Immobilization of laccase on magnetic PEGDA-CS inverse opal hydrogel for enhancement of bisphenol A degradation in aqueous solution.

Endocrine disruptors such as bisphenol A (BPA) adversely affect the environment and human health. Laccases are used for the efficient biodegradation of various persistent organic pollutants in an environmentally safe manner. However, the direct application of free laccases is generally hindered by short enzyme lifetimes, non-reusability, and the high cost of a single use. In this study, laccases were immobilized on a novel magnetic three-dimensional poly(ethylene glycol) diacrylate (PEGDA)-chitosan (CS) inverse opal hydrogel (LAC@MPEGDA@CS@IOH). The immobilized laccase showed significant improvement in the BPA degradation performance and superior storage stability compared with the free laccase. 91.1% of 100 mg/L BPA was removed by the LAC@MPEGDA@CS@IOH in 3 hr, whereas only 50.6% of BPA was removed by the same amount of the free laccase. Compared with the laccase, the outstanding BPA degradation efficiency of the LAC@MPEGDA@CS@IOH was maintained over a wider range of pH values and temperatures. Moreover, its relative activity of was maintained at 70.4% after 10 cycles, and the system performed well in actual water matrices. This efficient method for preparing immobilized laccases is simple and green, and it can be used to further develop ecofriendly biocatalysts to remove organic pollutants from wastewater.

A feasible approach for azo-dye (methyl orange) degradation by textile effluent isolate Serratia marcescens ED1 strain for water sustainability: AST identification, degradation optimization and pathway hypothesis.

Methyl orange (MO) is a dye commonly used in the textile industry that harms aquatic life, soil and human health due to its potential as an environmental pollutant. The present study describes the dye degradation ability of Serratia marcescens strain ED1 isolated from textile effluent and characterized by 16S rRNA gene sequence analysis. The laccase property of bacterial isolate was confirmed qualitatively. The effects of various factors (pH, temperature, incubation time, and dye concentration) were evaluated using Response Surface Methodology (RSM). The maximum dye (MO) degradation was 81.02 % achieved at 37 °C temperature and 7.0 pH with 200 mg/L dye concentration after 48 h of incubation. The beef extract, ammonium nitrate and fructose supplementation showed better response during bioremediation among the different carbon and nitrogen sources. The degree of pathogenicity was confirmed through the simple plate-based method, and an antibiotic resistance profile was used to check the low-risk rate of antibiotic resistance. However, the fate and extinct of degraded MO products were analysed through UV-Vis spectroscopy, FT-IR, and GC-MS analysis to confirm the biodegradation potential of the bacterial strain ED1 and intermediate metabolites were identified to propose metabolic pathway. The phytotoxicity study on Vigna radiata L. seeds confirmed nontoxic effect of degraded MO metabolites and indicates promising degradation potential of S. marcescens strain ED1 to successfully remediate MO dye ecologically sustainably.

Enhanced Interfacial Electron Transfer in Photocatalyst-Natural Enzyme Coupled Artificial Photosynthesis System: Tuning Strategies and Molecular Simulations.

Laccase is capable of catalyzing a vast array of reactions, but its low redox potential limits its potential applications. The use of photocatalytic materials offers a solution to this problem by converting absorbed visible light into electrons to facilitate enzyme catalysis. Herein, MIL-53(Fe) and NH2-MIL-53(Fe) serve as both light absorbers and enzyme immobilization carriers, and laccase is employed for solar-driven chemical conversion. Electron spin resonance spectroscopy results confirm that visible light irradiation causes rapid transfer of photogenerated electrons from MOF excitation to T1 Cu(II) of laccase, significantly increasing the degradation rate constant of tetracycline (TC) from 0.0062 to 0.0127 min-1. Conversely, there is only minimal or no electron transfer between MOF and laccase in the physical mixture state. Theoretical calculations demonstrate that the immobilization of laccase's active site and its covalent binding to the metal-organic framework surface augment the coupled system's activity, reducing the active site accessible from 27.8 to 18.1 Å. The constructed photo-enzyme coupled system successfully combines enzyme catalysis' selectivity with photocatalysis's high reactivity, providing a promising solution for solar energy use.

