Removal of Acetaminophen Using Enzyme ... - ACS Publications

Aug 13, 2009 - Griffin, Georgia 30223, and State Key Laboratory of Pollution. Control and Resource Reuse, School of the Environment,. Nanjing Universi...
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Environ. Sci. Technol. 2009, 43, 7062–7067

Removal of Acetaminophen Using Enzyme-Mediated Oxidative Coupling Processes: I. Reaction Rates and Pathways J U N H E L U , † Q I N G G U O H U A N G , * ,† A N D LIANG MAO‡ Department of Crop and Soil Sciences, University of Georgia, Griffin, Georgia 30223, and State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, P. R. China

Received January 23, 2009. Revised manuscript received July 30, 2009. Accepted July 30, 2009.

We found that acetaminophen could be effectively transformed and removed from water by laccase-mediated oxidative coupling processes. The removal of acetaminophen followed second-order kinetics with first-order to the concentrations of both the substrate and the enzyme. Mass spectrum analysis demonstratedthatpolymerizationthroughradical-radicalcoupling mechanism was the pathway leading to acetaminophen transformation. Coupling products thus formed are believed to be biologically inactive and more readily removable from water. Secondary mass spectra of the dimers in combination with molecular modeling analysis further elucidated that the coupling proceeded via covalent bonding between two molecules at their unsubstituted carbons in benzene rings. These findings demonstrated that laccase-mediated oxidative coupling can potentially serve as an alternative strategy to control certain micropollutants in water/wastewater treatment and reuse.

Introduction The widespread occurrence of human derived pharmaceuticals in the environment has raised growing concern to the public and regulatory agencies (1-3). These compounds pass through the human body and are partially metabolized with a significant fraction excreted via urine and feces, ending up in sewage. These pharmaceuticals are generally more hydrophilic than traditionally regulated pollutants and undergo incomplete biological transformation (3, 4). Consequently there is a greater likelihood for their transport to surface water, groundwater, and soil through discharge from wastewater treatment facilities, onsite septic systems, and land applications of biosolids. It was reported that at least 46 million Americans are exposed to trace amounts of pharmaceuticals in drinking water supplies (2). Among a vast array of reported pharmaceuticals, acetaminophen, a nonprescription analgesic, is one of the most frequently detected in surface water. In a national reconnaissance conducted by the U.S. Geological Survey during 1999-2000, acetaminophen was found in 24% of the 139 sampling streams in 30 states with median and maximum concentrations of 0.11 and 10 * Corresponding author phone: (770) 229-3302; fax: (770) 4124734; e-mail: [email protected]. † University of Georgia. ‡ Nanjing University. 7062

