The recombinant N-acyl-D-amino acid amidohydrolase (N-D-AAase) of Variovorax paradoxus Iso1 was unstable during protein purification and storage at 4 degrees C. Since the methionine oxidation might be the artificial factor leading to the inactivation of N-D-AAase, eight potential oxidation sensitive methionine residues of the enzyme were individually substituted with leucine utilizing site-directed mutagenesis. Among them, five mutants, M39L, M56L, M221L, M254L, and M352L remained at least 70% of wild-type specific activity. The enzyme kinetic parameters of M221L revealed a 44% decrease in K-m, and finally reflected a 2.4-fold increase in k(cat)/K-m. Moreover, its half-life at 4 degrees C increased up to 6-fold longer than that of the wild-type. Structural analysis of each methionine substitution was carried out based on the crystal structure of N-D-AAase from Alcaligenes faecalis DA1. Met(221) spatial closeness to the zinc-assistant catalytic center is highly potential as the primary site for oxidative inactivation. We conclude that the replacement of methionine M221 with leucine in N-D-AAase successfully enhances the oxidative resistance, half-life, and enzyme activity. This finding provides a promising basis for the engineering the stability and activity of N-D-AAase. (C) 2012 Elsevier Ltd. All rights reserved.