[Characterization of D-amino acid oxidase and its mutants from Arthrobacter protophormiae].

OBJECTIVE: To characterize D-amino acid oxidase from Arthrobacter protophormiae (DSM 20168). METHODS: Genes apdaao-1 and apdaao-2 from A. protophormiae (DSM 15035 & 20168) were cloned by PCR; expression vectors were constructed and expressed in E. coli BL21 (DE3). The mutant was constructed by site-directed mutagenesis using plasmid pET-ApDAAO-2 as the template. After Ni-NTA column chromatography purification, the protein was characterized. RESULTS: Protein ApDAAO-1, ApDAAO-2 and 4 mutants were expressed and purified successfully. The apparent molecular masses of all purified proteins were about 36 kDa by SDS-PAGE. The optimum temperature of ApDAAO-2 and 4 mutants was 30 degrees C similar to ApDAAO-1. ApDAAO-2 and its mutants exhibited much broader optimal pH than ApDAAO-1, and they revealed broad substrate specificity and high specificity to D-Met (100%) except T256K, which showed the substrate preference for D-Phe (108%). For substrates D-Met and D-Phe, the second-order rate constants k(cat)/Km of ApDAAO-2 and 4 mutants were several-fold higher than ApDAAO-1 and pKDAAO, respectively. CONCLUSION: Comparing with ApDAAO-1 and pKDAAO, ApDAAO-2 and its mutants had much broader substrate specificity and higher catalytic efficiency, which suggested that they might have much higher commercial value.