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This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

Laccases from white-rot fungi catalyze lignin depolymerization, a critical first step to upgrading lignin to valuable biodiesel fuels and chemicals. In this study, a wildtype laccase from the basidiomycete Fomitiporia mediterranea (Fom_lac) and a variant engineered to have a carbohydrate-binding module (Fom_CBM) were studied for their ability to catalyze cleavage of β-O-4 ether and C-C bonds in phenolic and non-phenolic lignin dimers using a nanostructure-initiator mass spectrometry-based assay. Fom_lac and Fom_CBM catalyze β-O-4 ether and C-C bond breaking, with higher activity under acidic conditions (pH < 6). The potential of Fom_lac and Fom_CBM to enhance saccharification yields from untreated and ionic liquid pretreated pine was also investigated. Adding Fom_CBM to mixtures of cellulases and hemicellulases improved sugar yields by 140% on untreated pine and 32% on cholinium lysinate pretreated pine when compared to the inclusion of Fom_lac to the same mixtures. Adding either Fom_lac or Fom_CBM to mixtures of cellulases and hemicellulases effectively accelerates enzymatic hydrolysis, demonstrating its potential applications for lignocellulose valorization. We postulate that additional increases in sugar yields for the Fom_CBM enzyme mixtures were due to Fom_CBM being brought more proximal to lignin through binding to either cellulose or lignin itself.

The ability of laccases to work effectively in properly selected aqueous-organic solutions significantly expands the scope of their application, making them commercially attractive and more competitive. However, it must be considered that molecules of organic solvent are very aggressive towards enzymes and destroy weak non-covalent interactions that maintain the spatial structure of the protein. In this work, we quantitatively described the influence of the nature of water-miscible organic solvents and their concentration on the activity and stability of Trametes versicolor laccase during the oxidation of ABTS (2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) through the maximum rates and the Michaelis constants KM. The obtained kinetic parameters make it possible to compare the effect of various organic solvents on laccase and select the most optimal one, which minimizes the decrease in enzyme activity, maintains its stability and allows repeated use. Among the solvents studied, polyhydroxy compounds have the least negative effect on laccase activity; however, in all cases, a high concentration of organic solvent (≥30% (v/v)) leads to suppression of enzymatic activity. It has been shown that the longer the alkyl chain (hydrophobic fragment of the structure), the stronger the negative effect of alcohols on enzymes. The most toxic solvent for Trametes versicolor laccase is isopropanol. In addition, numerous examples of various uses of Trametes versicolor in aquatic-organic environments are discussed here. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is a well-established potent food mycotoxin. We previously evidenced that laccase could restrain AFB1's genetic and oxidative toxicity in vitro conditions; nevertheless, there is no report about the in vivo toxicity of the laccase-mediated metabolites of aflatoxin B1 (LM-AFB1). In this investigation, we evaluated the in vivo safety of LM-AFB1 in comparison with the parent toxin using serum liver function tests (LFTs), oxidative stress biomarkers, histopathological examinations, and CYP1A2 and GSTA3 gene expression. Adult male mice were divided into four groups: (1) Control; (2) 4 mg/kg AFB1; (3) 4 mg/kg LM-AFB1; (4) 4 mg/kg intermediate transformational metabolites of AFB1 (CA-AFB1). In this study, AFB1's liver toxicity was confirmed but LM-AFB1 did not significantly change the values. Gene expression analysis indicates that the utilization of laccase for bioremediation does not result in the suppression of glutathione S-transferase, in contrast to AFB1. Our research findings demonstrate that laccase has the potential to mitigate AFB1-induced liver toxicity in mice by averting oxidative stress caused by AFB1. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application. Results: The rLac-HhC125 was partially purified by size exclusion chromatography and concentrated by ultrafiltration (10 kDa) with a yield of 57.6%. Oxidation reactions showed that adding 2 mM CuSO4 to the assay solution led to activating the laccase. To increase its initial activity, the rLac-HhC125 was treated at 50 °C for 20 min before the assays, improving its performance by fourfold using the syringaldazine as a substrate. When treated with EDTA, methanol, ethanol, and DMSO, the rLac-HhC125 maintained more than 80% of its original activity. Interestingly, the acetonitrile induced a twofold activity of the rLac-HhC125. The putative rLac-HhC125 demonstrated a capability of efficient transformation of different organic compounds at pH 6, known as dye precursors, into coloured molecules. Conclusion: The rLac-HhC125 was active at high temperatures and alkaline pH, exhibited tolerance to organic solvents, and efficiently transformed different hydroxy derivatives into coloured compounds, which indicates that it can be used in various biotechnological processes. [ABSTRACT FROM AUTHOR]

Background: The laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production. Results: This study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing PgpdA) mediated by the SPCAT, a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by PcitA-SPGlaA had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing PcitA-SPGlaA) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu2+ in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter. Conclusions: To our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SPCAT in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger. [ABSTRACT FROM AUTHOR]

Laccase immobilization is an effective way to realize the industrial application of laccase, but the loss of immobilized laccase activity is serious, which is an urgent problem to be solved. In this study, a novel laccase immobilization material was prepared by mixing waste Mn–Cu-loaded activated carbon with cellulose solution and regenerated in hydrochloric acid solution, which significantly improved the immobilized laccase activity. The optimum immobilization conditions (T, pH and initial laccase concentration) of Mn–Cu-AC/cellulose beads (Mn–Cu-ACC) for laccase were studied by the response surface method. Under the optimum conditions (T = 43 °C, pH = 4.0 and c = 6.8 g L−1), immobilized laccase (Laccase@Mn–Cu-ACC) showed excellent performance in terms of pH stability, thermostability and storage stability. And the enzyme activity of the laccase immobilized on the Mn–Cu-ACC carrier remarkably increased by 29.6% compared to that of the ACC. At last, the Laccase@Mn–Cu-ACC was used for the degradation of bisphenol A (BPA). Under the optimum conditions (t = 5.5 h, T = 45 °C, pH = 4.0 and initial cBPA = 70 mg L−1), the removal rate of BPA reached 72.3% in the first cycle, and after 5 cycles of reusing the immobilized laccase, the relative removal rate of BPA was retained at 53.2% of the initial removal rate. This study developed a new type of immobilized laccase carrier and provided a new path for the application of waste heavy metal loaded activated carbon. [ABSTRACT FROM AUTHOR]

Laccase is an enzyme known for its eco-friendly uses in environmental cleanup and biotechnology. However, it has limitations such as low stability, high cost, and complex recycling. So, there is a need for laccase mimics that can effectively imitate its properties. Herein, we created copper formate–lysine nanoparticles (Cuf-Lys) that mimic laccase's activity. The developed Cuf-Lys demonstrated remarkable polyphenol oxidase-like activity, stability, and recyclability, making them suitable for the fabrication of efficient colorimetric sensors for the detection of epinephrine. These sensors had a specific response and could accurately measure epinephrine concentrations ranging from 2.5 to 50 μM, with a detection limit as low as 1 μM. Furthermore, the biosensor demonstrated high sensitivity and selectivity when applied to the detection of rutin. The limit of detection for rutin was determined to be 0.16 μM while in the linear concentration range of 0.25 to 150.0 μM. We believe that Cuf-Lys provide a new route for the design of laccase mimics, showing potential applications for biomedical diagnosis and environmental monitoring. [ABSTRACT FROM AUTHOR]

