苯甘氨酸氨基转移酶基因<em>hpgt</em>的原核优化表达与酶动力学特性研究

中国生物工程杂志

2012, Vol. 32

Issue (05): 51-57

研究报告

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

王宗瑞, 赵广荣

天津大学化工学院制药工程系天津 300072

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

WANG Zong-rui, ZHAO Guang-rong

Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology of Tianjin University, Tianjin 300072, China

全文: PDF(1052 KB) HTML

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。

关键词： 基因hpgt; D-苯甘氨酸; 原核优化表达; 酶动力学特性

Abstract:

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.

Key words: hpgt gene D-phenylglycine Optimized prokaryotic expression Enzyme kinetic characteristics

收稿日期: 2012-02-20 出版日期: 2012-05-25

ZTFLH:

Q78

基金资助:

教育部博士点基金资助项目(20090032110015)

通讯作者: 赵广荣 E-mail: grzhao@tju.edu.cn

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引用本文:

王宗瑞, 赵广荣. 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[J]. 中国生物工程杂志, 2012, 32(05): 51-57.

WANG Zong-rui, ZHAO Guang-rong. Optimized Prokaryotic Expression of the Recombinant hpgt Gene and Enzyme Kinetic Characteristics. China Biotechnology, 2012, 32(05): 51-57.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I05/51

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