Spacer arm of ionic liquids facilitated laccase immobilization on magnetic graphene enhancing its stability and catalytic performance.

To fulfill the requirements of environmental protection, a magnetically recoverable immobilized laccase has been developed for water pollutant treatment. In order to accomplish this objective, we propose a polydopamine-coated magnetic graphene material that addresses the challenges associated with accumulation caused by electrostatic interactions between graphene and enzyme molecules, which can lead to protein denaturation and inactivation. To achieve this, we present a polydopamine-coated magnetic graphene material that binds to the enzyme molecule through flexible spacer arms formed by ionic liquids. The immobilized laccase exhibited a good protective effect on laccase and showed a high stability and recycling ability. Laccase-ILs-PDA-MGO has a wider pH and temperature range and retains about 80% of its initial activity even after incubation at 50 °C for 2 h, which is 2.2 times more active than free laccase. Furthermore, the laccase-ILs-PDA-MGO exhibited a remarkable removal efficiency of 97.0% and 83.9% toward 2,4-DCP and BPA within 12 h at room temperature. More importantly, laccase-ILs-PDA-MGO can be recovered from the effluent and used multiple times for organic pollutant removal, while maintaining a relative removal efficiency of 80.6% for 2,4-DCP and 81.4% for BPA after undergoing seven cycles. In this study, a strategy for laccase immobilization by utilizing ILs spacer arms to modify GO aims to provide valuable insights into the advancement of efficient enzyme immobilization techniques and the practical application of immobilized enzymes in wastewater treatment.

Safety evaluation of an extension of use of the food enzyme laccase from the non-genetically modified Trametes hirsuta strain AE-OR.

The food enzyme laccase (benzenediol:oxygen oxidoreductase, i.e. EC 1.10.3.2) is produced with the non-genetically modified Trametes hirsuta strain AE-OR by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in six food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of nine food manufacturing processes. Dietary exposure to the food enzyme-total organic solids (TOS) was calculated to be up to 0.030 mg TOS/kg body weight (bw) per day in European populations. Using the no observed adverse effect level previously reported (862 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 28,733. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Bioremediation of organic pollutants by laccase-metal-organic framework composites: A review of current knowledge and future perspective.

Immobilized laccases are widely used as green biocatalysts for bioremediation of phenolic pollutants and wastewater treatment. Metal-organic frameworks (MOFs) show potential application for immobilization of laccase. Their unique adsorption properties provide a synergic effect of adsorption and biodegradation. This review focuses on bioremediation of wastewater pollutants using laccase-MOF composites, and summarizes the current knowledge and future perspective of their biodegradation and the enhancement strategies of enzyme immobilization. Mechanistic strategies of preparation of laccase-MOF composites were mainly investigated via physical adsorption, chemical binding, and de novo/co-precipitation approaches. The influence of architecture of MOFs on the efficiency of immobilization and bioremediation were discussed. Moreover, as sustainable technology, the integration of laccases and MOFs into wastewater treatment processes represents a promising approach to address the challenges posed by industrial pollution. The MOF-laccase composites can be promising and reliable alternative to conventional techniques for the treatment of wastewaters containing pharmaceuticals, dyes, and phenolic compounds. The detailed exploration of various immobilization techniques and the influence of MOF architecture on performance provides valuable insights for optimizing these composites, paving the way for future advancements in environmental biotechnology. The findings of this research have the potential to influence industrial wastewater treatment and promoting cleaner treatment processes and contributing to sustainability efforts.

Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

Is Laccase derived from Pleurotus ostreatus effective in microplastic degradation? A critical review of current progress, challenges, and future prospects.

