CHARACTERIZATION OF D-ARABINONO-1,4-LACTONE OXIDASE FROM CANDIDA-ALBICANS ATCC-10231

D-Erythroascorbic acid was detected from the cell extracts of a dimorp hic fungus, Candida albicans. Its concentration in yeast cells grown a t 25 degrees C was estimated to be about 0.45 mu mol/ml cell water. D- Arabinono-1,4-lactone oxidase, which catalyses the final step in the b iosynthesis of D-erythroascorbic acid, was purified 639-fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Tri ton X-100 solubilisation, ammonium sulphate precipitation, anion-excha nge, hydrophobic-interaction, gel-filtration and dye-ligand chromatogr aphies. Gel-filtration chromatography and polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave apparent molecula r masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only o ne protein band corresponding to a molecular mass of 66.7 kDa. Conside ring the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spec trum of a flavin-containing enzyme. The flavin was not released by tre atment with SDS, CCl3CO2H or boiling, indicating that it may be covale ntly bound to the enzyme protein. The enzyme was;optimally active at 4 0 degrees C and at pH 6.1. The enzyme was stable in the range pH 7.5-1 0. An apparent K-m value for D-arabinono-1,4-lactone was 44.1 mM. L-Ga lactono-1,4-lactone, L-gulono-1,4-lactone and L-xylono-1,4-lactone cou ld also serve as substrates. Competitive inhibition was demonstrated w ith D-glucono-1,5-lactone, L-arabinono-1,4-lactone, D-galactono-1,4-la ctone and D-gulono-1,4-lactone. p-Chloromercuribenzoate, N-ethylmaleim ide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd 2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.