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ppb, respectively (5). The consequence of continuous exposure at such dosage levels remains unclear. A recent study indicated that trace acetaminophen could react with chlorine during drinking water disinfection to form hazardous products (6). Studies have shown that conventional water treatment procedures are only moderately effective in the removal of trace pharmaceuticals (3, 6-9). This becomes a particular issue in water reuse, because without an effective removal mechanism, micropollutants tend to concentrate when the water is recycled through the biosphere multiple times. Certain advanced treatment technologies, such as activated carbon, reverse osmosis, and advanced oxidation processes, appearviableforremovalofsomemicropollutants(3,7,8,10,11). Their applicability is, however, limited by the following two factors: (1) micropollutants are present in water at very low concentrations, much lower than many other competing or interfering impurities such as natural organic matter (12); and (2) significant reduction in concentration (orders of magnitude) is often required to achieve desirable safety concentrations for those micropollutants that are potent in biological/ecological effects. Consequently, it remains a scientific challenge to develop new technologies that can specifically and effectively remove micropollutants to address associated water-quality problems in a cost-effective and sustainable manner. Catalyzed oxidative coupling comprises a class of reactions that facilitate the polymerization of molecules having phenolic or anilidic features (13, 14). These reactions are in fact central to natural humification processes, leading to the formation and growth of soil organic matter from smaller building blocks (14-20). These reactions are catalyzed by a variety of naturally occurring extracellular enzymes, such as peroxidases and laccases, and by mineral oxides such as manganese dioxide (e.g., birnessite) (21-23). The reactions involve two stages (23), the first of which is oxidation of the substrates to produce highly reactive radicals or quinone intermediates, and the second is coupling of the oxidized intermediates to form polymerized products. In light of these facts, catalyzed oxidative coupling is highly likely to serve as an effective treatment or remediation strategy for removal of many micropollutants. Such reactions have been generally proven to be highly efficient, producing products of much larger molecular sizes as a result of coupling. The products are generally biologically inactive, and can readily settle from water or be immobilized in soil. In fact, it has been reported that some endocrine disrupting chemicals (EDCs), such as bisphenol A (24) and certain steroid estrogens (25-27), were able to participate in catalyzed coupling reactions resulting in their removal from water. Acetaminophen, containing both phenolic and anilidic groups, is a potential substrate for this reaction process. We hypothesize that it can actively participate in the catalyzed oxidative coupling process and consequently be removed. Laccases are a class of enzymes that are known to mediate effective oxidative coupling reactions. Laccases belong to a group of blue multicopper oxidases that catalyze one-electron oxidation of four substrate molecules concomitantly with four-electron reduction of molecular oxygen to water. A laccase usually contains four copper catalytic centers, including one T1, one T2, and two T3. The substrates are oxidized by the T1 copper. The electrons are transferred through a strongly conserved His-Cys-His tripeptide motif, to the T2/T3 copper where molecule oxygen is reduced (28). A sketch of laccase catalytic cycle is provided in Supporting Information (SI) Figure S1. In this study, the potential of 10.1021/es9002422 CCC: $40.75

 2009 American Chemical Society

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using laccase-mediated oxidative coupling processes to remove acetaminophen from water was explored. The reaction kinetics and mechanisms were systematically investigated. High performance liquid chromatography (HPLC) coupled tandem mass spectrometry (MS/MS) was employed to separate and characterize the main reaction products, whose structures were then determined by combining the MS/MS data with quantum chemistry analysis. This study provides fundamental information necessary for development of enzyme-based treatment processes to address acetaminophen and other trace contaminants of similar nature.

Experimental Section Materials. All reagents were ACS grade or better. Acetaminophen (98%) was obtained from Sigma-Aldrich Co (St. Louis, MO). A stock solution at the concentration of 1 mM was prepared and stored at 4 °C. Working solutions were made from the stock by dilution with deionized (DI) water. Laccase was also purchased from Sigma-Aldrich and laccase dosing solutions were prepared in DI water. Enzyme Assay. Laccase was freshly prepared and assayed for activity before each experiment. The activity was determined spectrometrically by oxidation of 1 mM 2,6-dimethoxyl phenol in citrate-phosphate buffer (pH 3.8) (23). One unit of laccase activity is defined as the amount of enzyme that causes a unit change per min in absorbance at 468 nm in 3.4 mL of this solution in cuvette with 1 cm light path. A Beckman DU640B spectrophotometer (Beckman, Fullerton, CA) was used to measure the absorbance. Coupling Reaction. Coupling reactions mediated by laccase were conducted in 50 mL glass flasks as completely mixed batch reactors (CMBRs). The reaction solution was prepared in 0.01 M phosphate buffer (pH 7.00 ( 0.02) containing 50 µM acetaminophen. The solution was magnetically stirred for 5 min to equilibrate oxygen gas-solution exchange before an appropriate amount of enzyme dosing solution was added to the reactor to initiate the reaction. At 10 min intervals over an hour, 0.5 mL of the reaction mixture was withdrawn from the reactor and immediately mixed with 0.5 mL methanol, which was found to be sufficient to result in denaturation of the enzyme and therefore stop the reaction. The residual acetaminophen and the reaction products in the solution were quantified using HPLC. A total of five enzyme dosages, from 0.2 to 1.0 unit/mL, were examined. Quantification. A Shimadzu LC 20AT HPLC equipped with a Shimadzu 20A photo diode array (PDA) detector (Shimadzu, Columbia, MD) was used to analyze the concentration of acetaminophen in solution. The separation was performed on an Ascentic C18 reversed phase column (250 × 4 mm, 5 µm, Supelco, St. Louis, MO). A binary gradient elution consisting of water containing 0.2% formic acid (A) and methanol containing 0.2% formic acid (B) at a flow-rate of 0.6 mL/min was used as mobile phase. Injection volume was 10 µL. The gradient spanned 55 min and was programmed as follows: 5% B increased linearly to 40% B at 30 min then 100% B at 40 min, and was then held at 100% B for additional 15 min. A 5 min run with 5% B was conducted prior to any sample measurement to equilibrate the HPLC column. Absorbance at 250 nm was used to quantify acetaminophen concentrations and a five-point external calibration was established for the quantification. The relative yields of major reaction products were also compared based on their absorbance at 260 nm. Characterization of the Coupling Products. 200 mL of 50 µM acetaminophen solution was treated with 1.0 unit/ mL laccase in a CMBR setup similar to that used in coupling reaction study described above. After 60 min of incubation, a few drops of concentrated H2SO4 were added to the solution to adjust the pH to below 2.0. The solution was then loaded

