Effect of Two Humic Acids on Laccase-Driven 17β-Estradiol Conversion Kinetics and Polymerization Products at Varying pH Levels

Estrogens with tremendous ecological risks are proverbially found in water. Laccase can drive humication of estrogens to reduce their ecotoxicity and removability, but little investigation existed in exploring the effect of humic acids (HAs) on E2 conversion kinetics and polymerization products at different pH conditions. Here, Trametes versicolor laccase (Tvlac) was capable of eciently converting a representative estrogen, 17β-estradiol (E2) with two different HAs, and the process followed a pseudo-rst-order kinetics. The velocity constants were respectively 0.048, 0.022, and 0.020 min − 1 for HA-free, peat-derived HA, and commercial HA at pH 5.0. The changing pH not only impacted E2 coupling kinetics in Tvlac-evoked humication, but altered the aromaticity and humication degrees of HAs. A total of ve intermediate species including estrone (E1), E2 dimer, trimer, and tetramer, as well as E1-E2 cross-linked products were tentatively detected, in which the dominating species were E2 self-oligomers resulting from radical-centered carbon-carbon/oxygen stepwise polymerization routes. Yields of dimeric, trimeric, and tetrameric species with increased molecular sizes were the highest at pH 5.0 in the given pH values, and they were readily handled by precipitation and ltration. Especially E2 was able to be covalently incorporated into humic constituents to generate new humied co-polymers, thereby accelerating E2 humication and detoxication. These ndings demonstrate that pH exhibits a far-reaching inuence on the conversion kinetics, humication degrees, and products distribution of E2 and HAs in Tvlac-reinforced polyreaction. Thus, there is need to reappraise the fate and transport of estrogens with HAs present in natural water at varying pH levels.


Introduction
Estrogens in essence are a category of steroid hormones, which have an extensive range of biological activities (Adeel et al., 2017;Yao et al., 2018). They not only act as a neuroprotective factor but as a signal, which regulate the function and organization of organisms (Dumas and Diorio, 2011;Brann et al., 2007). In general, the naturally-occurring estrogen such as 17β-estradiol (E2) is principally produced by animals and humans, it is continuously released into aquatic eco-environments owning to the incomplete elimination and conversion in sewage treatment plants (Bilal and Iqbal, 2019;Kolpin et al., 2002;Simpson, 2003). Currently, estrogens have drawn extensive concern because of their adverse effects on organisms, humans, and water ecosystems (da Silva et al., 2019). It is reported that the feminization of male sh was constantly observed in estrogen-polluted water even at an extremely low level (Harris et al., 2011;Vethaak et al., 2005). Therefore, it is particularly important to provide an approach with high e ciency and low cost for eliminating and converting estrogens in water (Bilal and Iqbal, 2019;Shrestha et al., 2012).
Humi cation refers to the process that the carbon of organic residues is converted into humus di cult to decompose by biochemical reactions (Chen et  . Humic acids (HAs) are omnipresent in natural aquatic environments, and natural enzyme-evoked humi cation coupling not only promotes oligomerization of estrogens but recombines humic constituents, which also contributes to the formation of new humi ed polymers between estrogens and HAs (Hur et al., 2013;Singh et al., 2015;Zhong et al., 2019). It follows that humi cation can serve as a vital natural decontamination and detoxi cation process of estrogens, and the core reaction is radical-centered oxidation and polymerization (Du et al., 2016;Feng et al., 2013). Thus, it is a novel strategy to utilize natural enzymecentered humi cation of estrogens and HAs for generating humi ed polymers . Nevertheless, how to select a high-e cient oxidation-reduction enzyme for participating in the humi cation coupling reaction is particularly important.
Laccase is a member of blue multicopper oxidases that can trigger a single-electron oxidation concomitantly with the four-electron reduction of O 2 to 2H 2 O (Barrios-Estrada et al., 2018; Bilal et al., 2019). Laccase generally has four copper ions, including one type-1 Cu (T1-Cu), one type-2 Cu (T2-Cu), and two type-3 Cu (T3-Cu), in which the T2-Cu and T3-Cu sites are believed to close together and thus form a trinuclear cluster (Garavaglia et al., 2004). The substrates are oxidized by the T1-Cu site, and the extracted electron is transferred via a conserved His-Cys-His pathway to the T2/T3-Cu sites, where O 2 as the second substrate binds to it. (Baldrian, 2006;Madhavi and Lele, 2009). Simultaneously, four unstable radicals from substrates are produced, and then they encounter a series of non-enzymatic coupling reactions to form a variety of dimers, trimers, tetramers, oligomers, and polymers (Chen et  Previous studies demonstrated that pH had a tremendous effect on laccase stability and activity (Baldrian, 2006;Schneider, et al., 1999;Xia et al., 2014). For instance, the enzyme activity at a greater pH is declined through the OH − binding to the T2/T3-Cu sites, thus blocking the internal-electron transfer from T1-Cu to T2/T3-Cu cores (Munoz et al., 1997). It is thus necessary to investigate how pH impacts the conversion kinetics and products distribution of E2 with HAs during laccase-reinforced humi cation. In this study, the effect of two HAs on E2 removal kinetics was systematically explored at different pH levels by Trametes versicolor laccase (Tvlac). Tvlac-driven the aromaticity and humi cation degrees of HAs regardless of E2 presence were respectively investigated by the absorbance ratios of A 280 nm /A 350 nm and A 250 nm /A 365 nm using a UV-Vis spectrophotometry. Particularly, the probable intermediate species of E2 with HAs in Tvlac-evoked humi cation were tentatively identi ed via Liquid Chromatography coupled with Triple Quadrupole Mass Spectrometry (LC-TQMS), and the impact of pH on the products distribution was assessed as well. Our results clearly revealed that HAs altered the conversion kinetics and products distribution of E2 at varying pH levels in Tvlac-centered humi cation processes. Thus, it is extremely important to understand the geochemical fate and transport of estrogens with HAs present in water ecosystems at different pH conditions.

