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