Kinetic insights into hydroxyindole oxidation and mediator-free dye decolorization by Trametes zonata 1525 laccase

Catalytic activity of fungal laccases toward biologically relevant indole metabolites remains insufficiently explored. In this study, extracellular laccase from Trametes zonata was characterized with particular focus on hydroxyindole oxidation and mediator-free dye decolorization. Among 38 screened basidiomycete strains, T. zonata exhibited the highest extracellular laccase production, while Cu²⁺ supplementation enhanced enzyme activity up to 38-fold. The purified enzyme (66 kDa) demonstrated high stability under acidic conditions, with optimal activity at pH 4.5 and 35 °C, and broad substrate specificity toward phenolic, synthetic, and indole compounds. Kinetic analysis revealed distinct substrate-dependent catalytic behavior toward hydroxyindoles. 4-Hydroxyindole exhibited higher catalytic efficiency (k_cat/K_M ≈ 1.1 × 10⁴ M^-1·s^-1), whereas 5-hydroxyindole-3-acetic acid showed lower turnover efficiency (k_cat/K_M ≈ 5.4 × 10³ M^-1·s^-1). The lower catalytic efficiency toward 5-hydroxyindole-3-acetic acid was associated primarily with turnover limitations rather than substrate binding, suggesting structure-dependent effects on electron transfer during catalysis. The enzyme was further applied for spectrophotometric detection of 5-hydroxyindole-3-acetic acid. In addition, T. zonata laccase efficiently decolorized synthetic azo dyes under mediator-free conditions, achieving up to 95% decolorization. These findings expand current understanding of fungal laccase activity toward hydroxyindole substrates and demonstrate the potential applicability of T. zonata laccase in environmentally relevant catalytic systems.