Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.     

Electrochemical evaluation of electron transfer kinetics of high and low redox potential laccases on gold electrode surface

Laccases and other multicopper oxidases are reported to be able to carry out direct electron transfer reactions when immobilized onto electrode surface. This allows detailed research of their electron transfer mechanisms. We have recently characterized the kinetic properties of four laccases in homogenous solution and immobilized onto an electrode surface with respect to a set of different redox mediators. In this paper we report the direct electron transfer of four purified laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL), by trapping the proteins within an electrochemically inert polymer of tributylmethyl phosphonium chloride coating a gold electrode surface. In particular, we have characterized the steps involved in the laccases electron transfer mechanism as well as the factors limiting each step. During the voltammetric experiments, non-turnover Faradic signals with midpoint potential of about 790 and 400 mV were observed for high potential laccases, ThL and TvL, corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively, whereas low redox potential laccases r-MaL and RvL shown a redox couple with a midpoint potential around 400 mV. The electrocatalytic properties of these laccase modified electrodes for the reduction of oxygen have been evaluated demonstrating significative direct electron transfer kinetics. The biocatalytic activity of laccases was also monitored in the presence of a well known inhibitor, sodium azide. On the basis of the experimental results, a hypothesis about the electronic pathway for intramolecular electron transfer characterizing laccases has been proposed.     

Laccase improvement in submerged cultivation: induced production and kinetic modelling

Improvement of laccase production by Trametes versicolor was made by employing different operational strategies. In the cell growth medium, various glucose concentrations were compared for improving laccase production. A clear and significant stimulation of enzyme production under carbon limitation was obtained. Copper, 2,5‐xylidine, and a phenolic mixture were also used as laccase inducers. A cooperative effect between the inducers on laccase production was identified. Mixtures of inducers produced higher laccase activities, reaching values of 5500 U dm^?3. Further productivity enhancement can be obtained using the inducers along with the carbon limitation strategy. It is shown that low laccase concentrations are obtained by a primary metabolism of T versicolor, and that phenolic compounds and carbon limitation induce a secondary metabolism, providing higher laccase concentrations. A mathematical model for laccase production based on a direct experimental measure of biomass, along with substrate consumption and enzymatic activity over time is proposed for non‐homogeneous fermentations of T versicolor. Copyright © 2005 Society of Chemical Industry     

Immobilization of the Laccases from Trametes versicolor and Streptomyces coelicolor on Single‐wall Carbon Nanotube Electrodes: A Molecular Dynamics Study

In this work, we investigate the immobilization of laccases from Trametes versicolor (TvL) and the small laccase (SLAC) from Streptomyces coelicolor on single‐wall carbon nanotube (SWCNT) surfaces. SLAC may potentially offer improved adsorption on the electrode, thus improving bioelectrocatalytic activity via direct electron transfer (DET). Laccase immobilization on SWCNTs is achieved non‐covalently with a molecular tether (1‐pyrene butanoic acid, succinimidyl ester) that forms an amide bond with an amine group on the laccase surface while the pyrene coordinates to the SWCNT by π–π stacking. In our approach, density functional theory calculations were first used to model the interaction energies between SWCNTs and pyrene to validate an empirical force field, thereafter applied in molecular dynamics (MD) simulations. In the simulated models, the SWCNT was placed near the region of the (type 1) Cu(T1) atom in the laccases, and in proximity to other regions where adsorption seems likely. Calculated interaction energies between the SWCNTs and laccases and distances between the SWCNT surface and the Cu(T1) atom have shown that SWCNTs adsorb more strongly to SLAC than to TvL, and that the separation between the SWCNTs and Cu(T1) atoms is smaller for SLAC than for TvL, having implications for improved DET.     

Additive effects of acyl-binding site mutations on the fatty acid selectivity of Rhizopus delemar lipase

