Purification and characterization of cellobiose dehydrogenase, a novel extracellular hemoflavoenzyme from the white-rot fungus Phanerochaete chrysosporium.
Cellobiose dehydrogenase (CDH), an extracellular hemoflavoenzyme produced by the cellulose-degrading cultures of Phanerochaete chrysosporium, oxidizes cellobiose to cellobionolactone. CDH has been purified to homogeneity by a five-step purification procedure. The homogeneous CDH is monomeric and has a relative molecular mass of 90,000. It is also a glycoprotein with a neutral carbohydrate content of 9.4%. Purified CDH contains one heme b and one flavin adenine dinucleotide per monomer. Homogeneous CDH has a specific activity of 10.3 mumol min-1 mg-1 for cytochrome c reduction, in the presence of cellobiose. Cellotriose, cellotetraose, cellopentaose, and lactose also serve as substrates for CDH, in addition to cellobiose. Cytochrome c, dichlorophenol-indophenol, Mn3+, and benzoquinones can function as electron acceptors in these oxidations. Kinetic studies suggest that cellobiose is the preferred substrate and cytochrome c is the preferred electron acceptor. In the absence of these electron acceptors, oxygen serves as a poor electron acceptor and is reduced to H2O2. CDH is very stable in the pH range 3-10 and up to 50 degrees C. At lower pH or at higher temperature, CDH is inactivated due to the release of flavin from the active site. The native ferric form of the enzyme has absorption maxima at 420, 529, and 570 nm. With the addition of cellobiose, these absorptions shift to 428, 534, and 564 nm. The ferric enzyme does not bind azide or cyanide, implying that the heme iron is probably hexacoordinate.
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Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton 97006-1999.
Usha
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Renganathan
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D000363:Agaricales / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006420:Hemeproteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013053:Spectrophotometry; D013379:Substrate Specificity; D013816:Thermodynamics
D006420:Hemeproteins; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase