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Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).

Many ligninolytic basidiomycete fungi have been shown to secrete a group of peroxidase isozymes whose sole function appears to be the peroxide-dependent oxidation of manganous [Mn(II)] to manganic [Mn(III)] ions. Manganic chelates and these Mn peroxidases have been implicated as central to the degradation of various natural and synthetic lignins and lignin-containing effluents by white rot (ligninolytic) fungi. Another group of enzymes, the laccases, are commonly secreted by wood-rotting fungi, but are generally regarded as being able to oxidize (and usually polymerize) only phenolic substrates. In this report it is shown that in the presence of appropriate oxidizable phenolic accessory substances or primary substrates, a variety of laccases and peroxidases catalyzing one-electron oxidations can also produce Mn(III) chelates from Mn(II).

Two cDNAs and two genomic DNAs coding for the allelic forms of the ligninolytic phenoloxidase were isolated from the white-rot fungus Coriolus hirsutus. The cloned genes were identified in genetic libraries by hybridization screening using four deoxyoligonucleotide probes which corresponded to the partial amino acid sequence of the purified enzyme. Each cDNA encoded the full-length of the phenoloxidase, a protein consisting of 499 amino acid residues, and its putative signal peptide of 21 amino acid residues. The nucleotide sequences of the two alleles differed by 18 single base changes within the open reading frames resulting in one amino acid substitution. Ten small introns interrupted both genomic DNAs as indicated by direct comparison with the corresponding cDNAs. Putative eukaryotic regulatory sequences, "CAAT" and "TATA," were observed in the 5'-flanking region of both genomic DNAs. Each of the phenoloxidase cDNAs was successfully expressed in an active form in Saccharomyces cerevisiae using the useful yeast expression vector YEp51.

In the presence of substrates such as Remazol Blue and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate) (ABTS), laccases Coriolus (Trametes) versicolor can also oxidize non-phenolic lignin model compounds. Veratryl alcohol (I) and 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-propane-1,3-diol (III) were oxidized by laccase and mediator to give the alpha-carbonyl derivatives. The beta-1 lignin model dimer, 1-(3,4-dimethoxyphenyl)-2-phenoxy-ethane-1,2-diol (II) was cleaved by laccase in the presence of ABTS to give veratraldehyde and benzaldehyde. On the basis of these observations, we propose that laccase is capable of oxidizing both phenolic and non-phenolic moieties of lignin but that the latter is dependent on the co-presence of primary laccase substrates.