title: [Characterization of D-amino acid oxidase and its mutants from Arthrobacter protophormiae].
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 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Liwei
authors.initials: L
authors.lastname: Feng
authors.forename: Jiaojie
authors.initials: J
authors.lastname: Guo
authors.forename: Huixin
authors.initials: H
authors.lastname: Li
authors.forename: Shujing
authors.initials: S
authors.lastname: Xu
authors.forename: Jiansong
authors.initials: J
authors.lastname: Ju
authors.forename: Baohua
authors.initials: B
authors.lastname: Zhao
mesh_terms: D001173:Arthrobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003605:D-Amino-Acid Oxidase