Exploration of Pleurotus ostreatus as a biological agent in the degradation of persistent plastics like polyethylene, polystyrene, polyvinyl chloride, and polyethylene terephthalate, revealing a promising avenue toward mitigating the environmental impacts of plastic pollution. Leveraging the intrinsic enzymatic capabilities of this fungus, mainly its production of laccase, presents a sustainable and eco-friendly approach to breaking down complex polymer chains into less harmful constituents. This review focused on enhancements in the strain's efficiency through genetic engineering, optimized culture conditions, and enzyme immobilization to underscore the potential for scalability and practical application of this bioremediation process. The utilization of laccase from P. ostreatus in plastic waste management demonstrates a vital step forward in pursuing sustainable environmental solutions. By using the potential of fungal bioremediation, researchers can move closer to a future in which the adverse effects of plastic pollution are significantly mitigated, benefiting the health of our planet and future generations.

Design of Mn-based nanozymes with multiple enzyme-like activities for identification/quantification of glyphosate and green transformation of organophosphorus.

A Mn-based nanozyme, Mn-uNF/Si, with excellent alkali phosphatase-like activity was designed by in-situ growth of ultrathin Mn-MOF on the surface of silicon spheres, and implemented as an effective solid Lewis-Brønsted acid catalyst for broad-spectrum dephosphorylation. H218O-mediated GC-MS studies confirmed the cleavage sites and the involvement of H2O in the new bonds. DRIFT NH3-IR and in-situ ATR-FTIR confirmed the coexistence of Lewis-Brønsted acid sites and the adjustment of adsorption configurations at the interfacial sites. In addition, a green transformation route of "turning waste into treasure" was proposed for the first time ("OPs→PO43-→P food additive") using edible C. reinhardtii as a transfer station. By alkali etching of Mn-uNF/Si, a nanozyme Mn-uNF with laccase-like activity was obtained. Intriguingly, glyphosate exhibits a laccase-like fingerprint-like response (+,-) of Mn-uNF, and a non-enzyme amplified sensor was thus designed, which shows a good linear relationship with Glyp in a wide range of 0.49-750 µM, with a low LOD of 0.61 µM, as well as high selectivity and anti-interference ability under the co-application of phosphate fertilizers and multiple pesticides. This work provides a controllable methodology for the design of bifunctional nanozymes, which sheds light on the highly efficient green transformation of OPs, and paves the way for the selective recognition and quantification of glyphosate. Mechanistically, we also provided deeper insights into the structure-activity relationship at the atomic scale.

Genome-wide analysis of the common bean (Phaseolus vulgaris) laccase gene family and its functions in response to abiotic stress.

BACKGROUND: Laccase (LAC) gene family plays a pivotal role in plant lignin biosynthesis and adaptation to various stresses. Limited research has been conducted on laccase genes in common beans. RESULTS: 29 LAC gene family members were identified within the common bean genome, distributed unevenly in 9 chromosomes. These members were divided into 6 distinct subclades by phylogenetic analysis. Further phylogenetic analyses and synteny analyses indicated that considerable gene duplication and loss presented throughout the evolution of the laccase gene family. Purified selection was shown to be the major evolutionary force through Ka / Ks. Transcriptional changes of PvLAC genes under low temperature and salt stress were observed, emphasizing the regulatory function of these genes in such conditions. Regulation by abscisic acid and gibberellins appears to be the case for PvLAC3, PvLAC4, PvLAC7, PvLAC13, PvLAC14, PvLAC18, PvLAC23, and PvLAC26, as indicated by hormone induction experiments. Additionally, the regulation of PvLAC3, PvLAC4, PvLAC7, and PvLAC14 in response to nicosulfuron and low-temperature stress were identified by virus-induced gene silence, which demonstrated inhibition on growth and development in common beans. CONCLUSIONS: The research provides valuable genetic resources for improving the resistance of common beans to abiotic stresses and enhance the understanding of the functional roles of the LAC gene family.

Micropollutants (ciprofloxacin and norfloxacin) remediation from wastewater through laccase derived from spent mushroom waste: Fate, toxicity, and degradation.

