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Although the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum catalyzed the reductive amination of pyruvic acid,with the formation of D-alanine,it showed low activities toward other larger 2-keto acids.In order to enlarge the substrate binding site for accommodating larger 2-keto acids,amino acid residues interacting with the L-center of meso-diaminopimelate dehydrogenase,Phe146,Thr171,Arg181 and His227,were selected for site-saturation mutagenesis.By enzymatic activity screening with phenylpyruvic acid as the substrate,several mutants were obtained with improved activities.Among them,the single mutation H227V had a specific activity of 2.39 ± 0.06U?mg-1,which was 35.1-fold enhancement over wild type enzyme.The positive mutants were combined to investigate the synergetic effects of different sites,but the resulting double and triple variants did not show higher activity.All the mutant enzymes did not show significant difference in Km comparing with wild type,but the kcat values ranged from about 110.5 to 3981.1s-1.Molecular dynamics simulations revealed that the distance between the Cα of imine intermediate and the C4 of NADPH nicotinamide ring in the wild type enzyme (4.8 ?) was longer than the mutant H227V (3.6 ?),consistent with the higher activity of H227V.