房鑫,田思思,姚忠,王浩绮,周治,熊强,孙芸,韦萍

• 1:南京工业大学食品与轻工学院

摘要(Abstract)：

γ-谷氨酰转肽酶(GGT)是生物体内谷氨酰循环的关键酶,在生物有机合成领域具有重要的应用价值。今建立了大肠杆菌和枯草芽孢杆菌GGT的克隆、重组及诱导表达方法。在此基础上,分别考察了重组大肠杆菌GGT(rE_GGT)和重组枯草芽孢杆菌GGT(rB_GGT)的催化特性和稳定性。结果表明,rE_GGT对供体对硝基苯胺(GpNA)的亲和力(Km)优于rB_GGT,但rB_GGT的催化常数(kcat)高达3.48×105 s?1,是rE_GGT的20倍。稳定性研究表明,rE_GGT在pH 6~9和T<45℃条件下的稳定性明显优于rB_GGT;但当温度超过45℃时,rB_GGT的稳定性更佳。通过对rE_GGT和rB_GGT的氨基酸组成及其大、小亚基界面相互作用力的分析表明,rE_GGT大小亚基间疏水键的数目明显多于rB_GGT,有助于稳定其四级结构,使其在中低温下具有良好的稳定性;而rB_GGT氨基酸组成中荷电残基和芳香族残基的比例较高,可有效维持其二级结构的稳定性。

关键词(KeyWords)： γ-谷氨酰转肽酶;重组;热失活;反应动力学;稳定性

Abstract:

Keywords:

基金项目(Foundation): 江苏省高校自然科学研究重大基金资助项目(10KJA530014);; 江苏省科技支撑计划(农业)基金资助项目(SBE 2012399);; 江苏省自然科学基金资助项目(BK2012825);; 国家自然科学基金(21206072)