Due to its poor hydration properties, oil palm kernel expeller dietary fiber (OPKEDF) is rarely used in the food industry, especially in hydrogels, despite its advantages of high availability and low cost. To address this situation, the effects of enzymolysis combined with hydroxypropylation or crosslinking on the structure and hydration properties of OPKEDF were investigated, and the impact of these modified OPKEDFs on the properties of egg white protein gel (EWPG) was studied. Enzymolysis combined with hydroxypropylation or phosphate crosslinking improved the soluble fiber content (5.25–7.79 g/100 g), water-retention and expansion abilities of OPKEDF (p < 0.05). The addition of unmodified OPKEDF or modified OPKEDF increased the random coil content of EWPG and increased the density of its microstructure. Moreover, enzymolysis combined with hydroxypropylation or crosslinking enhanced the effect of OPKEDF on the properties of EWPG, including improvements in its water-retention ability, pH, hardness (from 97.96 to 195.00 g), chewiness (from 78.65 to 147.39 g), and gumminess (from 84.63 to 152.27) and a reduction in its transparency (p < 0.05). Additionally, OPKEDF and enzymolysis and hydroxypropylated OPKEDF increased the resilience (0.297 to 0.359), but OPKEDF treated via enzymolysis and crosslinking reduced it. Therefore, OPKEDF modified by means of enzymolysis in combination with hydroxypropylation or crosslinking improved the gel properties of EWPG. However, further work is required to determine the effects of these modifications on the nutritional profile, scalability, and economic feasibility of OPKEDF and egg white gel. [ABSTRACT FROM AUTHOR]

Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study on six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted, and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acid side chains coordinate to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this experiment was to explore the effect of adding cellulase and laccase on fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains. Try to a new approach for the proper preservation and utilization of the agro-industrial by-products (corn stover and wet brewer's grains). Methods: The experiment was divided into four groups: CK (control), C (cellulase, 120 U/g fresh matter [FM]), L (laccase, 50 U/g FM), CL (cellulase 120 U/g FW and laccase 50 U/g FM), and the chemical composition, fermentation quality, microbial population and microbial community in mixed silage of corn stover and wet brewer's grains after 30 day's fermentation were determined. Results: Compared to control, the addition of cellulase significantly increased crude protein (CP), water-soluble carbohydrate (WSC), lactic acid bacteria (LAB) counts, while significantly decreased the neutral detergent fiber (NDF), acid detergent fiber (ADF) content (P < 0.05). Adding laccase significantly decreased the acid detergent lignin (ADL) content (P < 0.05). Combined application of cellulase and laccase significantly increased the CP, WSC content and LAB counts, while significantly decreased pH value, NDF, ADF and ADL content (P < 0.05), thereby improving fermentation quality. In addition, the application of cellulase and laccase increased the abundance of Firmicutes and LAB genera, and decreased microbial diversity level of the sample. Conclusion: The combined application of cellulase and laccase further improved fermentation quality and microbial community in mixed silage of corn stover and wet brewer's grains. [ABSTRACT FROM AUTHOR]

Enzymes secreted by white-rot fungi, such as laccase, offer a promising solution for treating xenobiotic compounds dangerous to the environment and human health. This study aimed to perform a comprehensive analysis of the tolerance of Pleurotus pulmonarius LBM 105 and its laccase activity toward the pesticides 2,4-D and chlorpyrifos both in vitro and in silico. The fungal strain was able to grow in different concentrations of the pesticides, showing evident morphological alterations. Laccase activity and a 53 kDa electromorph were present in all treatments, showing significant stability with peak activity achieved at a pH of 5.6 and within a temperature range of 50–60 °C. Three laccase genes were mapped, annotated, and characterized from the genome. PplacI obtained better structural validation and affinity energy of − 5.05 and − 7.65 kcal mol−1 with 2,4-D and chlorpyrifos, respectively. The Molecular Mechanics/Poisson-Boltzmann Surface Area analysis at 250 ns confirmed the docking results, revealing the existence of stronger hydrophobic interactions between laccase and chlorpyrifos and highlighting the importance of the Phe341 residue in stabilizing both complexes. Understanding the impact of pesticides on laccase's catalytic function is key to formulating and applying future biotechnological strategies with this enzyme. [ABSTRACT FROM AUTHOR]

White-rot fungi (WRF) are increasingly recognized for their biotechnological potential due to the wide range of applications of ligninolytic enzymes. The addition of different metals involved in the functioning of ligninolytic enzymes, mainly copper and manganese, has been widely studied to maximize the enzymatic activities of the WRF. This review aims to provide information on the effect of metal-fungi interaction mechanisms that justify the effects of enzymatic activity. The addition of copper is associated with increased laccase activity, with reported improvements in the laccase activity compared to controls without metal addition of up to 100% at doses between 0.5–1 mM. The addition of manganese resulted in an improvement in manganese peroxidase activity with respect to the control at the wide range of 1–18.2 mM. Furthermore, enzymatic activity was generally favored by using substrates with lignocellulosic fibers with respect to synthetic culture medium. Quantifying the concentration of metals in the substrate is required to monitor bioavailable metals for fungi in these assays accurately, making an external contribution less necessary. [ABSTRACT FROM AUTHOR]

Laccase is an enzyme that plays an important role in fungi, including lignin degradation, stress defense, and formation of fruiting bodies. Auricularia auricula-judae is a white-rot fungus in the Basidiomycota phylum, capable of delignifying wood. In this study, seven genes belonging to the laccase family were identified through de novo sequencing, containing Cu-Oxidase, Cu-Oxidase_2, and Cu-Oxidase_3 domains. Subsequently, the physical characteristics, phylogenetic relationships, protein secondary structure, and tertiary structure of the laccase family (AaLac1–AaLac7) were analyzed. Prediction of N-glycosylation sites identified 2 to 10 sites in the laccase family, with AaLac7 having the highest number of sites at 10. Sequence alignment and analysis of the laccase family showed high consistency in signature sequences. Phylogenetic analysis confirmed the relationship among laccases within the family, with AaLac3–AaLac4 and AaLac5–AaLac6 being closely positioned on the tree, exhibiting high similarity in tertiary structure predictions. This study identified and analyzed laccase family genes in Auricularia auricula-judae using de novo sequencing, offering a simple method for identifying and analyzing the laccase family in organisms with unknown genetic information. [ABSTRACT FROM AUTHOR]

Spent mushroom compost (SMC) is a waste byproduct generated by the mushroom industry, with the current disposal methods involving landfills or incineration, causing environmental problems. This study introduces a cascading valorization based on circular economy. A water-washing pretreatment is suggested to reduce the ash content and improve the calorific value. Furthermore, an oxidative torrefaction is proposed to enhance the physicochemical properties and evaluate the residence time, temperature, and initial weight effects. The investigation revealed that the calorific value increased by 49.46% in the best water washing scenario and the SMC contains laccase. During SMC torrefaction, mass and energy yields exceeded 80%, and the calorific value was improved. Finally, torrefaction in situ CO2 capture was achieved by forming CaCO3, with a mass fraction increase of up to 7.4%. The study describes the cascading innovation use of this residue on a TRL4 higher than previously reported studies, thereby contributing to gaining an understanding of sustainable industrial mushroom production. [ABSTRACT FROM AUTHOR]

Lignin is a phenolic polymer that is a major source of biomass. Oxidative enzymes, such as laccase and peroxidase, are required for lignin polymerisation. Laccase is a member of the multicopper oxidase family and has a high amino acid sequence similarity with ascorbate oxidase. However, the process of functional differentiation between the two enzymes remains poorly understood. In this study, the common ancestry sequence of laccase and ascorbate oxidase (AncMCO) was predicted via phylogenetic reconstruction, and its in vivo effect on lignin biosynthesis in Arabidopsis thaliana was assessed. The estimated AncMCO sequence conserved key residues that coordinate with copper ions, implying that the electron transfer system is likely to be conserved in AncMCO. However, multiple insertions/deletions corresponding to protein surface structures have been found between laccase, ascorbate oxidase, and AncMCO. The overexpression of canonical laccase (AtLAC4) and ascorbate oxidase (AtAAO1) in A. thaliana resulted in notable increases of syringyl/guaiacyl lignin unit ratio in stems, whereas, in contrast, the overexpression of AncMCO did not show any detectable change in lignin deposition. Transcriptomic analysis revealed that the AtAAO1-overexpressing line exhibited significant changes in the expression of a wide range of cell wall biosynthesis genes. These results highlight the importance of the molecular evolution of multicopper oxidase, which drives lignin biosynthesis during plant evolution. [ABSTRACT FROM AUTHOR]

