Transformation and toxicity evaluation of tetracycline in humic acid solution by laccase coupled with 1-hydroxybenzotriazole.

Enzyme-based catalyzed oxidative coupling reactions (E-COCRs) are considered as viable technologies to transform a variety of pharmaceutical antibiotics. This study indicated that the extracellular fungal laccase from Pleurotus ostreatus was effective in transforming tetracycline (TC) with 1-hydroxybenzotriazole (HBT) present at varying conditions during E-COCRs. The presence of humic acid (HA) showed suppressive effect on the transformation rate constants (k) of TC, and the k values for TC decreased as HA concentration increased. It was ascribed primarily to the covalent binding between TC and HA, which reduced the apparent concentration and availability of TC in water. It is noted that TC molecules from the cross-coupling products were likely re-released under extreme conditions (pH<2.0). The intermediate products were identified regardless of HA presence by high-resolution mass spectrometry (HRMS). A possible reaction pathway of TC in HA solution including electron transfer, hydroxylation, dehydrogenation, oxidation, radical reaction, decomposition, and covalent binding was proposed. The growth inhibition assays of Escherichia coli (E. coli) confirmed that the antimicrobial activity of TC was remarkably reduced with an increasing reaction time. These findings provide novel insights into the decomposition and cross-coupling of TC in a multi-solute natural aquatic environment by laccase-based catalyzed oxidative processes.