The fatty acid specificity and pH dependence of triacylglycerol hydrolysis by the Rhizopus delemar lipase acylbinding site mutant Val206Thr+Phe95Asp (Val, valine; Thr, threonine; Phe, phenylalanine; Asp, aspartic acid) were characterized. The activity of the double mutant prolipase was reduced by as much as 10-fold, compared to the wild-type prolipase. However, the fatty acid specificity profile of the enzyme was markedly sharpened and was dependent on the pH of the substrate emulsion. At neutral pH, strong preference (10-fold or greater) for hydrolysis of triacylglycerols of medium-chainlength fatty acids (C_8:0 to C_14:0) was displayed by the variant prolipase, with no hydrolysis of triacylglycerols of short-chain fatty acids (C_4:0 to C_6:0) and little activity manifested toward fatty acids with 16 or more carbons. At acidic pH values, the fatty acid selectivity profile of the double mutant prolipase expanded to include short-chain triacylglycerols (C_4:0, C_6:0). When assayed against a triacylglycerol mixture of tributyrin, tricaprylin and triolein, the Val206Thr+Phe95Asp prolipase displayed a high selectivity for caprylic acid and released this fatty acid at least 25-fold more efficiently than the others present in the substrate mixture. When presented a mixture of nine fatty acid methyl esters, the wild-type prolipase showed a broad substrate specificity profile, hydrolyzing the various methyl esters to a similar extent. Contrastingly, the double mutant prolipase displayed a narrowed substrate specificity profile, hydrolyzing caprylic methyl ester at nearly wild-type levels, while its activity against the other methyl esters examined was 2.5- to 5-fold lower then that observed for the wild-type enzyme.     

Trametes versicolor laccase immobilized poly(glycidyl methacrylate) based cryogels for phenol degradation from aqueous media

This study aims removal of phenols in wastewater by enzymatic oxidation method. In this study, Trametes versicolor laccase was covalently immobilized onto a cryogel matrix by the nucleophilic attack of amino groups of laccase to epoxy groups of matrix. Glycidyl methacrylate was chosen as functional monomer to prepare poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [p(HEMA‐co‐GMA)] cryogels. The enzyme immobilized matrix was characterized by FTIR, SEM, and swelling tests. The effect of pH, reaction time, temperature, substrate concentration, enzyme concentration, and storage period on immobilized enzyme activity was determined and compared with those of free enzyme. The model substrate was 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS). Lineweaver‐Burk plots were used to calculate K_m and V_m values. K_m values were 165.1 and 156.0 µM while V_m values were 55.2 µM min^?1 and 1.57 µM min^?1 for free and immobilized laccase, respectively. Immobilized enzyme was determined to retain 82.5% and 72.0% of the original activity, respectively, after 6 consecutive use and storage period of 4 weeks. The free enzyme retained only 24.0% of its original activity following the same storage period. Lastly, decomposition products resulting from enzymatic oxidation of a model phenolic compound (3,5‐dinitrosalicylic acid) in aqueous solution were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41981.     

Oxalate decarboxylase of Trametes versicolor: biochemical characterization and performance in bleaching filtrates from the pulp and paper industry

BACKGROUND: Oxalate decarboxylase (ODC) from acid‐induced cultures of the white‐rot fungus Trametes versicolor was purified and characterized with respect to its biochemical properties and the possibility to utilize the enzyme for treatment of process water with the intention to prevent problems with calcium‐oxalate scaling in the pulp and paper industry. RESULTS: Purified T. versicolor ODC was identified by tandem mass spectrometry. As estimated by using SDS‐PAGE, the molecular mass was 69 kDa, and 60 kDa after deglycosylation with N‐glycosidase F. The pH optimum was 2.5 and the temperature optimum was 40–45 °C. The effects of ten potential inhibitors in industrial filtrates were examined. The enzyme was sensitive to low concentrations (0.1 mmol L^?1) of chlorite and sulfite. T. versicolor ODC exhibited activity in 16 filtrates collected from mechanical pulping and kraft pulping. It had higher activity than ODC from Aspergillus niger in all of the filtrates and higher activity than oxalate oxidase from barley in all filtrates except two. CONCLUSIONS: The investigation shows basic biochemical properties of T. versicolor ODC and indicates that the enzyme may be useful for treatment of industrial filtrates under acidic conditions. Copyright © 2012 Society of Chemical Industry     

Decolorization of textile dyes by Bjerkandera sp. BOL 13 using waste biomass as carbon source

BACKGROUND: Phanerochaete chrysosporium, Trametes versicolor and Bjerkandera sp BOL13 were compared for decolorization of azo dyes supplied individually or as a mixture. The dye decolorization was also evaluated during continuous treatment under non‐sterile conditions using a lignocellulosic growth substrate. RESULTS: Bjerkandera sp BOL13 showed the highest dye decolorization potential. This fungus was also found to support high decolorization of Remazol Red RR at an initial pH of 4‐6 and when using straw as co‐substrate. The fungus was evaluated for Remazol Red RR decolorization in a continuously fed packed‐bed bioreactor operated under non‐sterile conditions with 3 days of hydraulic retention time. When glucose was supplied as growth‐substrate, decolorization efficiencies of 65‐90% were maintained for 12 days in a bioreactor packed with wooden material. The decolorization efficiency was lower when glucose was not fed to the fungus or when a plastic material was used as packing. Higher manganese peroxidase and laccase activities were also recorded when the wood packing was used. Contamination caused a drop in decolorization efficiency after 17‐19 days operation. CONCLUSIONS: The potential of Bjerkandera sp BOL13 for decolorization of azo dyes under non‐sterile conditions using lignocellulosic growth substrates was demonstrated. Research is needed to reduce contamination under non‐sterile conditions. © 2012 Society of Chemical Industry     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

