Isolation and characterization of the second extracellular peroxidase of the white-rot fungus Coriolus hirsutus

A second extracellular peroxidase of Coriolus hirsutus was purified to electrophoretic homogeneity using four chromatographic steps: DEAE-Sepharose, Sephacry S-200, Hitrap SP and Mono S column. The purified enzyme had a molecular mass of 52 kDa as determined by SDS-PAGE and an isoelectric point of about 7.6. The enzyme contained a negligible amount of carbohydrate. The N-terminal amino acid sequence of the enzyme was similar to those of manganese dependent peroxidases, but the internal peptide sequence differed from those of other fungal peroxidases. The absorption spectra of the native enzyme exhibited maxima at 407, 501, and 635 nm. In the reduced and cyano-adduct forms, the Sorer band was shifted to 438 and 422 nm, respectively. The optimum reaction temperature was between 35 and 40 C and optimum pH was 4.0. The enzyme catalyzed the oxidation of a variety of monomeric lignin model compounds, but not veratryl alcohol and Mn (II). Under standard assay conditions, the apparent Km values of the enzyme toward ferulic acid and H2O2 were 5.9 and 15.5 muM, respectively. The enzyme was completely inhibited by 0.1 mM L-cysteine and sodium bisulfite.