The development of a cost-effective and environment-friendly conversion method to overcome the recalcitrance of biomass is a challenging task for enhancing the value-added potential of lignocellulosic compounds. In this study, a photo-enzyme composite catalyst by immobilizing laccase on Fe 3 O 4 /TiO 2 (denoted as LC-Fe 3 O 4 /TiO 2) was applied to the pretreatment and enzymatic saccharification of Spartina alterniflora Loisel (SAL) under the action of alternating light/dark cycles. The effect of the pretreatment temperature, pretreatment time, LC-Fe 3 O 4 /TiO 2 concentration, mass concentration of straw particles, and hydrolysis time on delignification efficiency and reducing sugar yield was investigated by a central composite design (CCD). Under the selected conditions (48 ℃, pretreatment time of 70 min, LC-Fe 3 O 4 /TiO 2 concentration of 4.9 mg/mL, mass concentration of straw particles of 22 % (w/v), hydrolysis time of 55 h), 92.53 % delignification and 129.85 mg/g reducing sugar yield were achieved. The enzymatic hydrolysis efficiency was higher compared to conventional methods. Moreover, the LC-Fe 3 O 4 /TiO 2 showed good recyclability and reusability. Developed LC-Fe 3 O 4 /TiO 2 particles have a high potential for use in biomass utilization. [Display omitted] • Laccase-Fe 3 O 4 /TiO 2 integrated catalyst (LC-Fe 3 O 4 /TiO 2) is first synthesized. • Effects of LC-Fe 3 O 4 /TiO 2 on pretreatment and saccharification is studied. • LC-Fe 3 O 4 /TiO 2 can enhance sugar yield under the alternation of light and dark. • LC-Fe 3 O 4 /TiO 2 could be efficiently reused up to 3 cycles. [ABSTRACT FROM AUTHOR]

This study aimed to develop a nano-biosystem by covalently binding recombinant laccase from Bacillus licheniformis (rLAC BL) to PEGylated silica-coated magnetic nanoparticles (EMNPs-PEG) and evaluate its cytotoxicity and anti-inflammatory properties on a pro-monocytic cell line. The active rLAC BL was purified from the bacterial culture with a yield of approximately 14 mg/L and purity of about 91 %. The physicochemical characteristics of EMNP-PEG-rLAC BL were confirmed using DLS, UV-Vis spectrum, FTIR, EDS, and SEM. The immobilized rLAC BL exhibited improved kinetic parameters compared to the bare enzyme. The IC 50 value for EMNP-PEG-rLAC BL (58.7 μg/mL) was lower than that of the bare enzyme after 48 hours of treatment on U937 cells. Both EMNP-PEG-rLAC BL and rLAC BL significantly decreased the expression levels of key cytokines (TNF-α, IL-6, IL-17, and IL-23) in LPS-stimulated cells, indicating promising anti-inflammatory properties of rLAC BL. Given the association between chronic inflammation and cancer, this newly developed enzyme-based nanobiomedicine holds potential as a therapeutic option for inflammatory conditions and malignant diseases. However, further research is needed to validate these findings and fully assess the therapeutic potential of LACs before clinical translation can be considered. [Display omitted] • Recombinant laccase (rLAC BL) was produced from Bacillus licheniformis. • The rLAC B was immobilized on PEGylated silica-coated magnetic nanoparticles. • Immobilization of rLAC BL notably enhanced enzyme affinity for its substrate. • Immobilized rLAC BL exhibited anti-inflammatory effects on the U937 cell line. [ABSTRACT FROM AUTHOR]

Laccases are enzymes that catalyse oxidation of various aromatic compounds, with potential industrial and environmental applications. Here, a novel laccase, Cb Laccase, was purified to homogeneity from the medicinal wild mushroom Coriolus brevis using standard chromatographic procedures. The overall yield was approximately 11 %. Gel filtration and sodium dodecyl sulphate–polyacrylamide gel electrophoresis revealed that Cb Laccase is a monomer with a molecular mass of 51 kDa. The optimal reaction pH with 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) as a substrate was low (pH 2.5), and the optimal reaction temperature was relatively high (70 °C). The enzyme exhibited very low K m (0.02 mM) and high catalytic efficiency (7.2 × 106) with syringaldazine, and its substrate specificity followed the order: syringaldazine > o -dianisidine > 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) > 2,6-dimethoxyphenol > catechol. Addition of 1 mM Cu2+ enhanced the enzymatic activity, whereas dithiothreitol, sodium azide, and diethyldithiocarbamic acid significantly inhibited it. The enzyme was highly stable at high temperatures, over a wide range of pH values, and in the presence of various detergents. These findings suggest that Cb Laccase is distinct from laccases previously purified from other sources and may be useful for the degradation of environmental pollutants. [Display omitted] • Cb Laccase was purified from Coriolus brevis and then characterized. • It was a 51-kDa monomeric enzyme with varying specificities across substrates. • It was stable at high temperatures, over a wide pH range, and in detergents. • It could potentially be applicable in environmental pollutant degradation studies. [ABSTRACT FROM AUTHOR]

The rise in industrial establishments all around the globe has also enhanced the demand of enzymes for usage in processes. Laccases are one of the dominant enzymes which have been employed in industrial processes over the years. They are a type of multi-copper oxidases (MCOs) which is a category of enzymes having diverse substrate selectivity and varying biological activity. All multi-copper oxidases can convert oxygen into water without creating unwanted products owing to the existence of cupredoxin-like domains. This review article details up-to-date knowledge about laccases, their structures and various sources, the laccase mediator system, mechanism of action and applications of laccases in numerous biotechnological processes such as in food industry for wine and beer stabilization, synthesis of cosmetics and pharmaceutically important compounds, dye detoxification in industrial effluents, xenobiotic degradation, paper and pulp bleaching, biofuel cells and biosensor development for detection for various chemical compounds. [ABSTRACT FROM AUTHOR]

An intense black pigmented halotolerant yeast GUBPC1, was obtained from the solar salterns of Nerul, Goa-India. The isolate could tolerate 0 to 20 % NaCl. FE-SEM analysis revealed its polymorphic nature, exhibiting oval cells at higher salt concentrations and filamentous spindle like shapes at lower concentrations. Initially, the cells appear oval, yeast like in shape but gradually after 15 days of incubation, it becomes elongated and undergoes budding, exhibiting various budding patterns, from single polar bud to bipolar buds with annellidic ring, to lateral buds and eventually forming filamentous hyphae. The intracellular black pigment was identified as melanin based on ultraviolet–visible spectroscopy analysis. The molecular identification of the culture showed closest similarity with Hortaea werneckii. Plant polymer-degrading enzymatic activities such as cellulase, laccase, chitinase, xylanase, pectinase, amylase and protease were exhibited by the isolate GUBPC1. To further understand and explore its biotechnological potential, we performed whole-genome sequencing and analysis. The obtained genome size was 26.93 Mb with 686 contigs and a GC content of 53.24 %. We identified 9383 protein-coding genes, and their functional annotation revealed the presence of 435 CAZyme genes and 16 functional genes involved in secondary metabolite synthesis, thus providing a basis for its potential value in various biotechnological applications. [ABSTRACT FROM AUTHOR]

Wastewaters generated by the textile industry often contain significant amounts of harmful (carcinogenic and mutagenic) cationic dyes, whose efficient removal is of crucial importance. This study investigates the laccase immobilization on biochar obtained from sour cherry stones (SCS-B), as a cost effective adsorbent, and evaluates its application for brilliant green (BG) degradation. The successful immobilization of laccase on biochar was achieved via adsorption and confirmed using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and Fourier transform infrared spectroscopy (FTIR). An immobilization efficiency of 66% was achieved using 0.274 U/mL of laccase at pH 5 and a temperature of 40 °C. The adsorption kinetics of laccase followed a pseudo-second-order model, indicating that chemical adsorption plays a significant role in the immobilization process. The BG degradation by immobilized system was further optimized by evaluating effects of pH, temperature, dye concentration, and contact time. More than 92% of BG (50 mg/L) was removed within 4 h at pH 5 and temperature of 30 °C. These findings suggest that SCS-B can effectively be used as an enzyme carrier and be further utilized for the removal of emerging pollutants, positioning it as a sustainable solution for wastewater treatment. [ABSTRACT FROM AUTHOR]

