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Many protein-ionic liquid investigations have examined laccase interactions. Laccases are a class of poly-copper oxidoreductases that retain significant biotechnological relevance owing to their notable oxidative capabilities and their application in the elimination of synthetic dyes, phenolic compounds, insecticides, and various other substances. This study investigates the impact of surface active ionic liquids (SAILs), namely, decyltrimethylammonium bromide [N 10111 ][Br] and 1-decyl-3-methylimidazolium chloride [C 10mim ][Cl] as cationic surfactant ionic liquids and cholinium decanoate [Chl][Dec], an anionic surfactant ionic liquid, on the structure and function of laccase from the fungus Trametes versicolor (TvL) by the molecular dynamics (MD) simulation method. In summary, this study showed that laccase solvent-accessible surface area increased in the ionic liquid [Chl][Dec] while it decreased in the other two ionic liquids. Interestingly, [Chl][Dec] ionic liquid components formed hydrogen bonds with laccase, while [N 10111 ][Br] and [C 10mim ][Cl] components were unable to form hydrogen bonds with laccase. The quantity of hydrogen bonds formed between water molecules and the enzyme was also diminished in the presence of [Chl][Dec] in comparison to the other two ionic liquids. especially at a concentration of 250 mM. In 250 mM concentrations of [N 10111 ][Br] and [C 10mim ][Cl], clusters of long-chain cations are likely to form near the copper T1 site. However, even at low [Chl][Dec] concentrations, long [Dec] - chains were observed to penetrate the enzyme near the copper T1 site, and at 250 mM [Chl][Dec], a large cluster of anions occupied the opening of the active site. The results of the analysis also show that the interaction between the [Dec] - anion and the enzyme is stronger than the interaction between [N 10111 ] + and [C 10mim ] + with laccase; in addition, the [Dec] - anion, compared to [Br] - and [Cl] - has a much greater tendency to bind with the enzyme residues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Laccases hold great potential for biotechnological applications, particularly in environmental pollutant remediation. Laccase activity is governed by the solvent environment, and ionic liquids (ILs) emerge as a versatile solvent for activation or stabilization of enzymes. Herein, effects of cholinium-based ILs formulated with carboxylic acids, inorganic acid, and amino acids as anionic species, on the catalytic activity of laccase from Trametes versicolor were investigated by experimental and computational approaches. Experimental results showed that laccase activity was enhanced by 21.39 % in 0.5 M cholinium dihydrogen citrate ([Cho][DHC]), in relation to the laccase activity in phosphate buffer medium. However, cholinium aminoate ILs negatively affected laccase activity, as evidenced by the partial deactivation of laccase in both cholinium glycinate and cholinium phenylalaninate, at concentrations of 0.1 M and 0.5 M, respectively. Molecular dynamics studies revealed that the enhancement of laccase activity in [Cho][DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. Enhanced catalytic performance of laccase in [Cho][DHC] was postulated to be driven by the high accumulation level of dihydrogen citrate anions around laccase's surface. [Cho][DHC] holds great promise as a cosolvent in laccase-catalyzed biochemical reactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pellet production represents a critical step for several processes requiring fungal biomass, nevertheless, its optimization is seldom reported. The use of finely ground rice husk as a microcarrier and co-substrate permitted a marked increase (≈ 2.7×) in the productivity of fungal pellet production using Trametes versicolor compared to traditional production methods. The pellets show similar structure and smaller size compared to typical sole-mycelium pellets, as well as comparable laccase activity. The efficiency of the pellets for biodegradation was confirmed by the removal of the crystal violet dye, achieving significantly faster decolorization rates compared to the traditionally produced pellets. The use of these pellets during the continuous treatment of the dye in a stirred tank bioreactor resulted in 97% decolorization operating at a hydraulic residence time of 4.5 d., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

White rot fungi are promising organisms for the production of mycelial-based biofoams, providing a sustainable means of valorizing lignocellulosic wastes. This study explores the utilization of two indigenous fungal species, isolated from Argentina and belonging to the genera Trametes, for producing biofoams from brewery waste. The resulting biofoams exhibited an average density of 0.30 g cm -3 , a Young's modulus of approximately 1 MPa, and a compressive stress of around 19 MPa. Additionally, the variation of laccase activity throughout the biofoam production process was evaluated. Surprisingly, residual laccase activity was detected in the biofoams following oven drying at temperatures of 60, 80, and 100 °C. This detection highlights the untapped enzymatic potential of the biofoams and positions them as promising green catalysts for various biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o -toluidine exposure, in Trametes hirsuta AH28-2. Two Zn 2 Cys 6 -type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o -toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o -toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs.IMPORTANCEWhite-rot fungi differentially express laccase isoenzymes when exposed to aromatic compounds. Clarification of the molecular mechanisms underlying differential laccase expression is essential to elucidate how white-rot fungi respond to the environment. Our study shows that two Zn 2 Cys 6 -type transcription factors form heterodimers, interact with the promoters of laccase genes, and positively regulate laccase transcription in Trametes hirsuta AH28-2. Aromatic monomer addition induces faster heterodimer formation and rate of activity. These findings not only identify two new transcription factors involved in fungal laccase transcription but also deepen our understanding of the mechanisms underlying the response to aromatics exposure in white-rot fungi., Competing Interests: The authors declare no conflict of interest.

Laccases (EC 1.10.3.2) are multicopper oxidases with the capability to oxidize diverse phenolic and non-phenolic substrates. While the molecular mechanism of their activity towards phenolic substrates is well-established, their reactivity towards non-phenolic substrates, such as polycyclic aromatic hydrocarbons (PAHs), remains unclear. To elucidate the oxidation mechanism of PAHs, particularly the activation mechanism of the sp 2 aromatic C-H bond, we conducted a density functional theory investigation on the oxidation of two PAHs (anthracene and benzo[ a ]pyrene) using an extensive model of the T1 copper catalytic site of the fungal laccase from Trametes versicolor .

The present study explored the potential of leaf litter as a source of fungi able to produce ligninolytic enzymes for the biodegradation of anthraquinone dyes. Within the colonies isolated from the leaf litter, only three colonies of two species Trametes were selected based on the detection of oxidation and decolorization halos in Petri dishes with PDA (potato-dextrose-agar) + Guaicol and PDA + RBBR (Remazol Brilliant Blue R). The identification of the colonies was done through sequencing of the ITS region. The enzymatic activity of Lac (lacase), MnP (manganês peroxidase) and LiP (lignina peroxidase) was analyzed by spectrophotometry during fermentation in PD+RBBR imedium. Isolates A1SSI01 and A1SSI02 were identified as Trametes flavida, while A5SS01 was identified as Trametes sp. Laccase showed the highest enzymatic activity, reaching 452.13 IU.L-1 (A1SSI01, 0.05% RBBR) after 96h. Isolate A1SSI02 reached the highest percentage of decolorization, achieving 89.28% in seven days. The results imply that these Trametes isolates can be highly effective in waste treatment systems containing toxic anthraquinone dyes. Keywords: laccase, peroxidases, basidiomycete, litter and biodecolorization.

Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA) is an endocrine disruptor compound that has been detected in aquatic ecosystems. In this work, the development of an electrochemical biosensor for BPA determination based on laccase from Trametes versicolor is reported. A bioconjugate was optimized to maximize the biosensor electrocatalytic activity and stability, which for the first time involved the synergistic effect of this specific enzyme (6.8 UmL -1 ), chitosan (5 mgmL -1 ) and the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate in an optimum 5:5:2 (v/v/v) proportion. This bioconjugate was deposited onto a screen-printed carbon electrode previously modified with multi-walled carbon nanotubes (MWCNTs). Nanostructuration with MWCNTs enlarged the electrocatalytic activity and surface area, thus improving the biosensor performance. The BPA electrochemical reaction follows an EC mechanism at the optimum pH value of 5.0. Linearity up to 12 µM, a sensitivity of (6.59 ± 0.04) × 10 -2 μAμM -1 and a detection limit of 8.4 ± 0.3 nM were obtained coupled with high reproducibility (relative standard deviations lower than 6%) and stability (87% of the initial response after one month). The developed biosensor was employed to the analysis of BPA in river water displaying appropriate accuracy (94.6-97.9%) and repeatability (3.1 to 6% relative standard deviations) proving its high potential applicability for in situ environmental analysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Trametes hirsuta is able to secrete laccase isoenzymes including constitutive and inducible forms, and has potential application for bioremediation of environmental pollutants. Here, an inducible group B laccase from T. hirsuta MX2 was heterologously expressed in Pichia pastoris , and its yield reached 2.59 U/mL after 5 days of methanol inducing culture. The optimal pH and temperature of recombinant laccase (rLac1) to 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were 2.5 and 60 °C, respectively. Metal ions showed different effect on rLac1 which Mg 2+ , Cu 2+ , and K + increased enzyme activity as their concentration increased, whereas Zn 2+ , Na + , and Fe 2+ inhibited enzyme activity as their concentration increased. rLac1 showed good tolerance to organic solvents, and more than 42% of its initial activity remained in 10% organic solvents. Additionally, rLac1 exhibited a more efficient decolorization ability for remazol brilliant blue R (RBBR) than for acid red 1 (AR1), crystal violet (CV), and neutral red (NR). Molecular docking results showed RBBR has a stronger binding affinity with laccase than other dyes by interacting with substrate binding cavity of enzyme. The results indicated rLac1 may be a potential candidate for dye removal from textile wastewater.

In this study, the laccase obtained from Trametes versicolor was immobilized onto the chitosan(CTS)/halloysite (HNT) beads. In the immobilization step, the effects of chitosan (1-3% w/v), halloysite (0-2% w/v), glutaraldehyde (0.5-1.5% v/v) and enzyme concentrations (1-3%) on loading and immobilization efficiency were investigated. SEM, FT-IR, XRD, TGA and XPS analyses were performed to examine the structure of beads. In addition, the effects of parameters such as pH (4-10), temperature (25-55 °C), storage life on the activity of free and immobilized laccase were also investigated. The activities of free and immobilized laccase preserved 23% and 56% of its initial activity at the end of 59 days of storage. The effects of mediators such as 2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1-Hydroxybenzotriazole hydrate (HBT), 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) and violuric acid (VLA) on the dye removal efficiency were investigated. Reusability of the CTS/HNT/Lac in the presence of HBT and VLA mediators, which enable the highest dye removal, was tested. After 15 cycles, 42% and 54% dye removal were achieved with the CTS/HNT/Lac in the medium containing HBT and VLA, and 42% and 49% of the activity is preserved, respectively. This study showed that CTS/HNT/Lac can be used repeatedly for Remazol Red RR dye removal., (Copyright © 2021 Elsevier B.V. All rights reserved.)

(+)-Catechin-laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone- d 6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.

Two morphologies of laccase-mineral hybrid complexes, i.e., laccase-mineral hybrid nanoflowers (La-HNF) and nanopetals (La-HNP), were synthesized via biomineralization using Cu 3 (PO 4 ) 2 ·3H 2 O as the mineral for Evans Blue (EB) dye biodegradation. XRD patterns and FT-IR spectra results revealed the successful immobilization of laccase via in-situ formed Cu 3 (PO 4 ) 2 ·3H 2 O crystals. Compared with free laccase, laccase-mineral hybrid complexes showed higher enzymatic activity due to the activation effect induced by copper ions of Cu 3 (PO 4 ) 2 ·3H 2 O, further, the improved kinetic parameters of laccase-mineral hybrid complexes could be ascribed to nanoscale-dispersed laccase molecules within hybrid complexes. For EB dye biodegradation, the reason why the biodegradation efficiency (94.9%) of La-HNF was higher than that (86.8%) of La-HNP could be synergistic effect of immobilized laccase within 3D hierarchical structure of La-HNF. In addition, the optimized biodegradation conditions (pH 4.6 and 40 °C) of La-HNF were obtained, moreover, 93.2% and 48.1% of EB dye were biodegraded by La-HNF after stored for 30 days and reused for 10 cycles, respectively, demonstrating La-HNF have good practicability., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the present work, pharmaceutical micropollutant degradation by laccase immobilized on silica through an innovative process is proposed. The influence of different parameters on the immobilization conditions was evaluated by a 2 3 full factorial design, and parameters leading to the highest activity were identified. Under these conditions, laccase activity reached 14 ± 2 U g -1 of silica with a protein immobilization yield of 35%. The biocatalyst characterization did not show any change in pH and thermal stabilities but enhanced the long-term storage of laccases. Immobilized T. versicolor laccases were then tested to remove four pharmaceutical micropollutants (amoxicillin, ciprofloxacin, carbamazepine, and sulfamethoxazole) in the presence of redox mediators (syringaldehyde, p-coumaric acid, and ABTS). High removal yields (50-100% according to the pollutant) were obtained within 4 h of treatment due to the synergistic effect of laccase-mediator biotransformation and adsorption on the support. Overall, the pharmaceuticals' removal efficiency was highly influenced by their physicochemical properties; however, the presence of redox mediators impacted not only the oxidation mechanism but also the interactions between the biocatalyst and micropollutants. Finally, the reusability of the biocatalyst was proved during 7 degradation cycles.