onto a C18 solid-phase extraction (SPE) cartridge (Restek, 6 mL, 500 mg) preconditioned with 5 mL of methanol at a flow rate of 10 mL/min. After loading, the SPE cartridge was eluted with 5 mL of methanol and the eluent was collected, blown dry by gentle nitrogen flow, and then reconstituted to 3 mL with methanol. The sample was then analyzed using a Waters Micromass Quattra tandem mass spectrometer (MS/MS) (Waters, Milford, MA) with infusion injection. The mass spectrometer was operated in negative electronspray ionization mode (ESI-). The sample was also separated and analyzed using a Waters 2690 HPLC (Waters, Milford, MA) coupled to the MS (HPLC/MS). For optimum MS signals, the HPLC program used for HPLC/MS analysis (details provided in the SI) was different from the one used for acetaminophen and product quantification by HPLC/PDA as described above. MS was operated in positive electronspray ionization mode (ESI+), and the ions of interest were selectively monitored. Detailed operation conditions are presented in the SI. Molecular Modeling. All computations were performed using molecular and quantum mechanics algorithms available as part of HyperChem Molecular Modeling System, release 8.0 (Hypercube, Inc.). Substrate molecular structures were first optimized using the MM+ molecular mechanics force field and the Polak-Ribiere optimization algorithm set to a convergence gradient criterion less than 0.1 kcal/(Åmol). Then an electron was removed from the molecule by setting the total charge of the molecule as +1 and the spin multiplicity as 2. Subsequent quantum optimization for electronic structure was achieved using the AM1 semiempirical method and the Polake-Ribiere optimization set to the same convergence. After determination of the optimal quantum structure, partial charge and spin density distribution were computed.

