Enzyme-catalyzed oxidation of 17beta-estradiol using immobilized laccase from Trametes versicolor

Endocrine disruption is a problem of increasing environmental significance, as anomalies continue to be discovered in wildlife exposed to a variety of exogenous toxic compounds released into the aquatic environment through municipal and industrial effluents and agricultural runoff. The estrogens excreted by humans and entering aquatic systems via sewage treatment plants are of particular interest, as estrogen excretion cannot be feasibly controlled at the source and estrogens are among the most potent endocrine disruptors known. As phenolic compounds, estrogens are amenable to oxidation through the catalytic action of oxidative enzymes. Earlier work was directed toward characterizing the removal of estrogens using peroxidase enzymes as well as the fungal laccase Trametes versicolor in batch reactions. The ability of this laccase enzyme has been studied extensively and has demonstrated a very good ability to remove substrates such as phenol, bisphenol A and 17beta estradiol (E2) from aqueous solutions. In order to minimize the amount of enzyme required to achieve effective treatment, this study focuses on characterizing the removal of E2 using immobilized laccase. Through this approach, it is anticipated that treatment costs will be reduced since immobilization permits the re-use of the active enzyme, rather than discarding the enzyme with treated solutions. The enzyme was immobilized by covalent bonding onto silica beads and the reactions were conducted in a bench-scale continuous-flow packed bed reactor. The influent concentration of E2 was 10 muM for most studies.;The effects of mean residence time were determined for several influent E2 doses, and observable E2 transformation occurred under the reaction conditions employed. The stability and reactivity of the immobilized enzyme were observed over varying temperature and pH. As expected, conversion declined when the temperature of the system was changed from room temperature to near freezing at pH 5. However, this decline reflected a change in the rate of reaction and not an instability of the enzyme since it was found that conversion was restored to its original level when the system was brought back to room temperature. Likewise, conversion increased when the system was brought to warmer temperatures, and conversion levels were restored when the system was brought back to room temperature. Previous work conducted with aqueous laccase had demonstrated that the enzyme is more reactive toward E2 at pH 5, but the enzyme is slightly more stable over the long term at pH 7. As expected, in the present study, E2 removal increased when the pH of the influent to the immobilized laccase reactor was changed from pH 7 to pH 5. Also, studies aimed at observing the more long-term changes in reactivity for the system which is used only periodically for E2 removal and is stored at either pH 5 or pH 7 confirmed the enzyme to be more stable at pH 7 in the immobilized system. Studies also showed that the immobilized enzyme maintained a constant level of activity when treating a constant supply of aqueous E2 at a low mean residence time over a 12 hours period and when treating a constant supply of aqueous E2 at high mean residence time over a period of 9 days. Results from tracer studies suggest that the reactor used in this study was far from optimal; thus, the transformation of E2 under the studied conditions might be increased significantly simply by optimizing the reactor and its flow characteristics.