PRODUCTION, PURIFICATION AND CHARACTERIZATION OF AN ALCOHOL OXIDASE OF THE LIGNINOLYTIC FUNGUS PENIOPHORA-GIGANTEA

Following growth on complex medium with polyols, the white-rot fungus Peniophora gigantea produced an alcohol oxidase in the stationary grow th phase, with yields of 220 U per l of culture medium. The alcohol ox idase was purified from mycelium extracts to apparent homogeneity by a mmonium sulfate precipitation, chromatography on DEAE-Sephacel, Sephac ryl S-300, and Q-Sepharose with a yield of 60%. The native alcohol oxi dase had a relative molecular weight (M(r)) of 576 000 +/- 14 000 and a pI of 5.3. SDS-PAGE yielded one single polypeptide band of M(r) = 72 500, suggesting an octameric enzyme structure. The alcohol oxidase was shown to be a flavoprotein containing covalently bound FAD for which a stoichiometry of 7.2 molecules FAD per molecule of enzyme was determ ined. The alcohol oxidase was predominantly active with the short-chai ned primary alcohols methanol, ethanol, n-propanol and n-butanol, and to a lower extent with the polyols erythritol, ribitol, D-arabitol and D-ribose. For the conversion of ethanol a pH optimum ranging from pH 7.3 to 9.0, and an activation energy (DELTAH-degrees) of 63.9 kJ mol-1 was estimated. K(m) values were determined for methanol, ethanol, D-r ibose, ribitol, erythritol and dioxygen to be 1.8 mM, 2.9 mM, 71.5 mM, 250 mM, 670 mM, and 0.4 mM, respectively. The metal salts MgSO4, CaCl 2, MnSO4, ZnSO4 and CoCl2 each at a concentration of 1 mM had no signi ficant effects on the activity of the alcohol oxidase, but FeSO4, NiCl 2 and CuCl2 each at 1 mM, were almost completely inhibitory.