Abstract
Cellobiose dehydrogenase (CDH), a secreted flavocytochrome produced by a number of wood-degrading fungi, was detected in the culture supernatant of a biotechnologically important strain of Cerrena unicolor grown in a modified cellulose-based liquid medium. The enzyme was purified as two active fractions: CuCDH-FAD (flavin domain) (1.51-fold) with recovery of 8.35 % and CuCDH (flavo-heme enzyme) (21.21-fold) with recovery of 73.41 %. As CDH from other wood-rotting fungi, the intact form of cellobiose dehydrogenase of C. unicolor is a monomeric protein containing one flavin and one heme b with molecular mass 97 kDa and pI = 4.55. The enzyme is glycosylated (8.2 %) mainly with mannose and glucosamine residues. Moreover, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from the fungus C. unicolor were isolated, cloned, and characterized. The 2316-bp full-length cDNA of cdh1 encoded a mature CDH protein containing 771 amino acids preceded by a signal peptide consisting of 18 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.
Highlights
Fungi form an important group of microorganisms that have beneficial effects on the environment and human life
White-rot basidiomycetes are a group of fungi comprising from 1600 up to 1700 species characterized by the ability to depolymerize and mineralize lignin using a set of extracellular ligninolytic enzymes and low molecular compounds [2, 3]
Cellobiose dehydrogenase production by C. unicolor strain FLC139 was performed in shaking flasks on the cellulose-based medium
Summary
Fungi form an important group of microorganisms that have beneficial effects on the environment and human life. Fractions containing CDH activity (obtained from lactose-CPG) were collected, and a chromatofocusing analysis was performed on an Econo-chromatography column (Bio-Rad, Richmond, VA, USA; 130 cm, packed to a bed height of 20 cm) with a Polybuffer exchanger PBE 94 equilibrated with 250 ml of 0.025 M imidazole-HCl buffer (pH 7.4). Samples from lactose-CPG chromatography showing CDH activity (5 ml) were injected onto the column, and the enzyme was desorbed by elution with 200 ml Polybuffer 74-HCl (pH 3.0) at a flow rate of 0.5 ml/min.
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