Mediator-assisted tetracycline transformation and dye decolorization by a thermostable recombinant laccase from...

Pharmaceutical residues and synthetic dyes are among the most persistent environmental pollutants, creating an increasing demand for efficient and sustainable remediation strategies. In this study, a recombinant bacterial laccase from Yersinia enterocolitica 8081 (YeLac) was heterologously expressed in Escherichia coli BL21, purified, and biochemically characterized. The purified enzyme exhibited maximal catalytic activity at 65 °C and displayed substrate-dependent pH behavior, with optimal oxidation of ABTS at pH 3.5 and SGZ at pH 7.5. YeLac also showed broad pH stability, retaining more than 80% activity over a wide pH range after 24 h. Kinetic analysis revealed catalytic efficiencies (k_cat/K_m) of 0.58 µM⁻¹ s⁻¹ for ABTS and 1.38 µM⁻¹ s⁻¹ for SGZ. Beyond model substrate oxidation, YeLac was evaluated not only for pollutant transformation but also for its impact on biological activity. In synthetic dye decolorization assays, 1 U of YeLac achieved the highest removal for Rhodamine 6 G (up to 95%), while Nile Red and Malachite Green also showed notable decolorization after 24 h. The enzyme catalyzed tetracycline transformation, a frequently detected antibiotic contaminant. LC-MS/MS analysis confirmed that, in the presence of ABTS as a redox mediator, 1 U of YeLac completely transformed 10 mg L⁻¹ tetracycline at 65 °C. Importantly, antibacterial activity assays were used to assess whether this transformation corresponded to functional detoxification rather than simple chemical modification. These findings demonstrate that YeLac enables efficient high-temperature tetracycline transformation with reduced antibacterial activity, while also supporting dye decolorization, providing a more relevant evaluation of pollutant removal.