title: A Highly Stable D-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus.
abstract: d-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic d-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested d-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain d-amino acids, with the highest specific activity toward d-valine and catalytic efficiency (kcat/Km) toward d-leucine. Substrate inhibition was observed in the case of d-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain d-amino acids, and as a scaffold to generate a novel DAO via protein engineering.
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan shoutaka@vos.nagaokaut.ac.jp.
authors.forename: Shouji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Furukawara
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Omae
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Namiho
authors.initials: N
authors.lastname: Tadokoro
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Abe
authors.affiliation: Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kera
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D001565:Benzoates / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001565:Benzoates; D003067:Coenzymes; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D010664:Phenylmethylsulfonyl Fluoride; D003605:D-Amino-Acid Oxidase