Objective: Laccase is one of the best known biocatalysts which degrade wide varieties of complex molecules that are both non-cyclic and cyclic in structure. The study focused on enzyme kinetics of a purified laccase from Trametes hirsuta L. fungus and its application on biotransformation of a carcinogenic molecule 1,4-dioxane., Results: Laccase was purified from white-rot fungus T. hirsuta L. which showed specific activity of 978.34 U/mg after the purification fold of 54.08. The stable laccase activity (up to 16 h) is shown at 4-6 pH and 20-40 °C temperature range. The purified enzyme exhibited significant stability for 10 metal ions up to 10 mM concentration, except for Fe 2+ and Hg 2+ . The Cu 2+ ion induced laccase activity up to 142% higher than the control at 10 mM concentration. The laccase enzyme kinetic parameters K m was 20 ± 5 µM and 400 ± 60 µM, whereas K cat was 198.29 ± 0.18/s and 80.20 ± 1.59/s for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol respectively. The cyclic ether 1,4-dioxane (100 ppm) was completely degraded in presence of purified laccase within 2 h of incubation and it was confirmed by HPLC and GC analysis. The oxidation reaction was accelerated by 25, 22, 6 and 19% in presence of 1 mM syringaldehyde, vanillin, ABTS and guaiacol mediators respectively., Conclusions: In this study, fungal laccase (a natural biocatalyst) based degradation of synthetic chemical 1,4-dioxane was reported for the first time. This method has added advantages over the multiple methods reported earlier being a natural remedy.

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Laccases, oxidative copper-enzymes found in fungi and bacteria were used as the basis in the design of nona- and tetrapeptides. Laccases are known to be excellent catalysts for the degradation of phenolic xenobiotic waste. However, since solvent extraction of laccases is environmentally-unfriendly and yields obtained are low, they are less preferred compared to synthetic catalysts. The histidine rich peptides were designed based on the active site of laccase extracted from Trametes versicolor through RCSB Protein Data Bank, LOMETS and PyMol software. The peptides were synthesized using Fmoc-solid phase peptide synthesis (SPPS) with 30-40% yield. These peptides were purified and characterized using LC-MS (purities >75%), FTIR and NMR spectroscopy. Synthesized copper(II)-peptides were crystallized and then analyzed spectroscopically. Their structures were elucidated using 1D and 2D NMR. Standards (o,m,p-cresol, 2,4-dichlorophenol) catalysed using laccase from Trametes versicolor (0.66 U/mg) were screened under different temperatures and stirring rate conditions. After optimizing the degradation of the standards with the best reaction conditions reported herein, medications with phenolic and aromatic structures such as ibuprofen, paracetamol (acetaminophen), salbutamol, erythromycin and insulin were screened using laccase (positive control), apo-peptides and copper-peptides. Their activities evaluated using GC-MS, were compared with those of peptide and copper-peptide catalysts. The tetrapeptide was found to have the higher degradation activity towards salbutamol (96.8%) compared with laccase at 42.8%. Ibuprofen (35.1%), salbutamol (52.9%) and erythromycin (49.7%) were reported to have the highest degradation activities using Cu-tetrapeptide as catalyst when compared with the other medications. Consequently, o-cresol (84%) was oxidized by Tp-Cu while the apo-peptides failed to oxidize the cresols. Copper(II)-peptides were observed to have higher catalytic activity compared to their parent peptides and the enzyme laccase for xenobiotic degradation., Competing Interests: The authors have declared that no competing interests exist.

Fungal laccase has aroused great concern in rapidly removing estrogens because of its ability to accelerate humification and oligomerization. Here, the effect of two humic acids (HAs) on the reaction kinetics and products distribution of 17α-ethynylestradiol (EE2) in laccase-initiated humification and coupling was systematically elucidated. Laccase from Trametes versicolor exhibited over 98.3% removal rate for EE2 at pH 5.0 within 120 min, while HAs invariably restrained EE2 transformation by competing with target-substrate for the enzymatic catalytic center. EE2 removal followed pseudo-first-order kinetics, and the rate constant was decreased markedly with increasing concentration of two HAs (0-60 mg L -1 ). Additionally, laccase heightened the aromaticity and humification degrees (A 250 nm /A 365 nm ratio) of HAs probably due to the formation of new humic polymers such as (HA) m and/or (HA) m -(EE2) n (m and n represent the number of HA and EE2 units, respectively). Three major EE2 oligomers were identified, in accordance with a mechanism involving the phenoxy radical-driven polymerization to yield a wide variety of self-coupling products. Notably, HAs diminished the extent of EE2 self-coupling but aggrandized the cross-coupling between EE2 and HAs, and the inhibition degree of EE2 self-coupling increased with the concentration of HAs. One major reason is EE2 could be covalently incorporated into humic molecules to produce (HA) m -(EE2) n cross-coupling products via radical-caused C-C, C-O-C, and/or C-O-C bonds, thereby reducing EE2 self-oligomerization. These findings highlight that HAs play a vital role in the fungal laccase-induced humification and oligomerization of EE2, which obviously alter the geochemical fate and transport of EE2 in natural aquatic ecosystems., Competing Interests: Declaration of competing interest The authors declare no competing interests. Acknowledgments., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A ternary catalysis system was investigated to evaluate the comparative degradation of toxic fungicide metabolite 3,5-dichloroaniline (3,5-DCA) by laccase and MnO 2 with mediators. In this study, copper based fungal enzyme laccase (Trametes versicolor origin) and metal catalyst MnO 2 with various combinations of phenolic mediators (catechol, syringaldehyde, syringic acid, caffeic acid and gallic acid) were monitored to optimize and screen the better one for 3,5-DCA degradation assay. Catechol showed better potentiality in reduction of 3,5-DCA among the studied mediators. Catechol (2mM) showed the highest reduction rate (99-100%) followed by syringaldehyde (40.51%) with 2U/mL of laccase at 25 °C within 24 h reaction time. Similarly, complete degradation of 3,5-DCA was obtained by catechol (2mM) with 2 mg/mL of MnO 2 in MnO 2 -mediator assay. The notable finding of current study indicated the triggering of catechol for better 3,5-DCA degradation at higher pH condition but inertness in laccase-mediator assay due to laccase destabilization. The reaction pathways of optimized mediator-based catalysis for laccase and MnO 2 were proposed. Finally, the optimized laccase-catechol based degradation was considered as a pioneer green catalysis approach to reduce the toxic metabolite 3,5-DCA concentrations in aqueous medium as compared to MnO 2 -catechol catalysis., Competing Interests: Declaration of competing interest The authors declare that they have no potential conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A biosensor for phenolic compounds based on a chemically modified laccase from Coriolus hirsuta immobilized on functionalized screen-printed carbon electrodes (SPCEs) was achieved. Different enzyme modifications and immobilization strategies were analyzed. The electrochemical response of the immobilized laccase on SPCEs modified with carboxyl functionalized multi-walled carbon nanotubes (COOH-MWCNT) was the highest when laccase was aminated prior to the adsorption onto the working electrode. The developed laccase biosensor sensitivity toward different phenolic compounds was assessed to determine the biosensor response with several phenolic compounds. The highest response was obtained for ABTS with a saturation value of I max  = 27.94 μA. The electrocatalytic efficiency (I max /K app m ) was the highest for ABTS (5588 μA μM -1 ) followed by syringaldazine (3014 μA.μM -1 ). The sensors were considerably stable, whereby 99.5, 82 and 77% of the catalytic response using catechol as substrate was retained after 4, 8 and 10 successive cycles of reuse respectively, with response time average of 5 s for 12 cycles. No loss of activity was observed after 20 days of storage., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ecotoxicological evaluations require the use of assays with several bioindicators from different trophic levels. Only a few ecotoxicological tests using fungi have been developed, reason why, detection of adverse effects from compounds that exert fungicide action may be overlooked. This work developed a toxicity test based on the inhibition of laccase enzymatic activity in the fungus Trametes versicolor. The test was applied to several fungicides and succeeded to determine inhibition values (half maximum effective concentration, EC 50 ) for most of them (flusilazole, imazalil, pyrimethanil, tetraconazole), though a clear dose-response was not evident for others (thiabendazole, metalaxyl). The application on atrazine (herbicide), imidacloprid (insecticide) and oxytetracycline (antibiotic), proved the proposed test is suitable towards other agrochemicals. The test was also used to estimate the detoxification resulting from two different approaches employed in the removal of agrochemicals. (a) First, in the liquid-phase elimination by fungal biomass simultaneously removing atrazine, imazalil, tebuconazole and triadimenol, the test showed a significant decrease in toxicity by biodegradation (adsorption contribution to detoxification was negligible). (b) Second, a solid-phase biomixture (used for pesticide degradation from agricultural wastewater) partially removed atrazine, imazalil, metalaxyl and pyrimethanil after 33 d; nonetheless, this system could not reduce the toxicity of the matrix, and higher laccase inhibition was detected after the treatment. The design test increases the battery of available bioassays to determine the toxicity of agrochemicals, and provides an interesting tool to monitor biodegradation processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Laccases have been widely explored for their ligninolytic capability in bioethanol production and bioremediation of industrial effluents. However, low reaction rates have posed a major challenge to commercialization of such processes. This study reports the first evidence of laccase inhibition by two types of lignin degradation intermediates - fungal-solubilized lignin and alkali-treated lignin - thus offering a highly plausible explanation for low reaction rates due to buildup of inhibitors during the actual process. Reversed-phase high-performance liquid chromatography revealed the presence of similar polar compounds in both lignin samples. A detailed kinetic study on laccase, using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as the substrate, was used to calculate the Michaelis constant (K m ) and maximum reaction rate (V max ). With an increase in the concentration of lignin degradation intermediates, V max remained nearly constant, while K m increased from 1.3 to 4.0 times that of pure laccase, revealing that the inhibition was competitive in nature. The kinetic studies reported here and the insight gained into the nature of inhibition can help design process strategies to mitigate this effect and improve overall process efficiency. This work is applicable to processes that employ laccase for delignification of biomass, such as second-generation biofuels processes, as well as for industrial effluent treatment in paper and pulp industries.

