Approved Abstracts

Chlortetracycline degradation by an ascomycete laccase and analysis of transformation products by LC/MS



Author(s): Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas; Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas, Unidad de Espectrometría de Masas; University of California Santa Barbara, Department of Chemistry and Biochemistry; Universidade de São Paulo, Centro de Energia Nuclear na Agricultura; Universidad de Chile, Facultad de Ciencias, Departamento de Biología;
Presenter: Nadia Gavilán

Antibiotics are considered emerging pollutants and cause bacterial resistance, and currently, wastewater treatment plants cannot remove them efficiently. Tetracyclines (TCs) are broad-spectrum antibiotics used in veterinary medicine to prevent and avoid diseases and as promoting factors in animal husbandry for human consumption. However, almost 70% of the administrated dosage is excreted by urine and feces, reaching the soil, superficial, and groundwater, causing environmental pollution. Laccases are promising enzymes for bioremediation because they can oxidize a wide variety of substrates and produces water as a co-product of the oxidation reaction, which is considered ecofriendly. The aim of this study was to use a laccase from Botrytis aclada (Balac) in the bioremediation of chlortetracycline (CTC) and characterize its transformation products by LC-MS. The gene of Balac was expressed in Pichia pastoris, and after 7 days of incubation in BGM medium, it was purified by hydrophobic interaction chromatography (HIC) followed by gel filtration chromatography. The purified recombinant enzyme was incubated with a variety of concentrations of CTC ranging from 0.05 to 2.0 mM in a citrate-phosphate buffer at pH 6.0 and 25°C for 48 h. Samples were taken at different times and analyzed by LC-MS in negative ion mode. At these conditions, the enzyme oxidized about 95% of the substrate within 3 h for all the concentration range tested (0.05-2 mM) at pH 6. The LC-MS analysis showed the formation of products already at 30 min and the continuous appearance of new products during the 48h. It was observed that some products formed in the first hours and then disappear, while others accumulated during the assay. The formation of other products was also observed for higher concentrations of substrate. The spectrometric analysis of the products indicated the preservation of the Cl group, but probably other parts of the CTC structure were modified by the enzyme. One of the transformation products that formed fastest corresponds to (formula C22H23N2O9Cl). It would be formed by the incorporation of oxygen into CTC. Although this product has not been previously described for the oxidation of CTC by any laccase, a comparable product was observed in the degradation of TC by the laccase of Pleurotus ostreatus and T. versicolor. This product has been postulated as an unstable epoxy intermediate in the degradation of CTC by the enzymatic action of class A flavin monooxygenase or destructase. A second TP of rapid formation corresponded to the isomers m/z 491.0859 (formula C22H21N2O9Cl) and m/z 491.0836 (formula C22H21N2O9Cl) originated from different routes. Some of the described CTC transformation products were observed in the reactions and were also observed in the controls at later times. One of the products observed in control was m/z 493.1013, formed by incorporating oxygen into the CTC, most likely through a hydroxylation process. We observed the product m/z 465.1041 (formula C21H23N2O8Cl), which coincided with a degradation product described in the oxidation of CTC by destructase. This product would be formed by the elimination of carbon monoxide from m/z 493.1013. In resume, the laccase from B. aclada was able to oxidize CTC in the tested conditions, and the enzymatic oxidation of CTC formed a variety of transformation products. We thank to Anid Doctoral fellowship grant # 21160533, Centro de Estudios para el Desarrollo de la Química (CEPEDEQ) – University of Chile, and Food Research Center (FoRC) – University of São Paulo.

Keywords: Laccase; Chlortetracycline; Transformation products

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