- Email: [email protected]
Degradation of tetracycline by immobilized laccase and the proposed transformation pathway
Accepted Manuscript Title: Degradation of tetracycline by immobilized laccase and the proposed transformation pathway Author: Dr. Jie Yang Yonghui Lin Xiaodan Yang Tzi Bun Ng Xiuyun Ye Dr. Juan Lin PII: DOI: Reference:
S0304-3894(16)30916-5 http://dx.doi.org/doi:10.1016/j.jhazmat.2016.10.019 HAZMAT 18100
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
22-8-2016 6-10-2016 10-10-2016
Please cite this article as: Jie Yang, Yonghui Lin, Xiaodan Yang, Tzi Bun Ng, Xiuyun Ye, Juan Lin, Degradation of tetracycline by immobilized laccase and the proposed transformation pathway, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2016.10.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Degradation of tetracycline by immobilized laccase and the proposed transformation pathway
Jie Yang 1*, Yonghui Lin2, Xiaodan Yang 1, Tzi Bun Ng 3, Xiuyun Ye1, Juan Lin1*
1
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fujian
350116, China 2
Technical Center, Fujian Entry-Exit Inspection and Quarantine Bureau, Fuzhou,
Fujian 350001, China 3
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong
Kong, Shatin, New Territories, Hong Kong, China
*Corresponding Author: Dr. Jie Yang Phone: 86-591-22866376 Fax: 86-591-22866376 Email: [email protected] Dr. Juan Lin Phone: 86-591-22866376 Fax: 86-591-22866376 Email: [email protected]
Highlights
Laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) Laccase at 40 U/mL eliminated over 80 μg/mL TC at pH 6 and 25 ºC in 12 h
A mechanism of laccase-mediated tetracycline (TC) oxidation was proposed TC antibiotics treated with laccase showed reduced antimicrobial activity Laccase M-CLEAs are a green alternative in residual antibiotic removal in water
Abstract Magnetic cross-linked enzyme aggregates (M-CLEAs) were prepared for Cerrena laccase and used in antibiotic treatment. Of the seven antibiotics examined in this study, Cerrena laccase M-CLEAs were most effective in degradation of tetracycline
(TC)
and
oxytetracycline
(OTC),
followed
by
ampicillin,
sulfamethoxazole and erythromycin. The redox mediator ABTS was not able to improve degradation efficienciesefficiencies of degradation of TC and OTC. Cerrena laccase at 40 U/mL eliminated 100 μg/mL TC at pH 6 and 25 ºC in 48 h withoutin the absence of a redox mediator, with over 80% degradation occurring within the first 12 h. Laccase treatment also significantly loweredsuppressed the antimicrobial activity of TC and OTC. LC-TOF MS identified threeThree TC transformation products, the levels of which initially increased first and thensubsequently decreased during laccase treatment were identified by using LC-TOF MS. A proposedmechanism of laccase-mediated TC oxidation was proposed based on the identified intermediates.
Keywords: laccase, M-CLEAs, tetracycline, transformation products, antimicrobial activity
1. Introduction Antibiotics are commonly used in human and veterinary medicine and have attracted increasing attention as emerging contaminants. In addition to their ecotoxicity [1, 2], antibiotics in the environment may serve as selective pressure for antibiotic-resistant bacteria, thus imposing a threat to public health. Tetracycline antibiotics, such as tetracycline (TC) and oxytetracycline (OTC), are recalcitrant, broad-spectrum antimicrobial agents widely present in wastewater and natural water bodies [3, 4]. Conventional water treatment cannot effectively remove antibiotics [3], thereforehence, alternative methods shouldhave to be developed. Biodegradation of antibiotics has been explored. For example, crude manganese peroxidase and lignin peroxidase from Phanerochaete chrysosporium have been used to degrade TC and OTC [4, 5]. Mycelia of another white rot fungus, Pleurotus ostreatus, were employed in mycoremediation of OTC [6]. Laccase, along with manganese peroxidase and lignin peroxidase, comprise ligninolytic enzymes secreted by white rot fungi. Laccases are copper-containing oxidases that can catalyze the oxidation of a wide range of phenolic and non-phenolic compounds, including various dyestuffs and environmental pollutants [7-9]. In contrast to peroxidases, laccase does not require the presence of hydrogen peroxide or manganese for oxidation by using oxygen as the final electron receptor and offers a green alternative in wastewater treatment and bioremediation [10]. Oxidation of substrates with redox potentials higher than those of laccases, such as non-phenolic compounds, can be facilitated by small redox mediators, such as synthetic mediators 2,2’-azino-bis
(3-ethylbenzothiazoline-6-sulfonate)
(ABTS)
and
1-hydroxybenzotriazole (HBT). Laccase oxidation of the substrate may proceed differently with or without the involvement of a mediator. Furthermore, ABTS oxidizes the substrate via an electron transfer route, whereas HBT and natural phenolic mediators follow a hydrogen atom transfer mechanism [10-13]. Enzyme immobilization is used to improve enzyme stability and reusability and reduce application costs [14]. Enzymatic membrane reactors based on laccase immobilized on ceramic membranes have been evaluated for laccase-mediated TC degradation in wastewaters [15-18]. Magnetic cross-linked enzyme aggregates (M-CLEAs) represent a relatively new enzyme immobilization method, in which cross-linked enzyme aggregates (CLEAs) are attached to amino-functionalized magnetic nanoparticles (MNPs). Carrier-free CLEAs are easy and cheap to prepare; the general procedure consists of enzyme precipitation and subsequent cross-linking with a bifunctional agent [19]. M-CLEAs improve mechanical properties of CLEAs and allow easy separation and recycling of the immobilized enzyme from the reaction mixture with a magnetic field [20, 21]. Laccase M-CLEAs, which showed promise in dye decolorization [20] or elimination of pharmaceuticals [22, 23], have been prepared from Trametes versicolor. Recently, laccase has been proven useful in degradation of antibiotics such as TC, but such studies focused on laccase from T. versicolor [15-18, 24-26]. Although T. versicolor laccase is extensively studied, Cerrena laccase represents an attractive alternative with the high yields and application potentials [27-32]. However, the preparation of M-CLEAs from Cerrena laccase or utilization of Cerrena laccase in antibiotic treatment remains unexplored. In the present study, we investigated TC degradation by immobilized Cerrena laccase. Degradation products were identified
with LC-TOF MS, and a putative mechanism was deduced. The antimicrobial activity of laccase-treated TC was also analyzeddetermined.
2. Materials and methods 2.1. Strain and chemicals Cerrena unicolor [28] was maintained on potato dextrose agar (PDA) at 4 ºC in the culture collection of Fuzhou University, China. For laccase fermentation, 5 mycelial plugs (1 cm diameter) were removed from the peripheral region of a 4-d-old PDA plate stored at 30 ºC and inoculated in 50 mL potato dextrose broth (PDB) seed medium in a 250-mL Erlenmeyer flask. After allowing to grow for 2 d at 30 ºC and 200 rpm, an aliquot was transferred to a second PDB medium at the ratio of 8% (v/v). After growth for another 2 d, the fermentation medium (50 mL in 250 mL Erlenmeyer flasks) was inoculated with the second seed culture at the concentration of 8% (v/v). The fermentation medium contained (g L-1): maltodextrin, 60; peptone, 10; ammonium tartrate, 1.6; KH2PO4, 6; MgSO4·7H2O, 4.14; CaCl2, 0.3; NaCl, 0.18; CuSO4·5H2O, 0.0625; ZnSO4·7H2O, 0.018; and vitamin B1 0.015. Submerged fermentation was carried out for 6 d. Fermentation broth was collected by centrifugation at 8,000 g for 5 min and used directly for immobilization. Antibiotics
(tetracycline,
oxytetracycline,
ampicillin,
sulfamethoxazole,
erythromycin, chloramphenicol and trimethoprim) were purchased from Sangon Biotech (Shanghai, China). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Methanol and acetonitrile were of HPLC grade and obtained from Sinopharm Chemical Reagent (Beijing, China). All other chemicals were of analytical grade.
