Laccases stand out in the industrial context due to their versatile biotechnological applications. Although these enzymes are frequently investigated, currently, Pleurotus ostreatus laccase structural model is unknown. Therefore, this research aims to predict and validate a P. ostreatus laccase theoretical model by means of comparative homology. The laccase target’s primary structure (AOM73725.1) was obtained from the NCBI database, the model was predicted from homologous structures obtained from the PDB (PDB-ID: 5A7E, 2HRG, 4JHU, 1GYC) using the Swiss-Model and Modeller, and was refined in GalaxyRefine. The models were validated using PROCHECK, VERIFY 3D, ERRAT, PROVE and QMEAN Z-score servers. Moreover, molecular docking between the laccase model (Lacc4MN) and ABTS was performed on AutoDock Vina. The models that were generated by the Modeller showed superior stereochemical and structural characteristics to those predicted by the Swiss Model. The refinement made it difficult to stabilize the copper atoms which are typical of laccases. The Lacc4MN model showed the interactions between the amino acids in the active site of the laccase and the copper atoms, thereby hinting the stabilization of the metal through electrostatic interactions with histidine and cysteine. The molecular docking between Lacc4MN and ABTS showed negative free energy and the formation of two hydrogen bonds involving the amino acids ASP 208 and GLY 268, and a Pi–sulfur bond between residue HIS 458 and ABTS, which demonstrates the typical catalytic functionality of laccases. Furthermore, the theoretical model Lacc4MN presented stereochemical and structural characteristics that allow its use in silico tests. Communicated by Ramaswamy H. Sarma

Laccase is a superfamily of ligninolytic enzymes known to degrade a wide variety of xenobiotics, including synthetic dyes. Congo Red (CR) has a diazo dye function, carcinogenic and mutagenic potential, and is currently applied in clinical analysis. The objective of this work was to produce and characterize the crude extract of Lentinus sp. in semi-solid fermentation (FSS) and perform in vitro and in silico studies to assess the potential of the crude extract to discolor the CR dye. Laccase activity was determined using ABTS as substrate and characterized. The in vitro discoloration was carried out using experimental design 2(2) at room temperature and monitored at 340 nm for 24h. Molecular docking and molecular dynamics simulations were performed between laccase and CR. The maximum laccase activity production was 29.63 U L-1 with six days of FSS. The optimal temperature and pH were 50 degrees C and 3.0, respectively. Discoloration of the CR dye was obtained only in tests containing CuSO4. Laccase formed stable complexes with the dye, presenting negative binding energy values ranging from -70.94 to -63.16 kcal mol(-1) and the occurrence of seven hydrogen bonds. Molecular dynamics results showed the stability of the system (RMSD ranging from 1.0 to 2.5 a) and protein-ligand interaction along simulation. RMSF values pointed residues at the end of chains A (residues 300 to 305, 480 to 500) and B (residues 650 to 655 and 950 to 1000) as the most flexible regions of the laccase. This study highlighted the enzymatic action in the bioremediation of CR in vitro in agreement with the in silico simulations that demonstrate the enzyme potential.Communicated by Ramaswamy H. Sarma

Background: Azo dyes are widely used recalcitrant chemicals and may promote environmental hazards. Amongst the known azo dyes is CI Direct Red 28 (C₃₂H₂₂N₆Na₂O₆S₂) aka Congo Red (CR), which is a known mutagen and carcinogen. In this regard, this work aimed at the biodegradation of a CR-containing synthetic effluent, and the evaluation of the ecotoxicity of post-treatment residues. Methods: Lentinus sp. Laccase (Lac)-mediated bioremediation of CR was optimized upon added concentrations of sucrose and CuSO4, moreover a standard ecotoxicity assay was performed. Results: Results showed that the addition of 5% sucrose and 2 mM CuSO4 increased CR degradation, with Lac activity at 48 h of 30.2 U mL-1, and at 72 h of 31.9 U mL-1. Moreover, the ecotoxicity assay showcased that CR degradation by Lentinus sp. Lac seemingly generated low ecotoxic byproducts. Conclusion: Given that CR bioremediation byproducts were known to exhibit high toxicity, our results shed light on the use of Lentinus sp. catalytic arsenal to promote proper remediation of azo dyepolluted effluents.

Demand for natural red colorants has increased due to regulatory and consumer pressure to replace synthetic additives; however, their industrial application remains limited by challenges related to production, stability, and safety. In this study, the production, physicochemical stability, antimicrobial activity, and preliminary cytotoxicity of an azaphilone-type red colorant produced by Talaromyces sp. C1I3 under submerged fermentation were evaluated. Two sequential Box–Behnken designs identified optimal production conditions at approximately pH 5.8, 60 rpm, and 15.15 g·L⁻¹ monosodium glutamate. Under these conditions, colorant production increased from 1.46 AU_500nm on day 5 to 9.9 AU_500nm on day 10, with estimated kinetic parameters of µmax 0.0109 h⁻¹ and Yx/s 0.63 g·g⁻¹. The colorant showed high thermal stability, retaining approximately 95% of its color after heating at 80 °C for 10 min, and remained stable over a broad pH range, with only 18% degradation after 168 h of light exposure. No significant antimicrobial activity was detected, indicating that the colorant primarily functions as a color additive without additional bioactive effects. Furthermore, no acute cytotoxicity was observed in human neonatal dermal fibroblasts at concentrations ranging from 0.5 to 15 µg·mL⁻¹. These findings demonstrate the potential of this fungal colorant as a stable and safe natural colorant for future industrial applications.

Brazil, one of the largest producers and exporters of instant coffee, faces challenges in waste management, particularly due to the highly polluting wastewater generated. This study evaluated soluble coffee wastewater (SCWW) as a cultivation medium for laccase production by the fungus Pleurotus ostreatus and its application in degrading phenolic compounds in the effluent. Laccase production was monitored over 18 days at 28 °C using two media: a synthetic medium and one containing SCWW with a COD of 1000 mg L ⁻¹ . Results showed SCWW promoted a 22.3% increase in laccase activity compared to the synthetic medium and enabled degradation of pollutants. The enzyme had an optimal temperature of 40 °C, an ideal pH of 6.0, and high stability across broad temperature and pH ranges. Additionally, the enzymatic extract degraded 32.3% of phenolic compounds in SCWW without chemical mediators. These findings highlight SCWW’s dual potential as a low-cost substrate for enzyme production and as a target for fungal biocatalytic degradation. This offers a promising, sustainable strategy, especially considering the limitations of conventional biological methods inhibited by SCWW’s toxicity. Graphical Abstract

Natural colorants derived from filamentous fungi have gained increasing attention as safe, sustainable, and bioactive alternatives to synthetic counterparts. This study evaluated the red colorant production by Talaromyces sp. under solid and static submerged culture conditions, focusing on the selection of carbon and nitrogen sources and the influence of the inoculum’s physiological state. In solid-state cultivation, oat-based medium and Potato Dextrose Agar (PDA) supplemented with glycine (PDAG) promoted intense sporulation and red–yellow pigmentation, whereas sugar-rich media, specifically Yeast Extract Sucrose (YES) and Sabouraud Agar, led to rapid mycelial growth but reduced sporulation. Radial growth rates ranged from 0.295 to 0.391 cm/day, depending on the growth medium. YES medium supported the fastest growth; however, it was ineffective in promoting colorant induction when used as an inoculum source for liquid culture. Under static submerged fermentation conditions, inoculum derived from PDAG and PDA supplemented with yeast extract (PDA + YE) produced the highest colorant titers, highlighting that the inoculum developmental stage is a key determinant in activating azaphilone colorant biosynthesis. Additionally, commercial sucrose and sugarcane juice were identified as the most effective carbon sources, outperforming lignocellulosic hydrolysates from sugarcane bagasse, rice, and corn. A preliminary economic analysis estimated production costs ranging from US$ 3.11 to US$ 3.34 per liter of colorant. A Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis revealed key strengths such as high colorant productivity, alongside significant opportunities in the natural ingredients market. Overall, these findings underscore the technical and economic potential of Talaromyces-based biocolorant production, positioning it as a sustainable and competitive alternative to synthetic colorants.