Chemicals and materials
E2 (≥ 98%), 2,6-dimethoxyphenol (2,6-DMP, 99%), and Tvlac (≥ 0.5 U·mg − 1 ) were purchased from Sigma-Aldrich. Previous studies reported that Tvlac exhibited a higher redox potential (RP) than other laccases, which was better for humi cation coupling of substrates (Kurniawati and Nicell, 2008). In this experiments, the peat-derived HA (P-HA) was extracted using the sodium pyrophosphate extraction method suggested by the International Humic Substance Society (IHSS). The commercial HA (C-HA) (technical grade) was obtained from Sigma-Aldrich. The stock solutions of HAs were ltered by 0.45 µm membrane before utilization. The spectral characteristics of both P-HA and C-HA are shown in Fig. S1 (Supplementary Information). UV-Vis spectra of two HAs were characterless, which revealed a decrease in absorbance with increasing wavelength (Fig. S1a). Notably, C-HA presenting higher absorbance values disclosed a better linearity than P-HA with smaller absorbance. As displayed in UV-Vis assays, FTIR spectra of P-HA and C-HA also showed some visible structural differences (Fig. S1b). High-performance liquid chromatography (HPLC)-grade methanol and acetonitrile were obtained from Fisher Scienti c. All other reagents were analytical grade or higher and obtained from commercial suppliers.

UV-Vis spectrum analysis
Tvlac-triggered conversion of E2 was carried out in glass vials at room temperature (25°C) and 101.325 In this research, the apparent pseudo-rst-order velocity constant (k) and half-life (t 1/2 ) values of E2 regardless of HAs presence are calculated by the following equations: where C 0 is the initial level of E2, and C t represents the residual level of E2 at reaction time t.

Activity test of Tvlac
Activity of Tvlac was monitored at 25°C and 101.325 kPa by oxidizing 2,6-DMP to form visible chromogenic species (Sun et al., 2020a). The reaction mixture contained 3.4 mL of 10 mmol·L − 1 C-PBS (pH 3.8), 1.0 mmol·L − 1 2,6-DMP, and 20 µL of Tvlac. One unit of Tvlac activity (U·mL − 1 ) was de ned as the amount of enzyme that induced a unit change per minute in absorbance at 468 nm.