Ganoderma, a well-known genus in the Ganodermataceae family, has caused the extinction of several tree species due to its pathogenicity. This study explored the pathogenic effect of a newly identified Ganoderma species on trees and its competitive efficiency against Trichoderma species. Ganoderma camelum sp. nov. is characterized by small sessile basidiomata and a velvety, soft, camelbrown pileus. Phylogenetic analysis and ITS rDNA sequences indicated that the species were Trichoderma and Ganoderma camelum. Both fungal species competed antagonistically by secreting laccase. The laccase activity of G. camelum, with a value of 8.3 ± 4.0 U/mL, demonstrated the highest competitive activity against Trichoderma species. The laccase produced by T. atroviride (2.62 U/mL) was most effective in countering the pathogenic action of the novel G. camelum. The molecular weights of laccase were determined using SDSPAGE (62.0 kDa for G. camelum and 57.0 kDa for T. atroviride). Due to the white rot induced by this Ganoderma species in the host tree, G. camelum showed the highest percentage inhibition of radial growth (76.3%) compared to T. atroviride (28.7%). This study aimed to evaluate the competitive antagonistic activity of Ganoderma and Trichoderma on malt extract agar media in the context of white rot disease in the host tree. This study concluded that the laccase from G. camelum caused weight loss in rubber wood blocks through laccase action, indicating tissue injury in the host species. Therefore, it was also concluded that G. camelum was more effective in pathogenic action of the host and resisted the biological action of T. atroviride. In principal components analysis (PCA), all the species associated with laccase exhibited a very strong influence on the variability of the system. The PIRG rate (percentage inhibition of radial growth) was strongly and positively correlated with laccase activity. [ABSTRACT FROM AUTHOR]

Nanozymes based on metal-organic frameworks (MOFs) have been concentrated on due to their naturally high-disperse metal active sites and the adjustable coordination chemistry. In this work, an N-rich melamine (Mel) was introduced into the Cu-MOF composed of copper(II) nitrate and 2-aminoterephthalic acid (Cu-NH2-BDC-Mel) to mimic the laccase, which enzyme-like activities were assessed and applied in sensing analyses toward several phenols and amines. Compared to unmodified Cu-NH2-BDC, the resulting Cu-NH2-BDC-Mel exhibits enhanced laccase-like activity, superior stability and catalytic kinetics. It is demonstrated that melamine-doping has increased nitrogen content as well as the surface area, as a result, exhibits a lower Michaelis–Menten constant (Km) (0.1877 mM) and higher maximum reaction rate (Vmax) (1.7933 × 10−3 mM·min−1) in comparison with that of natural laccase. Based on that, an efficient colorimetric sensing strategy for several phenols and amines was built up with excellent selectivity and anti-interference by using the laccase-like Cu-NH2-BDC-Mel, the detection limits are 3.51 µM of adrenaline and 4.41 µM of dopamine. The work broadens the prospect development of bio-colorimetric sensing based on the ligand-modified Cu-MOFs nanozymes catalysis. [ABSTRACT FROM AUTHOR]

A variety of important agricultural crops host fungi from the Aspergillus genus can produce cancerogenic secondary metabolites such as aflatoxins. Consequently, novel strategies for detoxification and their removal from food and feed chains are required. Here, detoxification of Aflatoxin B1 (AFB1) by the Bacillus subtilis multi-copper oxidase CotA (BsCotA) was investigated. This laccase was recombinantly produced in E. coli while codon optimization led to duplication of the amount of active protein obtained. CuCl2 was added to the cultivation medium leading to a 25-fold increase of Vmax corresponding to improved incorporation of Cu2+ into the enzyme protein which is essential for the catalytic reaction. To avoid potential cytotoxicity of Cu2+, cultivation was performed at microaerobic conditions indeed leading to 100x more functional protein when compared to standard aerobic conditions. This was indicated by an increase of Vmax from 0.30 ± 0.02 to 33.56 ± 2.02 U/mg. Degradation kinetics of AFB1 using HPLC with fluorescence detection (HPLC-FLD) analysis indicated a theoretical substrate saturation above solubility in water. At a relatively high concentration of 500 μg/L, AFB1 was decomposed at 10.75 μg/Lh (0.17 nmol*min−1*mg−1) at a dosage of 0.2 μM BsCotA. AFQ1 and epi-AFQ1 were identified as the initial oxidation products according to mass spectrometry (i.e., HPLC-MS, HPLC-QTOF). None of these molecules were substrates for laccase but both decomposed in buffer. However, decomposition does not seem to be due to hydration of the vinyl ether in the terminal furan ring. Genotoxicity of the formed AFB1 was assessed in several dilutions based on the de-repression of the bacterial SOS response to DNA damage indicating about 80-times reduction in toxicity when compared to AFQ1. The results of this study indicate that BsCotA has high potential for the biological detoxification of aflatoxin B1. [ABSTRACT FROM AUTHOR]

Laccase, the selectively lignin degrader, vital to the initiation of lignocellulosic deconstruction was immobilized onto activated agarose beads to increase its reuse potential. Laccase cross-linked beads (~ 3.42 mm) recorded a specific activity of 23 Umg− 1, retaining about 80.43% enzyme activity after 45 days of storage. The immobilization yield and efficiency were 89% and 97% respectively. The equilibrium data fitted the Freundlich equation (R2 = 0.9987) demonstrating multilayer adsorption and the presence of Cu, Fe, and S in the elemental analysis of immobilized beads established effective binding between activated agarose beads and the laccase protein. Characterization studies of the immobilized laccase-treated crop residues revealed significant differences in the lignin polymer after each treatment cycle. An increase in digestibility of 26.21% and 7.62% was observed in paddy and finger millet-treated straws respectively, over the controls corroborating efficient lignin depolymerization. The propitious performance of laccase beads authenticated in the batch enzymatic reactor to treat crop residues paves headway as a sustainable green technology in the deconstruction of crop residues for use as ruminant feed, augmenting productivity. [ABSTRACT FROM AUTHOR]

AbstractOwing to the production of lignin-modifying enzymes (LMEs), white-rot fungi (WRF) such as polypores are potent organisms in the biodegradation of xenobiotic pollutants. The nonspecific function of LMEs including laccase and manganese peroxidase (MnP), has enabled the use of WRF in biotechnological applications, particularly in bioremediation. In this study, 12 strains from nine white-rot basidiomycete genera viz., Ganoderma, Inocutis, Irpex, Lentinus, Lenzites, Oxyporus, Peniophora, Sanghuangporus, and Trametes were isolated from Iran and identified using morphological and molecular tools. The enzyme activity of laccase and manganese peroxidase that directly correlated with the biodegradation were determined, and the strains with the highest enzyme activities were evaluated for their ability to degrade 400 mg/L of anthracene over 28 days. Gas chromatography with flame ionization detector (GC-FID) revealed that four polypores viz., Trametes versicolor v21te, T. versicolor v22da, T. hirsuta, and Oxyporus sp. degraded 64%, 52%, 34%, and 20% of the anthracene, respectively. According to our analysis, the examined indigenous WRF are potentially useful candidates for the development of new mycoremediation techniques to degrade polycyclic aromatic hydrocarbons (PAHs). [ABSTRACT FROM AUTHOR]

