A moderately thermostable pyranose oxidase (PROD) was purified to apparent homogeneity with a yield of 71% from mycelium extracts of the white rot fungus Phlebiopsis gigantea by an efficient three-step procedure that included heat treatment, immunoaffinity chromatography, and gel filtration on Superdex 200. PROD of P. gigantea is a glycoprotein with a pI between pH 5.3 and 5.7. The relative molecular weight (M r) of native PROD is 295,600 ؎ 5% as determined by four independent methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PROD revealed two distinct but similar stained bands corresponding to polypeptides with M r s of 77,000 and 70,000, suggesting a heterotetrameric enzyme structure. The tetrameric structure of PROD was confirmed by electron microscopic examinations, which additionally showed the ellipsoidal shape (4.6 by 10 nm) of each subunit. Spectral analyses and direct determinations showed the presence of covalently bound flavin adenine dinucleotide with a stoichiometry of 3.12 mol/mol of enzyme. A broad pH optimum was determined in the range pH 5.0 to 8.0 in 100 mM sodium phosphate, and the activation energy for D-glucose oxidation was 24.7 kJ/mol. The main substrates of PROD are D-glucose, L-sorbose, and D-xylose, for which K m values 1.2, 16.5, and 22.2 mM were determined, respectively. PROD showed high stability during storage. In 100 mM sodium phosphate (pH 6.0 to 8.0), the half-life of PROD activity was >300 days at 40؇C, >110 days at 50؇C (pH 7.0), and 1 h at 65؇C.
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