Quanti cation of E2 by HPLC
The level of E2 was analyzed and quanti ed by a Waters HPLC system equipped with a Waters 600 pump, a Waters 2707 autosampler, and a Waters 2998 photodiode array detector. An Agilent ZORBAX Eclipse Plus C18 column (4.6 mm × 150 mm, 5 µm particle size) was used for chromatographic separation at 40°C. The mobile phase consisting of acetonitrile and water (70: 30, v/v) was pumped via the column at a ow rate of 1.0 mL·min − 1 . The detection wavelength of E2 was set at 280 nm, which was the maximum absorption wavelength observed by UV-Vis spectrophotometer. 20 µL of injection volume was introduced onto the HPLC system every 10 min. The retention time of E2 was 3.529 min, and its level was quanti ed using a calibration curve. In this study, the quanti cation limit of E2 was below 10 nmol·L − 1 , and its recoveries in the presence of HA-free, P-HA, and C-HA were respectively 100.4%, 99.8%, and 101.3% (n = 5). . The injection volume was 5.0 µL. Mass Spectrometry analysis was operated in the negative scan mode with the following parameters: drying gas temperature 300°C, sheath gas temperature 300°C, drying gas ow 6.0 Lžmin − 1 , sheath gas ow 11 Lžmin − 1 , nebulizer 45 psi, capillary entrance voltage 3.5 kV. The total ionization chromatography was collected in the m/z range of 50-1500. In this study, the polymerized species of E2 were not con rmed because of lack of analytical standards.

Impact of pH on E2 UV-Vis spectrum in Tvlac-evoked reactions
Conversion of E2 was qualitatively analyzed at pH 4.0, 5.0, 6.0, and 7.0 during Tvlac-triggered humi cation processes at 25°C and 101.325 kPa by UV-Vis absorption spectra. As shown in Fig. 1, the changes in the absorption spectra were a direct result of pH, and the maximum absorption peak of E2 was displayed at 280 nm (A 280 nm ). 3.2. Tvlac-evoked humi cation kinetics of E2 with HAs at varying pH levels pH is a crucial factor governing the conversion kinetics of phenolic pollutants with HAs in fungal laccasetriggered humi cation systems (Gan et al., 2019;Spina et al., 2020;Wang et al., 2018). In this study, the effect of varying pH levels on the conversion kinetics of E2 was quantitatively analyzed in Tvlaccatalyzed humi cation processes by HPLC. As presented in Fig. 2, it is obvious that the level of residual E2 decreased with reaction time increasing. Tvlac-caused the conversion e ciency of E2 followed the order of pH 5.0 > pH 6.0 > pH 4.0 > pH 7.0. For example, Tvlac was capable of eliminating 99.3% E2 at pH 5.0 for a 120-min incubation, however, the removal e ciencies of E2 respectively were 94.9%, 98.0%, and 35.2% at pH 4.0, 6.0, and 7.0. Both P-HA and C-HA exhibited obviously inhibitory effects on the removal and conversion of E2 within 60 min. For instance, compared with HA-free, the conversion e ciencies of E2 reduced 24.9% and 27.4% in the presence of P-HA and C-HA for 60 min incubation, respectively. It can be due to the competition occurred between HA and E2 for the catalytic site of Tvlac (Dou et al., 2018;Sun et al., 2017). Moreover, the inhibitory effect of C-HA on E2 conversion was mildly higher than that of P-HA, implying that P-HA had a stronger binding ability with E2. Notably, overall the removal rate of E2 per unit time decreased with the increase of the reaction course. For example, the removal rates of E2 per minute without HA respectively reached 1.7%, 2.6%, 1.8%, and 0.5% for 30 min reaction at pH 4.0, 5.0, 6.0, and 7.0. However, the removal rates of E2 per minute reduced to 1.3%, 1.6%, 1.5%, and 0.4% after 60 min . It is because Tvlac remained relatively stable and its activity was hardly changed in this reaction process. As illustrated in Table 1, the k and t 1/2 values at pH ranged from 4.0 to 7.0 were respectively 0.025, 0.048, 0.032, and 0.004 min − 1 , and 27.73, 14.56, 21.58, and 173.29 min in Tvlac-evoked E2 reactions without HAs. The highest k value was obtained at pH 5.0, which was 12fold higher than the reaction at pH 7.0. It follows that Tvlac had a higher reactivity to catalyze E2 conversion in acidic solution (pH 4.0, 5.0, and 6.0) than neutral or alkaline solution (pH ≥ 7.0, Tvlacevoked E2 removal was thoroughly intercepted in pH ≥ 8.0 solution), which accorded with the results shown in Fig. 1. Compared with HA-free, the k values for P-HA and C-HA were much lower at the all measured pH conditions, demonstrating that the presence of HAs markedly restrained the conversion kinetics of E2 by Tvlac. . It is suggested that the bell-shaped pH pro le of phenolic compounds was produced by two opposite ways. On the one hand, the enzymatic reaction of phenolic contaminants is reduced at low pH (generally, pH was less than 5.0) due to the inhibition of the electron transfer between substrate and laccase T1-Cu site (Margot et al., 2013). Researchers pointed out that the RP of phenolic compound decreased with the increasing of pH, owning to the transfer of proton during the catalytic reaction . However, the RP of T1-Cu site in laccase had no signi cant effect by pH (Xu, 1997). The RP difference value between phenolic compound and T1-Cu site augmented with pH, thus improving the conversion kinetics of substrate (Mateljak et al., 2019). On the other hand, the enzymatic reaction of phenolic contaminants is also impeded at high pH (usually, pH was greater than 6.0) because of the limitation of the intramolecular electron transfer (Solomon et al., 2015). Some reports stated that OH − was bene cial to bind with T2/T3-Cu sites in laccase, which terminated the internal-electron transfer and thus suppressed the reactivity of laccase (Xu, 1997;Wang et al., 2018). Additionally, pH can also change the ionization and charge of substrate, and impact the electrostatic interactions of laccase and substrate (Su et al., 2019). This shows we need to make a concession to seek the optimal pH that allows the highest conversion e ciencies of estrogens and other contaminants.