Results Reaction Rates. The rates of laccase-mediated acetaminophen removal were explored in batch systems at varying initial enzyme activity. As shown in Figure 1a, the concentration of acetaminophen in the solution followed first-order decay at each enzyme dosage investigated (29). This suggests that laccase activity remained relatively constant during the reaction. The pseudo first-order kinetic constants were determined by fitting the data to the first-order kinetic equation and are plotted in Figure 1b against the enzyme dosage used in the reactions. It is evident in Figure 1b that the first-order rate constants are proportional to the enzyme activity in the solution. It can thus be concluded that laccase mediated removal of acetaminophen is a second-order reaction, first-order in both the enzyme and the substrate. Based on the linear correlation shown in Figure 1b, the second-order rate constant k is determined to be 4.0 × 10-4 unit-1mLs-1. Product Identification and Reaction Pathways. Reaction samples containing mixtures of products were extracted using SPE and analyzed by MS in negative ESI mode. This technique generates deprotonated negative ions with m/z values of M-1 (M is the molecular weight of the analyte) (30). Figure 2 illustrates a typical mass spectrum of the extracted reaction products. The peak with m/z value of 150 corresponds to the residual acetaminophen (M0 ) 151) in the solution. In addition to this peak, the peaks with m/z equals to 299, 448, 597, and 746 are prominent. The respective masses of the parent molecules of these ions follow a pattern of nM0 - 2 (n - 1), where n is a natural number. We hypothesize that these reaction products result from the coupling of 2-5 molecules of acetaminophen. The pattern of the molecular masses of the coupling products suggests that they are formed through radical-radical coupling mechanism; i.e., laccase mediates one-electron oxidation of the substrates to generate VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Distribution of charge and spin densities of an acetaminophen radical obtained by semiempirical calculation. The values reflect the status of a single acetaminophen molecule with a specific configuration. Values between C2 and C6, C3 and C5 exhibit minor differences due to the asymmetry of this specific molecular configuration. However, these pairs of carbon atoms are equivalent macroscopically with mixtures of configurations.

FIGURE 1. Rates of the removal of acetaminophen catalyzed by laccase. (a) Pseudo first-order rate plots for the decay of acetaminophen at different enzyme dosages; (b) the dependence of the pseudo first-order rate constants on the enzyme dosage. ([acetaminophen]0 ) 50 µM, pH 7.0, error bars representing 95% confidence intervals). free radicals, which couple to each other subsequently to form dimers as illustrated in SI Figure S2. Radical-radical coupling results in covalent bonding of two parent molecules with two hydrogen atoms eliminated, one from each molecule (17, 25). Such a coupling product is still, in most cases, a substrate of the oxidative coupling enzymes, and can thus undergo further coupling reactions yielding higher level oligomer products (17, 18, 20, 25) (SI Figure S2). Molecular weights of the coupling products thus generated have a pattern of nM0 - 2(n - 1). Determining the reactive sites in acetaminophen molecules between which the covalent bonds formation occurs is critical to elucidate the reaction pathways. An acetami-

nophen molecule (Figure 3) consists of a benzene ring core, substituted by one hydroxyl group and the nitrogen atom of an acetamino group in the para pattern. It is an extensively conjugated system, as the lone pairs on the hydroxyl and carbonyl oxygens, the benzene π cloud, the nitrogen lone pair, and the p orbital on the carbonyl carbon are all conjugated. Potentially, such a system is susceptible to oxidation via losing an electron from either the hydroxyl or amide group to form a free radical. The unpaired electron may delocalize through resonance to the respective conjugated positions therefore lowering the overall energy state of the free radical (31). Molecular modeling provides hints as to how the single electron tends to resonate in the system. Figure 3 presents the results of semiempirical characterization of the acetaminophen free radical that was generated by abstracting an electron from the molecule as described in the Experimental Section. “Spin density” designates the probability of the presence of an unpaired electron, and “charge density” indicates net charges associated with each atom. The computation results for spin density shown in Figure 3 reveal that the single electron has the greatest probability for distribution to the carbon atoms on the benzene ring in addition to the oxygen and nitrogen atoms. The acetamino nitrogen and the carbons at the conjugated ortho (C2 and C6) and para (C4) sites of the acetamino group possess higher spin density than those of the hydroxyl oxygen. This seems to be consistent to the classic view that the hydroxyl group tends to be more resistant to loss of an electron than the amide group, because oxygen is more electronegative than nitrogen (31). According to the radical-radical coupling mechanism, the unsubstituted carbon atoms C2, C3, C5, C6,

