A Highly Stable D-Amino Acid Oxidase of the Thermophilic Bacterium Rubrobacter xylanophilus

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 (k(cat)/K-m) 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 degrees C, respectively, and was stable between pH 5.0 and pH 8.0, with a T-50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64 degrees C. No loss of enzyme activity was observed after a 1-week incubation period at 30 degrees 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.