Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of pyranose oxidases is D-glucose which is converted to 2-keto-D-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-D-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-D-glucose can serve as an intermediate in an industrial process for the conversion of D-glucose into D-fructose has stimulated research on the use of pyranose oxidases in biotechnical applications. Unique catalytic potentials of pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs.

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Pyranose oxidases are known to oxidise d-glucose, d-xylose and lsorbose to keto-aldoses, biochemically interesting compounds that may also be used for synthetic purposes in a variety of reactions. In this study pyranose oxidase from the basidiomycete Peniophora gigantea was investigated, and it was found that this enzyme is able to oxidise a broad variety of substrates very effectively. In analogy to its natural mode of action, most substrates are oxidised regioselectively in position 2. Certain compounds, however, are converted into 3-keto derivatives, and the enzyme even exhibits transfer potential, that is, disaccharides are formed from bglycosides of higher alcohols. Substrates that may be oxidised at C-2 in yields between 40 ± 98 % are d-allose, d-galactose, 6-deoxy-d-glucose, d-gentiobiose, a-d-glucopyranosyl fluoride and the very interesting 3-deoxy-d-glucose. 1,5-Anhydro-d-glucitol (1-deoxy-d-glucose) is very effectively oxidised in position 2 in 98 % yield and additionally gives a product of dioxidation at C-2 and C-3 upon prolonged reaction time. Selective oxidation at C-3 was found for 2-deoxyd-glucose in very good yields and for methyl b-d-gluco-and methyl b-galactopyranoside in lower yields. All oxidation products were unequivocally characterised by NMR spectroscopy and/or chemical derivatisation. In addition, the kinetic data of the enzymatic reactions were determined for all substrates. On the basis of these data and the structural characteristics of the substrates, a model for the minimal structural requirements of the enzyme ± substrate interaction is suggested. The enzyme presumably uses two different binding modes for the regioselective C-2 and the C-3 oxidations, which are described.

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen. The enzyme forms a D(2) symmetric homotetramer and contains one covalently bound FAD per subunit. The structure of the enzyme from Peniophora sp. was determined by multiwavelength anomalous diffraction (MAD) based on 96 selenium sites per crystallographic asymmetric unit and subsequently refined to good-quality indices. According to its chain fold, the enzyme belongs to the large glutathione reductase family and, in a more narrow sense, to the glucose-methanol-choline oxidoreductase (GMC) family. The tetramer contains a spacious central cavity from which the substrate enters one of the four active centers by penetrating a mobile barrier. Since this cavity can only be accessed by glucose-sized molecules, the enzyme does not convert sugars that are part of a larger molecule. The geometry of the active center and a comparison with an inhibitor complex of the homologous enzyme cellobiose dehydrogenase allow the modeling of the reaction at a high confidence level.

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 (MJ of 322800 It 18300 as determined on the basis of its Stokes' radius (rs = 6.2 nm) and sedimentation coefficient = 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDSPAGE resulted in one single polypeptide band of Mr76000 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 P-D-glucoside, maltose and D-fucose. Regioselective oxidation of D-glucose, L-sorbose and D-xylose to 2-keto-~-glucose, 5-keto-~-fructose and 2-keto-D-xylose, was demonstrated by identifying the reaction products by mass spectroscopy I3C-NMR spectroscopy and '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 ( A H") for the conversion of D-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the K, 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.