Here, molecular docking simulation was used to predict and compare interactions between a recombinant Trametes sp. C30 laccase from Saccharomyces cerevisiae and four aflatoxins (AFB 1 , AFB 2 , AFG 1 , and AFG 2 ) as well as their degradation at a molecular level. The computational result of docking simulation indicates that each of the aflatoxins tested can interact with laccase with a binding ability of AFB 1 >AFG 2 >AFG 1 >AFB 2 . Simultaneously, it also demonstrated that aflatoxin B 1 ， B 2 ， G 1 ， G 2 may interact near the T1 copper center of the enzyme through H-bonds and hydrophobic interactions with amino acid residues His481 and Asn288; His481； Asn288, and Asp230; His481 and Asn288. Biological degradation test was performed in vitro in the presence of a recombinant laccase. Degradation increased as incubation time increased from 12 to 60 hr and the maximum degradation obtained for AFB 1 , AFB 2 , AFG 1 , and AFG 2 was 90.33%, 74.23%, 85.24%, and 87.58%, respectively. Maximum degradation of aflatoxins was determined with a total activity 3 U laccase at 30 °C in 0.1 M phosphate buffer, pH 5.7 after 48-hr incubation. The experimental results are consistent with that of docking calculation on the biological degradation test of four aflatoxins by laccase. PRACTICAL APPLICATION: In this study, the degradation efficiencies of laccase for B and G series of aflatoxins were determined by computer simulation and verified by performing in vitro experiments. It can provide reference for rapid screening of aflatoxin degradation-related enzymes., (© 2020 Institute of Food Technologists®.)

The azo dye Congo red is heavily used in textile industries and is actively present in the wastewater run-offs. Its structural complexity and physical characteristics make it resistant to the physicochemical treatments employed by the industry. Over time, application of the enzyme laccase has proved to be quite useful due to its ability to oxidize and eventually decolorize the dye. Moreover, the use of ABTS as the electron mediator also helps in enhancing the oxidizing capability of the enzyme with congo red. The present study involves establishing the role of the individual components i.e. laccase, ABTS and the dye, in the LMS electrochemically. Congo red doesn't have any form of electrochemical activity by itself, but the enzyme brings about a substantial change by increasing the rate of reduction. The effect of ABTS, though same, is concentration-dependent. For LMS, laccase helps in bringing about the rate of reduction much faster in the presence of the mediator, initiating the decolorization of the dye., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bisphenol A (BPA) is an environmental pollutant with adverse effects on different ecosystems. In this study, immobilized laccase enzymes onto inorganic supports were used to remove BPA. Laccase was successfully immobilized on sodium zeolite Y (NaY) and its modified desilicated (DSY) and dealuminated (DAY) forms. NaY-based supports were instrumentally characterized. The immobilized laccase on NaY (laccase@NaY), desilicated (laccase@DSY), and dealuminated (laccase@DAY) forms showed significant improvement on immobilization yield (IY%) and efficiency (IE%). Laccase@DSY and laccase@NaY showed IY% = 73.18 ± 3.33 % and 46.23 ± 1.81 % and IE% = 94.50 ± 1.86 %, and 74.39 ± 1.41 %, respectively, whereas IY% and IE% for laccase@DAY were achieved as 81.12 ± 1.32 % and 98.56 ± 2.93 %, respectively. The supports also increased the enzyme characteristics such as pH-temperature range, catalytic stability, and reusability. K m values were 0.73 ± 0.05, 0.26 ± 0.09, 0.31 ± 0.5, and 1.01 ± 0.03 mM for laccase@NaY, laccase@DAY, laccase@DSY, and the free enzyme, respectively. The enzyme demonstrated higher biodegradation ability of bisphenol A upon immobilization on the supports compared to that of the soluble enzyme. A bio-removal yield of 86.7 % was obtained considering three parameters including amount of laccase@DAY (8 U mg -1 ), concentration of BPA (0.5 mM), and treatment time (1 h) based on response surface methodology (RSM). Biodegradation metabolites (49 ± 5.8 %) and unconverted BPA (14 ± 5.2 %) were analyzed by gas chromatography-mass spectrometry., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Laccases are very interesting biocatalysts of recognized importance for several industrial applications. Its production by Trametes versicolor, a white-rot fungus, was induced by a combination of cotton gin wastes (1%), a lignocellulosic waste, and vinasse (15%), an industrial by-product from sugarcane industry. The use of these agro-industrial wastes are interesting, since it helps in reducing the enzyme production costs, due to their low cost and wide availability, as well as the environmental contamination issues, due to their improper disposal. Thus, laccase production was studied in submerged fermentation of T. versicolor using these agro-industrial wastes (cotton gin waste and vinasse) as carbon source and an additional nitrogen source (0.1% peptone). Three different bioreactors were evaluated for laccase production, such as BioFlo 310 bioreactor, aluminium tray and Erlenmeyer flasks to achieve high levels of laccase production. The highest specific production of laccase was found in BioFlo 310 bioreactor with 12 days of fermentation (55.24 U/mg prot.), which has been shown to be closely related to the oxygen supply to the microorganism through aeration of the fermentation medium. This study brings new insights into green biotechnology regarding vinasse utilization, which is frequently discharged in soils, rivers, and lakes causing adverse effects on agricultural soils and biota, as well as the cotton gin waste recovery.

