Oxidation of chlortetracycline and its isomers by Botrytis aclada laccase in the absence of mediators: pH dependence and identification of transformation products by LC-MS.

Abstract

Tetracyclines are antibiotics considered emerging pollutants and currently, wastewater treatment plants are not able to remove them efficiently. Laccases are promising enzymes for bioremediation because they can oxidize a wide variety of substrates. The aim of this study was to evaluate the Botrytis aclada laccase for the oxidation of chlortetracycline and its isomers in the absence of a mediator molecule, at a pH range between 3.0 to 7.0, and to characterize the transformation products by LC-MS. Chlortetracycline and three isomers were detected in both, controls and reaction mixtures at 0h and in controls after 48h of incubation but in different proportions depending on pH. An additional isomer was also detected, but only in the presence of BaLac. Based on the transformation products identified in the enzymatic reactions and information from literature, we assembled a network of transformation pathways starting from chlortetracycline and its isomers. The spectrometric analysis of the products indicated the probable occurrence of oxygen insertion, dehydrogenation, demethylation and deamination reactions. Four new products were identified, and we also described a novel transformation product without the chloro group. We observed that increasing pH led to higher diversity of main products. This is the first study using the laccase from fungi Botrytis aclada to oxidate chlortetracycline and its isomers and it can be considered as an ecological alternative to be used in bioremediation processes such as wastewater.