Donoso, M.; Munoz-Gacitua, D.; Salfate, G.; Toledo-Neira, C.; Blamey, J. M.

Using an extremophilic laccase with a phenolic redox mediator, we rapidly abolished doxycycline antibacterial activity and achieved near-complete chemical degradation (>97%) within 2 h under mild conditions. Antibiotic residues in aquatic and terrestrial environments drive the emergence and spread of microbial resistance genes, yet many treatment strategies are limited by slow kinetics or incomplete transformation. Here, we evaluated doxycycline degradation by the recombinant FNT laccase across pH and temperature conditions, comparing direct enzymatic oxidation with a laccase-mediator system. RP-HPLC quantified doxycycline removal. Residual toxicity was assessed using growth inhibition assays with Bacillus subtilis and Escherichia coli BL21. In the absence of a mediator, oxidation produced only partial loss of function, indicating that structural modification alone does not guarantee pharmacological inactivation. In contrast, the addition of acetosyringone markedly increased biodegradation efficiency, yielding >97% doxycycline depletion and eliminating antibacterial activity. To rationalize condition-dependent performance, we complemented experiments with molecular simulations of doxycycline binding at the laccase active site, revealing pH-dependent differences in stability and substrate interactions consistent with enhanced turnover under alkaline conditions. These results demonstrate that an extremophilic laccase-mediator system provides rapid, effective detoxification of doxycycline, supporting its potential for treatment of antibiotic-contaminated effluents.