title: Overproduction and characterization of a recombinant D-amino acid oxidase from Arthrobacter protophormiae.
abstract: A screening of soil samples for D-amino acid oxidase (D-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type D-AAO, the gene sequence was determined and designated dao. An alignment of the deduced primary structure with eukaryotic D-AAOs and D-aspartate oxidases showed that the D-AAO from A. protophormiae contains five of six conserved regions; the C-terminal type 1 peroxisomal targeting signal that is typical for D-AAOs from eukaryotic origin is missing. The dao gene was cloned and expressed in Escherichia coli. The purified recombinant D-AAO had a specific activity of 180 U mg protein(-1) for D-methionine and was slightly inhibited in the presence of L-methionine. Mainly, basic and hydrophobic D-amino acids were oxidized by the strictly enantioselective enzyme. After a high cell density fermentation, 2.29 x 10(6) U of D-AAO were obtained from 15 l of fermentation broth.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Research Centre Jülich, 52426 Jülich, Germany. birgit.geueke@eawag.ch
authors.forename: Birgit
authors.initials: B
authors.lastname: Geueke
authors.forename: Andrea
authors.initials: A
authors.lastname: Weckbecker
authors.forename: Werner
authors.initials: W
authors.lastname: Hummel
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003605:D-Amino-Acid Oxidase