Characterization of cellobiose dehydrogenase and its FAD-domain from the ligninolytic basidiomycete Pycnoporus sanguineus

• The first purified cellobiose dehydrogenase and separately FAD-domain from Pycnoporus sanguineus.
• Characterization of both proteins comprising kinetics, pH and temperature dependence.
• First analysis of prooxidant and antioxidant properties of fungal CDH and its FAD-domain.

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in the culture supernatant of the selective delignifier Pycnoporus sanguineus maintained on a cellulose-based liquid medium. Cellobiose dehydrogenase was purified as two active fractions: CDH1-FAD (flavin domain) (40.4 fold) with recovery of 10.9% and CDH1 (flavo-heme enzyme) (54.7 fold) with recovery of 9.8%. As determined by SDS-PAGE, the molecular mass of the purified enzyme was found to be 113.4 kDa and its isoelectric point was 4.2, whereas these values for the FAD-domain were 82.7 kDa and pI = 6.7. The carbohydrate content of the purified enzymes was 9.2%. In this work, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from fungus P. sanguineus were isolated, cloned, and characterized. The 2310 bp full-length cDNA of cdh1 encoded a mature CDH protein containing 769 amino acids, which was preceded by a signal peptide of 19 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.