生物学杂志 ›› 2021, Vol. 38 ›› Issue (6): 31-.doi: 10.3969/j.issn.2095-1736.2021.06.031

• 研究报告 • 上一篇    下一篇

NADPH依赖型谷氨酸脱氢酶的辅酶特异性改造

  

  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
  • 出版日期:2021-12-18 发布日期:2021-12-15
  • 通讯作者: 杨立荣,教授,研究方向为生物催化与转化,E-mail: lryang@zju.edu.cn
  • 作者简介:陆利兵,硕士研究生,研究方向为生物催化与转化,E-mail: 21728084@zju.edu.cn
  • 基金资助:
    国家自然科学基金面上项目( 21476199) ; 国家“973”计划项目( 2011CB710800)

Coenzyme specific modification of NADPH-dependent glutamatedehydrogenase

  1. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University,Hangzhou 310027, China
  • Online:2021-12-18 Published:2021-12-15

摘要: 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 kcat/Km)增强了1 607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,T10 min1/2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。

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

Abstract: 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 1 607 times(Ratio of kcat/Km), 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 T10 min1/2 reached 58.1 ℃, and the PpGDH-D264V had good stability under neutral and slightly alkaline environment, which laid a foundation for subsequent application.

Key words: proteinogenic amino acids, glutamate dehydrogenase, nicotinamide coenzyme, semirational design, homologus recombination

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