Effect of pH on Tvlac-triggered HAs aromaticity and humi cation degrees
HAs are complex mixtures of large-to small-molecular-weight species held together through supramolecular interactions. Infrared spectra had testi ed that both P-HA and C-HA constituents presented a variety of functional groups such as -OH, -COOH, and C = O (Fig. S1b) , 2020b). Generally, the spectral change of a speci c band can be used to describe the aromaticity of HAs in enzyme-driven humi cation reactions (Deng, et al., 2019). In this study, we conducted batch tests to investigate how pH would impact the aromaticity of HAs without or with E2 in Tvlac-evoked reaction systems via evaluating the ratio of A 280 nm /A 350 nm . As shown in Fig. 3, the ratio of A 280 nm /A 350 nm increased gradually as the reaction went on, indicating that the enzymatic reaction augmented the aromaticity of HAs by generating new stable macromolecular structures. An earlier report also stated that the ratio of A 280 nm /A 350 nm displayed outstanding correlations for the aromaticity and molecular weight of HA (Rodríguez et al., 2016). Moreover, the change rate of A 280 nm /A 350 nm ratio (ΔA 280 nm /A 350 nm ) at pH 5.0 was larger than that of pH 4.0, 6.0, and 7.0, implying that pH 5.0 was the optimal reaction condition for increasing the aromaticity of HAs by Tvlac in all the tested pH values. Notably, the treatment groups with E2 presented higher aromaticity than those of E2-free at varying pH levels.