Laccase, a multi‐copper oxidase, is limited by its optimal temperature range and isolation costs. To overcome these challenges, we synthesized copper‐doped zeolitic imidazolate framework‐67 (Cu‐doped ZIF‐67) with 16 mol % Cu as an artificial laccase catalyst. The introduced Cu site acts as the phenol oxidation site, and Co‐based ZIF‐67 is the four‐electron oxygen reduction site. Laccase also employs this division of oxidation and reduction sites. Cu‐doped ZIF‐67 demonstrated significant catalytic activity, superior to natural laccase, especially at elevated temperatures, and maintained stability across multiple reaction cycles. These findings suggest that Cu‐doped ZIF‐67 is a robust, reusable alternative for industrial applications requiring high thermal stability and efficient catalysis. [ABSTRACT FROM AUTHOR]

In the current investigation, crude laccase was utilized to decolorize the leather azo dyes (AB 113 and AB 52). The crude laccase was extracted from Penicillium chrysogenum strain TSV1 pH 5.5, temp. at 32°C for 7 days in a liquid culture medium, and shows maximum enzyme activity of 7.92 U/mL. The crude laccase has been applied and achieved maximum decolorization efficiency up to 83% and 69% respectively for AB 113 and AB 52 within 48-h duration. Furthermore, the addition of 1 mM HOBT mediator has enhanced the dye decolorization rate up to 98% and 85% within 24-h duration respectively for both dyes. Furthermore, the decolorization was confirmed by UV-Vis spectra analysis and has showed peaks at 570 nm and 580 nm which corresponded to chromophores respectively for both dyes and disappeared after decolorization. The bands were observed at the bending region from 1561 to 1494 cm−1 and from 1344 to 1031 cm−1 by FTIR analysis corresponding to an aromatic ring of AB 113. Similarly, the peaks observed at 1589cm−1 and 1451cm−1 corresponded to the aromatic region of AB 52 and indicated the presence of -N=N-. These bands disappeared and new bands appeared after decolorization. The UPLC analysis has shown different peaks at various retention times for both dyes and these peaks disappeared after decolorization. Furthermore, the COD (86.5 ± 5% and 83.52 ± 3%) and TOC (87.86% and 83.82%) have been reduced for both dyes. The toxicity of the dyes also has been reduced after decolorization. The laccase was a more potential enzyme for the decolorization of azo dyes. [ABSTRACT FROM AUTHOR]

Podaxis pistillaris is neutraceutically, cosmoceutically and medicinally recognised macrofungus. During this research work, this edible mushroom was systematically characterised. Its culturability, laccase production, and dye decolorisation potential were evaluated and optimised. Among the different media tested, PDA proved as most efficient medium for culturability of P. pistillaris. Conditions for laccase production were optimised in submerged state fermentation. Maximum laccase secretion was noted after 14th day of Incubation at 35 °C with 130 rpm and 5 pH of medium. Fructose and ammonium-phosphate was found as best carbon and nitrogen source, while wheat straw revealed as good ligno-cellulosic source for strengthening laccase production. Congo-red dye decolorisation capability by crude laccase enzyme was evaluated and found maximum decolorisation potential (92.2%) after 288h of incubation. From this pilot study, it was confirmed that this edible macrofungus has culturability, laccase production and dye decolorisation attributes that will further contribute in delignification, biosorption and bioremediation. [ABSTRACT FROM AUTHOR]

Background: Hazardous synthetic dye wastes have become a growing threat to the environment and public health. Fungal enzymes are eco-friendly, compatible and cost-effective approach for diversity of applications. Therefore, this study aimed to screen, optimize fermentation conditions, and characterize laccase from fungal endophyte with elucidating its ability to decolorize several wastewater dyes. Results: A new fungal endophyte capable of laccase-producing was firstly isolated from cladodes of Opuntia ficus-indica and identified as T. harzianum AUMC14897 using ITS-rRNA sequencing analysis. Furthermore, the response surface methodology (RSM) was utilized to optimize several fermentation parameters that increase laccase production. The isolated laccase was purified to 13.79-fold. GFC, SDS-PAGE revealed laccase molecular weight at 72 kDa and zymogram analysis elucidated a single band without any isozymes. The peak activity of the pure laccase was detected at 50 °C, pH 4.5, with thermal stability up to 50 °C and half life span for 4 h even after 24 h retained 30% of its activity. The Km and Vmax values were 0.1 mM, 22.22 µmol/min and activation energy (Ea) equal to 5.71 kcal/mol. Furthermore, the purified laccase effectively decolorized various synthetic and real wastewater dyes. Conclusion: Subsequently, the new endophytic strain produces high laccase activity that possesses a unique characteristic, it could be an appealing candidate for both environmental and industrial applications. [ABSTRACT FROM AUTHOR]

Fungal infection in agricultural grains is a global problem, particularly if it is accompanied by mycotoxin production. In this study, the degradation of mycotoxins by laccase and manganese peroxidase was investigated. Aspergillus flavus, Aspergillus fumigatus, and Fusarium graminearum were recorded in infected maize grains. Aflatoxin B1 (AF B1) was detected (from 3.38 to 2.60 ppm) on the infected samples by fungi compared to other detected aflatoxins. Trichothecene (T-2) toxin and deoxynivalenol (DON) were recorded with concentrations ranging from 0.464 to 0.184 ppm and 0.370 to 0.214 ppm, respectively. The addition of laccase and manganese peroxidase to the inoculated medium with A. flavus and F. graminearum individually degraded the produced AF B1, B2, G1, G2, T-2 toxin, and DON from 5.0, 1.33, 0.76, 0.61, 0.63, and 0.38 ppm to 2.77, 0.66, 0.37, 0.15, 0.45, and 0.38 ppm using laccase, to 3.08, 1.25, 0.61, 0.39, 0.55, and 0.36 ppm using manganese peroxidase. The computational technique (docking) demonstrated the laccase and manganese peroxidase activities on aflatoxin and DON degradation. Consequently, the results suggested that laccase (PDB ID: 1HFU) and manganese peroxidase (PDB ID: 1MNP) promise innovative activity toward aflatoxin degradation, while 1HFU has more effect than 1MNP on DON degradation. [ABSTRACT FROM AUTHOR]

The laccase activity of a low-temperature strain of Volvariella brumalis was studied using different lignocellulosic biomasses for liquid fermentation. The results showed that the laccase activity induced by wheat bran or pressed rapeseed residue was higher than the other auxiliary cultivation substrates. Compared to other primary cultivation substrates, Quercus acutissima sawdust and rice straw not only stimulated V. brumalis to produce more laccase in liquid fermentation, but also promoted better mycelial growth in solid-state fermentation. Therefore, these agricultural and forestry wastes should be prioritized as culturing materials of V. brumalis for laccase high-secretion and hyphal growth to increase production of V. brumalis. [ABSTRACT FROM AUTHOR]

Analysis of the structure of two-domain laccase ScaSL from Streptomyces carpinensis VKM Ac-1300 (with a middle-redox potential) revealed determinants that could affect the increased potential of ScaSL. Site-directed mutagenesis of the ScaSL laccase was carried out, and mutants H286A, H286T, H286W, and F232Y/F233Y were obtained. Replacement of His 286 with Ala led to a decrease in redox potential (0.45 V) and an increase in stability at pH 9 and 11; replacement with Thr led to an increase in redox potential (0.51 V) but to a decrease in the thermal stability of the protein; replacement with Trp did not affect the enzyme properties. Replacement of Phe residues 232 and 233 with Tyr led to a shift in enzyme activity to the acidic pH range without changing the redox potential and a decrease in the thermostability and pH stability of the enzyme. All mutants more efficiently oxidized phenolic substrate 2,6-DMP and were able to participate in direct electron transfer (DET) with MWCNT-modified electrodes. The F232Y/F233/Y mutant was unable to degrade triphenylmethane dyes without a mediator but showed a greater degree of decolorization of azo dyes in the presence of the mediator. The crystal structure of laccase with the highest potential was determined with high resolution. [ABSTRACT FROM AUTHOR]