From Diols to Lactones under Aerobic Conditions using a Laccase/TEMPO Catalytic System in Aqueous Medium

An efficient catalytic system to oxidize quantitatively aliphatic diols using Trametes versicolor laccase and TEMPO has been developed in aqueous medium. Oxidations have occurred in a non‐stereoselective fashion but with complete regio‐ and/or monoselectivity, obtaining lactones with excellent purity after simple extraction. This catalytic system has been demonstrated to be scalable, compatible with the presence of a variety of functionalities, and also allowed the successful enzyme recycling using a laccase‐cross‐linked enzyme aggregates (CLEA) preparation.     

Surface characterization and direct bioelectrocatalysis of multicopper oxidases

Multicopper oxidases (MCO) have been extensively studied as oxygen reduction catalysts for cathodic reactions in biofuel cells. Theoretically, direct electron transfer between an enzyme and electrode offers optimal energy conversion efficiency providing that the enzyme/electrode interface can be engineered to establish efficient electrical communication. In this study, the direct bioelectrocatalysis of three MCO (Laccase from Trametes versicolor, bilirubin oxidase (BOD) from the fungi Myrothecium verrucaria and ascorbate oxidase (AOx) from Cucurbita sp.) was investigated and compared as oxygen reduction catalysts. Protein film voltammetry and electrochemical characterization of the MCO electrodes showed that DET had been successfully established in all cases. Atomic force microscopy imaging and force measurements indicated that enzyme was immobilized as a monolayer on the electrode surface. Evidence for three clearly separated anodic and cathodic redox events related to the Type 1 (T1) and the trinculear copper centers (T2, T3) of various MCO was observed. The redox potential of the T1 center was strongly modulated by physiological factors including pH, anaerobic and aerobic conditions and the presence of inhibitors.     

Laccase-initiated reaction between phenolic acids and chitosan

Phenolic acids are known to possess antioxidant activities whilst chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. Merging both types of molecules could therefore provide several potential applications. In this work, antioxidant properties of phenolic acid–functionalized-chitosan were investigated after being prepared from structurally-different phenolic acids (caffeic and gallic acids) and chitosan using the laccase from Trametes versicolor as the reaction initiator. A laccase-mediated oxidation kinetic of phenolic acids was monitored by UV–vis spectroscopy and cyclic voltammetry, as well as spin-trapping electron paramagnetic resonance spectroscopy (ST-EPR). The pH was shown to have a significant effect on the degree of phenolic acid self-polymerization, indicating the involvement of phenolate anions within the formations of coupled polyphenol products, and their functionalities, i.e. antioxidant activity. All the phenolic acid-functionalized-chitosans displayed greatly improved ABTS radical cation scavenging capacities, compared with the untreated chitosan.     

Inoculation of Filamentous Fungi in Manufactured Gas Plant Site Soils and PAH Transformation

Solid carriers have been developed to inoculate Trametes versicolor and Cunninghamella elegans into manufactured gas plant site soils. Pelleted wheat bran carriers were very efficient in stimulating the growth of fungi in an industrial soil containing about 2800 mg kg^?1 PAHs. Fungal biomass and activity of extracellular laccases, enzymes produced by T. versicolor as markers of metabolic activity in the contaminated soil, both decreased after 2 weeks of incubation. Supplementing the soil with a mixture of carbon, nitrogen and phosphorus enhanced the fungal activity period. A 38% decrease of solvent extractable PAHs was observed in manufactured gas plant site soils when supplemented with T. versicolor, Glucidex 19^TM, ammonium nitrate, lime phosphate and Montanox 80^TM, after 20 weeks. Then, the degradation proceeded more slowly during the following 30 weeks, and reached 43% of initial extractable PAHs. Some factors governing a limited PAH biotransformation in the soil are discussed.     