2.2. Preparation of M-CLEAs Magnetic nanoparticles (MNPs) were synthesized and amino-functionalized as previously described [20]. Fermentation broth was adjusted to pH 8.0 and MNPs were added. Saturated ammonium sulfate solution was added to the mixture at the ratio of 5:1 to precipitate laccase at 25 ºC for 2 h. Glutaraldehyde was used as the cross-linking agent. Laccase:MNPs ratios (0.4:1-1.4:1), glutaraldehyde concentrations (5-40 mM) and cross-linking time (1-6 h) were sequentially examined for maximum recovery of enzyme activity. M-CLEAs were recovered with a magnet and washed several times with distilled water until no free laccase activity was detected. Prepared M-CLEAs were visualized with a Nova NanoSEM 230 (FEI, Hillsboro, Oregon, USA) field-emission scanning electron microscope (SEM) at 6 kV in low vacuum mode at the Testing Center of Fuzhou University.
2.3. Enzyme activity assay Laccase activity was assayed based on a previously published method [33] at pH 3.0 and 30 ºC with ABTS (ε = 36,000 M-1 cm-1) as the substrate. Absorbance change at 420 nm was followed for 5 min. One unit of enzyme activity was defined as the amount of enzyme needed to oxidize 1 μmol ABTS in 1 min. All measurements were carried out in triplicate.
2.4. Laccase treatment of antibiotics Individual antibiotics (100 mg/L) were incubated with M-CLEAs (20 U/mL) in the presence or absence of 5 μM ABTS at pH 7.0 and 25 ºC for 48 h. Controls
included heat-inactivated M-CLEAs. Residual antibiotic concentrations were determined by employing an ACQUITY UHPLC system (Waters, Milford, MA, USA) with API 5500 Mass spectrometry (MS) equipped with electrospray (ESI) LC interface (AB SCIEX, Toronto, Canada). Separations were carried out in an Acquity UPLC HSS T3 column of 100×2.1 mm, 1.8 μm. The column oven was set at 30 °C. Elution solvents included 0.2% formic acid solution (A) and acetonitrile (B). The mobile phase gradient used (A/B; v/v) was 85:15 at 0 to 1 min, 30:70 at 2 to 6.5 min, and 85:15 at 7 to 10 min. Flow rate was set at 0.4 mL/min and the injection volume was 10 μL. The entire column effluent was directed into the mass spectrometer interface. The optimized source parameters for sample analysis were set as follows: ion spray voltage, 5500 V; curtain gas, 35 Psi; Gas 1, 55 Psi; Gas 2, 55 Psi; turbo heater temperature, 500 ºC; collision activation dissociation, 7 Psi. The compound dependent mass parameters and multiple reaction monitoring (MRM) transitions used for quantitation of antibiotics are summarized in Table 1. The data were acquired two times respectively by positive ions mode and negative ions mode. Analyst classic software version 1.5.2 was used to control all parameters of LC and MS. Furthermore, the effects of pH values (2.0-10.0) and laccase activity (5-50 U/mL) on TC (100 mg/L) degradation were investigated.
2.5. Identification of TC transformation products Effect of pH values (2.0-10.0) and laccase activity (5-50 U/mL) on TC (100 mg/L) degradation was investigated. For identification of TC transformation products, TC treatment was carried out with 40 U/mL M-CLEAs at pH 6 and 25 ºC for 48 h. Samples were taken at different time intervals. Liquid chromatography (LC) was
performed by using an Agilent RRLC 1200 rapid resolution LC system (Agilent, Santa Clara, CA, USA). Separations were carried out in a Phenomenex Luna C18 column of 150×3 mm, 3 μm id (Phenomenex, Torrance, CA, USA). The column oven was set at 35 ºC. Elution solvents were 0.2% formic acid (A) and acetonitrile (B). The mobile phase gradient used (A/B; v/v) was 90:10 at 0 to 2 min, 60:40 at 3 to 10 min, 90:10 at 11 to 13 min. Flow rate was set at 0.4 mL/min and the injection volume was 5 μL. The entire column effluent was directed into an Agilent 6224 Accurate - Mass Time of Fight system (Agilent, Santa Clara, CA, USA) equipped with an electrospray interface. Positive ions were acquired in accurate mass mode. The optimal conditions for sample analysis were as follows: capillary voltage: 3.5 kV, sheath gas temperature: 360 ºC, sheath gas flow: 11 mL/min, nebulizer pressure: 50 psi, skimmer voltage: 60 V, cone voltage: 110 V, and MS scanning range: 100-600. Agilent MassHunter Workstation software version B.0301 was used for the instrument control, data acquisition and data analysis.
2.6. Antimicrobial activity of TC and OTC before and after laccase treatment transformation products Growth inhibition tests by using gram-negative E. coli and gram-positive B. licheniformis were performed as described [25] with minor modifications. Bacterial cultivationculture was performed in Luria-Bertani (LB) medium on 96-well plates at 37 ºC with shaking at 180 rpm. Absorbance at 600 nm was measured with a Multiskan GO spectrophotometer (ThermoFisher) after cultivationculture for 24 h in the presence of various concentrations of TC (0-5 mg/L) or OTC (0-3 mg/L) for 24 h. Bacterial growth inhibition (%) was calculated based on the OD600 of control culture
(supplemented with 1% NaCl) versus antibiotic-supplemented cultures, and the 24-h IC50 values of TC and OTC against E. coli and B. licheniformis were determined (Table 2). TC or OTC at 100 mg/L were treated with immobilized laccase and then diluted for growth inhibition tests so that the TC or OTC concentration of the untreated solution corresponded to 4 or 3 mg/L.Antibiotic solutions (100 mg/L TC or OTC) with or without laccase treatment were diluted identically for microbial growth inhibition tests. The final antibiotic concentrations in diluted, untreated TC/OTC solutions were 3.75 mg/L for E.coli inhibition tests and 3 mg/L for B. licheniformis inhibition tests. All tests were carried out in triplicate.
3. Results and discussion 3.1. Laccase treatment of antibioticsPreparation of laccase M-CLEAs A maximal activity recovery of 46.8% was obtained at the laccase/MNPs ratio (w/w) of 0.8:1 and after cross-linking with 40 mM glutaraldehyde for 2 h (Fig. 1). A SEM image of prepared Cerrena laccase M-CLEAs is shown in Fig. 2. Our recovery rate (46.8%) was higher than the recovery rates of M-CLEAs prepared from T. versicolor laccase in two previous studies (31.8% and 39.0%, respectively) [20, 23]. On the other hand, an activity recovery of 62.2% was achieved with T. versicolor laccase by using chitosan, instead of glutaraldehyde, as the cross-linking agent [22]. Activity recovery of immobilized enzymes seemed to rely on the nature of the protein as well as the immobilization method and parameters. In addition to possible enzyme and inactivation during immobilization, reduced enzyme flexibility, steric hindrance and diffusion limitations might also lead to loss of enzyme activity [19, 21].
