The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was prepared by reverse transcriptase-polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector, pTrc99A, under the control of tac promoter. Expression of daao gene significantly affected the growth and morphology of E. coli. The highest D-amino acid oxidase (DAAO) activity was 705 U (mg of protein)(-1), which was about 12-fold higher than that of D-alanine-induced T. variabilis. The DAAO protein exhibited activity on native-PAGE and had a M-r value of 39.3 kDa. We also constructed an expression plasmid, pKm-DAAO, in which kanamycin instead of ampicillin was used as the selective marker. High-performance liquid chromatography (HPLC) analysis demonstrated that cephalosporin C could be converted to 7-glutarylcephalosporanic acid by cell-free extract of E. coti harboring pKm-DAAO. Four inactive DAAO mutants were obtained by error-prone PCR. Sequence analysis of these four DAAO mutants indicated the occurrence of mutations at Val-167, Pro-291, Pro-309, and Ala-343 residues. The His(6)-tagged DAAOs were expressed in E. coli and purified by nickel ion affinity chromatography. The results showed that all DAAO mutants lost their enzymatic activities and characteristic adsorption spectra for flavoenzyme. Based on the crystal structure of a homologous protein, pig DAAO, it is suggested that these four residues may play essential structural roles in DAAO conformation, thereby influencing DAAO's catalytic activity.