The immobilization of enzymes on an electrode surface is critical in preserving enzyme activity and providing a sufficient electron transfer pathway for bioelectrocatalysis. Here, we present a novel single-step, cross-linker free immobilization for direct bioelectrocatalysis using an ionic strength induced phase inversion of oppositely charged polyelectrolytes. Cationic poly-guanidinyl-propyl-methacrylate (pGPMA, PG) and anionic inorganic polyphosphate, sodium hexametaphosphate (P6) were used to make an electrostatically condensed phase (PGP6). A mixture of PGP6 and laccase (LAC) from Tramates versicolor or HRP (HRP) from Armoracia rusticana were deposited on the electrode surface and were submerged in DI water to form white porous electrode coatings. Each electrode showed a current generation corresponding to the respective substrates via direct bioelectrocatalysis.

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H 2 O 2 . POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (I max app /K M app ) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Methodology was developed to expand the range of benign alkyl glycoside surfactants to include also anionic types. This was demonstrated possible through conversion of the glycoside to its carboxyl derivative. Specifically, octyl β-D-glucopyranoside (OG) was oxidised to the corresponding uronic acid (octyl β-D-glucopyranoside uronic acid, OG-COOH) using the catalyst system T. versicolor laccase/2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and oxygen from air as oxidant. The effects of oxygen supply methodology, concentrations of laccase, TEMPO and OG as well as reaction temperature were evaluated. At 10 mM substrate concentration, the substrate was almost quantitatively converted into product, and even at a substrate concentration of 60 mM, 85% conversion was reached within 24 h. The surfactant properties of OG-COOH were markedly dependent on pH. Foaming was only observed at low pH, while no foam was formed at pH values above 5.0. Thus, OG-COOH can be an attractive low-foaming surfactant, for example for cleaning applications and emulsification, in a wide pH range (pH 1.5-10.0).

Agricultural practices generate lignocellulosic waste that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, fungal enzymes are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The aim of this work was to characterize LBM 033 strain and to analyze its efficiency in the hydrolysis of cellulosic substrates, including barley straw. LBM 033 strain was identified as Trametes villosa by molecular techniques, through the use of the ITS and rbp2 markers and the construction of phylogenetic trees. The cell-free supernatant of T. villosa LBM 033 showed high titers of hydrolytic enzymatic activities, necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing pure cellulose to cellobiose and glucose and also degraded the polysaccharides contained in barley straw to short soluble oligosaccharides. These results indicate that macro fungi from tropical soil environments, such as T. villosa LBM 033 can be a valuable resource for in-house, cost effective production of enzymes that can be applied in the hydrolysis stage, which could reduce the total cost of bioethanol production.

In this study, two laccase isoenzymes (Lac1 and Lac2) from the culture supernatant of Trametes hirsuta MX2 were purified, and the genes (Lac1 and Lac2) coding the isoenzymes were cloned. Both Lac1 and Lac2 contained an open reading frame of 1563 bp with an identity of 79%. The two isoenzymes showed significant biochemical differences. The maximal activities of Lac1 and Lac2 were at pH 2.5 with 2-2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and the optimal temperatures for the activities of Lac1 and Lac2 were 60 and 50 °C, respectively. Lac1 exhibited excellent resistance to acidic conditions and retained 62.17% of its initial activity at pH 2.5 after a 72-h incubation. Lac2 was more thermostable than Lac1 with half-lives (t 1/2 ) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t 1/2 of Lac1 were only 4.19 and 0.88 h, respectively. Both Lac1 and Lac2 isoenzymes have a strong tolerance to Mg 2+ , Mn 2+ , Cu 2+ , and EDTA (50 mM). At a low concentration of 0.05 U mL -1 , the enzymes could decolorize towards Remazol Brilliant Blue R, Acid Red 1, Crystal Violet, and Neutral Red in the presence of ABTS. These unusual properties demonstrated that the two laccases have strong potential for specific industrial applications.