作者(Author): 房鑫,田思思,姚忠,王浩绮,周治,熊强,孙芸,韦萍

参考文献(References)：

• [1]Meister A,Suresh S T,Owen W G.γ-Glutamyltranspeptidase[J].Methods in Enzymology,1981(77):237-253.
• [2]Keillor J W,Castonguay,Lherbet C.Gamma-Glutamyl transpeptidase substrate specificity and catalytic mechanism[J].Method Enzymol,2005(401):449-467.
• [3]Suzuki H,Izuka S,Miyakawa N,et al.Enzymatic production of theanine,an“umami”component of tea,from glutamine and ethylamine with bacterialγ-glutamyltranspeptidase[J].Enzyme Microb Tech,2002(32):884-889.
• [4]Kumagai H,Echigo T,Suzuki H,et al.Synthesis of gamma-glutamyl-DOPA from L-glutamine and L-DOPA by gamma-glutamyltranspeptidase of Escherichia coli K-12[J].Agr BiolChem,1988(52):1741-1745.
• [5]Suzuki H,Miyakawa N,Kumagai H,et al.Enzymatic production ofγ-L-glutamyltaurine through the transpeptidation reaction ofγ-glutamyltranspeptidase from Escherichia coli K-12[J].Enzyme Microb Tech,2002(30):883-888.
• [6]Wallace John B,Hani Z M.Process for the production ofγ-glutamylcysteine:WO,2006102722[P].2006-03-31.
• [7]Suzuki H,Kato K.Kumagai H.Development of an efficient enzymatic production of D-glutamyl-L-tryptophan(SCV-07),a prospective medicine for tuberculosis,with bacterialγ-glutamyltranspeptidase[J].J Biotechnol,2004(111):291-295.
• [8]Suzuki,H,Izuka S,Minami H,et al.Use of bacterialγ-glutamyltranspeptidase for enzymatic synthesis ofγ-D-glutamyl compounds[J].Appl Environ Microb,2003(69):6399-6404.
• [9]Suzuki H,KajimotoY,KumagaiH.Improvement of the bitter taste of amino acids through the transpeptidation reaction of bacterialγ-glutamyltranspeptidase[J].Agric Food Chem,2002,50(2):313-318.
• [10]Jun H,Makoto I,Hideyuki S.Identification of catalytic nucleophile of escherichia coliγ-glutamyltranspeptidase by mechanism-based affinity label[J].ICR Annual Report,2000(7):44-45.
• [11]Suzuki H,Kumagai H,Tochikura T.Gamma-glutamyl-transpeptidase from Escherichia coli k-12:purification and properties[J].Journal of Bacteriology,1986,168(3):1325-1331.
• [12]Yao Y F,Weng Y M,Hu H Y,et al.Expression optimization and biochemical characterization of a recombinant gammaglutamyltranspeptidase from Escherichia coli Novablue[J].Protein Journal,2006,25(6):431-441.
• [13]Wu Q,Xu H,Zhang L J,et al.Production,purification and properties of gamma-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2[J].Journal of Molecular Catalysis B-Enzymatic,2006,43(1-4):113-117.
• [14]Ogawa Y,Hosoyama H,Hamano M,et al.Purification and properties of gamma-glutamyltranspeptidase from Bacillus subtilis(natto)[J].Agricultural and Biological Chemistry,1991,55(12):2971-2977.
• [15]Xu K,Strauch M A.Identification,sequence,and expression of the gene encoding gamma-glutamyltranspeptidase in Bacillus subtilis[J].Journal of Bacteriology,1996,178(14):4319-4322.
• [16]Abe K,Ito Y,Ohmachi T,et al.Purification and properties of two isozymes of gamma-glutamyltranspeptidase from Bacillus subtilis TAM-4[J].Bioscience Biotechnology and Biochemistry,1997,61(10):1621-1625.
• [17]Okada T,Suzuki H,Wada K,et al.Crystal structures of gamma-glutamyltranspeptidase from Escherichia coli,a key enzyme in glutathione metabolism,and its reaction intermediate[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(17):6471-6476.
• [18]Suzuki H,Kumagai H,Tochikura T.Isolation,genetic-mapping,and characterization of Escherichia coli K-12 mutants lacking gamma-glutamyltranspeptidase[J].Journal of Bacteriology,1987,169(9):3926-3931.
• [19]Wada K,Irie M,Suzuki H,et al.Crystal structure of the halotolerant gamma-glutamyltranspeptidase from Bacillus subtilis in complex with glutamate reveals a unique architecture of the solvent-exposed catalytic pocket[J].Febs Journal,2010,277(4):1000-1009.
• [20]JIA Xiao-he(贾晓鹤),CHEN Li(陈莉),ZHAO Ning-wei(赵宁伟),et al.Production of L-theanine by bioconversion with recombinantγ-Glutamyltranspeptidase(生物转化法应用重组谷氨酞转肤酶合成L-茶氨酸)[J].Science and Technology of Food Industry(食品工业科技),2008,29(2):166-169.
• [21]WANG Na(王娜),ZHANG Jie(张洁),SHEN Wei(沈微),et al.Construction fermentation of engineered strain and properties of recombinantγ-glutamyltranspeptidase(γ-谷氨酰基转移酶(GGT)基因工程菌的构建及其发酵条件的初步研究)[J].China Biotechnology(生物工程杂志),2006,26(11):48-53.
• [22]SHI Lin-mei(施林姝),WANG Dong-ming(王东明),ZHAN Zha-jun(占扎君),et al.Expression ofγ-gGlutamyltranspeptisase(γ-ggt)from pET plasmid using lactose as inducer(乳糖诱导γ-ggt基因在大肠杆菌中的表达)[J].Pharmaceutical Biotechnology(药物生物技术),2010,17(4):287-290.
• [23]Taniguchi N,Ikeda Y.Gamma-glutamyltranspeptidase catalytic mechanism and gene expression[J].Adv Enzymol Relat Areas Mol Biol,1998,72:239-278.
• [24]Roberto Fernandez-Lafuente.Stabilization of multimeric enzymes:Strategies to prevent subunit dissociation[J].Enzyme and Microbial Technology,2009,45:405-418.
• [25]Liskin S K,Brown C S.Resmap:automated representation of macromolecular interfaces as two-dimensional networks[J].Bioinformatics,2005,21(15):3327-3328.
• [26]Liskin S K.Using networks to identify fine structural differences between functionally distinct protein states[J].Biochemistry,2004,43(34):10886-10895.