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

Abstract:

Abstract:［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℃ 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 secondorder rate constants kcat/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.