Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants

The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was pre pared by reverse transcriptase-polymerase chain reaction (PCR) and cloned i nto Escherichia coli expression vector, pTrc99A, under the control of tac p romoter. Expression of daao gene significantly affected the growth and morp hology 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-alani ne-induced T. variabilis. The DAAO protein exhibited activity on native-PAG E and had a M-r value of 39.3 kDa. We also constructed an expression plasmi d, pKm-DAAO, in which kanamycin instead of ampicillin was used as the selec tive marker. High-performance liquid chromatography (HPLC) analysis demonst rated 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. co li and purified by nickel ion affinity chromatography. The results showed t hat all DAAO mutants lost their enzymatic activities and characteristic ads orption spectra for flavoenzyme. Based on the crystal structure of a homolo gous protein, pig DAAO, it is suggested that these four residues may play e ssential structural roles in DAAO conformation, thereby influencing DAAO's catalytic activity. (C) 2000 Elsevier Science Inc. All rights reserved.