Fluoroquinolone antibiotics frequently found in environmental matrices (wastewater treatment plants, hospital wastewater, industrial wastewater and surface wastewater) causes potential threat to the environment. Enzymatic treatment for degradation of antibiotics from environmental matrices is a green and sustainable approach. Focusing on this, this study aimed to degrade two frequently found fluroquinolone emergent pollutants, ciprofloxacin and norfloxacin from wastewater. The trinuclear cluster of copper ions present in laccase has the ability to effectively remove organic micropollutants (OMPs). The uniqueness of this study is that it utilizes laccase enzyme extracted from spent mushroom waste (SMW) of P. florida for degradation of ciprofloxacin and norfloxacin and to achieve highest degradation efficiency various parameters were tweaked such as pH (3-6), temperature (30 °C and 50 °C), and ABTS (0.05, 0.6, and 1 mM) concentration. The results showed that the most effective degradation of ciprofloxacin (86.12-75.94%) and norfloxacin (83.27-65.94%) was achieved in 3 h at pH 4.5, temperature 30 °C, and 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 0.05 mM concentration. Nevertheless, achieving degradation at 50 °C for both antibiotics, indicates thermostability nature of laccase (P. florida). Further, the fate of transformed products obtained from laccase mediated degradation was confirmed by liquid chromatography (LC-MS). Both the antibiotics undergo decarboxylation, depiperylyzation, dealkylation and defluorination as a result of laccase-mediated bond breakage. Anti-microbial activity of the biodegraded products was monitored by residual anti-bacterial toxicity test (E. coli and Staphylococcus aureus). The biodegraded products were found to be non-toxic and resulted in the growth of E. coli and Staphylococcus aureus, as determined by the agar-diffusion method. Moreover, the storage stability of laccase was determined for 28-day duration at varying pH (3-10) and temperature (4-50 °C). The maximum storage stability was obtained at pH 4.5 and temperature 30 °C. Therefore, utilizing SMW for the degradation of OMPs from wastewater not only benefits in degradation but also reuses SMW agro waste, shedding light on agro waste management. Thus, SMW is a one-pot solution for both OMPs biodegradation and circularity in the economy.

Effects of Laccase and Transglutaminase on the Physicochemical and Functional Properties of Hybrid Lupin and Whey Protein Powder.

Plant-based protein is considered a sustainable protein source and has increased in demand recently. However, products containing plant-based proteins require further modification to achieve the desired functionalities akin to those present in animal protein products. This study aimed to investigate the effects of enzymes as cross-linking reagents on the physicochemical and functional properties of hybrid plant- and animal-based proteins in which lupin and whey proteins were chosen as representatives, respectively. They were hybridised through enzymatic cross-linking using two laccases (laccase R, derived from Rhus vernicifera and laccase T, derived from Trametes versicolor) and transglutaminase (TG). The cross-linking experiments were conducted by mixing aqueous solutions of lupin flour and whey protein concentrate powder in a ratio of 1:1 of protein content under the conditions of pH 7, 40 °C for 20 h and in the presence of laccase T, laccase R, or TG. The cross-linked mixtures were freeze-dried, and the powders obtained were assessed for their cross-linking pattern, colour, charge distribution (ζ-potential), particle size, thermal stability, morphology, solubility, foaming and emulsifying properties, and total amino acid content. The findings showed that cross-linking with laccase R significantly improved the protein solubility, emulsion stability and foaming ability of the mixture, whereas these functionalities were lower in the TG-treated mixture due to extensive cross-linking. Furthermore, the mixture treated with laccase T turned brownish in colour and showed a decrease in total amino acid content which could be due to the enzyme's oxidative cross-linking mechanism. Also, the occurrence of cross-linking in the lupin and whey mixture was indicated by changes in other investigated parameters such as particle size, ζ-potential, etc., as compared to the control samples. The obtained results suggested that enzymatic cross-linking, depending on the type of enzyme used, could impact the physicochemical and functional properties of hybrid plant- and animal-based proteins, potentially influencing their applications in food.

