Isolation and characterization of the cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana (Schum ex Fr.) Karst.
The cellobiose dehydrogenase secreted by Coniophora puteana (Schum ex Fr) Karsten during growth on cellulose was isolated by successive anion-exchange chromatography on Q Sepharose fast flow and on TSK DEAE-650S and gel filtration on Superose 12. The enzyme was recovered at a 41% yield with a 43-fold increase in specific activity. The purified sample was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium lauryl sulfate (SDS)-PAGE, and electrophoretic titration curve analysis and stained positively for glycoprotein (periodic acid/Schiff base reaction) and hemoprotein (peroxidase reaction). By isoelectric focusing over a narrow pH range two distinct bands were observed: a major band (pI 3.9) flanked by a minor band on its acidic side. FPLC gel filtration on TSK G3000 SW revealed a M(r) of 192,000, whereas on SDS-PAGE a single band, corresponding to a M(r) of 111,000, was observed. The enzyme contained 13% sugar as mannose and upon digestion with endoglycosidase H, its molecular weight was lowered by 11 kDa. The enzyme showed a visible spectrum compatible with that of a b-type cytochrome containing a flavin cofactor. It was able to oxidize cellobiose, cellodextrins, and lactose at their C1-reducing group, with dichlorophenol indophenol as oxidant. Oxygen consumption (oxidase reaction in a Clark electrode) was not at a detectable rate. Km and Vmax for cellobiose oxidation were 84 microM and 2.98 mumol mg-1 min-1, respectively, but the enzyme was strongly substrate (cellobiose) inhibited (Kis 5.4 mM).
Schmidhalter
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DR
Institut de Biologie végétale et de Phytochimie, Université de Fribourg, Switzerland.
Canevascini
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D000363:Agaricales / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism*; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D006420:Hemeproteins / Q000302:isolation & purification / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight
D002241:Carbohydrates; D006023:Glycoproteins; D006420:Hemeproteins; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase