Laccase Characterization and Discovering Metabolites of Laccase-Catalyzed Degradation of 17α- Ethynylestradiol Using Fungi from Oak Mountain State Park

Endocrine disrupting chemicals (EDC’s) are industrial and pharmaceutical pollutants, however some EDC’s (estrogens) are naturally synthesized by humans and animals. EDCs can reach waterways through wastewater facility effluent since many treatment plants are not yet capable of removing these pollutants. Research has shown that white rot fungi have the ability to break down wastewater contaminants such as EDC’s. These contaminants are broken down by lignolytic fungal enzymes, specifically lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase. Laccase has the advantage of catalyzing oxidation reactions with a concomitant reduction of oxygen to water. The use of oxygen as a second substrate (electron acceptor), rather than the peroxide required by MnP and LiP, makes laccase a more attractive enzyme for use in applications such as removal of wastewater contaminants. Even though removal of estrogenic activity in the presence of laccase has been demonstrated, little is known about the metabolites of laccase-catalyzed degradation of estrogens, and the mechanism of estrogen breakdown has not been determined. Likewise, there is not yet a consensus on what fungal source is the best producer of laccase. For this project, an optimal Oak Mountain State Park (OMSP) fungal source for laccase production was determined. Additionally, laccase from each source was purified and kinetically characterized with a natural substrate analog. We also began development of a new assay making use of a ruthenium-containing fluorescent probe to directly monitor the oxidation of estrogen. Finally, we identified the metabolites of laccase-catalyzed estrogen degradation. Turkey tail exhibited the best laccase activity. EE2 degradation resulted in the formation of dimers, trimers, tetramers, pentamers, and hexamers. We were unable to purify the cultured laccase.