Purine metabolite inosine induced by transforming growth factor­ß promotes epithelial­mesenchymal transition in colorectal cancer.

Transforming growth factor-ß (TGF-ß) signaling pathway serves a pivotal role in the pathogenesis of colorectal cancer (CRC). However, the specific molecular mechanisms by which the TGF-ß signaling pathway regulates CRC are still not fully understood. In the present study, metabolomics and transcriptomics were used to screen for key metabolites and regulatory genes most related to the regulation of the TGF-ß signaling pathway in CRC. Additionally, reverse transcription-quantitative PCR, western blotting and Transwell assays were performed to assess the process of epithelial-mesenchymal transition (EMT). Metabolomics analysis indicated that TGF-ß1 has an impact on purine metabolism, leading to an increase in the purine metabolite inosine. The increase of inosine is essential for facilitating EMT and cell migration in CRC cells. Furthermore, the integrated analysis of metabolomics and transcriptomics data revealed that TGF-ß1 induces the expression of laccase domain-containing 1 (LACC1), an enzyme involved in the regulation of inosine. Knockdown of LACC1 resulted in a reduction of TGF-ß1-induced alterations in inosine levels, EMT and cell migration in CRC cells. The results of the present study suggest that the TGF-ß signaling pathway is involved in the regulation of purine metabolism in CRC through the modulation of LACC1 expression. Furthermore, LACC1 appears to influence EMT and cell migration by elevating the levels of the purine metabolite inosine.

Transformation of antibiotics to non-toxic and non-bactericidal products by laccases ensure the safety of Stropharia rugosoannulata.

The substantial use of antibiotics contributes to the spread and evolution of antibiotic resistance, posing potential risks to food production systems, including mushroom production. In this study, the potential risk of antibiotics to Stropharia rugosoannulata, the third most productive straw-rotting mushroom in China, was assessed, and the underlying mechanisms were investigated. Tetracycline exposure at environmentally relevant concentrations (<500 µg/L) did not influence the growth of S. rugosoannulata mycelia, while high concentrations of tetracycline (>500 mg/L) slightly inhibited its growth. Biodegradation was identified as the main antibiotic removal mechanism in S. rugosoannulata, with a degradation rate reaching 98.31 % at 200 mg/L tetracycline. High antibiotic removal efficiency was observed with secreted proteins of S. rugosoannulata, showing removal efficiency in the order of tetracyclines > sulfadiazines > quinolones. Antibiotic degradation products lost the ability to inhibit the growth of Escherichia coli, and tetracycline degradation products could not confer a growth advantage to antibiotic-resistant strains. Two laccases, SrLAC1 and SrLAC9, responsible for antibiotic degradation were identified based on proteomic analysis. Eleven antibiotics from tetracyclines, sulfonamides, and quinolones families could be transformed by these two laccases with degradation rates of 95.54-99.95 %, 54.43-100 %, and 5.68-57.12 %, respectively. The biosafety of the antibiotic degradation products was evaluated using the Toxicity Estimation Software Tool (TEST), revealing a decreased toxicity or no toxic effect. None of the S. rugosoannulata fruiting bodies from seven provinces in China contained detectable antibiotic-resistance genes (ARGs). This study demonstrated that S. rugosoannulata can degrade antibiotics into non-toxic and non-bactericidal products that do not accelerate the spread of antibiotic resistance, ensuring the safety of S. rugosoannulata production.

Effects of Orange Peel Extract on Laccase Activity and Gene Expression in Trametes versicolor.