Laccase is a copper‐containing polyphenol oxidase that can oxidize phenolic and non‐phenolic organic substrates. In the past decades, laccases had received considerable attention because of the ability to degrade various organic substances. Based on the codon preference of the Pichia pastoris expression system, this study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea through synthetic biology methods. A new gene Lcc1I was synthesized and heterologously expressed in P. pastoris. After 3 days of cultivation in a shake flask at 30°C, the transformants produced at a yield of 890 mg L−1protein. The highest production level of the recombinant laccase was 2760 U L−1. The molecular mass of the recombinant laccase was estimated at 60 kDa. The enzyme showed highest activity at pH 3.4 and 45°C. It possessed better stability at higher pH and lower temperature condition. Using 2,2'‐azino‐bis‐(3‐ethylbenzothiazoline)−6‐sulphonate (ABTS) as the substrate, the Km and Vmax values were 0.136 mM and 9778 μM min−1 mg−1, respectively. The recombinant laccase could directly oxidize some triphenylmethane dyes like leuco‐crystal violet (LCV) and leuco‐malachite green (LMG). With the help of ABTS mediator, it could oxidize and degrade 77.7% crystal violet (CV) and 79.2% malachite green (MG) within 1 h. Our results indicate that optimization of the laccase gene achieves good expression results in the host system. The dye degradation model constructed in this study may also be applied to the degradation of other organic pollutants and toxic substances, providing new solutions for environmental remediation against the increasingly severe environmental pollution. Summary: This study optimized the gene structure of the laccase gene Lcc1 from Coprius cinerea using synthetic biology methods, which could be successfully expressed in Pichia pastoris.The recombinant laccase possessed better substrate affinity, reaction rate and capable of efficient degradation of triphenylmethane dyes.In the current context of increasingly severe organic pollution, the degradation model of dyes constructed in this study can be widely applied to environmental remediation dealing with various pollutantsCompared with traditional methods, the advantages including higher efficiency, lower cost, easier operation, and without secondary pollution, providing a new solution for environmental pollution. [ABSTRACT FROM AUTHOR]

Laccase, a copper-containing oxidoreductase, has close links with secondary metabolite biosynthesis in plants. Its activity can affect the synthesis and accumulation of secondary metabolites, thereby influencing plant growth, development, and stress resistance. This study aims to identify the grape laccases (VviLAC) gene family members in grape (Vitis vinifera L.) and explore the transcriptional regulatory network in berry development. Here, 115 VviLACs were identified and divided into seven (Type I–VII) classes. These were distributed on 17 chromosomes and out of 47 VviLACs on chromosome 18, 34 (72.34%) were involved in tandem duplication events. VviLAC1, VviLAC2, VviLAC3, and VviLAC62 were highly expressed before fruit color development, while VviLAC4, VviLAC12, VviLAC16, VviLAC18, VviLAC20, VviLAC53, VviLAC60 and VviLAC105 were highly expressed after fruit color transformation. Notably, VviLAC105 showed a significant positive correlation with important metabolites including resveratrol, resveratrol dimer, and peonidin-3-glucoside. Analysis of the transcriptional regulatory network predicted that the 12 different transcription factors target VviLACs genes. Specifically, WRKY and ERF were identified as potential transcriptional regulatory factors for VviLAC105, while Dof and MYB were identified as potential transcriptional regulatory factors for VviLAC51. This study identifies and provides basic information on the grape LAC gene family members and, in combination with transcriptome and metabolome data, predicts the upstream transcriptional regulatory network of VviLACs. [ABSTRACT FROM AUTHOR]

Laccases are extracellular multicopper oxidases that react with a broad range of aromatic substrates and are used for many biotechnological applications. Recently, microalgal laccases were discovered and started to be characterized in some Chlorophyceae (Tetracystis aeria and a few other species). A volumetric activity of maximum 27 U L-1 could be reached with Chlamydomonas moewusii, which is largely insufficient to compete with fungi and heterologous expression systems. Here, the acidophilic green microalga Chlamydomonas pitschmannii was shown to be a new promising source of laccase competing with fungi in terms of secretion yields. By modulating culture pH and CO2 supply, laccase volumetric activity in the supernatant could be increased from 12 to 459 U L-1. Laccase-containing supernatants were found to oxidize many phenolic and non-phenolic substrates with an efficiency that was pH-dependent. 1D SDS-PAGE was achieved and lighted up that C. pitschmannii laccase is a glycosylated protein. Functional measurements pointed out the participation of glycosylation in functional enzymatic regulation and integrity. Two bands of about 50 and 45 kD were detected that presumably corresponded to laccase. In agreement with previous studies in T. aeria, we hypothesize that C. pitschmannii laccase is a heterodimer, a feature that denotes with most known laccases, essentially monomeric. Altogether, those results demonstrate that efforts of discovery and characterization of microalgal laccases must be continued with regards to the amazing potential they offer for biotechnological applications. [ABSTRACT FROM AUTHOR]

Synthetic dyes such as azo dyes are significant pollutants in the wastewater released from various textile industries. The low biodegradability and production from synthetic sources with high shelf life make azo dyes a challenging material for degradation. This study used chemically mutated Aspergillus terrus in the laccase production under solid-state fermentation using sugarcane bagasse. Initially, the wild-type strain produced a laccase activity of 4.12 U/mL. Later, the alkaline pretreatment of sugarcane bagasse showed a significant increase in laccase activity by 38.9%. Further, random mutagenesis treatment with 100 mM EMS generated a hyper laccase-producing strain with a 2.3-fold increment in laccase activity compared to the wild-type strain. The enzyme displayed optimal activity at pH 6.5 and 35 °C. The metal ions such as Fe3+ (29.4 U/mL), Fe2+ (20.8 U/mL) and Cu2+ (18.05 U/mL) showed positive effects on laccase activity. The crude laccase was used to bioremediate Congo red, a prominent azo dye used in textile and pharmaceutical industries. The preliminary studies with a crude enzyme displayed 68.86% dye decolourization after 24 h of incubation. Additionally, with Taguchi orthogonal array optimization experiments, the maximal dye decolorization of 78.24% was achieved by maintaining crude enzyme concentration (20 U), dye concentration (25 mg/L) and pH 4.5. [ABSTRACT FROM AUTHOR]

Bisphenols as an important industrial raw material has endocrine disruption, reproductive toxicity, and neurotoxicity. Laccase (Lac) has demonstrated excellent catalytic oxidation performance for phenolic compounds. Micro-nano bubbles (MNB) have been extensively used in wastewater treatment, and the high efficiency of MNB-assisted laccase (MNB-Lac) as a hybrid technique for removing bisphenols from water has been established. In this study, MNB-Lac system was used to degrade bisphenols including bisphenol A (BPA), bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), and bisphenol F (BPF). A comparative analysis was conducted to evaluate the degradation of individual and mixed bisphenols by MNB-Lac. The results indicated that the degradation effect on all individual bisphenols was superior. Notably, BPC consistently demonstrated higher selectivity in the mixed degradation process. In the mixed bisphenol degradation process, a distinct degradation sequence between BPE and BPF was observed. The affinity order of BPA and BPE was found to be inconsistent with their individual degradation order, and the degradation reaction was found to conform to second-order kinetics. By gas chromatography mass spectrometry detection, it was found that the degraded products of BPA, BPB, BPC, BPE, and BPF were ring and chain compounds, and the degradation pathways of the five substances were also maintenance ring structure and direct ring opening. The possible transformation relationship of mixed bisphenols during degradation by MNB-Lac was proposed. Moreover, the industrial application of MNB-Lac was evaluated by increasing the wastewater volume of BPA. [ABSTRACT FROM AUTHOR]

