The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds (dihydroxybenzyl alcohol and hydroxytyrosol) from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.

Fungal laccases oxidize a wide range of substrates with a diverse spectrum of subsequent non-specific free radical reactions, leading to the production of unwanted byproducts. This work describes a unique recombinant alkaliphilic laccase from Paramyrothecium roridum VKM F-3565 capable of performing specific oligomerization of phenylpropanoids (precursors of natural lignin and lignans) in a neutral environment, thus preventing the reverse reaction of depolymerization which occurs in an acidic environment. The recombinant alkaliphilic laccase from P. roridum VKM F-3565 with a specific enzyme activity of about 154.0 U/mg (in the reaction with 1 mM ABTS) was obtained using a Komagataella phaffii transformant with a yield of 20 ± 1.5 mg/L. The recombinant laccase had an increased degree of N-glycosylation (MW = 97 kDa), higher pH optimum in reaction with phenylpropanoids and a decreased temperature optimum, compared to the wild-type laccase. The enzyme exhibited great resistance to surfactants and the EDTA in the neutral conditions rather than the acidic ones, whereas its tolerance to mono- and divalent-metal ions was high at acidic conditions. This work demonstrates the important role of N-glycosylation of the alkaliphilic laccase of P. roridum VKM F-3565 in its functional activity. The presence of pH-dependent reactions makes the studied laccase attractive for the phenylpropanoid oligomerization with the production of novel oligomeric phenylpropanoid derivatives for industrial and pharmacological purposes.