苯甘氨酸氨基转移酶基因hpgt的原核优化表达与酶动力学特性研究  

Optimized Prokaryotic Expression of the Recombinant hpgt Gene and Enzyme Kinetic Characteristics

作　　者：王宗瑞[1] 赵广荣[1] 

机构地区：[1]天津大学化工学院制药工程系,天津300072

出　　处：《中国生物工程杂志》2012年第5期51-57,共7页China Biotechnology

基　　金：教育部博士点基金资助项目(20090032110015)

摘　　要：苯甘氨酸氨基转移酶(4-Hydroxyphenylglycine aminotransferase)是假单胞菌所产生的一种能够合成D-苯甘氨酸的重要转氨酶。利用密码子优化技术,合成苯甘氨酸转移酶基因。构建原核重组质粒pCDF-hpgt,转入感受态细胞E.coli BL21(DE3),优化表达His-HpgT蛋白。利用Ni-NTA柱纯化技术获得高纯度的His-HpgT融合蛋白。分别测定融合蛋白在正反向反应中的酶活力单位及最佳的反应温度、pH值及其他动力学参数,并对该酶特性作相关的机理分析。测定结果表明,正向反应和反向反应的酶比活力分别为749mU/mg、2 257mU/mg,此酶分解苯甘氨酸的能力要强于合成苯甘氨酸;正向反应的最适温度与pH分别是35℃和8.0;由米氏方程得出该酶对苯甘氨酸的亲和力远大于谷氨酸;较低浓度的苯乙醛酸即可抑制反应的进行。4-Hydroxyphenylglycine aminotransferase which can synthesize D-phenylglycine transaminase is produced by Pseudomonas.The hpgt gene was synthesized through the codon optimization technology.The recombinant prokaryotic plasmid pCDF-hpgt was obtained.The plasmid was transformed into the competent cell E.coli BL21（DE3）.The recombinant His-HpgT protein was obtained after the optimized expression and purified by nickel chelate affinity chromatography method.The enzyme activity of the forward and reverse reactions was measured and the activity of the forward reaction reached 749mU/mg which was lower than the reverse,2 257mU/mg.Also the optimized temperature and pH were measured,with the result of 35℃and 8.0.Other kinetic parameters and the mechanism analysis of enzyme characteristics were explained.The enzyme affinity to phenylglycine was higher than the glutamate which obtained by the Michaelis-Menten equation;and the reaction was inhibited by the lower concentration of phenylglyoxylic acid.

关 键 词：基因hpgt D-苯甘氨酸 原核优化表达 酶动力学特性 

分 类 号：Q78[生物学—分子生物学]