Mechanistic and Molecular Docking Insights into Laccase-Mediated Methyl Orange Decolorization for Wastewater Treatment

Laccases are environmentally friendly biocatalysts capable of oxidizing a broad range of organic pollutants by using molecular oxygen as the sole electron acceptor, producing water as the only by-product. The cofactor-independent activity makes them attractive for sustainable wastewater treatment, particularly for the removal of synthetic dyes. In this study, laccase from Trametes versicolor was evaluated for the decolorization of the azo dye Methyl Orange, with emphasis on the effects of redox mediators and metal ions. Laccase alone exhibited maximum activity at pH 3.0, achieving 40.5% decolorization after 24 h. The addition of redox mediators markedly enhanced dye removal. Both synthetic mediators (ABTS, HBT, and TEMPO) and natural mediators (p-coumaric acid, vanillin, gallic acid, ascorbic acid, and syringaldehyde) improved decolorization in a concentration-dependent manner. Among them, ABTS and syringaldehyde were the most effective, achieving 98.8% and 96.9% decolorization, respectively, at 0.2 mM after 24 h, with syringaldehyde offering the advantage of natural origin. Metal ions also modulated laccase activity, with several ions enhancing decolorization at 0.5 mM, whereas higher concentrations were generally inhibitory; potassium ions showed the strongest enhancement (80.3%). Molecular docking analysis suggested favorable binding of Methyl Orange within the laccase active pocket, supported by predicted hydrogen-bonding and hydrophobic interactions. The molecular docking analysis was performed using a representative T. versicolor laccase structure and provides supportive computational insight into plausible enzyme–dye interactions rather than isoform-specific mechanistic validation. Overall, these findings demonstrate that optimization of mediators and metal ion concentrations significantly improve laccase-mediated dye decolorization, while docking provides supportive computational insight into possible enzyme–dye interactions relevant to sustainable wastewater treatment.