Open AccessPurification and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products
Author(s) -
DANNEEL HansJürgen,
RÖSSNER Ellen,
ZEECK Axel,
GIFFHORN Friedrich
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb17982.x
Subject(s) - chemistry , botany , pyranose , stereochemistry , biology
A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea . This enzyme was purified 104‐fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE‐Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass ( M r ) of 322800 ± 18300 as determined on the basis of its Stokes' radius ( r s = 6.2 nm) and sedimentation coefficient ( s 20,w = 10.6), dynamic light‐scattering experiments, gradient‐gel electrophoresis and cross‐linking studies. SDS/PAGE resulted in one single polypeptide band of M r 76000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates d ‐glucose, d ‐xylose, l ‐sorbose, d ‐galactose, methyl β‐ d ‐glucoside, maltose and d ‐fucose. Regioselective oxidation of d ‐glucose, l ‐sorbose and d ‐xylose to 2‐keto‐ d ‐glucose, 5‐keto‐ d ‐fructose and 2‐keto‐ d ‐xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13 C‐NMR spectroscopy and 1 H‐NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0–6.5 in 0.1 M potassium phosphate, and its activation energy ( H°) for the conversion of d ‐glucose was 34.6 kJ/mol. The reactions with the sugars exhibited 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 reagents and bivalent cations each at 1 mM.