THE LIGNINOLYTIC SYSTEM OF THE WHITE-ROT FUNGUS PYCNOPORUS CINNABARINUS - PURIFICATION AND CHARACTERIZATION OF THE LACCASE

The white rot fungus Pycnoporus cinnabarinus was characterized with re spect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with lo w amounts of an unusual peroxidase. Neither lignin peroxidase nor mang anese peroxidase was detected, Laccase was produced constitutively dur ing primary metabolism, Addition of the most effective inducer, 2,5-xy lidine, enhanced laccase production ninefold without altering the isoe nzyme pattern of the enzyme, Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,0 00 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major b and (pI, 3.7) and several minor bands of decreasing intensity which ap peared at regular, closely spaced intervals toward the alkaline end of the gel, Repeated electrophoresis of the major band under identical c onditions produced the same pattern, suggesting that the laccase was s ecreted as a single acidic isoform with a pi of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This a ppeared to be confirmed by N-terminal amino acid sequencing of the pur ified enzyme, which yielded a single sequence for the first 21 residue s, Spectroscopic analysis indicated a typical laccase active site in t he P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indi cated the enzyme to be a typical fungal laccase, The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versico lor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The pr incipal features of the P. cinnabarinus enzyme system, a single predom inant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible al ternative pathways of lignin degradation by white rot fungi.