3.2. Laccase treatment of antibiotics The resulting laccase M-CLEAs were used for treatment of seven antibiotics. In the absence of a redox mediator, the highest degradation efficiency was observed with TC and OTC, followed by ampicillin, erythromycin and sulfamethoxazole. The mediator ABTS increased degradation efficiencies of sulfamethoxazole, ampicillin and trimethoprim, but decreased degradation efficiencies of TC and OTC. Laccase was not able to transform chloramphenicol even with the help of ABTS (Fig. 23). Ampicillin, chloramphenicol, erythromycin and TC have previously been found to affect fungal growth or laccase production of Cyathus bulleri or Pycnoporus cinnabarinus [34], but laccase degradation of ampicillin, chloramphenicol and erythromycin has not been previously reported. Since TC was the most suitable laccase substrate in our screening, it was chosen for subsequent studies. The optimal pH for laccase treatment of TC was pH 6.0 (Fig. 4A). Enzyme activity also played a role on TC transformation efficiency, which increased with increasing laccase activity up to 40 U/mL (Fig. 4B). Under the optimal conditions of TC treatment, LC-TOF MS was employed to detect TC (m/z 445.16, elution time 2.77 min) at different time intervals (Fig. 5A). Degradation of over 80% of TC by Cerrena laccase occurred within the first 12 h (Fig. 6). A long duration of 14 d is needed for P. ostreatus mycelia to remove 50-100 mg/L OTC by absorption. Interestingly, although P. ostreatus laccase has been extensively studied, it was not involved in OTC degradation [6], reinforcing the importance of ligninolytic enzymes in efficient TC treatment. Similarly, T. versicolor laccase, regardless of mediator presence, did not participate in OTC degradation [35],
which was opposite to our observation that OTC was transformed by Cerrena laccase. Cerrena laccase was also more effective in TC removal compared with T. versicolor laccase. Free and immobilized T. versicolor laccase on a ceramic membrane degraded 20 mg/L TC with an efficiency of 30% and 56%, respectively, after 24 h at pH 6 and 25 ºC [17]. It has also been reported that T. versicolor laccase removed 78% of 100 mg/L TC after 18 h in the absence of a mediator [24]. In our hands, the mediator ABTS was not useful in improving degradation efficiencies of TC or OTC although ABTS was the best mediator for decolorization of Coomassie Brilliant Blue R-250 by Cerrena laccase [36]. The effect of a mediator on laccase oxidation varies with the laccase and substrate and depends on the radicals formed, recyclability of the mediator and stability of the laccase [9, 12, 37-39]. In many cases, redox mediators speed up laccase oxidation [12, 37-40]. For example, HBT promotes TC oxidation by T. versicolor laccase [25, 26], and ABTS assisted in sulfamethoxazole transformation by our laccase and T. versicolor laccase [38]. In other cases, mediators might not stimulate or even hamper laccase oxidation. For instance, sinapic acid significantly lowered anthracene removal by P. cinnabarinus laccase, which was opposite to other tested synthetic and natural mediators including HBT and p-coumaric acid [41]. Another example comes from ABTS-assisted laccase decolorization of flexographic inks. ABTS increased decolorization of the monoazo dyes Red 48:4 and Magenta HX-E by Coriolopsis rigida laccase, but not Myceliophthora thermophile laccase, for which two syringyl-type natural phenolic mediators (i.e., acetosyringone and methyl syringate) were helpful. For triarylmethane dyes Blue 1 and Violet 3:1, laccase/ABTS promoted decolorization during the first hours, but at the end of the 48-h incubation, decolorization efficiencies by
laccase/ABTS were similar to or even lower than those attributed to C. rigida laccase alone [40]. Considering the fact that a laccase that can effectively catalyze without the help of a mediator is advantageous and preferable because mediators may be costly and toxic and can inactivate laccases [9, 12, 37, 39], we did not pursue further on laccase mediators for TC conversion. Next, we identified TC conversion products upon laccase oxidation and assessed microbial toxicity of the reaction mixture.
3.3.
Identification of
TC
transformation products and the proposed
transformation pathwayLaccase-mediated TC transformation Optimal pH of laccase treatment of TC was pH 6.0 (Fig. 3A). Enzyme activity also played a role on TC transformation efficiency, which increased with increasing laccase activity up to 40 U/mL. Next, TC treatment was performed at pH 6 with 40 U/mL laccase, and LC-TOF MS was employed to detect TC (m/z 445.16, elution time 2.77 min) and its transformation products. Three TC transformation products, with m/z 459.13, 431.11 and 396.07 (Figs. 45B-D) and elution time 2.69, 6.01 and 6.35 min, were identified with LC-TOF MS. These three transformation products were designated as TP 459, TP 431 and TP 396, respectively. Relative abundance of TC and the transformation products during laccase treatment is shown in Fig. 56. Accompanying diminishing TC levels, the levels of transformation product levels increased first and then decreaseddeclined. A longer period of 14 d is needed for P. ostreatus mycelia to remove 50-100 mg/L OTC by absorption. Interestingly, although P. ostreatus laccase has been extensively studied, it was not involved in OTC degradation, reinforcing the
importance of ligninolytic enzymes in efficient TC treatment. Indeed, over 80% of TC degradation by Cerrena laccase occurred within the first 12 h. Trametes versicolor laccase removed 78% of 100 mg/L TC after 18 h in the absence of a mediator. It has also been reported that free and immobilized T. versicolor laccase degraded 20 mg/L TC with an efficiency of 30% and 56%, respectively, after 24 h at pH 6 and 25 ºC. In these two studies, toxicity of TC degradation products generated by T. versicolor laccase was not analyzed. In our hands, the mediator ABTS was not useful in improving degradation efficiencies of TC or OTC although ABTS was the best mediator for decolorization of Coomassie Brilliant Blue R-250 by Cerrena laccase. In contrast, use of redox mediators could speed up TC oxidation by T. versicolor laccase or P. chrysosporium lignin peroxidase despite the fact that mediators such as 1-hydroxybenzotriazole (HBT) may be costly and potentially toxic. Furthermore, although laccase oxidation of the substrate may proceed differently with or without a mediator, the TC degradation pathway with laccase/mediator was not elucidated. A mechanism of TC transformation by laccase was proposed based on the three identified products (Fig. 67). TC was first oxidized by laccase inat position 5 to the corresponding ketone (TP 459) by laccase, and then the amino group is bi-demethylated inat position 4 iswas bi-demethylated to form TP 431. Subsequently, oxidation inat position 4, water elimination inat position 6 and dehydrogenation inat position 12 resulted in TP 396. A similar metabolic pathway was suggested by Llorca et al., although the authors did not detect the ketone intermediate (TP 459) by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass
spectrometer LTQ-Orbitrap [24]. TP 459 was a necessary intermediate for the formation of TP 431, which was confirmed in this study.
3.4. Bacteria growth inhibition of TC antibiotics after laccase treatment It has been reported that TC degradation products may be ofdemonstrate the same potency toward bacteria as TC [1]. Therefore, aAntimicrobial activity of TC and OTC tetracycline transformation products rendered bybefore and after Cerrena laccase treatment was evaluated. TC and OTC displayed IC50 values of 2.2 and 2.5 mg/L against E. coli. In comparison, B. licheniformis was more sensitive, and the IC50 values of TC and OTC against B. licheniformis were 1.4 and 1.2 mg/L, respectively. Inhibition of growth of the tested bacteria by TC and OTC werewas alleviated after 24 h of laccase treatment and diminished after 48 h (Table 3). Therefore, Cerrena laccase was effective in detoxification of tetracycline antibiotics.
4. Conclusions Cerrena laccase was immobilized as M-CLEAs and used in antibiotic degradation. At pH 6.0 and 25 ºC, 40 U/mL Cerrena laccase removed 100 mg/L TC in the absence of a redox mediator. Laccase treatment also significantly reduced antimicrobial activity of TC. Three TC transformation intermediates were determined by LC-TOF MS, and a laccase-mediated TC conversion pathway was hypothesized. Laccase M-CLEAs represent a green and promising approach for antibiotic removal in water treatment.
Competing interests
The authors declare no competing interests.
Acknowledgments This study was funded by Natural Science Foundation of China (41306120, 31671795), Oceanic Public Welfare Industry Special Research Project of China (201305015) and Fujian Guidance Project (2016Y0059).