The Deuterium Isotope Effect as a Tool to Investigate Enzyme Catalysis: Proton-Transfer Control Mechanisms in Cytochrome c Oxidase

Cytochrome c oxidase is a membrane-bound redox-driven proton pump. The coupling of the exergonic electron-transfer reactions from cytochrome c to oxygen to proton translocation across the membrane requires control of internal electron- and proton-transfer reactions. In this work, we focus on the kinetics of electron and proton transfer during those reaction steps that are coupled to proton pumping in cytochrome c oxidase. The results show that during the first pumping step (peroxy → oxoferryl transition), proton transfer regulates intramolecular electron transfer. The proton transfer takes place in two steps: (1) Internal proton transfer from a protonatable group, proposed to be Glu(I-286), in the so-called D-pathway, to an oxygen intermediate at the binuclear center (τ ≅ 100 μs); (2) Rapid re-protonation of Glu(I-286) from the bulk solution (τ < 100 μs). Only after proton uptake from solution the last (fourth) electron is transferred “one step closer” towards the binuclear center (from Cu_A to heme a). During the second proton-pumping step (oxoferryl → oxidized), this electron is transferred to the binuclear center, linked to the uptake of a proton through the D-pathway. The electron-transfer rate displays a kinetic-isotope effect (k_H/k_D) of 6 ± 1 (in a pH range in which the pH dependence of the rate is small), which indicates that the electron transfer is rate-limited by the proton transfer. The entry into the D-pathway (around Asp(I-132)) is composed of a cluster of negatively-charged amino acid residues together with a number of histidines, forming a so-called proton-collecting antenna designed to allow rapid protonation of groups within the proton-transfer pathway.     

Enzymatic transformation of sinapine using polyphenol oxidase from Trametes versicolor. Effect of pH and temperature and model development

Sinapine, a choline ester of sinapic acid and a main component of the phenolic fraction of rapeseed meals, was enzymatically transformed by an enzyme secreted by a white rot fungus Trametes versicolor. A model based on the Theorell-Chance Bi-Bi mechanism that describes the effect of pH, temperature, substrates and enzyme concentrations on the initial reaction rate was developed. The model parameters were estimated from the data regarding the effect of pH and temperature on initial reaction rates using a two-step estimation procedure that was developed in this work. The model predicts experimental data fairly well, and is valid for any pH and temperatures ranges. The optimum pH and temperature of reaction determined experimentally and confirmed by the model are 4.24 and 50 °C, respectively. However, when the effect of temperature on the oxygen solubility is not considered, i.e. oxygen is not the limiting substrate, the model shows that the optimum temperature of reaction is 60 °C. A relation between the temperature and the optimum pH of reaction was proposed. The developed model was used to predict the dynamics of sinapine transformation. The results showed that the investigated enzymatic system includes additional enzymatic reactions between oxygen and the products of sinapine transformation.     

Controlled release of biocides in solid wood. III. Preparation and characterization of surfactant‐free nanoparticles

Polymeric nanoparticles containing the fungicides tebuconazole and chlorothalonil were prepared by a simple, surfactant‐free method and found to have significantly smaller median particle diameters and more stable aqueous suspensions than their surfactant‐stabilized counterparts. These more stable suspensions were delivered into southern yellow pine and birch wood with greater efficiency than the equivalent surfactant‐stabilized nanoparticle suspensions. We found that the suspensions protected the treated wood against fungal attack by Gloeophyllum trabeum, a common brown rot wood decay fungus, and Trametes versicolor, a common white rot wood decay fungus, at low tebuconazole and chlorothalonil contents in the wood. Southern pine lost 5% or less of its mass after 55 days of exposure to G. trabeum when the tebuconazole or chlorothalonil content in the wood was only 0.4 kg/m³, while a tebuconazole or chlorothalonil content of 0.8 kg/m³ in birch wood was sufficient to bring its mass loss to less than 5% after 55 days of exposure to T. versicolor. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 615–621, 2002     

Effects of reaction conditions on laccase‐catalyzed α‐naphthol polymerization

Enzymatic oxidative polymerization of α‐naphthol was carried out batch‐wise with the laccase enzyme, produced by Trametes versicolor (ATCC 200801). The polymerization reaction was conducted in a closed, temperature controlled system containing acetone (solvent) and sodium acetate buffer for pH control. The effects of the organic solvent (acetone) composition, monomer (α‐naphthol) and enzyme concentrations, buffer pH and temperature on the polymerization rate were investigated with respect to initial reaction conditions and depletion rate of dissolved oxygen. The optimum acetone composition, pH, monomer, dissolved oxygen and enzyme concentrations were determined as 50% (v/v), 5, 3409 gm⁻³, 20.3 gm⁻³ and 0.173 U cm⁻³, respectively; these values provided the most desirable conditions for initial reaction rate. Temperature rise supported the rate increase up to 37 °C, after which the rate tended to be stable due to a drop in dissolved oxygen concentration. The product polymer, poly(α‐naphthol), with an average molecular weight of 4920 Da was soluble in common organic solvents. © 2000 Society of Chemical Industry     

