Purification by immunoaffinity chromatography, characterization, and structural analysis of a thermostable pyranose oxidase from the white rot fungus Phlebiopsis gigantea

• Published in: Applied and Environmental Microbiology Vol. 62; no. 7; pp. 2586 - 2592
• Main Authors: SCHÄFER, A, BIEG, S, HUWIG, A, KOHRING, G.-W, GIFFHORN, F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.07.1996
• Subjects: actividad enzimatica; activite enzymatique; alcohol dehydrogenase; alcohol deshidrogenasa; alcool deshydrogenase; basidiomycotina; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; carie du bois; champignon; chromatographie; chromatography; cromatografia; degradacion; degradation; Enzymes; enzymic activity; Fundamental and applied biological sciences. Psychology; Fungi; hongos; Microbiology; Miscellaneous; Mission oriented research; molecular weight; Oxidation; peso molecular; Phlebiopsis gigantea; poids moleculaire; pudricion de la madera; purificacion; purification; wood decay; Purification; Enzyme; Immunoaffinity chromatography; Environmental factor; Fungi; Characterization; Enzymatic activity; Substrate specificity; Thermostable; Oxidoreductases; Physicochemical properties; Thallophyta; Structural analysis; Pyranose oxidase
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.62.7.2586-2592.1996

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 (Mr) 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 Mrs 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 Km 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 degrees C, >110 days at 50 degrees C (pH 7.0), and 1 h at 65 degrees C.