References [1] B. Halling-Sørensen, G. Sengeløv, J. Tjørnelund, Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria, Arch. Environ. Contam. Toxicol., 42 (2002) 263-271. [2] B. Halling-Sørensen, Algal toxicity of antibacterial agents used in intensive farming, Chemosphere, 40 (2000) 731-739. [3] J. Jeong, W. Song, W.J. Cooper, J. Jung, J. Greaves, Degradation of tetracycline antibiotics: Mechanisms and kinetic studies for advanced oxidation/reduction processes, Chemosphere, 78 (2010) 533-540. [4] X. Wen, Y. Jia, J. Li, Degradation of tetracycline and oxytetracycline by crude lignin peroxidase prepared from Phanerochaete chrysosporium – A white rot fungus, Chemosphere, 75 (2009) 1003-1007. [5] X. Wen, Y. Jia, J. Li, Enzymatic degradation of tetracycline and oxytetracycline by crude manganese peroxidase prepared from Phanerochaete chrysosporium, J. Hazard. Mater., 177 (2010) 924-928. [6] L. Migliore, M. Fiori, A. Spadoni, E. Galli, Biodegradation of oxytetracycline by
Pleurotus ostreatus mycelium: a mycoremediation technique, J. Hazard. Mater., 215– 216 (2012) 227-232. [7] P. Baldrian, Laccases-occurrence and properties, FEMS Microbiol. Rev., 30 (2006) 215-242. [8] D.S. Arora, R.K. Sharma, Ligninolytic fungal laccases and their biotechnological applications, Appl. Biochem. Biotechnol., 160 (2010) 1760-1788. [9] B. Ashe, L.N. Nguyen, F.I. Hai, D.-J. Lee, J.P. van de Merwe, F.D.L. Leusch, W.E. Price, L.D. Nghiem, Impacts of redox-mediator type on trace organic contaminants degradation by laccase: Degradation efficiency, laccase stability and effluent toxicity, Int. Biodeterior. Biodegradation, 113 (2016) 169-176. [10] D.W. Wong, Structure and action mechanism of ligninolytic enzymes, Appl. Biochem. Biotechnol., 157 (2009) 174-209. [11] R. Pogni, M.C. Baratto, A. Sinicropi, R. Basosi, Spectroscopic and computational characterization of laccases and their substrate radical intermediates, Cell. Mol. Life Sci., 72 (2015) 885-896. [12]
O.V.
Morozova,
G.P.
Shumakovich,
S.V.
Shleev,
Y.I.
Yaropolov,
Laccase-mediator systems and their applications: A review, Appl. Biochem. Microbiol., 43 (2007) 523-535. [13] E.I. Solomon, P. Chen, M. Metz, S.-K. Lee, A.E. Palmer, Oxygen binding, activation, and reduction to water by copper proteins, Angew. Chem. Int. Ed., 40 (2001) 4570-4590. [14] J.D. Cui, S.R. Jia, Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges, Crit. Rev. Biotechnol., 35 (2015) 15-28.
[15] R. Abejón, M.P. Belleville, J. Sanchez-Marcano, Design, economic evaluation and optimization of enzymatic membrane reactors for antibiotics degradation in wastewaters, Sep. Purif. Technol., 156 (2015) 183-199. [16] R. Abejón, M. De Cazes, M.P. Belleville, J. Sanchez-Marcano, Large-scale enzymatic membrane reactors for tetracycline degradation in WWTP effluents, Water Res., 73 (2015) 118-131. [17] M. de Cazes, M.P. Belleville, E. Petit, M. Llorca, S. Rodríguez-Mozaz, J. de Gunzburg, D. Barceló, J. Sanchez-Marcano, Design and optimization of an enzymatic membrane reactor for tetracycline degradation, Catal. Today, 236 (2014) 146-152. [18] M. de Cazes, M.P. Belleville, M. Mougel, H. Kellner, J. Sanchez-Marcano, Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation, J. Membrane Sci., 476 (2015) 384-393. [19] R.A. Sheldon, Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs), Appl. Microbiol. Biotechnol., 92 (2011) 467-477. [20] V.V. Kumar, S. Sivanesan, H. Cabana, Magnetic cross-linked laccase aggregates — Bioremediation tool for decolorization of distinct classes of recalcitrant dyes, Sci. Total Environ., 487 (2014) 830-839. [21] S. Talekar, A. Joshi, G. Joshi, P. Kamat, R. Haripurkar, S. Kambale, Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs), RSC Adv., 3 (2013) 12485-12511. [22] V.V. Kumar, H. Cabana, Towards high potential magnetic biocatalysts for on-demand elimination of pharmaceuticals, Bioresour. Technol., 200 (2016) 81-89. [23] A. Arca-Ramos, V.V. Kumar, G. Eibes, M.T. Moreira, H. Cabana, Recyclable
cross-linked laccase aggregates coupled to magnetic silica microbeads for elimination of pharmaceuticals from municipal wastewater, Environ. Sci. Pollut. Res., 23 (2016) 8929-8939. [24] M. Llorca, S. Rodríguez-Mozaz, O. Couillerot, K. Panigoni, J. de Gunzburg, S. Bayer, R. Czaja, D. Barceló, Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap, Chemosphere, 119 (2015) 90-98. [25] T. Suda, T. Hata, S. Kawai, H. Okamura, T. Nishida, Treatment of tetracycline antibiotics by laccase in the presence of 1-hydroxybenzotriazole, Bioresour. Technol., 103 (2012) 498-501. [26] H. Ding, Y. Wu, B. Zou, Q. Lou, W. Zhang, J. Zhong, L. Lu, G. Dai, Simultaneous removal and degradation characteristics of sulfonamide, tetracycline, and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorption, J. Hazard. Mater., 307 (2016) 350-358. [27] G. Songulashvili, G.A. Jimenéz-Tobón, C. Jaspers, M.J. Penninckx, Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants, Fungal Biol., 116 (2012) 883-889. [28] J. Yang, Q. Lin, T.B. Ng, X. Ye, J. Lin, Purification and characterization of a novel laccase from Cerrena sp. HYB07 with dye decolorizing ability, PLoS ONE, 9 (2014) e110834. [29] S.-C. Chen, P.-H. Wu, Y.-C. Su, T.-N. Wen, Y.-S. Wei, N.-C. Wang, C.-A. Hsu, A.H.-J. Wang, L.-F. Shyur, Biochemical characterization of a novel laccase from the basidiomycete fungus Cerrena sp. WR1, Protein Eng. Des. Sel., 25 (2012) 761-769.
[30] A. Michniewicz, S. Ledakowicz, R. Ullrich, M. Hofrichter, Kinetics of the enzymatic decolorization of textile dyes by laccase from Cerrena unicolor, Dyes Pigm., 77 (2008) 295-302. [31] A. Jarosz-Wilkołazka, T. Ruzgas, L. Gorton, Use of laccase-modified electrode for amperometric detection of plant flavonoids, Enzyme Microb. Technol., 35 (2004) 238-241. [32] D.T. D'Souza, R. Tiwari, A.K. Sah, C. Raghukumar, Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes, Enzyme Microb. Technol., 38 (2006) 504-511. [33] R. Bourbonnais, M.G. Paice, Demethylation and delignification of kraft pulp by Trametes
versicolor
laccase
in
the
presence
of
2,2
′
-azinobis-(3-ethylbenzthiazoline-6-sulphonate), Appl. Microbiol. Biotechnol., 36 (1992) 823-827. [34] S. Dhawan, R. Lal, M. Hanspal, R.C. Kuhad, Effect of antibiotics on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus, Bioresour. Technol., 96 (2005) 1415-1418. [35] J.A. Mir-Tutusaus, M. Masis-Mora, C. Corcellas, E. Eljarrat, D. Barcelo, M. Sarra, G. Caminal, T. Vicent, C.E. Rodriguez-Rodriguez, Degradation of selected agrochemicals by the white rot fungus Trametes versicolor, Sci. Total Environ., 500-501 (2014) 235-242. [36] J. Yang, X. Yang, X. Ye, J. Lin, Optimal parameters for laccase-mediated destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels, Data Brief, 7 (2016) 1-7. [37] M.R. Hu, Y.P. Chao, G.Q. Zhang, Z.Q. Xue, S. Qian, Laccase-mediator system in
the decolorization of different types of recalcitrant dyes, J. Ind. Microbiol. Biotechnol., 36 (2009) 45-51. [38] J. Margot, P.-J. Copin, U. von Gunten, D.A. Barry, C. Holliger, Sulfamethoxazole and isoproturon degradation and detoxification by a laccase-mediator system: Influence of treatment conditions and mechanistic aspects, Biochem. Eng. J., 103 (2015) 47-59. [39] S. Kurniawati, J.A. Nicell, Efficacy of mediators for enhancing the laccase-catalyzed oxidation of aqueous phenol, Enzyme Microb. Technol., 41 (2007) 353-361. [40] U. Fillat, A. Prieto, S. Camarero, Á.T. Martínez, M.J. Martínez, Biodeinking of flexographic inks by fungal laccases using synthetic and natural mediators, Biochem. Eng. J., 67 (2012) 97-103. [41] A.I. Cañas, M. Alcalde, F. Plou, M.J. Martínez, Á.T. Martínez, S. Camarero, Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil, Environ. Sci. Technol., 41 (2007) 2964-2971.