Laccase mediator system (LMS), a very attractive candidate for refractory organics biodegradation, harbors tremendous potential on industry application. However, the performance of LMS usually varies with the discrepancy of mediators and substrates in their chemical structures. Here, we adopt electrochemical analysis that is able to assess the degradation performance of various LMS on three different dyes by quantitative analysis of reaction outcome. Two mechanisms were suggested to explain the grafting of three mediators (1-Hydroxybenzotriazole, Violuric Acid and Acetosyringone), involving the transformation of proton or electron to produce active moieties, which subsequently react with target substrates. A thorough electrochemical insight into the redox features of mediators and its change in the presence of laccase and substrates were carried out using electrochemical analysis. The effectiveness of each kind of LMS on substrates was preliminarily evaluated by analyzing the change of the peak current and potential of mediators. The actual conversion rate of dyes was used to verify the analysis results, which confirms the important role of the stability of the oxidized form as well as their redox potential of the mediators in determining the mechanism of substrate oxidation. The application of electrochemical analysis in efficiency evaluation of LMS shed new light on effective selection of suitable mediators for degradation of refractory organics. It was therefore possible to prejudge the efficacy of LMS by analyzing the electrochemical parameters of target substances and mediators, which undoubtedly has broad further application prospects of LMS., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Polysaccharopeptides (PSPs) extracted from Trametes versicolor show antitumor, anti-inflammatory, and immunomodulation effects. According to our previous report, the enzymatic hydrolysates obtained from T versicolor PSPs by 80 U/mL β-1,3-D-glucanase (PSPs-EH80) did not change the functional groups of PSPs but enhanced their antioxidative activities. However, the mechanism elevating the antioxidant and anti-inflammatory effect of PSPs-EH80 is not clear., Aims: This research focused on the protective mechanism(s) of PSPs-EH80 against free radical and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative damage in human keratinocyte (HaCaT) cells., Methods: We evaluated the anti-inflammatory potential of PSPs-EH80 by assessing its free radical-induced oxidative damage. Using the HaCaT cell as the experimental system, we tested the protective effects of PSPs-EH80 on a model of AAPH-induced cellular oxidative damage through the assessment of cell survival rate. Heme oxygenase 1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), cyclooxygenase-2 (COX-2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase were determined using MTT assays and Western blotting., Results: We demonstrated that PSPs-EH80 significantly enhanced keratinocyte viability, and augmented the antioxidant HO-1 expressions through upregulation of the Nrf2, compared with PSPs. Furthermore, PSPs-EH80 significantly reduced AAPH-induced COX-2 expressions through downregulation of the ERK, p38, and NF-κB signaling pathways., Conclusion: The PSPs-EH80 exhibits a stronger antioxidant and anti-inflammatory capacity than PSPs. Therefore, PSPs-EH80 could be effective for attenuating free radical-induced oxidative damage in human skin and can be applied widely in the fields of cosmetics and medicine., (© 2019 Wiley Periodicals, Inc.)

Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3-4), higher thermo-stability (60 °C), and broad enzyme kinetics (K m ) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Laccases have attracted a great deal of interest because of their remarkable ability for the degradation of synthetic dyes present in wastewaters. New laccase producing sources with robust operational and functional properties are being continuously explored. In this work, the potential for the decolorization and detoxification of synthetic dyes was evaluated in two Mexican strains of the genus Trametes . The decolorization capacity of Trametes maxima LE130 and Trametes sp. LA1 was tested in solid and liquid media. The phytotoxicity of the degradation products was determined using Raphanus sativus and Pisum sativum seeds. In solid media, both strains showed a higher decolorization capacity ( p ≤ 0.05) than Phanerochaete chrysosporium ATCC 24725, which is known to be very efficient in lignin and dye-degradation. They produced laccase as the main ligninolytic enzyme; T. maxima LE130 secreted a single isoform of 43.9 kDa, while Trametes sp. LA1 produced three isoforms of 67.3, 58.6 and 52.7 kDa, respectively. Trametes sp. LA1 culture fluids were capable of decolorizing and detoxifying chemically diverse dyes (anthraquinonic dye Remazol Brilliant Blue R, azoic Reactive Black 5 and triphenylmethane Crystal Violet) without the addition of redox mediators. Therefore, this could be considered as a new laccase source which could be potentially competitive in the bioremediation of dye-containing wastewaters., Competing Interests: The authors declare no conflict of interests.

In this study, we investigated the transformation of atenolol (ATL) by the naturally occurring laccase from Trametes versicolor in aqueous solution. Removal efficiency of ATL via laccase-catalyzed reaction in the presence of various laccase mediators was examined, and found that only the mediator 2, 2, 6, 6-tetramethyl-1-piperidinyloxy (TEMPO) was able to greatly promote ATL transformation. The influences of TEMPO concentration, laccase dosage, as well as solution pH and temperature on ATL transformation efficiency were tested. As TEMPO concentrations was increased from 0 to 2000 μM, ATL transformation efficiency first increased and then decreased, and the optimal TEMPO concentration was determined as 500 μM. ATL transformation efficiency was gradually increased with increasing laccase dosage. ATL transformation was highly pH-dependent with an optimum pH of 7.0, and it was almost constant over a temperature range of 25-50 °C. Humic acid inhibited ATL transformation through competition reaction with laccase. The presence of anions HCO 3 - and CO 3 2- reduced ATL transformation due to both anions enhanced solution pHs, while Cl - , SO 4 2- , and NO 3 - at 10 mM showed no obvious influence. The main transformation products were identified, and the potential transformation pathways were proposed. After enzymatic treatment, the toxicity of ATL and TEMPO mixtures was greatly reduced. The results of this study might present an alternative clean strategy for the remediation of ATL contaminated water matrix., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The purpose of current research is to design and acquire novel biological macromolecule materials with enhanced functional properties. Chitosan-ferulic acid binary conjugate (CFC) was synthesized based on the carbodiimide-mediated coupling reaction, and then β-lactoglobulin-ferulic acid-chitosan ternary conjugate (BFCC) was fabricated by laccase induction. Furthermore, the impact of laccase concentration on the formation mechanism of BFCC was investigated by the analyses of reaction group content, ultraviolet-visible (UV-vis) absorption, circular dichroism (CD), and fluorescence spectroscopy. Results showed that hetero- and homo-conjugates between CFC and β-lactoglobulin (β-LG) were achievable at the low concentration (≤4 U/mL) and high concentration (≥6 U/mL) of laccase, respectively. The CD spectrum indicated that the interaction with CFC made β-LG more disorderly. Functional evaluation results revealed that the antioxidant activity and thermal stability of BFCC were improved compared with β-LG. The knowledge obtained in the present study provided an effective method to acquire innovative biological macromolecule materials with desirable functional characteristics.

