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

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 Y-n and Y-g. Peptide fingerprinting analysis suggested that Y-n was constituted mainly of laccase Q02497 and Y-g 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 (K-M), catalytic constant (k(cat)) and kinetic efficiency (K=k(cat)/K-M)) for the transformation of synthetic hormone 17 alpha-ethynylestradiol and the anti-inflammatory agent diclofenac indicates a lower K-M and k(cat) for laccase Y-n but relative similar K constant compared to Y-g. Synergistic effects were observed for the transformation of diclofenac, unlike 17 alpha-ethynylestradiol. Transformation studies of 17 alpha-ethynylestradiol at different temperatures (4 and 21 degrees C) indicate a transformation rate reduction of approximately 75-80% at 4 degrees C against 25% for diclofenac in less than an hour. Finally, the classification of laccases Y-g and Y-n into one of eight groups (group A-H) suggests that laccase Y-g belongs to group A (constitutive laccase) and laccase Y-n belongs to group B (inducible laccase).