Figure legends Fig. 1 Preparation of M-CLEAs from Cerrena laccase. (A) Effect of enzyme loading on M-CLEA activity recovery. Fermentation broth and functionalized were mixed in different w:w ratios. Laccase was precipitated with ammonium sulfate and cross-linked with glutaraldehyde. (B) Effect of glutaraldehyde concentration and cross-linking time on M-CLEA activity recovery.
Fig. 2 SEM image of M-CLEAs of Cerrena laccase.
Fig. 3 Degradation of antibiotics by laccase. Individual antibiotics at 100 mg/L were treated with 20 U/mL immobilized laccase in the presence or absence of 5 μM ABTS at pH 7.0 and 25 ºC for 48 h. Residual antibiotic concentrations were determined by LC-MS/MS. Error bars represent standard deviation.
Fig. 4 Effect of pH (A) and enzyme activity (B) on TC (100 mg/L) degradation by laccase. Residual antibiotic concentrations were determined by LC-MS/MS.
Fig. 5 Mass spectra of TC transformation products uponafter laccase treatment. TC (100 mg/L) treatment was performed at pH 6 and 25 ºC with 40 U/mL laccase. TC (A, m/z 445.16) and TC transformation products (B-D, m/z 459.13, 431.11 and 396.07) were identified with LC-TOF MS.
Fig. 6 Formation of detected transformation products during TC degradation. TP, transformation product.
Fig. 7 Proposed degradation pathway of TC by laccase.
Tables Table 1 Optimized mass parameters and MRM transitions for antibiotics. Analytes
Tetracycline
Oxytetracycline
Ampicillin
Electrospray
MRM
Dwell
Declustering
Collision
mode
transition
time
potential
energy
(ms)
(V)
(V)
445>410
10
50
28
445>427
10
50
20
461>443
10
50
20
461>426
10
50
30
350>160
10
80
24
350>192
10
80
20
254>156
10
65
23
254>108
10
65
35
734>158
10
80
41
734>576
10
80
43
291>230
10
110
32
291>261
10
110
33
Negative
321>152
10
-85
-25
ions
321>257
10
-85
-16
Positive ions
Positive ions
Positive ions
Sulfamethoxazole Positive ions
Erythromycin
Trimethoprim
Chloramphenicol
Positive ions
Positive ions
The major MRM transition for quantification is indicated in boldface.
Table 2 IC50 values of TC and OTC for E. coli and B. licheniformis. IC50 (mg/L) TC
OTC
E. coli
2.2
2.5
B. licheniformis
1.4
1.2
Table 2 Antimicrobial activity of TC and OTC before and after laccase treatment.
TC
OTC
Growth inhibition (%)
Treatment time (h)
E.coli
B. licheniformis
0
99.5 ± 1.1
100.0 ± 0.4
24
27.8 ± 6.2
78.8 ± 3.5
48
5.8 ± 6.1
8.6 ± 4.6
0
98.4 ± 1.8
99.7 ± 0.4
24
34.9 ± 7.4
72.2 ± 4.7
48
7.3 ± 5.8
7.4 ± 6.5
Values represent means ± standard deviations (n=3).
Relative activity (%)
(A) 50 40 30 20 10 0 0.4:1
0.6:1
0.8:1
1.0:1
1.2:1
1.4:1
Laccase:MNPs (w/w) 60 Relative activity (%)
(B)
50 40 30 10 mM 20 mM 30 mM 40 mM
20 10 0 0
1
2
3
Time (h) Fig. 1
4
5
6
Fig. 2
Degradation (%)
80 Without ABTS
70 60 50 40 30 20
With ABTS
10 0
Antibiotics Fig. 3
(A) Degradation (%)
100 80 60 40 20 0 1
2
3
4
5
6
7
8
9 10
pH (B) Degradation (%)
120 100 80 60 40 20 0 0
10
20
30
Laccase (U/mL) Fig. 4
40
50
Fig. 5
Tetracycline TP 459 TP 431 TP 396
Relative abundance (%)
120 100 80 60 40 20 0 0
6
12
18
24
30
Time (h) Fig. 6
36
42
48
Fig. 7
S0304-3894(16)30916-5 http://dx.doi.org/doi:10.1016/j.jhazmat.2016.10.019 HAZMAT 18100
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
22-8-2016 6-10-2016 10-10-2016
Please cite this article as: Jie Yang, Yonghui Lin, Xiaodan Yang, Tzi Bun Ng, Xiuyun Ye, Juan Lin, Degradation of tetracycline by immobilized laccase and the proposed transformation pathway, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2016.10.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Degradation of tetracycline by immobilized laccase and the proposed transformation pathway
Jie Yang 1*, Yonghui Lin2, Xiaodan Yang 1, Tzi Bun Ng 3, Xiuyun Ye1, Juan Lin1*
1
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fujian
350116, China 2
Technical Center, Fujian Entry-Exit Inspection and Quarantine Bureau, Fuzhou,
Fujian 350001, China 3
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong
Kong, Shatin, New Territories, Hong Kong, China
*Corresponding Author: Dr. Jie Yang Phone: 86-591-22866376 Fax: 86-591-22866376 Email: [email protected] Dr. Juan Lin Phone: 86-591-22866376 Fax: 86-591-22866376 Email: [email protected]
Highlights
Laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) Laccase at 40 U/mL eliminated over 80 μg/mL TC at pH 6 and 25 ºC in 12 h
A mechanism of laccase-mediated tetracycline (TC) oxidation was proposed TC antibiotics treated with laccase showed reduced antimicrobial activity Laccase M-CLEAs are a green alternative in residual antibiotic removal in water
Abstract Magnetic cross-linked enzyme aggregates (M-CLEAs) were prepared for Cerrena laccase and used in antibiotic treatment. Of the seven antibiotics examined in this study, Cerrena laccase M-CLEAs were most effective in degradation of tetracycline
(TC)
and
oxytetracycline
(OTC),
followed
by
ampicillin,
sulfamethoxazole and erythromycin. The redox mediator ABTS was not able to improve degradation efficienciesefficiencies of degradation of TC and OTC. Cerrena laccase at 40 U/mL eliminated 100 μg/mL TC at pH 6 and 25 ºC in 48 h withoutin the absence of a redox mediator, with over 80% degradation occurring within the first 12 h. Laccase treatment also significantly loweredsuppressed the antimicrobial activity of TC and OTC. LC-TOF MS identified threeThree TC transformation products, the levels of which initially increased first and thensubsequently decreased during laccase treatment were identified by using LC-TOF MS. A proposedmechanism of laccase-mediated TC oxidation was proposed based on the identified intermediates.
