The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase

Abstract

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 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 band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by Nterminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated 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 versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin-or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.

Physiology of exolaccase production by Thelephora terrestris

“…The laccase activity was 50% inhibited by 1 mM hydroxyquinoline but not by 1 mM EDTA like the enzymes of Trametes sanguinea [22] and Pycnoporus coccineus [25]. The enzyme was inactivated by either 1 mM cysteine, 1 mM mercaptoethanol or 1 mM sodium azide as observed with other fungal enzymes [21,22]. Copper atoms participate in the catalysis and dithiocarbamate, a speci¢c copper chelator, inactivated the exolaccase activity of T. terrestris only at 7.5 mM, suggesting that the copper atom might be inaccessible to the chelator.…”

“…The expression of laccase in T. terrestris was regulated by a low C/N ratio in spite of the growth rate. High C/N ratios increased the production of laccase in Pycnoporus cinnabarinus [21] whereas in Lentinus edodes [24], Phanerochaete chry-soporium [6] and Phanerochaete £avido-alba [7] the opposite was observed.…”

Veratryl alcohol stimulates fruiting body formation in the oyster mushroom, Pleurotus ostreatus

“…Enzyme extraction of the solid medium and assay of laccase PPO-I activity showed that laccase production increased as the concentration of veratryl alcohol in the solid media increased up to 20 Wmol g 31 substrate ( Table 1). Several basidiomycetes are known to produce veratryl alcohol, albeit at low levels [4], which induced laccase production [8]. Pleurotus spp.…”

“…The chief function of veratryl alcohol is as a redox mediator between LiP and lignin degradation [6], and in protecting LiP during the redox cycle from metabolically produced H 2 O 2 [7]. It can also act as an inducer of laccases in basidiomycetes [8] and ascomycetes [9], and hence promote lignocellulose degradation.…”

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I‐937 and expression in Pichia pastoris

“…However, recent studies suggest that in white-rot fungi the combination of laccase with either lignin peroxidase and/or manganese peroxidase are responsible for the lignin degradation. In the strain Pycnoporus cinnabarinus I-937 (ATCC 200478) neither lignin peroxidase nor manganese peroxidase were detected in lignin degradation conditions [4]. The blue multicopper oxidases, like laccases, are in the ascorbate oxidase and mammalian plasma ceruloplasmin family, which have been subjected to extensive biochemical and structural characterization [5].…”