Pyranose 2-oxidase from Phanerochaete chrysosporium--further biochemical characterisation

• Published in: Applied microbiology and biotechnology 1997-05, Vol.47 (5), p.508-514
• Main Authors: Artolozaga, M.J, Kubatova, E, Volc, J, Kalisz, H.M
• Format: Article
• Language: English
• Subjects: Basidiomycetes; Basidiomycota - enzymology; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Carbohydrate Dehydrogenases - isolation & purification; Carbohydrate Dehydrogenases - metabolism; Enzymes; Fundamental and applied biological sciences. Psychology; Glucose; Hydrogen-Ion Concentration; Kinetics; Liquid chromatography; Miscellaneous; Mission oriented research; Phanerochaete; Phanerochaete chrysosporium; plant biochemistry; plant physiology; Temperature
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530050964

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 degrees C. It is active over a broad pH range (5.0-9.0) with maximum activity at pH 8.0-8.5 and at 55 degrees C, and a broad substrate specificity. D-Glucose is the preferred substrate, but 1-beta-aurothioglucose, 6-deoxy-D-glucose, L-sorbose, D-xylose, 5-thioglucose, D-glucono-l,5-lactone, maltose and 2-deoxy-D-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0 with a catalytic constant (kcat) of 111.0 s-1 and an affinity constant (Km) of 1.43 millimole for D-glucose and 83.2 micromolar for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH greater than or equal to 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the kcat/Km value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the kcat decreased to 60.9 s-1 and the Km increased to 240 millimole. The activity of P2O was completely inhibited by 10 millimole HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.