Keywords: laccase, M-CLEAs, tetracycline, transformation products, antimicrobial activity
1. Introduction Antibiotics are commonly used in human and veterinary medicine and have attracted increasing attention as emerging contaminants. In addition to their ecotoxicity [1, 2], antibiotics in the environment may serve as selective pressure for antibiotic-resistant bacteria, thus imposing a threat to public health. Tetracycline antibiotics, such as tetracycline (TC) and oxytetracycline (OTC), are recalcitrant, broad-spectrum antimicrobial agents widely present in wastewater and natural water bodies [3, 4]. Conventional water treatment cannot effectively remove antibiotics [3], thereforehence, alternative methods shouldhave to be developed. Biodegradation of antibiotics has been explored. For example, crude manganese peroxidase and lignin peroxidase from Phanerochaete chrysosporium have been used to degrade TC and OTC [4, 5]. Mycelia of another white rot fungus, Pleurotus ostreatus, were employed in mycoremediation of OTC [6]. Laccase, along with manganese peroxidase and lignin peroxidase, comprise ligninolytic enzymes secreted by white rot fungi. Laccases are copper-containing oxidases that can catalyze the oxidation of a wide range of phenolic and non-phenolic compounds, including various dyestuffs and environmental pollutants [7-9]. In contrast to peroxidases, laccase does not require the presence of hydrogen peroxide or manganese for oxidation by using oxygen as the final electron receptor and offers a green alternative in wastewater treatment and bioremediation [10]. Oxidation of substrates with redox potentials higher than those of laccases, such as non-phenolic compounds, can be facilitated by small redox mediators, such as synthetic mediators 2,2’-azino-bis
(3-ethylbenzothiazoline-6-sulfonate)
(ABTS)
and
1-hydroxybenzotriazole (HBT). Laccase oxidation of the substrate may proceed differently with or without the involvement of a mediator. Furthermore, ABTS oxidizes the substrate via an electron transfer route, whereas HBT and natural phenolic mediators follow a hydrogen atom transfer mechanism [10-13]. Enzyme immobilization is used to improve enzyme stability and reusability and reduce application costs [14]. Enzymatic membrane reactors based on laccase immobilized on ceramic membranes have been evaluated for laccase-mediated TC degradation in wastewaters [15-18]. Magnetic cross-linked enzyme aggregates (M-CLEAs) represent a relatively new enzyme immobilization method, in which cross-linked enzyme aggregates (CLEAs) are attached to amino-functionalized magnetic nanoparticles (MNPs). Carrier-free CLEAs are easy and cheap to prepare; the general procedure consists of enzyme precipitation and subsequent cross-linking with a bifunctional agent [19]. M-CLEAs improve mechanical properties of CLEAs and allow easy separation and recycling of the immobilized enzyme from the reaction mixture with a magnetic field [20, 21]. Laccase M-CLEAs, which showed promise in dye decolorization [20] or elimination of pharmaceuticals [22, 23], have been prepared from Trametes versicolor. Recently, laccase has been proven useful in degradation of antibiotics such as TC, but such studies focused on laccase from T. versicolor [15-18, 24-26]. Although T. versicolor laccase is extensively studied, Cerrena laccase represents an attractive alternative with the high yields and application potentials [27-32]. However, the preparation of M-CLEAs from Cerrena laccase or utilization of Cerrena laccase in antibiotic treatment remains unexplored. In the present study, we investigated TC degradation by immobilized Cerrena laccase. Degradation products were identified
with LC-TOF MS, and a putative mechanism was deduced. The antimicrobial activity of laccase-treated TC was also analyzeddetermined.
2. Materials and methods 2.1. Strain and chemicals Cerrena unicolor [28] was maintained on potato dextrose agar (PDA) at 4 ºC in the culture collection of Fuzhou University, China. For laccase fermentation, 5 mycelial plugs (1 cm diameter) were removed from the peripheral region of a 4-d-old PDA plate stored at 30 ºC and inoculated in 50 mL potato dextrose broth (PDB) seed medium in a 250-mL Erlenmeyer flask. After allowing to grow for 2 d at 30 ºC and 200 rpm, an aliquot was transferred to a second PDB medium at the ratio of 8% (v/v). After growth for another 2 d, the fermentation medium (50 mL in 250 mL Erlenmeyer flasks) was inoculated with the second seed culture at the concentration of 8% (v/v). The fermentation medium contained (g L-1): maltodextrin, 60; peptone, 10; ammonium tartrate, 1.6; KH2PO4, 6; MgSO4·7H2O, 4.14; CaCl2, 0.3; NaCl, 0.18; CuSO4·5H2O, 0.0625; ZnSO4·7H2O, 0.018; and vitamin B1 0.015. Submerged fermentation was carried out for 6 d. Fermentation broth was collected by centrifugation at 8,000 g for 5 min and used directly for immobilization. Antibiotics
(tetracycline,
oxytetracycline,
ampicillin,
sulfamethoxazole,
erythromycin, chloramphenicol and trimethoprim) were purchased from Sangon Biotech (Shanghai, China). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Methanol and acetonitrile were of HPLC grade and obtained from Sinopharm Chemical Reagent (Beijing, China). All other chemicals were of analytical grade.
2.2. Preparation of M-CLEAs Magnetic nanoparticles (MNPs) were synthesized and amino-functionalized as previously described [20]. Fermentation broth was adjusted to pH 8.0 and MNPs were added. Saturated ammonium sulfate solution was added to the mixture at the ratio of 5:1 to precipitate laccase at 25 ºC for 2 h. Glutaraldehyde was used as the cross-linking agent. Laccase:MNPs ratios (0.4:1-1.4:1), glutaraldehyde concentrations (5-40 mM) and cross-linking time (1-6 h) were sequentially examined for maximum recovery of enzyme activity. M-CLEAs were recovered with a magnet and washed several times with distilled water until no free laccase activity was detected. Prepared M-CLEAs were visualized with a Nova NanoSEM 230 (FEI, Hillsboro, Oregon, USA) field-emission scanning electron microscope (SEM) at 6 kV in low vacuum mode at the Testing Center of Fuzhou University.
2.3. Enzyme activity assay Laccase activity was assayed based on a previously published method [33] at pH 3.0 and 30 ºC with ABTS (ε = 36,000 M-1 cm-1) as the substrate. Absorbance change at 420 nm was followed for 5 min. One unit of enzyme activity was defined as the amount of enzyme needed to oxidize 1 μmol ABTS in 1 min. All measurements were carried out in triplicate.
2.4. Laccase treatment of antibiotics Individual antibiotics (100 mg/L) were incubated with M-CLEAs (20 U/mL) in the presence or absence of 5 μM ABTS at pH 7.0 and 25 ºC for 48 h. Controls
included heat-inactivated M-CLEAs. Residual antibiotic concentrations were determined by employing an ACQUITY UHPLC system (Waters, Milford, MA, USA) with API 5500 Mass spectrometry (MS) equipped with electrospray (ESI) LC interface (AB SCIEX, Toronto, Canada). Separations were carried out in an Acquity UPLC HSS T3 column of 100×2.1 mm, 1.8 μm. The column oven was set at 30 °C. Elution solvents included 0.2% formic acid solution (A) and acetonitrile (B). The mobile phase gradient used (A/B; v/v) was 85:15 at 0 to 1 min, 30:70 at 2 to 6.5 min, and 85:15 at 7 to 10 min. Flow rate was set at 0.4 mL/min and the injection volume was 10 μL. The entire column effluent was directed into the mass spectrometer interface. The optimized source parameters for sample analysis were set as follows: ion spray voltage, 5500 V; curtain gas, 35 Psi; Gas 1, 55 Psi; Gas 2, 55 Psi; turbo heater temperature, 500 ºC; collision activation dissociation, 7 Psi. The compound dependent mass parameters and multiple reaction monitoring (MRM) transitions used for quantitation of antibiotics are summarized in Table 1. The data were acquired two times respectively by positive ions mode and negative ions mode. Analyst classic software version 1.5.2 was used to control all parameters of LC and MS. Furthermore, the effects of pH values (2.0-10.0) and laccase activity (5-50 U/mL) on TC (100 mg/L) degradation were investigated.
