PURIFICATION AND CHARACTERIZATION OF A PYRANOSE OXIDASE FROM THE BASIDIOMYCETE PENIOPHORA-GIGANTEA AND CHEMICAL-ANALYSES OF ITS REACTION-PRODUCTS

A pyranose oxidase was isolated from mycelium extracts of the basidiom ycete Peniophora gigantea. This enzyme was purified 104-fold to appare nt homogeneity with a yield of about 75 % by steps involving fractiona ted ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxid ase has a relative molecular mass (M(r)) of 322 800 +/- 18 300 as dete rmined on the basis of its Stokes' radius (r(s) = 6.2 nm) and sediment ation coefficient (s20,w = 10.6), dynamic light-scattering experiments , gradient-gel electrophoresis and cross-linking studies. SDS/PAGE res ulted in one single polypeptide band of M(r) 76 000 indicating that th e enzyme consists of four subunits of identical size. The pyranose oxi dase was shown to be an extremely stable glycoprotein with an isoelect ric point of pH 5.3. It contains covalently bound FAD with an estimate d stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates D-glucose, D-xylose, L-sorbose, D-gala ctose, methyl beta-D-glucoside, maltose and D-fucose. Regioselective o xidation Of D-glucose, L-sorbose and D-xylose to 2-keto-D-glucose, 5-k eto-D-fructose and 2-keto-D-Xylose, was demonstrated by identifying th e reaction products by mass spectroscopy C-13-NMR spectroscopy and H-1 -NMR spectroscopy after purification and derivatization. The pH optimu m of the pyranose oxidase was in the range pH 6.06. 5 in 0.1 M potassi um phosphate, and its activation energy (DELTA H-degrees) for the conv ersion Of D-glucose was 34.6 kJ/mol. The reactions with the sugars exh ibited Michaelis-Menten kinetics, and the K(m) values determined for D -glucose, L-sorbose, D-Xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reag ents and bivalent cations each at 1 mM.