FIGURE 2. Mass spectrum of the coupling products of acetaminophen mediated by laccase. Initial acetaminophen concentration 50 µM, laccase 1.0 unit/mL, pH 7.0, reaction time 60 min. 7064

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FIGURE 4. Secondary mass spectra of the dimer and trimer of acetaminophen upon oxidative coupling mediated by laccase. Initial acetaminophen concentration 50 µM, laccase 1.0 unit/mL, pH 7.0, reaction time 60 min. as well as the hydroxyl oxygen and nitrogen atoms from which monitored the ions with m/z of 301, 450, and 599, correa proton can be potentially dissociated are presumed to be sponding to the protonated dimer, trimer, and tetramer, the possible reactive sites. However, all of these sites are respectively. The chromatograph is given in Figure 5, which partially negatively charged, which prevents them from clearly indicates multiple peaks for each oligomer. The approaching to each other. Among them the oxygen and number of isomer species increases with the degree of nitrogen atoms have higher charges than carbon atoms, polymerization. For example, two dimer species with retenmaking bond formation at these sites kinetically less favortion times of 23.2 and 27.1 min, respectively, were detected. able. Taking both charge and spin into account, acetamiUnfortunately, secondary fragmentations of these species nophen radicals are likely to couple between the unsubstiwere not achieved which prevented further structural tuted carbon atoms (C2, C3, C5, and C6) on the aromatic analysis. Nonetheless, the higher abundance of the latter ring. Among the four possible sites, atom C3 possesses the peak indicates that it may correspond to the 3-3′ coupling lowest charge. Therefore, two acetaminophen radicals are species, which was shown to be the primary reaction pathway most likely to couple between each of the C3 atoms to form based on the quantum chemistry analysis described above. a 3-3′ coupling species. Although this does not exclude the The peak at 23.2 min thus may correspond to the 2-3′ possibility of forming a bond at other potential sites, the coupling species. spin and charge distribution does suggest that the possibility The main products were also identified in HPLC/PDA for radical formation and coupling at C3, the sites ortho to along with the residual acetaminophen. Because there are the hydroxyl group, tends to be greater. no standards available, it was impossible to quantify the To further elucidate the structures of the coupling yields. Yet, their relative abundance in solution can be products, secondary mass spectra of the dimer and trimer determined by normalizing their peak areas to that of were measured. Both of them show similar pattern as acetaminophen at time 0. In this way, the relative concenillustrated in Figure 4. The principal fragmentation pathways tration profiles of the principal dimer, trimer, and one of the were the elimination of either a water or an acetic acid tetramer species (respectively corresponding to the species molecule, yielding the fragment with m/z value of (M-1 -18) at the retention times of 27.1, 27.3, and 28.1 min in Figure or (M-1 -60), respectively. We speculate that the neutral acetic 5) over time were obtained and given in Figure 6. This is a acid molecule (M ) 60) is eliminated from a hydroxyl group typical profile of stepwise reactions, with lower degree and an acetamino group via the formation of a 5-member oligomers serving as substrates to form higher degree ring, as shown in SI Figure S3. This corresponds to a oligomers under the studied reaction condition, and the yield deprotonated 2-3′ coupling species. This suggests that the of 3-3′ dimer reached its maximum at around 30 min. dimer had been formed by covalent bonding between the ortho site of the hydroxyl group of one parent acetaminophen Discussion molecule and the ortho site of the acetamino group of the Acetaminophen can be effectively transformed via laccase other. Similarly, the ion m/z 281 is postulated to be generated catalyzed polymerization. The fact that the removal rate of from the parent ion by eliminating a water molecule (M ) acetaminophen exhibited first-order with respect to both 18), this could serve as an indication of 3-3′ or 2-3′ coupling the substrate concentration and the enzyme dosage indicated pathway (SI Figure S3). There is however no direct evidence that the oxidation of substrate by the enzyme was the ratethat supports the existence of 2-2′ coupling products. limiting step, implying that the supply of the enzyme cofactor, Otherwise, a fragment with m/z of 240, which corresponds dissolved oxygen, was sufficient and that the laccase activity to an elimination of ammonium acetate (M ) 59), would be remained constant during the reaction. With the enzyme formed. The electrostatic repulsion (Figure 3) as well as the activity at 1.0 unit/mL, the half-life time of acetaminophen steric hindrance generated when two aceticamino groups was 0.78 h. This may not be as rapid as some other enzymatic approach to each other apparently does not favor this reactions (19, 32, 33), but it is compensated by the fact that pathway. Hence, the coupling processes took place prethis enzyme remains stable for long time while many other dominantly via 2-3′ and 3-3′ pathways as schematically enzymes inactivate quickly during reactions. Other factors, indicated in SI Figure S4. such as the presence of colloids and dissolved natural organic The products were further separated and analyzed with matter, may also influence the enzyme activity under field HPLC/MS under positive ESI mode. We used positive conditions, which remains to be explored. ionization because formic acid was present in the mobile The enzymatic polymerization does not end in the phase of HPLC, which suppressed the deprotonation of the formation of dimers, because the dimers still possess phenolic analytes but enhanced the protonation therefore generating and anilidic groups to enable them serving as active substrates relatively strong positive ions (M + 1)+ (30). We selectively VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. HPLC/MS chromatograph of the coupling products of acetaminophen catalyzed by laccase. Initial acetaminophen concentration was 50 µM, laccase 1.0 unit/mL, pH 7.0, reaction time 60 min, ESI-. molecules of contaminants into polymers with larger molecular sizes and therefore leads to deactivation or removal. Such a treatment strategy minimizes the carbon footprint associated with water/wastewater treatment operations (35). Laccases utilize dissolved oxygen as the ultimate electron acceptor, and thus do not require additional input of oxidation reagents and energy. This makes it possible to readily incorporate this treatment process into currently existing treatment courses without adding extra facilities. The long-term stability of laccase during the reaction is another advantage of this process as a treatment strategy, potentially reducing significant operational cost. This process can be particularly useful when potable water reuse is demanded. In such a case, laccase-mediated coupling process can be integrated into wastewater treatment plant (WWTP) as an additional measure to control micropollutants, which may build up through multiple reuse cycles.

