NADPH依赖型谷氨酸脱氢酶的辅酶特异性改造  

Coenzyme specific modification of NADPH-dependent glutamate dehydrogenase

作　　者：陆利兵 周海胜[1] 吴坚平[1] 杨立荣[1] LU Libing;ZHOU Haisheng;WU Jianping;YANG Lirong(Institute of Bioengineering,College of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China)

机构地区：[1]浙江大学化学工程与生物工程学院生物工程研究所,杭州310027

出　　处：《生物学杂志》2021年第6期31-35,共5页Journal of Biology

基　　金：国家自然科学基金面上项目(21476199);国家“973”计划项目(2011CB710800)。

摘　　要：基于结构信息及保守序列比对,通过半理性设计和高通量筛选,将来源于Pseudomonas putida的NADPH依赖型谷氨酸脱氢酶PpGDH改造为NADH依赖型。与原始PpGDH相比,突变体PpGDH-D264V对NADH的偏好性(Ratio of k_(cat)/K_(m))增强了1607倍,并具有对多种酮酸底物的催化活力,可用于制备多种非蛋白质氨基酸。对PpGDH-D264V进行酶学性质表征,其最适温度为45℃,最适pH值8.0,T_(1/2)^(10 min)温度达到58.1℃,在中性及弱碱性环境下具备良好的稳定性,为后续应用奠定基础。Based on structural information and conservative sequence alignment,this study modified the NADPH-specific glutamate dehydrogenase,from Pseudomonas putida,to NADH-dependent form in the future through semi-rational design and high-throughput screening.Compared that with the original PpGDH,the preference of the mutant PpGDH-D264V to NADH was increased by 1607 times(Ratio of k_(cat)/K_(m)),and it had the catalytic activity of various keto acids,which could be used to prepare various non-protein amino acids.The enzymic properties of PpGDH-D264V were characterized.The optimal temperature was 45℃,the optimal pH was 8.0,the temperature of T_(1/2)^(10 min) reached 58.1℃,and the PpGDH-D264V had good stability under neutral and slightly alkaline environment,which laid a foundation for subsequent application.

关 键 词：非蛋白质氨基酸 谷氨酸脱氢酶 烟酰胺辅酶 半理性设计 同源重组 

分 类 号：Q936[生物学—微生物学] TQ925[轻工技术与工程—发酵工程]