Lignin and lignin components of woody biomass have been identified as an attractive alternative to fossil fuels. However, the complex composition of this plant polymer is one of the drawbacks that limits its exploitation. Biocatalysis of lignin to produce platform chemicals has been receiving great attention as it presents a sustainable approach for lignin valorisation. Aligned with this area of research, in the present study we have applied ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to identify the preferred lignin substrates of a ligninolytic enzyme, a laccase produced by the terrestrial fungus Trametes versicolor. A commercial lignin was incubated with the laccase and acetosyringone (a laccase mediator) for up to 168 h and direct infusion electrospray FT-ICR MS enabled the identification of thousands of molecular species present in the complex lignin sample at different incubation time points. Significant changes in the chemical composition of lignin were detected upon laccase treatment, which resulted in a decrease in the molecular mass distribution of assigned species, consistent with laccase lytic activity. This reduction was predominantly in species classified as lignin-like (based on elemental ratios) and polymeric in nature (>400 Da). Of particular note was a fall in the number of species assigned containing sulfur. Changes in the chemical composition/structure of the lignin polymer were supported by FT-IR spectroscopy. We propose the use of FT-ICR MS as a rapid and efficient technique to support the biotechnological valorisation of lignin as well as the development and optimization of laccase-mediator systems for treating complex mixtures., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Utilization of polyphenol oxidases (laccase, tyrosinase) in biosensor technology is an efficient approach towards phenol detection, which is significant in numerous fields such as environmental monitoring, food industry etc. The use of crude extract instead of pure enzyme eliminates the need for costly and laborious processes of enzyme separation and purification. This study employs polyphenol oxidase extract, biosynthesized by white-rot fungi Trametes pubescens (TP) for the development of amperometric biosensors for catechol detection. The catalytic activity of the crude extract was firstly used to induce the bio-synthesis of conducting polymer - polypyrrole (Ppy), resulting in the self-encapsulation of the enzyme extract within the conducting material. The viability and biological integrity of the enzyme extract was preserved after the synthesis and was able to efficiently detect phenolic compounds such as catechol. Comparative evaluations between the biosynthesized Ppy based biosensor (bio-Ppy) and the biosensor based on bio-PPy with additional enzyme extract (bio-Ppy-TP) were performed. Lastly, the performance of these two biosensors was compared with that of a third one, based on chemically synthesized Ppy with enzyme extract (chem-Ppy-TP). All three types of biosensors proved high efficiency for catechol detection at low concentration (1-60 μM) and were employed for real sample detection in fruit wines showing linear correlation with the spectrophotometric results obtained with the Folin-Ciocalteau standard test., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hypothesis: The development of enzymatic conjugates with industrial applications require approaches with good scalability and batch-to-batch reproducibility. Hereof, nearly monodisperse iron oxide nanoparticles can be synthesized by thermal decomposition with high yields. A mixture of gallic and polyacrylic acid is used for the direct water transfer and later immobilization of laccase (Trametes versicolor)., Experiments: Nanoparticles were synthesized by thermal decomposition (13.1 nm by TEM, 50 nm by DLS) and later transferred to water by a ligand exchange method with polyacrylic acid and a polyacrylic acid/gallic acid mixture. Laccase was immobilized on water dispersions of both nanoparticles via a carbodiimide coupling., Findings: The nanoparticles exhibited superparamagnetic behavior with insignificant values of iH c . The presence of gallic acid hindered the formation of multiple polyacrylic acid layers, therefore improving the colloidal stability of the nanoparticles (100 nm by DLS) after weeks of storage. Nanoparticles containing only polyacrylic acid showed poor activity (60% loading, 4.5% activity), while nanoparticles with both polyacrylic and gallic acids showed enzymatic activity values 4.4 times higher than the free enzyme (13% loading, 57% activity). The nanoparticles improved the storage stability (8 times) of the enzyme, its thermoresistance (4 times), and its reactivity against azo dyes Camalgite and Congo Red (21 and 27% increase, respectively). In addition to some improved catalytic properties in comparison to similar works, this is the first report of the use of gallic acid for both the direct transfer to water and enzyme immobilization on highly monodisperse, batch-to-batch reproducible superparamagnetic nanoparticles., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Untethered actuation is important for robotic devices to achieve autonomous motion, which is typically enabled by using batteries. Using enzymes to provide the required electrical charge is particularly interesting as it will enable direct harvesting of fuel components from a surrounding fluid. Here, a soft artificial muscle is presented, which uses the biofuel glucose in the presence of oxygen. Glucose oxidase and laccase enzymes integrated in the actuator catalytically convert glucose and oxygen into electrical power that in turn is converted into movement by the electroactive polymer polypyrrole causing the actuator to bend. The integrated bioelectrode pair shows a maximum open-circuit voltage of 0.70 ± 0.04 V at room temperature and a maximum power density of 0.27 µW cm -2 at 0.50 V, sufficient to drive an external polypyrrole-based trilayer artificial muscle. Next, the enzymes are fully integrated into the artificial muscle, resulting in an autonomously powered actuator that can bend reversibly in both directions driven by glucose and O 2 only. This autonomously powered artificial muscle can be of great interest for soft (micro-)robotics and implantable or ingestible medical devices manoeuvring throughout the body, for devices in regenerative medicine, wearables, and environmental monitoring devices operating autonomously in aqueous environments., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High potential multicopper oxidases (MCOs) have T1 reduction potentials >600 mV (vs normal hydrogen electrode), making them important catalysts for O 2 reduction in various biotechnological applications. The oxygen reduction mechanism for the low potential MCOs is well-characterized; however, O 2 reactivity of high potential MCOs is not well understood. In this study, we have shown that laccase from Trametes versicolor , where the T1 redox potential is increased by ∼350 mV over that of the low potential MCOs corresponding to an 8 kcal/mol decrease in the driving force, exhibits a slower intramolecular electron transfer (IET) rate to the trinuclear Cu cluster (TNC) in the native intermediate (NI), relative to the low potential MCO from Rhus vernicifera laccase. This IET rate is, however, >10 2 times faster than the decay rate of the NI, demonstrating that this intermediate form of the enzyme is catalytically relevant enabling fast turnover. However, in contrast to the low potential MCOs where T1 reduction by substrate is rate limiting, the rate limiting step in turnover of high potential MCOs is the first IET to NI. Part of the reduction potential difference of the T1 sites in high vs low potential MCOs is balanced by an ∼100 mV higher reduction potential of NI due to the more positive protein environment in the vicinity of the TNC.

Trametes versicolor glutathione transferase Omega 3S (TvGSTO3S) catalyzes the conjugation of isothiocyanates (ITC) with glutathione (GSH). Previously, this isoform was investigated in depth both biochemically and structurally. Structural analysis of complexes revealed the presence of a GSH binding site (G site) and a deep hydrophobic binding site (H site) able to bind plant polyphenols. In the present study, crystals of apo TvGSTO3S were soaked with glutathionyl-phenethylthiocarbamate, the product of the reaction between GSH and phenethyl isothiocyanate (PEITC). On the basis of this crystal structure, we show that the phenethyl moiety binds in a new site at loop β 2 -α 2 while the glutathionyl part exhibits a particular conformation that occupies both the G site and the entrance to the H site. This binding mode is allowed by a conformational change of the loop β 2 -α 2 at the enzyme active site. It forms a hydrophobic slit that stabilizes the phenethyl group at a distinct site from the previously described H site. Structural comparison of TvGSTO3S with drosophila DmGSTD2 suggests that this flexible loop could be the region that binds PEITC for both isoforms. These structural features are discussed in a catalytic context., (© 2019 The Protein Society.)

