Characterization and high-level production of D-amino acid oxidase in Candida boidinii

Abstract

D-Amino acid oxidase (DAO, EC 1.4.3.3) from a methylotrophic yeast, Candida boidinii, was produced at a high level under the control of the alcohol oxidase gene promoter in the original host. The enzyme was a peroxisomal and monomeric enzyme, and contained noncovalently-bound FAD as a cofactor. The enzyme was active toward several n-amino acids such as D-Ala, D-Met, and D-Ser. An alcohol oxidase-depleted strain (aod1Δ) was found to be a more suitable host for DAO production than the wild-type strain. Several post-translational effects may be responsible for the improvement of the DAO productivity by the aod1Δ strain. Finally, an aod1Δ strain transformant having multi-copies of an expression plasmid on its chromosome could produce DAO amounting up to 30% of the total soluble proteins.