Laccases act as green catalysts for oxidative cross-coupling of phenolic antioxidnt compounds, but low stability and non-recyclability limit its application. To address that, metal–organic frameworks Cu-BTC and Cr-MOF were synthesized as supports to immobilize the efficient laccase from Cerrena sp. HYB07. The Brunauer–Emmett–Teller surface area of Cu-BTC and Cr-MOF were 1213.2 and 907.1 m2/g, respectively. The two carriers respectively presented pore diameters of 1.2–10 nm and 1.4–12 nm as octahedron, indicating nano-scale mesoporosity. These Cu-BTC and Cr-MOF carriers could adsorb laccase with enzyme loading of 1933.2 and 1564.4 U/g carrier, respectively. The stability and organic solvent tolerance of Cu-BTC-laccase and Cr-MOF-laccase were both obviously improved compared to free laccase. Thermal inactivation kinetics showed that both the two immobilized laccases displayed lower thermal inactivation rate constants. Importantly, the Cu-BTC-laccase and Cr-MOF-laccase both showed much higher activity for cross-coupling of ethyl ferulate than free laccase, which had 2.5-fold higher cross-coupling efficiency than that by free laccase. The ethyl ferulate coupling product was also analyzed by mass spectroscopy and the synthesis pathway of ethyl ferulate dimer was proposed. The cross coupling of ethyl ferulate required the formation of radical intermediates of ethyl ferulate generated by laccase mediated oxidation. This work paved the way for MOFs immobilized laccase for cross coupling of antioxidant phenols. [ABSTRACT FROM AUTHOR]

Polyethylene, one of the most used petroleum-derived polymers, causes serious environmental pollution. The ability of Pleurotus ostreatus to degrade UV-treated and untreated recycled and unused (new) low-density polyethylene (LDPE) films was studied. We determined the fungal biomass production, enzyme production, and enzyme yield. Changes in the chemical structure and surface morphology of the LDPE after fungal growth were analyzed using FTIR spectroscopy and SEM. Functional group indices and contact angles were also evaluated. In general, the highest Lac (6013 U/L), LiP (2432 U/L), MnP (995 U/L) and UP (6671 U/L) activities were observed in irradiated recycled LDPE (IrRPE). The contact angle of all samples was negatively correlated with fermentation time; the smaller the contact angle, the longer the fermentation time, indicating effective biodegradation. The IrRPE samples exhibited the smallest contact angle (49°) at 4 weeks, and the samples were fragmented (into two pieces) at 5 weeks. This fungus could degrade unused (new) LDPE significantly within 6 weeks. The biodegradation of LDPE proceeded faster in recycled than in unused samples, which can be enhanced by exposing LDPE to UV radiation. Enzymatic production during fungal growth suggest that LDPE degradation is initiated by laccase (Lac) followed by lignin peroxidase (LiP), whereas manganese peroxidase (MnP) and unspecific peroxygenase (UP) are involved in the final degradation process. This is the first experimental study on the fungal growth and its main enzymes involved in LDPE biodegradation. This fungus has great promise as a safe, efficient, and environmentally friendly organism capable of degrading LDPE. [ABSTRACT FROM AUTHOR]

Laccases are enzymes with low substrate specificity that in certain cases require the presence of a mediator in order to work properly. Therefore, this paper investigated the degradation of Orange G, Ponceau 4 R and Methyl Red in the presence of laccase isolated from Trametes versicolor and laccase-ABTS system. Laccase, in high concentration, was able to oxidize only Orange G and Ponceau 4 R, with a degree of decolorization less than 10 % in 20 min. In the presence of laccase-ABTS system, the reaction is much faster, and at least 40 % of dyes were degraded in 15 min. A kinetic model accounting for enzymatic oxidation of all dyes, including a step for enzyme operational inactivation, was proposed. This study proves that we can use a single kinetic model for the oxidation of all dyes in the laccase-ABTS system. [Display omitted] • Kinetics of Orange G, Ponceau 4 R, and Methyl Red oxidation by laccase and laccase-ABTS system was studied. • Operational inactivation of laccase was highlighted for all systems. • Kinetic models for dye degradation by laccase, and laccase-ABTS system were proposed. • The dyes oxidation can be attributed both to the oxidation by a complex between laccase and ABTS, and by ABTS ∙ + . [ABSTRACT FROM AUTHOR]

This study explores the Laccase gene (AaLac) family along with AaLac1 expression in hairy roots of A. annua. 42 AaLacs were identified by detecting three conserved domains: Cu-oxidase, Cu oxidase-2, and Cu oxidase-3. The physicochemical properties show that AaLacs are proteins with 541–1075 amino acids. These proteins are stable, with an instability index less than 40. Phylogenetic and motif studies have shown structural variants in AaLacs, suggesting functional divergence. 22 AaLac cis-regulatory elements were selected for their roles in drought stress, metabolic modulations, defense, and stress responses. A comparison of AtLac and AaLac proteins showed that 11 AtLacs mitigates stress reactions. In silico expression, analysis of 11 AtLacs showed that AtLac84 may function under osmotic stress. Thus, the Homolog AaLac1 was selected by expression profiling. The real-time PCR results showed that AaLac1 enhances osmotic stress tolerance in shoot and root samples. It was also used to analyze AaLac1, ADS, and CYP71AV1 gene expression in hairy roots via induction. The transformed hairy roots exhibited a greater capacity for PEG-induced osmotic stress tolerance in contrast to the untransformed roots. The gene expression analysis also depicted a significant increment in expression of AaLac1, ADS, and CYP71AV1 genes to 3.8, 6.9, and 3.1 folds respectively. The transformed hairy roots exhibited a significant increase of 2.2 and 1.4 fold in flavonoid and phenolic content respectively. Also, lignin content and artemisinin content increased by 7.05 folds and 95.6% with respect to the control. Thus, transformed hairy roots of A. annua under PEG-induced osmotic stress demonstrate the involvement of the AaLac1 gene in stress responses, lignin biosynthesis, and secondary metabolism production. [ABSTRACT FROM AUTHOR]

This study investigated the decolorization and detoxification of a dye originating from a feather-dyeing house using the laccase enzyme. Efficient decolorization was achieved under a wide range of environmental conditions, including pH, temperature, enzyme loading, and dye concentration. The decolorization rate reached up to 98% within 10 min when the mediator acetosyringone was present. Decolorization was attributed to the degradation of the dye by laccase, as analyzed using liquid chromatography-mass spectrometry (LC–MS). At least three dominant intermediates (m/z 167.993, 194.475, 118.040) were identified, deriving from the original dye (m/z 521.044). Compared to the non-decolorized dye solution, the decolorized dye ones at various concentrations exhibited lower toxicity towards aerobic activated sludge (AS). This was evident from the significantly higher respiratory intensity of AS, with an O₂ uptake of 35 mg for the decolorized solution versus 11 mg for the non-decolorized solution after 12 h at a 100 mg/L dye concentration. The results suggest that laccase-catalyzed decolorization could serve as an effective pretreatment method for traditional activated sludge (AS)-based processes, thereby enhancing the overall treatment performance of dye wastewater. [ABSTRACT FROM AUTHOR]

Laccase is a multicopper oxidase enzyme that oxidizes a variety of substrates, including polyphenols and polycyclic aromatic hydrocarbons (PAHs). It catalyzes the four-electron reduction of molecular oxygen that results in the production of water as a by-product. Thus, laccase can play an important role in environmental care. Previously, we have successfully expressed Trametes trogii laccase (TtLcc1) in the yeast Saccharomyces cerevisiae. In this work, we have expressed in yeast another laccase, LacA from Trametes sp. AH28-2, and tested its function on PAHs. Yeast cells engineered to produce the two laccases performed efficient PAH degradation. Both TtLcc1 and LacA led to the construction of spatiotemporal fluorescence-pulse generators when combined with a benzoate/salicylate yeast biosensor in a two-population system. Moreover, laccases returned a visual output signal in yeast synthetic circuits—upon reacting with ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)). Thus, in S. cerevisiae, laccases are a powerful alternative to fluorescent reporter proteins.