Removal of aqueous phenolic compounds from a model system by oxidative polymerization with turnip (Brassica napus L var purple top white globe) peroxidase

Turnip roots, which are readily available in Mexico, are a good source of peroxidase, and because of their kinetic and biochemical properties have a high potential as an economic alternative to horseradish peroxidase (HRP). The efficiency of using turnip peroxidase (TP) to remove several different phenolic compounds as water‐insoluble polymers from synthetic wastewater was investigated. The phenol derivatives studied included phenol, 2‐chlorophenol, 3‐chlorophenol, o‐cresol, m‐cresol, 2,4‐dichlorophenol and bisphenol‐A. The effect of pH, substrate concentration, amount of enzyme activity, reaction time and added polyethylene glycol (PEG) was investigated in order to optimize reaction conditions. A removal efficiency ≥85% was achieved for 0.5 mmol dm^?3 phenol derivatives at pH values between 4 and 8, after a contact time of 3 h at 25 °C with 1.28 U dm^?3 of TP and 0.8 mmol dm^?3 H₂O₂. Addition of PEG (100–200 mg dm^?3) significantly reduced the reaction time required (to 10 min) to obtain >95% removal efficiency and up to 230% increase in remaining TP activity. A relatively low enzyme activity (0.228 U dm^?3) was required to remove >95% of three phenolic solutions in the presence of 100–200 mg dm^?3 PEG. TP showed efficient and fast removal of aromatic compounds from synthetic wastewaters in the presence of hydrogen peroxide and PEG. These results demonstrate that TP has good potential for the treatment of phenolic‐contaminated solutions. © 2002 Society of Chemical Industry     

Detoxification of mercury by immobilized mercuric reductase

Mercuric reductase which originated from a recombinant Escherichia coli PWS1 was purified and immobilized on a chemically modified diatomaceous earth support. The mercury reduction kinetics, pH dependence, storage stability, and reusability of the immobilized enzyme were investigated. Four dyes were examined for their electron transfer efficiency with the soluble and bound mercuric reductase. Continuous mercury detoxification by the immobilized mercuric reductase was also performed in fixed‐bed processes. The effects of bed‐length, mercury loading rate, and electron donor on the performance of the fixed beds were assessed. Immobilized mercuric reductase exhibited substrate‐inhibition‐type kinetics with a maximal activity (1.2 nmol Hg mg⁻¹ protein s⁻¹) occurring at an initial Hg²⁺ concentration of 50 µmol dm⁻³. The optimal pH was 7.0 for the soluble and immobilized mercuric reductase, but the immobilized enzyme maintained higher relative activity for less favorable pH values. Immobilization of the enzyme appeared to significantly enhance its storage stability and reusability. Of four artificial electron donors tested, azure A (5 mmol dm⁻³) demonstrated the highest relative activity (78%) for soluble mercuric reductase. For the immobilized enzyme, neutral red (5 mmol dm⁻³) gave a relative activity of nearly 82%. With a fixed‐bed, the mercury‐reducing efficiency of using neutral red was only 30–40% of that obtained using NADPH. Fixed‐bed operations also showed that increased bed length facilitated mercury reduction rate, and the optimal performance of the beds was achieved at a flow rate of approximately 100–200 cm³ h⁻¹. © 1999 Society of Chemical Industry     

The Laccase of Myrothecium roridum VKM F-3565: A New Look at Fungal Laccase Tolerance to Neutral and Alkaline Conditions**

Most of the currently known fungal laccases show their maximum activity under acidic environmental conditions. It is known that a decrease in the activity of a typical laccase at neutral or alkaline pH values is the result of an increase in the binding of the hydroxide anion to the T2/T3 copper center, which prevents the transfer of an electron from the T1 Cu to the trinuclear copper center. However, evolutionary pressure has resolved the existing limitations in the catalytic mechanism of laccase, allowing such enzymes to be functionally active under neutral/alkaline pH conditions, thereby giving fungi an advantage for their survival. Combined molecular and biochemical studies, homological modeling, calculation of the electrostatic potential on the Connolly surface at pH 5.0 and 7.0, and structural analysis of the novel alkaliphilic laccase of Myrothecium roridum VKM F-3565 and alkaliphilic and acidophilic fungal laccases with a known structure allowed a new intramolecular channel near the one of the catalytic aspartate residues at T2-copper atom to be found. The amino acid residues of alkaliphilic laccases forming this channel can presumably serve as proton donors for catalytic aspartates under neutral conditions, thus ensuring proper functioning. For the first time for ascomycetous laccases, the production of new trimeric products of phenylpropanoid condensation under neutral conditions has been shown, which could have a potential for use in pharmacology.