Characterization of laccases from Trametes hirsuta in the context of bioremediation of wastewater treatment plant effluent.

ABSTRACT

The bioremediation of pharmaceutical compounds contained in wastewater, in an ecological and sustainable way, is possible via the oxidative action of fungal laccases. The discovery of new fungal laccases with unique physico-chemical characteristics pushes researchers to identify suitable laccases for specific applications. The aim of this study is to purify and characterize laccase isoenzymes produced from the Trametes hirsuta IBB450 strain for the bioremediation of pharmaceutical compounds. Two main laccases mixtures were observed and purified in the extracts and were called Yn and Yg. Peptide fingerprinting analysis suggested that Yn was constituted mainly of laccase Q02497 and Yg of laccase A0A6M5CX58, respectively. Robustness tests, based on tolerance and stability, showed that both laccases were affected in a relatively similar way by salts (KCl, NaCl), organic solvents (ACN, MeOH), denaturing compounds (urea, trypsin, copper) and were virtually unaffected and stable in wastewater. Determination of kinetic constants (Michaelis (KM), catalytic constant (kcat) and kinetic efficiency (K=kcat/KM)) for the transformation of synthetic hormone 17α-ethynylestradiol and the anti-inflammatory agent diclofenac indicates a lower KM and kcat for laccase Yn but relative similar K constant compared to Yg. Synergistic effects were observed for the transformation of diclofenac, unlike 17α-ethynylestradiol. Transformation studies of 17α-ethynylestradiol at different temperatures (4 and 21 °C) indicate a transformation rate reduction of approximately 75-80% at 4 °C against 25% for diclofenac in less than an hour. Finally, the classification of laccases Yg and Yn into one of eight groups (group A-H) suggests that laccase Yg belongs to group A (constitutive laccase) and laccase Yn belongs to group B (inducible laccase).