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.

We present the cloning and heterologous expression of the α-amylase gene amyBL159 from a thermophilic strain Bacillus licheniformis MGMM159, which was isolated from wastewater sediments self-heated to 70 °C. The gene was successfully cloned into the pET22b and pHT01 vectors, expressed and AmyBL159_Ec and AmyBL159_Bs recombinant α-amylases were purified from Escherichia coli BL21(DE3)pLys and Bacillus subtilis 168 strains, respectively. The AmyBL159_Ec enzyme was most active in the range of 75–95 °C, while AmyBL159_Bs showed maximum activity at temperatures from 45 to 75 °C. AmyBL159_Bs was shown to be more thermostable. Both enzymes were active over a broad pH range of 4.0–12.0. It was shown that Mn²⁺ ions enhanced the activity of both enzymes (up to 163% for AmyBL159_Ec and 142% for AmyBL159_Bs). These results highlight the importance of choosing an expression system for modulating the functional characteristics of recombinant α-amylase. The obtained AmyBL159_Ec and AmyBL159_Bs enzymes are promising for biotechnological applications under extreme conditions. The structure of the α-amylase was generated using the AlphaFold 3 web service. A structure–function analysis of this enzyme and previously studied α-amylases from B. licheniformis identified significant amino acid substitutions at positions 134(133) and 210(209) of the amino acid chain which may contribute to enhanced enzyme thermostability.

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.