γ-谷氨酰转肽酶的酶学性质及其转肽反应机制  

Properties and Catalytic Mechanism of γ-Glutamyltranspeptidase from B.subtilis NX-2

作　　者：汪前[1] 姚忠[1] 荀志金[1] 徐晓滢[1] 徐虹[1] 韦萍[1] 

机构地区：[1]南京工业大学制药与生命科学学院,江苏南京210009

出　　处：《高校化学工程学报》2008年第2期288-293,共6页Journal of Chemical Engineering of Chinese Universities

基　　金：国家973项目(2003CB7160004)资助;江苏省高校"青蓝工程"项目资助;南京工业大学国家自然科学基金预研项目

摘　　要：γ–谷氨酰转肽酶(γ-glutamyltranspeptidase,GGT)能专一地催化γ-谷氨酰基的转移,在γ-谷氨酰基类衍生物的合成方面具有重要的应用价值。今利用Bacillus subtilis NX-2发酵生产GGT,上清液中的GGT经硫酸铵沉淀后,以DEAE Sepharose FF和Source15Q两步离子交换进行纯化。以γ-D-Gln-L-Trp(SCV-07)为目的产物,研究了GGT的基本酶学性质,确定了其最适反应温度为40℃,最适pH值10.0,供体/受体浓度为5:7,最适反应时间为4h,产物转化率可高达42%。结合反应进程曲线,分析了GGT的作用机制,并通过实验证实了GGT不仅具有转肽活性,还可催化产物的不可逆水解,这是导致产物回收率下降的关键原因。经测定得到GGT的转肽反应米氏常数(Km)为5.08mmol·L-1,最大反应速率(rmax)为0.034mmol·(min·L)-1;对γ-D-Gln-L-Trp的水解反应米氏常数(Km’)为2.267mmol·L-1,最大反应速率(rmax’)为0.012mmol·(min·L)-1。Since it can specially catalyze the transfer of γ-glutamyl moiety,the γ-glutamyltranspeptidase（GGT） has important practical value for the synthesis of analogues of γ-glutamyl-containing compounds.In order to obtain high purity GGT,the GGT produced from Bacillus subtilis NX-2 was purified by ammonium sulfate fractional precipitation and then two-step ion exchange chromatography of DEAE Sepharose FF and Source 15Q.The enzymology properties of GGT in the synthesis of γ-D-Gln-L-Trp（SCV-07） were investigated,and the optimal synthesis conditions of pH 10.0,the γ-glutamyl donor（D-Gln） to acceptor（L-Trp） ratio of 5：7,reaction temperature of 40℃ and reaction time of 4 h were found,under these optimal conditions,high conversion ratio reached 42%.According to the conversion process curve of γ-D-Gln-L-Trp,the catalytic mechanism of GGT was discussed.It was demonstrated that the GGT can catalyze not only the reaction of transpeptidation,but also the irreversible hydrolysis of the products,which results in the decrease of the yield of the products.The Michaelis constant of transpeptidation reaction and its maximum reaction rate were determined as Km=5.08 mmol·L^-1 and rmax=0.034 mmol·（min·L）^ -1,respectively,while the Michaelis constant of hydrolysis reaction and its maximum reaction rate were measured respectively as Km＇=2.267 mmol·L^-1 and rmax＇=0.012 mmol·（min·L）^ -1.

关 键 词：Γ-谷氨酰转肽酶 纯化 性质 γ-D-谷氨酰-L-色氨酸 转肽反应机制 

分 类 号：Q555.3[生物学—生物化学]