Back
Isolation and characterization of the cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana (Schum ex Fr.) Karst.
Journal article

Isolation and characterization of the cellobiose dehydrogenase from the brown-rot fungus Coniophora puteana (Schum ex Fr.) Karst.

  • 1993-02-01
Published in:
  • Archives of biochemistry and biophysics. - 1993
Agaricales
Carbohydrate Dehydrogenases
Carbohydrates
Chromatography, Gel
Chromatography, High Pressure Liquid
Chromatography, Ion Exchange
Electrophoresis, Polyacrylamide Gel
Glycoproteins
Hemeproteins
Isoelectric Focusing
Kinetics
Molecular Weight
English 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).
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/250843
Statistics
Document views: 29 File downloads: