Cancer represents the main cause of morbidity and mortality worldwide, constituting a serious health problem. In this context, melanoma represents the most aggressive and fatal type of skin cancer, with death rates increasing every year. Scientific efforts have been addressed to the development of inhibitors targeting the tyrosinase enzyme as potential anti-melanoma agents due to the importance of this enzyme in melanogenesis biosynthesis. Coumarin-based compounds have shown potential activity as anti-melanoma agents and tyrosinase inhibitors. In this study, coumarin-based derivatives were designed, synthesized, and experimentally evaluated upon tyrosinase. Compound FN-19, a coumarin–thiosemicarbazone analog, exhibited potent anti-tyrosinase activity, with an IC₅₀ value of 42.16 ± 5.16 µM, being more active than ascorbic acid and kojic acid, both reference inhibitors. The kinetic study showed that FN-19 acts as a mixed inhibitor. Still, for this compound, molecular dynamics (MD) simulations were performed to determine the stability of the complex with tyrosinase, generating RMSD, RMSF, and interaction plots. Additionally, docking studies were performed to elucidate the binding pose at the tyrosinase, suggesting that the hydroxyl group of coumarin derivative performs coordinate bonds (bidentate) with the copper(II) ions at distances ranging from 2.09 to 2.61 Å. Then, MM/PBSA calculations revealed that van der Waals interactions are the most relevant intermolecular forces for complex stabilization. Furthermore, it was observed that FN-19 has a binding energy (ΔE_MM) value similar to tropolone, a tyrosinase inhibitor. Therefore, the data obtained in this study will be useful for designing and developing novel coumarin-based analogs targeting the tyrosinase enzyme.

The increasing generation of agro-industrial waste and its improper disposal have raised significant environmental concerns, highlighting the urgent need for sustainable alternatives which would repurpose these materials. In this context, enzymes such as endoglucanase play a critical role in degrading lignin–cellulose biomass by catalyzing the breakdown of β-1,4-glycosidic bonds in cellulose, thereby converting it into fermentable sugars with diverse industrial applications. This study aimed to investigate the production, purification, and characterization of an endoglucanase produced by the fungus Pleurotus djamor PLO13, using coconut fiber, sugarcane bagasse, wheat bran, and pineapple crown as substrates. Endoglucanase activity was measured by the Miller method (1959), using 2% (w/v) carboxymethyl cellulose (CMC) as substrate. Solid-state fermentation (SSF) was found to be highly efficient for enzyme synthesis, with wheat bran emerging as the most effective substrate, yielding an enzyme production of 7.19 U after 120 h of cultivation. The endoglucanase was purified through ethanol precipitation and ion-exchange chromatography using DEAE-Sepharose, achieving a recovery rate of 110%, possibly due to removal of inhibitors present in the crude extract. The purified enzyme exhibited stability across a broad pH range and thermostability, with optimal activity at pH 5.0 and 50 °C. Furthermore, the enzyme was activated by EDTA, Mn²⁺, and Ca²⁺, while being inhibited by Mg²⁺. Notably, the enzyme demonstrated halotolerance, with activity increasing by 60% upon the addition of 3 M NaCl. Kinetic analysis revealed that the purified enzyme showed affinity to the CMC substrate at the analyzed parameters (pH 5.0 and 50 °C), with Km and Vmax values of 0.0997 mg/mL and 112.2 µg/min/mL, respectively. These findings suggest that the endoglucanase from P. djamor PLO13 has promising potential for biotechnological applications, underscoring the feasibility of the use of lignocellulosic waste as sustainable substrates in industrial processes.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in peptides and proteins, have widespread industrial applications, particularly in milk coagulation for cheese production. Microbial enzymes have been employed as alternatives to animal rennet, offering advantages such as cost-effectiveness, availability, and compliance with dietary, cultural, and religious requirements. Solid-state fermentation (SSF) is widely employed for microbial enzyme production because of its low operational costs, reduced water and energy requirements, high product concentrations, and the ability to utilize agro-industrial residues as low-cost substrates, thereby contributing to both process sustainability and waste valorization. We report the production and characterization of a novel milk-clotting protease produced by Trichoderma koningii FFT13. The protease was produced by SSF using wheat bran as the substrate, an agro-industrial residue. It was classified as a chymotrypsin-like serine protease and exhibited a specific caseinolytic activity of 9861 U/mg. The enzyme coagulated both reconstituted skim milk and pasteurized whole milk in the presence or absence of calcium. Coagulation was enhanced by increasing temperature, reaction time, enzyme concentration, and calcium levels. Scanning electron microscopy revealed destabilization of casein micelles, their progressive aggregation, and the formation of a well-defined gel network, confirming the effectiveness of the protease in milk coagulation. Therefore, these results demonstrate that the chymotrypsin-like protease from T. koningii is a promising enzyme for milk coagulation, with potential application in cheese production. The enzyme obtained constitutes an alternative to traditional coagulants, overcoming limitations related to animal rennet while potentially offering additional advantages in terms of process sustainability and industrial scalability.