reduction of brain serotonin concentration in rats[J].Bioscience Biotechnology & Biochemistry,1998,62(4):816-817.
[8] Kakuda T,Nozawa A,Unno T,et al. Inhibiting effects of theanine on caffeine stimulation evaluated by EEG in the rat[J].Journal of the Agricultural Chemical Society of Japan,2000,64(2):287-293.
[9] 帅玉英. γ-谷氨酰转肽酶的纯化和性质及其用于L-茶氨酸的生物制备研究[D].扬州:江南大学,2011.
[10] 王风青,梁金钟,付大伟,等. γ-PGA降解酶系基因pgdS和ggt的克隆及生物信息分析[J].食品工业科技,2016,37(16):190-194.
[11] Suzuki H,Izuka S,Miyakawa N,et al. Enzymatic production of theanine,an “umami” component of tea,from glutamine and ethylamine with bacterial γ-glutamyltranspeptidase[J].Enzyme & Microbial Technology,2002,31(6):884-889.
[12] Boanca G,Sand A,Okada T,et al. Autoprocessing of Helicobacter pylori gamma-glutamyltranspeptidase leads to the formation of a threonine-threonine catalytic dyad[J].Journal of Biological Chemistry,2007,282(1):534.
[13] Takahashi H,Watanabe H. Post‐translational processing of Neisseria meningitidisγ‐glutamyl aminopeptidase and its association with inner membrane facing to the cytoplasmic space[J].Fems Microbiology Letters,2004,234(1):27-35.
[14] 杨运桂,徐京宁,胡泰山,等.信号肽疏水性的提高促进青霉素G酰化酶分泌[J].生物化学与生物物理学报(英文),2000(2):163-168.
[15] Izard J W,Doughty M B,Kendall D A. Physical and Conformational Properties of Synthetic Idealized Signal Sequences Parallel Their Biological Function[J].Biochemistry,1995,34(31):9904.
[16] 郭亮,沈微,王正祥,等. 生物转化法生产茶氨酸的重组大肠杆菌的构建[J].食品与生物技术学报,2005,24(2):41-45.
[17] Suzuki H,Kumagai H,Tochikura T. γ-Glutamyltranspeptidase from Escherichia coli K-12:Formation and location [J].Journal of Bacteriol,1986,168 (3):1332-1335.
[18] Hiromichi Minami,Hideyuki Suzuki,Hidehiko Kumagai. Salt-tolerant γ-glutamyltranspeptidase from Bacillus subtilis,168 with glutaminase activity[J].Enzyme & Microbial Technology, 2003,32(3):431-438.
[19] 郭建华. 蔗糖浓度对渗透休克法提取细胞周质重组蛋白的影响[J].安徽农业科学,2017,45(4):147-148.
[20] 贾晓鹤,陈莉,赵宁伟,等. 生物转化法应用重组谷氨酰转肽酶合成L-茶氨酸[J].食品工业科技,2008(2):166-169.
[21] Suzuki H,Kumagai H. Autocatalytic Processing of γ-Glutamyltranspeptidase[J].Journal of Biological Chemistry,2002,277(45):43536-43543.
[22] Ishiye M, Yamashita M, Niwa M. Molecular cloning of the γ-glutamyltranspeptidase gene from a Pseudomonas strain [J].Biotechnol ogy Progree,1993,9 (3):323-331.
[23] Suzuki H,Kumagai H. Gamma-glutamyltranspeptidase from Escherichia coli K-12[J].Seikagaku the Journal of Japanese Biochemical Society,1989,61(6):491-496.
[24] Chevalier C,Thiberge J M,Ferrero R L,et al. Essential role of Helicobacter pylori γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice[J].Molecular Microbiology,2010,31(5):1359-1372.
[25] Xu K,Strauch MA. Identification,sequence,and expression of the gene encoding gamma-glutamyltranspeptidase in Bacillus subtilis[J].Journal of Bacteriology,1996,178(14):4319-4322.
[26] Lin LL,Chou PR,Hua YW,et al.Overexpression,one-step purification,and biochemical characterization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis[J].Applied Microbiology and Biotechnology,2006,73 (1):103-112.
[27] JeanF. Tomb,Owen White,Anthony R. Kerlavage,et al. The complete genome sequence of the gastric pathogen Helicobacter pylori[J].Nature,1997,388(6642):539.