D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic d-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic d-amino acids and a neutral d-amino acid, d-Gln, with the highest activity toward d-Glu. The K-m and k(cat) values for d-Glu were 2.16mM and 217s(-1), respectively. The enzyme had an optimum pH and temperature 8.0 and 60 degrees C, respectively, and was stable between pH 5.0 and 10.0, with a T-50 of ca. 51 degrees C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and d-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.