In this study we constructed an artificial flavohemoprotein by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) x min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H2O2 and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by high-performance liquid chromatography (HPLC) analysis. Relative specificity of immobilized VHb-DAO on D-alpha-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C. (C) 2003 Wiley Periodicals, Inc.