Characterization of a D-amino acid oxidase with high activity against cephalosporin C from the yeast Trigonopsis variabilis.

We describe an improved method to purify D-amino acid oxidase with activity towards cephalosporin C. The protein has a carbohydrate content of 1.3% and two molecules of non-covalently bound flavin cofactor per protein molecule. HPLC profiles and enzymatic analysis have indicated that the cofactor is FAD, even though fluorescence spectroscopy shows a slightly altered spectral profile in the 400-500 nm range compared to authentic FAD. N-terminal sequencing of the protein revealed a high level of similarity (56% identity in 25 amino acids) between the fungal and mammalian oxidase, and probably represents a "Rossman fold" with a beta-alpha-beta structure for the binding of the adenosyl moiety of the cofactor.