Yellow laccases of white rot fungi Pleurotus ostreatus var. Florida and Schizophyllum commune: Catalytic properties and biotechnological potential

Laccases are the most widespread enzymes found in lignin-degrading fungi, occurring in both aquatic and terrestrial species. They are involved in lignin degradation, spore production, the formation of fruiting bodies and rhizomorphs, as well as in defense mechanisms against oxidative stress and heavy metals. Furthermore, they participate in the degradation of various types of pollutants, thereby playing an undeniably significant role in fungal biology. Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2) belong to a family of multicopper oxidases that catalyze the oxidation of a wide range of compounds, coupled with the reduction of O2 to H2O. Typical "blue" laccases catalyze the oxidation of polyphenols, methoxysubstituted phenols, aromatic diamines, and various other compounds. The substrate specificity of these enzymes can be significantly broadened to include non-phenolic aromatic compounds through the use of so-called redox mediators. Unusual "yellow" laccases, by contrast, catalyze the oxidation of a broad spectrum of compounds including both phenolic and non-phenolic aromatic structures directly, without of exogenous redox mediators. This makes them biotechnologically attractive enzymes for the processing of lignocellulosic waste (due to their delignification capabilities) and for applications in environmental biotechnology (such as the treatment of contaminated water). The search for new fungal producers, along with the study of the nature and catalytic properties of yellow laccases, is of both theoretical and practical interest.

For the production of yellow laccases of the white-rot fungus Pleurotus ostreatus var. Florida and Schizophyllum commune, we have developed a semi-submerged cultivation system consisting of a natural substrate (sorghum straw and pine sawdust, respectively) and a basidiomycetes rich medium. By periodically replacing the culture medium while maintaining a single initial inoculation of the plant substrates, this system can be used to produce crude enzyme preparations of yellow laccases up to seven times. A purification protocol for these enzymes was developed, yielding three isoforms of yellow laccase from P. ostreatus and one from S. commune. The enzymes were characterized based on their primary catalytic properties, including the determination of pH optima, KM and Vmax values for different substrates: ABTS, 2,6-dimethoxyphenol, syringaldazine, 2,7-diaminofluorene, guaiacol, pyrocatechol, and o-dianisidine. Notably, the laccase from S. commune was found to be stable at pH 8.5-9.0 a characteristic unusual for fungal laccases, which typically exhibit maximum activity and stability under slightly acidic conditions. Furthermore, unlike their common "blue" counterparts, all the isolated yellow laccases were shown to oxidize polycyclic aromatic hydrocarbons (fluorene, phenanthrene, anthracene, fluoranthene, and pyrene), anthraquinone-type dyes (Basic Blue 22, Acid Blue 62, and Reactive Blue 4), and a lignin-like compound (veratryl alcohol) without the exogenous mediator in the reaction mixture. These properties make the yellow laccases from P. ostreatus and S. commune highly promising for biotechnological applications.