As a typical class of environmental endocrine disruptors, bisphenol A poses a potential threat to the sustainable survival and reproduction of living beings and human beings. In this study, the interaction between Trametes versicolor laccase and bisphenol A (BPA) was studied by molecular docking simulation, and the catalytic degradation of BPA was verified by experiments. The conditions for the laccase production of T. versicolor were optimized by orthogonal design, and the degradation of BPA was studied using its crude enzyme solution. The optimum degradation conditions were obtained by response surface methodology (RSM). Ultimately, the transformation products after 3 and 6 h of reaction were detected by gas chromatography-mass spectrometry. Docking results demonstrated that the reaction between laccase and BPA was spontaneous, and the degradation rate in 24 h reached 88.76%. RSM results showed that the highest BPA degradation rate of 97.68% was reached after 1 h reaction at 44.6 °C, 5 mg/L initial BPA concentration, and pH 5.20. The intermediate products of BPA catalyzed by laccase included ethylbenzene, p-xylene, and cyclohexanone 1-methyl-4-isopropenyl-2-cyclohexenol. This finding reveals that BPA degradation by the crude laccase from T. versicolor starts from the C atoms between two benzene rings that connect BPA. Compared with expensive pure enzyme, the crude laccase solution prepared by T. versicolor showed greater efficiency in BPA degradation. This work provides theoretical references and experimental methods for the biological processing of harmful pollutants., (Copyright © 2019. Published by Elsevier Ltd.)

The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L -1 on day 15) and MnP (17,027.4 ± 87.2 IU L -1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L -1 ) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L -1 ). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bentonite is a natural and environmentally clay mineral, and bentonite-derived mesoporous materials (BDMMs) were obtained conveniently from the alkali and acid treatment of bentonite. In the present study, BDMMs were explored for immobilization of laccase obtained from Trametes versicolor. As a result, bentonite-derived mesoporous materials-Laccase (BDMMs-Lac) was developed for the removal of tetracycline (TC). The enzyme immobilization process was carried out through physical adsorption contact (ion exchange adsorption, hydrogen bond adsorption, and Van der waals adsorption) between the BDMMs and laccase. The process of immobilization remarkably increased its operating temperature. The BDMMs-Lac exhibited over 60% removal efficiency for TC within 3 h in the presence of 1-hydroxybenzotriazole (HBT). In conclusion, BDMMs-Lac showed more promising potential than free laccase for practical continuous applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This investigation may be of interest for researchers working on the determination of several biocatalytic properties of the laccase from Trametes versicolor. So, We will treated the effects of pH, temperature, several organic components and heavy metals by performing enzyme assays in the presence of a 2,6 dimethoxyphenol (DMP) as substrate on the laccase activity. The optimum activity and temperature are 4 and 40 °C, respectively. The maximum rate of the reaction is 124.53 U/mg and the Michaelis constant is in order of 1.23 mM. The effect of metal ions on the laccase activity with a final concentrations range varying from 1 to 5 mM show that the Cu 2+ ions increase the activity for concentration inferiors to 4 mM and the other metal ions have a relative influence on the laccase activity. Four tri-block copolymers based on poly(ethylene oxide) and poly(propylene oxide) and two polyethylene glycols are used to study the synthetic polymers effects on the enzymatic activity. Also, we have demonstrated that the laccase keeps 95% of its initial activity at 60 °C in the PEGDA8000 and PEGDA6000 gel matrix. The maximum rate of the immobilized laccase is approximately around 21.03 and 47.22% smaller than the free one., (Copyright © 2019. Published by Elsevier B.V.)

Peptide-based supramolecular hydrogels are promising scaffold materials and have been utilized in many fields. The mechanical properties of peptide hydrogels are usually enhanced by synthetic or natural polymers to expand their application scope. In this study, antioxidant supramolecular hydrogels based on feruloyl-modified peptide and glycol chitosan were fabricated via a mild laccase-mediated crosslinking reaction. A natural polysaccharide derivative, feruloyl glycol chitosan (GC-Fer), was used to enhance the mechanical properties of peptide hydrogels. Feruloyl groups were introduced into the gel matrix via covalent bonds, which endowed the hydrogels with inherent antioxidant properties. This was beneficial for their in vivo application via scavenging harmful free radicals existing in a cutaneous wound. Further in vivo experiments demonstrated that the feruloyl-containing antioxidant hydrogel can improve the cutaneous wound healing process. The regeneration process of mature epithelium and connective tissues was accelerated in a full-thickness skin defect model.

Trametes versicolor can degrade bark as a source for carbon necessity. Therefore, it secretes lignin peroxidase, mangan peroxidase, and laccase. The laccase enzyme was produced in high yield at pH of 5 and glucose concentration of 10 g L -1 . In optimized medium, the enzyme activity was between 200 and 250 U L -1 when an inducer was absent. It was seen that the activity reached 400 U L -1 when phenol was used as an inducer. The molecular weight of purified laccase was found to be 80 kDa with SDS-PAGE, and kinetic constant K m and V max values for 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonate were determined to be 3.66 × 10 -4 μM and 1652 U L -1 , respectively. Because of these properties, these enzymes are widely used, free or immobilized, in industrial areas. Laccase enzyme decolorization of six different dyes was carried out. A decolorization capacity of 50-99% was achieved by cultivation for 20 days using a beginning dye concentration of 20 ppm. The removal of color with an active enzyme was obtained around 90%. Also, the laccase enzyme was conjugated, amine-functionalized, low-symmetry phthalocyanine. This conjugate was examined by both photodynamic therapy and chemosensor application. This conjugate fluorescence had a quantum yield of 0.32 (lifetime 3.59 ns) and efficiently generated singlet oxygen (quantum yield 0.4). The conjugate successfully displayed photodamage in HeLa and HuH-7 cells in the photodynamic therapy application. These results indicate that the conjugate represents an interesting agent with potential applications in photodynamic therapy. In addition, the chemosensor behavior of this compound to different metal ions has been studied, and this conjugate is displayed as a fluorescence chemosensor for the determination of Fe 3+ ions.

Aerogels are obtained by laccase/2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-oxidation of galactomannans (GMs) from the leguminous plants fenugreek (Trigonella foenum-graecum), sesbania (Sesbania bispinosa) and guar (Cyamopsis tetragonolobus). GM oxidation in aqueous solutions causes a viscosity increase, resulting in structured and stable hydrogels. Upon lyophilization, water-insoluble aerogels are obtained, capable of water uptake up to several times their own weight. The materials derived from the three gums have been analyzed and compared. Chemical modifications have been studied by electrospray-ionization mass spectrometry (ESI-MS) and Fourier-transform infrared spectroscopy (FTIR). Polymer structure has been determined by liquid-state and semi-quantitative solid-state nuclear magnetic resonance (NMR) spectroscopy and chemical stability by incubation under variable conditions of pH, ionic strength and solvent nature. The results show that hydrogel formation is due to oxidation of primary hydroxyl groups to carbonyl and carboxyl groups and subsequent formation of hemiacetal and ester bonds. Fenugreek displays the highest stability, compared to guar and sesbania rehydrated aerogels. This feature could be interpreted by its higher degree of substitution (Gal:Man = 1:1) and consequently higher amount of galactose primary alcohols, leading to more extensive crosslinking., (Copyright © 2018 Elsevier Ltd. All rights reserved.)