Purification and characterization of dehydroascorbate reductase from Pleurotus ostreatus

Dehydroascorbate reductase was purified 93-fold relative to the elude cell extracts from the cytosolic fraction of the mycelia of Pleurotus ostreatus with an overall yield of 5%. The molecular mass of the native enzyme determined by gel filtration chromatography was 86 kDa. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the enzyme is a single polypeptide. Dehydroascorbate reductase from P. ostreatus contained relatively quite a lot of lysine and a relatively small amount of glutamate/glutamine. The enzyme was optimally active at pH 7.5 and at 45 degrees C. Apparent K-m values of dehydroascorbate reductase were 2.5 mM and 0.7 mM for dehydroascorbate and glutathione. The enzyme was significantly unstable under acidic and highly alkaline conditions. The absorption spectrum of the purified enzyme showed an unusual flavin peak, the result of which suggests that the enzyme might form flavin adduct.