Radical coupling mechanisms of E2 and/or HAs in Tvlac-triggered humi cation
Tvlac-evoked conversion species of E2 were tentatively identi ed by LC-TQMS. As displayed in Table 2 where 272.18 is the MM of E2, 2.02 is the atomic mass of 2H, and n corresponds to the number of E2 units (n is integer, and n ≥ 1).
Additionally, estrone (E1) and E1-E2 cross-linked species were also detected in Tvlac-evoked conversion of E2 according to the peaks of m/z = 269.15 and 539.31, respectively. Even so, the yields of these two products were very low. As summarized in Fig. 4, we proposed the possible humi cation and oligomerization pathways for Tvlacevoked conversion of E2 and HAs. In the initial stage, Tvlac triggered the one-electron oxidation of E2 and/or HAs to produce abundant unstable phenoxy radical intermediates (Bilal et  by electro-enzymatic oxidative polyreaction. They pointed out the presence of HA could present chain reactions from estrogen self-coupling to co-polymerization. The formation of the polymerized precipitates dramatically reduced the ecotoxicity and removability of parent compound that had been con rmed by previous researchers (Feng et al., 2013;Lu et al., 2009), and these highly self/cross-linked precipitates were easily removed by precipitation and ltration. For example, the solubility of E2 oligomers in water was > 10 7 times lower than E2 monomer, and thus the bioavailability and transferability of these oligomers were much less than parent compound (Qin et al., 2015). Note that the functional substituents on HA might prominently impact the cross-linking with phenolic pollutants, as well as the type and stability of the polymerized species (Du et al., 2016). These results clearly demonstrated that Tvlac reinforced the humi cation and oligomerization of E2, and the presence of HAs changed the distribution of E2 self-linked products by generating E2-HA co-polymerization species in radical-centered cross-linking . In this study, we calculated the relative abundance ratios (RARs) of E2 and its dimer, trimer, and tetramer at different pH conditions, due to lacking of analytical standard samples, these oligomer levels were not quanti ed. As shown in Fig. 5, the relative abundance of E2 dimer was higher relative to trimer and tetramer in all the treatment groups for a 60-min incubation, implying that E2 dimer was the most high-producing oligomeric species in Tvlac-evoked humi cation. Similar results were also obtained by Qin et al. (2015), they documented that the sum of dimer peak areas was much larger than that of trimer and tetramer, and the yield of dimer aggrandized eetly and reached the maximum value for a 1-h incubation. Amazingly, the relative abundances of all oligomers in the presence of HAs were signi cantly lower than those of the HA-free. Such as the relative abundances of dimer, trimer, and tetramer were respectively 66.55%, 16.94%, and 2.30% in Tvlac-evoked E2 coupling without HA at pH 5.0, which were greater than that of the reaction with P-HA (or C-HA). These results revealed that HAs lowered the degree of E2 self-linking likely due to the proportional rise of E2-HA co-polymerization species. For instance, the RARs of E2 and its dimer, trimer, and tetramer at pH 5. HAs presence. These results revealed that pH presented a remarkable in uence on the distribution of E2 and/or HAs self/co-polymerization species in water during Tvlac-evoked humi cation and oligomerization.

Conclusions
In this study, Tvlac was able to cause completely the single-electron transfer of four E2 molecules with the concomitant reduction of O 2 to 2H 2 O regardless of HAs presence, thus accomplishing the decontamination and carbon sequestration of organic pollutants. Notably, HAs exhibited a strong effect on the conversion kinetics, humi cation degrees, and products distribution of E2 at different pH conditions during Tvlac-evoked reactions at 25°C and 101.325 kPa. The optimal pH for the catalytic systems was 5.0 in the speci ed pH values, with slow conversion kinetics of E2 and humi cation degrees of HAs for pH 7.0. Additionally, Tvlac accelerated the self-linking of E2 to form a variety of E2 oligomeric species such as dimer, trimer, and tetramer by radical-centered carbon-carbon/oxygen covalent binding.
However, the presence of HAs altered the distribution of E2 self-oligomeric products by producing E2-HA co-polymerization species. The degree of oligomerization was highest at pH 5.0 during Tvlac-triggered humi cation coupling of E2 and HAs under all the experimental pH levels. These ndings are bene cial to understand the pH dependent conversion kinetics and products distribution of E2 with HAs in laccasedriven humi cation processes.