Acknowledgments FIGURE 6. Time-dependent formation of major products of acetaminophen upon oxidative coupling mediated by laccase. Initial acetaminophen concentration was 50 µM, laccase 1.0 unit/mL, pH 7.0. Yields of the products were determined according to their respective HPLC peak area measured as absorbance at 260 nm normalized to that of acetaminophen before the reaction. to laccase and therefore the coupling process proceeds continuously to form higher degree oligomers (SI Figure S2) (20, 34). The physicochemical properties of the oligomers as well as their behavior in natural and engineered water systems are subjected to further investigation, but the coupling products tend to be less hydrophilic with the increase of the degree of polymerization, and consequently more readily removable from aqueous phase through precipitation, coagulation, and/or adsorption processes. This notion is indirectly corroborated by the longer retention times of the higher degree coupling products in the reverse-phase HPLC column as shown in Figure 5. However, the reaction pathways and formation of products at environmentally relevant levels need to be further explored. Laccase-mediated coupling process provides a novel strategy to control certain emerging trace-level contaminants in water treatment practice. Opposite to degradation, it mimics the humification process and incorporates small 7066

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The study was supported in part by U.S. EPA STAR grant G6M10518 and by HATCH funds. The content of the paper does not necessarily represent the views of the funding agencies. L.M. thanks China Scholarship Council for supporting him as a visiting student to participate in this study at UGA.

Supporting Information Available (i) Laccase catalytic cycle; (ii) possible reaction pathways of acetaminophen coupling and (iii) possible fragmentation pathways of acetaminophen dimer in MS/MS; (iv) reaction pathways leading to acetaminophen dimer species; and (v) HPLC/MS operation conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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