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 elec trophoretic homogeneity using four chromatographic steps: DEAE-Sepharose, S ephacry S-200, Hitrap SP and Mono S column. The purified enzyme had a molec ular mass of 52 kDa as determined by SDS-PAGE and an isoelectric point of a bout 7.6. The enzyme contained a negligible amount of carbohydrate. The N-t erminal amino acid sequence of the enzyme was similar to those of manganese dependent peroxidases, but the internal peptide sequence differed from tho se 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 opti mum reaction temperature was between 35 and 40 C and optimum pH was 4.0. Th e enzyme catalyzed the oxidation of a variety of monomeric lignin model com pounds, but not veratryl alcohol and Mn (II). Under standard assay conditio ns, 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.