The genome of Trametes versicolor encodes multiple laccase isozymes, the expression of which is responsive to various conditions. Here, we set out to investigate the potential of orange peel extract as an inducer of laccase production in this white-rot fungus, in comparison to the previously identified inducing chemical compound, veratryl alcohol. For four geographically distinct T. versicolor strains, a positive correlation has been observed between their oxidative activity and incubation time in liquid cultures. The addition of 20% orange peel extract or 5 mM veratryl alcohol caused a rapid increase in the oxidative potential of T. versicolor M99 after 24 h, with a more pronounced effect observed for the orange peel extract. To elucidate the underlying molecular mechanisms of the induced laccase activity, a transcriptional gene expression analysis was performed for the seven individual laccase genes in T. versicolor, revealing the upregulation of several laccase genes in response to the addition of each inducer. Notably, the gene encoding TvLac5 demonstrated a substantial upregulation in response to the addition of 20% orange peel extract, likely contributing to the observed increase in its oxidative potential. In conclusion, our results demonstrate that orange peels are a promising agro-industrial side stream for implementation as inducing agents in large-scale laccase production with T. versicolor.

Polydopamine bridging encapsulated laccase on MOF-based mixed-matrix membrane for selective dye/salt separation.

Mixed-matrix membranes (MMMs) exhibit significant potential for dye/salt separation. However, overcoming the "trade-off" between permeability and selectivity, as well as membrane fouling, remains a formidable task. In this work, a biocatalytic membrane was prepared using polydopamine (PDA) as a "bridge" connecting the metal-organic framework (MOF)-based MMM and immobilized laccase. The MOF-based MMM featured an interconnected MOF anchoring on the polyvinylidene fluoride (PVDF) skeleton structure, effectively mitigating the "trade-off" phenomenon and enabling efficient separation of dyes and salts. Enzyme-MOF was in situ grown on the MOF-based MMM via coordination reactions between PDA and metal ion, effectively degrading the adhesion of organic pollutants and fouling, ensuring the long-term stable operation of the membrane. The Lac-MOF@PDA MMM exhibited excellent water permeability of 142.4 L·m-2·h-1, 100 % rejection for dye, and less than 10 % rejection for NaCl. Furthermore, the separation mechanism of Lac-MOF@PDA MMM was systematically investigated, and the results suggested a synergistic combination of rejection, adsorption and catalysis processes. This biocatalytic membrane with multiple sieving and biological catalysis is expected to pave a promising way for efficient wastewater treatment applications.

Two Zn2Cys6-type transcription factors respond to aromatic compounds and regulate the expression of laccases in the white-rot fungus Trametes hirsuta.

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 Zn2Cys6-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 Zn2Cys6-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.

Study of phenoxy radical couplings using the enzymatic secretome of Botrytis cinerea.

Phenoxy radical coupling reactions are widely used in nature for the synthesis of complex molecules such as lignin. Their use in the laboratory has great potential for the production of high value compounds from the polyphenol family. While the enzymes responsible for the generation of the radicals are well known, the behavior of the latter is still enigmatic and difficult to control in a reaction flask. Previous work in our laboratory using the enzymatic secretome of B. cinerea containing laccases has shown that incubation of stilbenes leads to dimers, while incubation of phenylpropanoids leads to dimers as well as larger coupling products. Building on these previous studies, this paper investigates the role of different structural features in phenoxy radical couplings. We first demonstrate that the presence of an exocyclic conjugated double bond plays a role in the generation of efficient reactions. In addition, we show that the formation of phenylpropanoid trimers and tetramers can proceed via a decarboxylation reaction that regenerates this reactive moiety. Lastly, this study investigates the reactivity of other phenolic compounds: stilbene dimers, a dihydro-stilbene, a 4-O-methyl-stilbene and a simple phenol with the enzymatic secretome of B. cinerea. The observed efficient dimerization reactions consistently correlate with the presence of a para-phenol conjugated to an exocyclic double bond. The absence of this structural feature leads to variable results, with some compounds showing low conversion or no reaction at all. This research has allowed the development of a controlled method for the synthesis of specific dimers and tetramers of phenylpropanoid derivatives and novel stilbene derivatives, as well as an understanding of features that can promote efficient radical coupling reactions.

Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

A food color-based colorimetric assay for Cryptococcus neoformans laccase activity.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.