2.5. Identification of TC transformation products Effect of pH values (2.0-10.0) and laccase activity (5-50 U/mL) on TC (100 mg/L) degradation was investigated. For identification of TC transformation products, TC treatment was carried out with 40 U/mL M-CLEAs at pH 6 and 25 ºC for 48 h. Samples were taken at different time intervals. Liquid chromatography (LC) was
performed by using an Agilent RRLC 1200 rapid resolution LC system (Agilent, Santa Clara, CA, USA). Separations were carried out in a Phenomenex Luna C18 column of 150×3 mm, 3 μm id (Phenomenex, Torrance, CA, USA). The column oven was set at 35 ºC. Elution solvents were 0.2% formic acid (A) and acetonitrile (B). The mobile phase gradient used (A/B; v/v) was 90:10 at 0 to 2 min, 60:40 at 3 to 10 min, 90:10 at 11 to 13 min. Flow rate was set at 0.4 mL/min and the injection volume was 5 μL. The entire column effluent was directed into an Agilent 6224 Accurate - Mass Time of Fight system (Agilent, Santa Clara, CA, USA) equipped with an electrospray interface. Positive ions were acquired in accurate mass mode. The optimal conditions for sample analysis were as follows: capillary voltage: 3.5 kV, sheath gas temperature: 360 ºC, sheath gas flow: 11 mL/min, nebulizer pressure: 50 psi, skimmer voltage: 60 V, cone voltage: 110 V, and MS scanning range: 100-600. Agilent MassHunter Workstation software version B.0301 was used for the instrument control, data acquisition and data analysis.
2.6. Antimicrobial activity of TC and OTC before and after laccase treatment transformation products Growth inhibition tests by using gram-negative E. coli and gram-positive B. licheniformis were performed as described [25] with minor modifications. Bacterial cultivationculture was performed in Luria-Bertani (LB) medium on 96-well plates at 37 ºC with shaking at 180 rpm. Absorbance at 600 nm was measured with a Multiskan GO spectrophotometer (ThermoFisher) after cultivationculture for 24 h in the presence of various concentrations of TC (0-5 mg/L) or OTC (0-3 mg/L) for 24 h. Bacterial growth inhibition (%) was calculated based on the OD600 of control culture
(supplemented with 1% NaCl) versus antibiotic-supplemented cultures, and the 24-h IC50 values of TC and OTC against E. coli and B. licheniformis were determined (Table 2). TC or OTC at 100 mg/L were treated with immobilized laccase and then diluted for growth inhibition tests so that the TC or OTC concentration of the untreated solution corresponded to 4 or 3 mg/L.Antibiotic solutions (100 mg/L TC or OTC) with or without laccase treatment were diluted identically for microbial growth inhibition tests. The final antibiotic concentrations in diluted, untreated TC/OTC solutions were 3.75 mg/L for E.coli inhibition tests and 3 mg/L for B. licheniformis inhibition tests. All tests were carried out in triplicate.
3. Results and discussion 3.1. Laccase treatment of antibioticsPreparation of laccase M-CLEAs A maximal activity recovery of 46.8% was obtained at the laccase/MNPs ratio (w/w) of 0.8:1 and after cross-linking with 40 mM glutaraldehyde for 2 h (Fig. 1). A SEM image of prepared Cerrena laccase M-CLEAs is shown in Fig. 2. Our recovery rate (46.8%) was higher than the recovery rates of M-CLEAs prepared from T. versicolor laccase in two previous studies (31.8% and 39.0%, respectively) [20, 23]. On the other hand, an activity recovery of 62.2% was achieved with T. versicolor laccase by using chitosan, instead of glutaraldehyde, as the cross-linking agent [22]. Activity recovery of immobilized enzymes seemed to rely on the nature of the protein as well as the immobilization method and parameters. In addition to possible enzyme and inactivation during immobilization, reduced enzyme flexibility, steric hindrance and diffusion limitations might also lead to loss of enzyme activity [19, 21].
3.2. Laccase treatment of antibiotics The resulting laccase M-CLEAs were used for treatment of seven antibiotics. In the absence of a redox mediator, the highest degradation efficiency was observed with TC and OTC, followed by ampicillin, erythromycin and sulfamethoxazole. The mediator ABTS increased degradation efficiencies of sulfamethoxazole, ampicillin and trimethoprim, but decreased degradation efficiencies of TC and OTC. Laccase was not able to transform chloramphenicol even with the help of ABTS (Fig. 23). Ampicillin, chloramphenicol, erythromycin and TC have previously been found to affect fungal growth or laccase production of Cyathus bulleri or Pycnoporus cinnabarinus [34], but laccase degradation of ampicillin, chloramphenicol and erythromycin has not been previously reported. Since TC was the most suitable laccase substrate in our screening, it was chosen for subsequent studies. The optimal pH for laccase treatment of TC was pH 6.0 (Fig. 4A). Enzyme activity also played a role on TC transformation efficiency, which increased with increasing laccase activity up to 40 U/mL (Fig. 4B). Under the optimal conditions of TC treatment, LC-TOF MS was employed to detect TC (m/z 445.16, elution time 2.77 min) at different time intervals (Fig. 5A). Degradation of over 80% of TC by Cerrena laccase occurred within the first 12 h (Fig. 6). A long duration of 14 d is needed for P. ostreatus mycelia to remove 50-100 mg/L OTC by absorption. Interestingly, although P. ostreatus laccase has been extensively studied, it was not involved in OTC degradation [6], reinforcing the importance of ligninolytic enzymes in efficient TC treatment. Similarly, T. versicolor laccase, regardless of mediator presence, did not participate in OTC degradation [35],
which was opposite to our observation that OTC was transformed by Cerrena laccase. Cerrena laccase was also more effective in TC removal compared with T. versicolor laccase. Free and immobilized T. versicolor laccase on a ceramic membrane degraded 20 mg/L TC with an efficiency of 30% and 56%, respectively, after 24 h at pH 6 and 25 ºC [17]. It has also been reported that T. versicolor laccase removed 78% of 100 mg/L TC after 18 h in the absence of a mediator [24]. In our hands, the mediator ABTS was not useful in improving degradation efficiencies of TC or OTC although ABTS was the best mediator for decolorization of Coomassie Brilliant Blue R-250 by Cerrena laccase [36]. The effect of a mediator on laccase oxidation varies with the laccase and substrate and depends on the radicals formed, recyclability of the mediator and stability of the laccase [9, 12, 37-39]. In many cases, redox mediators speed up laccase oxidation [12, 37-40]. For example, HBT promotes TC oxidation by T. versicolor laccase [25, 26], and ABTS assisted in sulfamethoxazole transformation by our laccase and T. versicolor laccase [38]. In other cases, mediators might not stimulate or even hamper laccase oxidation. For instance, sinapic acid significantly lowered anthracene removal by P. cinnabarinus laccase, which was opposite to other tested synthetic and natural mediators including HBT and p-coumaric acid [41]. Another example comes from ABTS-assisted laccase decolorization of flexographic inks. ABTS increased decolorization of the monoazo dyes Red 48:4 and Magenta HX-E by Coriolopsis rigida laccase, but not Myceliophthora thermophile laccase, for which two syringyl-type natural phenolic mediators (i.e., acetosyringone and methyl syringate) were helpful. For triarylmethane dyes Blue 1 and Violet 3:1, laccase/ABTS promoted decolorization during the first hours, but at the end of the 48-h incubation, decolorization efficiencies by
laccase/ABTS were similar to or even lower than those attributed to C. rigida laccase alone [40]. Considering the fact that a laccase that can effectively catalyze without the help of a mediator is advantageous and preferable because mediators may be costly and toxic and can inactivate laccases [9, 12, 37, 39], we did not pursue further on laccase mediators for TC conversion. Next, we identified TC conversion products upon laccase oxidation and assessed microbial toxicity of the reaction mixture.
3.3.
