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Laccases are copper-containing oxidases that catalyze a one-electron abstraction from various phenolic and non-phenolic compounds with concomitant reduction of molecular oxygen to water. It is well-known that laccases from various sources have different substrate specificities, but it is not completely clear what exactly provides these differences. The purpose of this work was to study the features of the substrate specificity of four laccases from basidiomycete fungi Trametes hirsuta, Coriolopsis caperata, Antrodiella faginea, and Steccherinum murashkinskyi, which have different redox potentials of the T1 copper center and a different structure of substrate-binding pockets. Enzyme activity toward 20 monophenolic substances and 4 phenolic dyes was measured spectrophotometrically. The kinetic parameters of oxidation of four lignans and lignan-like substrates were determined by monitoring of the oxygen consumption. For the oxidation of the high redox potential (>700 mV) monophenolic substrates and almost all large substrates, such as phenolic dyes and lignans, the redox potential difference between the enzyme and the substrate (∆E) played the defining role. For the low redox potential monophenolic substrates, ∆E did not directly influence the laccase activity. Also, in the special cases, the structure of the large substrates, such as dyes and lignans, as well as some structural features of the laccases (flexibility of the substrate-binding pocket loops and some amino acid residues in the key positions) affected the resulting catalytic efficiency.

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Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy – AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family.

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White-rot basidiomycetes from the poorly studied residual polyporoid clade of Polyporales order Junghuhnia nitida (Pers.) Ryvarden and Steccherinum bourdotii Saliba & A. David grow as secondary xylotrohps on well decomposed woody materials. The main objective of the current study was to compare oxidative potential, growth, production of oxidative enzymes and laccase properties of J. nitida and S. bourdotii with that of typical primary xylotrohps Trametes hirsuta (Wulfen) Lloyd and Coriolopsis caperata (Berk.) Murrill, belonging to the core polyporoid clade. For the first time we report species J. nitida and S. bourdotii as active laccase producers. New laccases from J. nitida and S. bourdotii were purified and characterized. They had an identical molecular weight of 63 kDa and isoelectric points of 3.4 and 3.1, respectively. However, the redox potential of the T1 copper site for both J. nitida (610 mV) and S. bourdotii (640 mV) laccases was lower than those for T. hirsuta and C. caperata laccases. The new laccases showed higher temperature optima and better thermal stability than T. hirsuta and C. caperata laccases. Their half-lives were more than 40 min at 70 °C. The laccases from J. nitida and S. bourdotii showed higher affinity to syringyl-type phenolic compounds than T. hirsuta and C. caperata laccases. The oxidative potential of studied fungi as well as the properties of their laccases are discussed in terms of the fungal life-style.

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.