Purification by Immunoaffinity Chromatography, Characterization, and Structural Analysis of a Thermostable Pyranose Oxidase from the White Rot Fungus Phlebiopsis gigantea.

Applied and Environmental Microbiology
A SchaferF Giffhorn

Abstract

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(infr)) of native PROD is 295,600 (plusmn) 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(infr)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-gl...Continue Reading

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Citations

Jan 22, 2005·Applied Microbiology and Biotechnology·Sabine BastianFriedrich Giffhorn
Jul 12, 2012·Applied Microbiology and Biotechnology·Christoph A GasserPhilippe F-X Corvini
Jul 27, 2001·Applied and Environmental Microbiology·C LeitnerD Haltrich
Dec 19, 2008·Preparative Biochemistry & Biotechnology·Nurdan Kasikara PazarliogluDilek Tahsinsoy
Sep 21, 2006·The Journal of Biological Chemistry·Magdalena KujawaChristina Divne
Sep 23, 2020·Biotechnology Advances·Simone Savino, Marco W Fraaije
Dec 1, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Annabelle T AbreraDietmar Haltrich
Sep 30, 2021·Proceedings of the National Academy of Sciences of the United States of America·Takuto KumanoMichihiko Kobayashi
Jan 17, 2009·Archives of Biochemistry and Biophysics·Kunchala Rungsrisuriyachai, Giovanni Gadda

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