Elsevier

Journal of Hazardous Materials

Volume 356, 15 August 2018, Pages 9-16
Journal of Hazardous Materials

Influence of natural organic matter on horseradish peroxidase-mediated removal of 17α-ethinylestradiol: Role of molecular weight

https://doi.org/10.1016/j.jhazmat.2018.05.032Get rights and content

Highlights

  • Both pristine and Mf-NOMs restrained the removal of EE2.

  • Low Mf-NOMs were competitive substrates for HRP.

  • High Mf-NOMs could sequester substrate from the attack of HRP.

  • The contribution of each inhibitory process was related to reaction conditions.

Abstract

Ubiquitous natural organic matter (NOM) plays a crucial role in the peroxidase-mediated transformation of phenolic pollutants in aquatic environment. As a poorly defined polydispersed mixture of assorted organic substances with wide molecular weight (MW) distribution, NOM has far prevented researchers from finding out the primarily responsible components for the specific effect. In this work, MW fractionated NOMs (Mf-NOMs) were used to investigate their roles on horseradish peroxidase (HRP)-mediated transformation of 17α-ethinylestradiol (EE2). The removal rate of EE2 was restrained in the presence of pristine or Mf-NOMs, and the inhibitory mechanism was MW-dependent. Low Mf-NOMs restrained the enzymatic reaction by acting as competitive substrates, while high Mf-NOMs retained freely dissolved EE2 which reduced its availability for enzymatic reaction. The contribution of these two processes to the inhibition induced by pristine NOM was further quantified and found to be relevant to the reaction conditions, especially EE2 concentration. The findings of this work reveal more complex influences of NOM on the enzymatic reaction than ever demonstrated, which aids in understanding the fate of EE2 and other congener contaminants in natural and municipal water.

Introduction

Endocrine disrupting chemicals (EDCs) are attracting public concern and growing research interest due to their potent and detrimental effects on human and wildlife [[1], [2], [3], [4]]. This broad class of pollutants are generally persistent and environmentally mobile, and a variety of studies have reported their occurrence in aquatic environment [5]. 17α-ethinylestradiol (EE2), the major component of the contraceptive pill, is among the most commonly detected endocrine disruptors [6]. It is more stable than natural estrogens and contributes to a lager extent to the estrogenicity in surface water [7].

The fate of EDCs is mainly governed by two intricate counter-directional processes: biochemical degradation and humification [8]. Degradation results in the decrease of contaminant sizes, while the latter are processes where small molecules are aggregated into macromolecular structures for eventual formation of humic substances. Oxidative coupling process mediated by extracellular enzymes, like horseradish peroxidase (HRP), is a class of humification process, and has been shown to be highly efficient and specific in the removal of phenolic compounds [9]. In this sense, enzymatic reaction is also supposed to be promising alternative to address EDCs and other micropollutants that are sensitive to peroxidases, as reported previously [10].

However, either in natural or water treatment process, such potentially important reaction is strongly impacted by natural organic matter (NOM) which is ubiquitous in aquatic environment. Recently, it is becoming generally recognized that NOM is supramolecular association of relatively small heterogeneous molecules by weak dispersive forces (van der Waals, CH/π) [11], which indicates NOM may serve as a substrate of peroxidase since it is partially comprised of phenolic compounds. This assumption could lead to highly variable results with different substrates. For instance, the removal efficiency of substrates which are comparatively less sensitive to peroxidase, like polychlorinated biphenyls (PCBs), is enhanced, because more nonspecific free radicals are generated in the reaction between enzyme and NOM compared to enzyme and substrate [8,12]. Conversely, inhibitory effect of NOM was observed, perhaps by the same reason, for the reaction system with substrates which are sensitive to peroxidase, like estrogens [1]. The availability of substrates may also be modified by the noncovalent interactions with NOM, which is highlighted by noting the effect of NOM on the adsorption or biodegradation process of EDCs [13,14]. Gadad et al. directly demonstrated this effect by using steady-state fluorescence spectroscopy and further distinguished the interaction type of NOMs with varying aromaticity [15]. It’s thus hypothesized that NOM may sequester EDCs from the oxidation process mediated by peroxidase.

The molecular weight (MW) of the polydispersed organic substances in NOM ranges from less than 100 Da to over 300 kDa, and the chemical complexity of NOM has far prevented researchers from finding out the primarily responsible components for the specific effect on enzymatic reactions [16]. MW fractionated NOMs (Mf-NOMs) usually differed greatly in biochemical properties. Low MW fractions are found to contain more carboxylic groups, while aromatic groups are concentrated in high MW fractions. This indicates the effect of NOM on the biochemical reactions may further diverge as a function of MW. Unfortunately, no relevant research in terms of the effect of the Mf-NOMs on the enzymatic reactions is found. In previous researches, the influence of NOM was usually investigated by considering it as simple macromolecular polymers as polysaccharide, and the results were explained with single and ambiguous mechanism, which actually were the combined effects of various components in NOM [[17], [18], [19], [20]]. In another word, the role of NOM was not differentiated.

The objective of this study is to illustrate the different mechanisms by which pristine and Mf-NOM impact the HRP-mediated transformation of EE2 and to analyze the relative importance of these mechanisms to the reaction. Suwannee River Natural Organic Matter (SRNOM) was selected and fractionated by ultrafiltration (UF) into four fractions (<1 kDa, <10 kDa, <30 kDa, Bulk). The fractionated NOMs were then applied to explore their underlying roles on the enzymatic reactions.

Section snippets

Materials

HRP (type 2, units 250 U mg−1), 2, 2′- azino - bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (98% in diammonium salt form), EE2 (purity > 99%), H2O2 (30% wt), bovine serum albumin (BSA), dextran and sodium polystyrenesulfonate (PSS) were purchased from Sigam-Aldrich Corporation (St. Louis, MO, USA). Suwannee River Natural Organic Matter (SRNOM) (2R101 N) was obtained from the International Humic Substance Society (IHSS) (St. Paul, MN, USA). A solvent-resistant stir cell with 1 kDa, 10 kDa

Characteristics of pristine and Mf-NOM

Three fractions of NOM with MW <30 kDa, <10 kDa and <1 kDa were obtained by UF. Table 1 shows the characterization of pristine and Mf-NOM. From the TOC values of each fraction, it can be seen that small (<1 kDa) and large (>30 kDa) MW components were relatively abundant. It should be noted that considering only filtrate was collected, high MW NOM fraction was actually a mixture containing low, medium and high MW components. The value of SUVA254 for pristine NOM was much larger than that for

Conclusions

This study reveals that NOM can strongly inhibit the HRP-mediated transformation of EE2, and the effects of NOMs are MW-dependent. Low Mf-NOMs restrains the enzymatic reaction by acting as competitive substrates, while the inhibition from high Mf-NOMs are primarily attributed to the sequestration of EE2 from the HRP attack. The contribution of each inhibitory process to the inhibition induced by pristine NOM is quantified and found to be related to reaction conditions, especially EE2

Acknowledgement

This work was supported by the National Natural Science Foundation of China (21577059)

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