PURIFICATION AND CHARACTERIZATION OF D-GLUCOSE OXIDASE FROM WHITE-ROTFUNGUS PLEUROTUS-OSTREATUS

D-Glucose oxidase was purified 27.5-fold to apparent homogeneity with an overall yield of 23.8%, from Pleurotus ostreatus, through a purific ation procedure of ammonium sulphate precipitation, gel-permeation, an ion-exchange and hydrophobic-interaction chromatography. The molecular mass determined by gel filtration was found to be 290 kDa. SDS/PAGE r evealed that the enzyme consists of four subunits with a molecular mas s of 70 kDa. The absorption spectra of the enzyme exhibit maxima at 28 0, 360 and 460 nm. The enzyme shows a fluorescence spectrum with an ex citation maximum at 470 nm and an emission maximum at 530 nm. These re sults indicate that the prosthetic group of the enzyme is flavin and t hat the enzyme contains 4 mol flavin/mol enzyme. The enzyme is optimal ly active at 50-degrees-C and at pH 5.5-6.0. It exhibits broad affinit y for various sugars and specificity for D-glucose with K(m) value of 1.34 mM. 2,6-Dichloroindophenol, Wurster's blue, and 4-benzoquinone ca n function as electron acceptors but phenazine methosulphate cannot fu nction as an electron acceptor. The enzyme is inhibited completely by mercuric chloride and partially by silver sulphate, sodium azide and 8 -hydroxyquinoline.