Laccase-Driven $17\beta$-Estradiol Humification in the Presence of Humic Acids at Varying pH Levels

Estrogens posing huge eco-environmental risks are universally found in water ecosystems. Laccase, as a multicopper oxidoreductase, can evoke humification and polymerization of estrogens to reduce their biotoxicity and removability, but little information exists in investigating the influence of humic acids (HAs) on E2 conversion kinetics, humification degree, and oligomer distribution at varying pH values. Herein, Trametes versicolor laccase (Tvlac) was able to efficiently convert $17\beta \text{-}\mathrm{estradiol}$ (E2) in the presence of two different HAs, and the process fitted a pseudo-first-order kinetic model ($R^2=0.8474–0.9952$). The kinetic constants were 0.048, 0.022, and $0.020{\min }^{-1}$ for HA-free, peat-derived HA, and commercial HA at pH 5.0, respectively. The changing pH not only affected E2 conversion kinetics, but altered the aromaticity and humification degrees of HAs. A total of five humified products including estrone (E1) and E2 self-oligomers (i.e., dimer, trimer, and tetramer), as well as E1-E2 copolymeric species, were tentatively identified, in which the dominant intermediates were E2 self-oligomers resulting from radical-based C─ C and/or C─ O bonds. Productions of E2 dimer, trimer, and tetramer with increased molecular sizes were the highest at pH 5.0 in the given pH conditions, and they were easily handled by centrifugation and filtration. In particular, E2 was capable of being covalently bound into HAs to form new humified supramolecular polymers, thus promoting E2 copolymerization and detoxification. Our results disclose that HAs exhibit a vitally important influence on the conversion kinetics and product distribution of E2 in Tvlac-started humification. Consequently, there is need to reunderstand the fate and geochemical behavior of estrogens with HAs present in the aquatic eco-environments at different pH conditions.