Abstract Background The laccase Lcc9 from Coprinopsis cinerea has optimal catalytic activity at moderate to alkaline pH conditions, making it invaluable for industrial applications. However, C. cinerea naturally secretes Lcc9 at low expression levels, which limits the industrial application of Lcc9 on a large scale. Recombinant production of Lcc9 using Aspergillus niger would be an effective way to achieve its high production. Results This study achieved the secretory production of Lcc9 in A. niger and established an efficient transformation procedure for A. niger by optimizing its protoplast preparation system. The transformation efficiency of A. niger was increased 3.8-fold under the optimal system (cell wall digestion enzyme solution: 2% cellulase, 1% snailase, 1% lyticase, and 0.5% lysozyme; incubation time: 3 h; incubation temperature: 37 ℃; culture time: 48 h). The extracellular yield of Lcc9 was enhanced by optimizing gene expression cassette and bioprocess. First, the strain AnGgcL (containing P gpdA ) mediated by the SPCAT, a signal peptide of the extracellular high abundance protein catalase, had an extracellular laccase activity of 10 U/L after shake flask fermentation. Then, by optimizing promoter and signal peptide combinations that regulate lcc9 expression, the strain AnGcgL mediated by P citA -SPGlaA had an extracellular laccase activity of 20 U/L. Subsequently, the strain AnRcgL1 (containing P citA -SPGlaA) obtained by random integration had an extracellular laccase activity of 86 U/L. Sequencing revealed that the lcc9 expression cassette was integrated into the citrate synthase gene locus in the AnRcgL1 genome in a 9-copy form. By optimizing the microparticle, osmolyte, and Cu2+ in the fermentation medium, the AnRcgL1 extracellular laccase activity was further increased to 1566.7 U/L, which was 156.7-fold higher than that of AnGgcL. Furthermore, its extracellular laccase activity was increased to 1961 U/L in a 1-L fermenter. Conclusions To our knowledge, this study is the first to report the recombinant extracellular production of the C. cinerea laccase Lcc9 in A. niger and to use SPCAT in the A. niger expression system. The results of this study will help accelerate the industrial application of Lcc9. Moreover, the strategy used in this work provides methodological guidance for increasing other exogenous protein yields in A. niger.

Background and purpose: A biosensor is composed of two main components: a biological sensing element and a transducer, which together determine the concentration of the analyte by facilitating a biological reaction near the transducer to improve detection capabilities. Compared to traditional analyte measurement and biochemical control methods, biosensors offer advantages such as reusability, fast response times, and high specificity. Laccase is a critical enzyme within the oxidoreductase family, with catalytic properties particularly effective toward ortho- and para-diphenols, depending on the producing organism. Unlike many enzymes that act exclusively on a specific substrate, laccase catalyzes the oxidation of a broad range of substrates, including diphenols, polyphenols, diamines, methoxyphenols, aromatic amines, and ascorbate. Protein tyrosine phosphatases are a class of enzymes that remove phosphate groups from tyrosine residues in proteins. This project aimed to design and develop an enzyme-based biosensor using modified screen-printed carbon electrodes with high sensitivity and selectivity for accurate and rapid dephostatin measurement. Materials and methods: The biosensor was fabricated by immobilizing laccase on the electrode surface. First, the surface of the screen-printed carbon electrode was modified with a graphene oxide/polyaniline nanocomposite, after which the laccase enzyme was immobilized on the modified surface using EDC/NHS linkers. Electrochemical characteristics of the electrodes were assessed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Optimal parameters, including oxidation potential, pH, temperature, and concentration, were calibrated. At an oxidation potential of 0.45 V relative to the Ag/AgCl electrode, the current response for each sample was measured with the biosensor. Calibration curves were generated, and measurements were obtained by interpolating the amperometric signals from dephostatin solutions. Dephostatin standards were then introduced to the diluted Nescafe sample (phosphate buffer, 0.05 M, pH 6.5) without any pretreatment, and recovery rates were calculated. Results: By immobilizing graphene oxide and laccase on the electrode surface, the modified electrode exhibited increased cathodic and anodic currents compared to the unmodified electrode in K₄Fe(CN)₆/K₃Fe(CN)₆ solution. Due to the presence of graphene nanoparticles on the electrode surface, the electron transfer rate improved, as indicated by a 20 µA increase. The electrode response was linear across a concentration range of 10 to 900 nM, with a detection limit of 6 nM. Conclusion: Since protein tyrosine phosphatases play essential roles in cell signaling pathways and disease development, the use of dephostatin as an inhibitor has gained interest. Therefore, an accurate and rapid method for dephostatin measurement is crucial in the pharmaceutical industry. The prepared biosensor, which incorporates laccase enzyme and graphene nanoparticles stabilized with a chitosan nanocomposite, allows for precise dephostatin measurement. The laccase enzyme immobilized on the electrode surface can specifically oxidize the dephostatin molecule, producing a unique electrode response for this molecule.

Abstract Laccase, the selectively lignin degrader, vital to the initiation of lignocellulosic deconstruction was immobilized onto activated agarose beads to increase its reuse potential. Laccase cross-linked beads (~ 3.42 mm) recorded a specific activity of 23 Umg− 1, retaining about 80.43% enzyme activity after 45 days of storage. The immobilization yield and efficiency were 89% and 97% respectively. The equilibrium data fitted the Freundlich equation (R2 = 0.9987) demonstrating multilayer adsorption and the presence of Cu, Fe, and S in the elemental analysis of immobilized beads established effective binding between activated agarose beads and the laccase protein. Characterization studies of the immobilized laccase-treated crop residues revealed significant differences in the lignin polymer after each treatment cycle. An increase in digestibility of 26.21% and 7.62% was observed in paddy and finger millet-treated straws respectively, over the controls corroborating efficient lignin depolymerization. The propitious performance of laccase beads authenticated in the batch enzymatic reactor to treat crop residues paves headway as a sustainable green technology in the deconstruction of crop residues for use as ruminant feed, augmenting productivity.

Owing to the production of lignin-modifying enzymes (LMEs), white-rot fungi (WRF) such as polypores are potent organisms in the biodegradation of xenobiotic pollutants. The nonspecific function of LMEs including laccase and manganese peroxidase (MnP), has enabled the use of WRF in biotechnological applications, particularly in bioremediation. In this study, 12 strains from nine white-rot basidiomycete genera viz., Ganoderma, Inocutis, Irpex, Lentinus, Lenzites, Oxyporus, Peniophora, Sanghuangporus, and Trametes were isolated from Iran and identified using morphological and molecular tools. The enzyme activity of laccase and manganese peroxidase that directly correlated with the biodegradation were determined, and the strains with the highest enzyme activities were evaluated for their ability to degrade 400 mg/L of anthracene over 28 days. Gas chromatography with flame ionization detector (GC-FID) revealed that four polypores viz., Trametes versicolor v21te, T. versicolor v22da, T. hirsuta, and Oxyporus sp. degraded 64%, 52%, 34%, and 20% of the anthracene, respectively. According to our analysis, the examined indigenous WRF are potentially useful candidates for the development of new mycoremediation techniques to degrade polycyclic aromatic hydrocarbons (PAHs).

A novel, simple and low-cost colorimetric sensor has been developed for the detection of bisphenol A (BPA). In the assay, laccase catalyses the reaction between BPA and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) [ABTS] radicals, resulting in a colored product showing λmax at 560 nm. Raman spectroscopy and mass spectra analysis of the product suggests the possible formation of ABTS dication radicals. This assay could detect BPA up to 2 µM by the naked eye, which is lower than the European Union specific migration limit of 2.6 µM. UV–visible spectroscopy showed a linear response in the concentration range of 1.0–10.0 µM with a lower detection limit of 0.5 µM. Sensitivity of the assay was enhanced by 40 folds when laccase and ABTS were fabricated on polyvinyl alcohol (PVA) hydrogel beads, allowing BPA to be detected at concentrations as low as 50 nM. These hydrogel beads were highly stable for long-term storage. Screening of different phenolic contaminants showed that laccase along with ABTS exhibits propensity for para-substituted alkyl-phenols that are considered as xenoestrogens and are highly toxic, estrogenic and carcinogenic in nature. Thus, the assay developed may be utilized for in-situ monitoring of BPA and other toxic xenoestrogens present in the environment. Additionally, its applicability can be extended towards other potential laccase mediators. [ABSTRACT FROM AUTHOR]