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The N-carbamoyl-D-amino acid amidohydrolase (Nca) is an important enzyme used in the bioconversion of N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG-) to D-p-hydroxyphenylglycine (D-p-HPG). The nca gene of Agrobacterium radiobacter was cloned and expressed in Escherichia coli. The Nca is a homodimeric enzyme with subunit molecular mass of 36 kDa. Enzymatic activity of Nca is inactivated after 5 h of incubation at 50℃. To examine the oxidation stability of Nca, the enzyme was incubated at 37℃ in the presence of different concentrations of H2O2. Mass spectrum revealed that artificial oxidation of Nca with 0.4 mM H2O2 was too harsh to discriminate oxidized residues. Molecular mass of Nca inactivatede under natural oxidation was increased by 27 Da compared to that of native enzyme. Therefore, the methionine residues of Nca seem to be oxidized by oxygen. To verify whether methionine residues were oxidized, all methionine residues in Nca were substituted by leucine via site-directed mutagenesis. The results revealed that mutant Met-239-Leu/Met-244-Leu retained its activity after H2O2 treatment, indicating that Met-239 or Met-244 might be involved in enzyme oxidation. To improve the Nca activity, PCR-based random mutation coupled plasmid multimerization was applied to nca gene evolution. Of one thousand mutants, the Nca activities of four mutants were increased by 1.5-fold compared to that of the parent strain, but specific activity remained unchanged. Amino acid sequence alignment of A. radiobacter Nca and Pseudomonas sp. Nca displayed 58.6% identity. Based on this identity, a number of aspartic acid and histidine residues in conserved regions of A. radiobacter Nca were selected for site-directed mutagenesis to evalute the effect of these amino acid residues on Nca activity. Nca mutants, Asp-209-Ala and His-129-Ala, lost their activities completely, suggesting that Asp-209 and His-129 residues might be important in catalysis. The Asp-209-Glu mutant retained 30% activity, indicating that carboxyl group of Asp-209 participated in catatlysis. Substitution of His-129 by asparagine or arginine abolished enzyme activity completely, suggesting that His-129 might be involved in proton transfer, but not in hydrogen bonding.
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