Identification of
TC
transformation products and the proposed
transformation pathwayLaccase-mediated TC transformation Optimal pH of laccase treatment of TC was pH 6.0 (Fig. 3A). Enzyme activity also played a role on TC transformation efficiency, which increased with increasing laccase activity up to 40 U/mL. Next, TC treatment was performed at pH 6 with 40 U/mL laccase, and LC-TOF MS was employed to detect TC (m/z 445.16, elution time 2.77 min) and its transformation products. Three TC transformation products, with m/z 459.13, 431.11 and 396.07 (Figs. 45B-D) and elution time 2.69, 6.01 and 6.35 min, were identified with LC-TOF MS. These three transformation products were designated as TP 459, TP 431 and TP 396, respectively. Relative abundance of TC and the transformation products during laccase treatment is shown in Fig. 56. Accompanying diminishing TC levels, the levels of transformation product levels increased first and then decreaseddeclined. A longer period of 14 d is needed for P. ostreatus mycelia to remove 50-100 mg/L OTC by absorption. Interestingly, although P. ostreatus laccase has been extensively studied, it was not involved in OTC degradation, reinforcing the
importance of ligninolytic enzymes in efficient TC treatment. Indeed, over 80% of TC degradation by Cerrena laccase occurred within the first 12 h. Trametes versicolor laccase removed 78% of 100 mg/L TC after 18 h in the absence of a mediator. It has also been reported that free and immobilized T. versicolor laccase degraded 20 mg/L TC with an efficiency of 30% and 56%, respectively, after 24 h at pH 6 and 25 ºC. In these two studies, toxicity of TC degradation products generated by T. versicolor laccase was not analyzed. In our hands, the mediator ABTS was not useful in improving degradation efficiencies of TC or OTC although ABTS was the best mediator for decolorization of Coomassie Brilliant Blue R-250 by Cerrena laccase. In contrast, use of redox mediators could speed up TC oxidation by T. versicolor laccase or P. chrysosporium lignin peroxidase despite the fact that mediators such as 1-hydroxybenzotriazole (HBT) may be costly and potentially toxic. Furthermore, although laccase oxidation of the substrate may proceed differently with or without a mediator, the TC degradation pathway with laccase/mediator was not elucidated. A mechanism of TC transformation by laccase was proposed based on the three identified products (Fig. 67). TC was first oxidized by laccase inat position 5 to the corresponding ketone (TP 459) by laccase, and then the amino group is bi-demethylated inat position 4 iswas bi-demethylated to form TP 431. Subsequently, oxidation inat position 4, water elimination inat position 6 and dehydrogenation inat position 12 resulted in TP 396. A similar metabolic pathway was suggested by Llorca et al., although the authors did not detect the ketone intermediate (TP 459) by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass
spectrometer LTQ-Orbitrap [24]. TP 459 was a necessary intermediate for the formation of TP 431, which was confirmed in this study.
3.4. Bacteria growth inhibition of TC antibiotics after laccase treatment It has been reported that TC degradation products may be ofdemonstrate the same potency toward bacteria as TC [1]. Therefore, aAntimicrobial activity of TC and OTC tetracycline transformation products rendered bybefore and after Cerrena laccase treatment was evaluated. TC and OTC displayed IC50 values of 2.2 and 2.5 mg/L against E. coli. In comparison, B. licheniformis was more sensitive, and the IC50 values of TC and OTC against B. licheniformis were 1.4 and 1.2 mg/L, respectively. Inhibition of growth of the tested bacteria by TC and OTC werewas alleviated after 24 h of laccase treatment and diminished after 48 h (Table 3). Therefore, Cerrena laccase was effective in detoxification of tetracycline antibiotics.
4. Conclusions Cerrena laccase was immobilized as M-CLEAs and used in antibiotic degradation. At pH 6.0 and 25 ºC, 40 U/mL Cerrena laccase removed 100 mg/L TC in the absence of a redox mediator. Laccase treatment also significantly reduced antimicrobial activity of TC. Three TC transformation intermediates were determined by LC-TOF MS, and a laccase-mediated TC conversion pathway was hypothesized. Laccase M-CLEAs represent a green and promising approach for antibiotic removal in water treatment.
Competing interests
The authors declare no competing interests.
Acknowledgments This study was funded by Natural Science Foundation of China (41306120, 31671795), Oceanic Public Welfare Industry Special Research Project of China (201305015) and Fujian Guidance Project (2016Y0059).
References [1] B. Halling-Sørensen, G. Sengeløv, J. Tjørnelund, Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria, Arch. Environ. Contam. Toxicol., 42 (2002) 263-271. [2] B. Halling-Sørensen, Algal toxicity of antibacterial agents used in intensive farming, Chemosphere, 40 (2000) 731-739. [3] J. Jeong, W. Song, W.J. Cooper, J. Jung, J. Greaves, Degradation of tetracycline antibiotics: Mechanisms and kinetic studies for advanced oxidation/reduction processes, Chemosphere, 78 (2010) 533-540. [4] X. Wen, Y. Jia, J. Li, Degradation of tetracycline and oxytetracycline by crude lignin peroxidase prepared from Phanerochaete chrysosporium – A white rot fungus, Chemosphere, 75 (2009) 1003-1007. [5] X. Wen, Y. Jia, J. Li, Enzymatic degradation of tetracycline and oxytetracycline by crude manganese peroxidase prepared from Phanerochaete chrysosporium, J. Hazard. Mater., 177 (2010) 924-928. [6] L. Migliore, M. Fiori, A. Spadoni, E. Galli, Biodegradation of oxytetracycline by
Pleurotus ostreatus mycelium: a mycoremediation technique, J. Hazard. Mater., 215– 216 (2012) 227-232. [7] P. Baldrian, Laccases-occurrence and properties, FEMS Microbiol. Rev., 30 (2006) 215-242. [8] D.S. Arora, R.K. Sharma, Ligninolytic fungal laccases and their biotechnological applications, Appl. Biochem. Biotechnol., 160 (2010) 1760-1788. [9] B. Ashe, L.N. Nguyen, F.I. Hai, D.-J. Lee, J.P. van de Merwe, F.D.L. Leusch, W.E. Price, L.D. Nghiem, Impacts of redox-mediator type on trace organic contaminants degradation by laccase: Degradation efficiency, laccase stability and effluent toxicity, Int. Biodeterior. Biodegradation, 113 (2016) 169-176. [10] D.W. Wong, Structure and action mechanism of ligninolytic enzymes, Appl. Biochem. Biotechnol., 157 (2009) 174-209. [11] R. Pogni, M.C. Baratto, A. Sinicropi, R. Basosi, Spectroscopic and computational characterization of laccases and their substrate radical intermediates, Cell. Mol. Life Sci., 72 (2015) 885-896. [12]
O.V.
Morozova,
G.P.
Shumakovich,
S.V.
Shleev,
Y.I.
Yaropolov,
Laccase-mediator systems and their applications: A review, Appl. Biochem. Microbiol., 43 (2007) 523-535. [13] E.I. Solomon, P. Chen, M. Metz, S.-K. Lee, A.E. Palmer, Oxygen binding, activation, and reduction to water by copper proteins, Angew. Chem. Int. Ed., 40 (2001) 4570-4590. [14] J.D. Cui, S.R. Jia, Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges, Crit. Rev. Biotechnol., 35 (2015) 15-28.
[15] R. Abejón, M.P. Belleville, J. Sanchez-Marcano, Design, economic evaluation and optimization of enzymatic membrane reactors for antibiotics degradation in wastewaters, Sep. Purif. Technol., 156 (2015) 183-199. [16] R. Abejón, M. De Cazes, M.P. Belleville, J. Sanchez-Marcano, Large-scale enzymatic membrane reactors for tetracycline degradation in WWTP effluents, Water Res., 73 (2015) 118-131. [17] M. de Cazes, M.P. Belleville, E. Petit, M. Llorca, S. Rodríguez-Mozaz, J. de Gunzburg, D. Barceló, J. Sanchez-Marcano, Design and optimization of an enzymatic membrane reactor for tetracycline degradation, Catal. Today, 236 (2014) 146-152. [18] M. de Cazes, M.P. Belleville, M. Mougel, H. Kellner, J. Sanchez-Marcano, Characterization of laccase-grafted ceramic membranes for pharmaceuticals degradation, J. Membrane Sci., 476 (2015) 384-393. [19] R.A. Sheldon, Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs), Appl. Microbiol. Biotechnol., 92 (2011) 467-477. [20] V.V. Kumar, S. Sivanesan, H. Cabana, Magnetic cross-linked laccase aggregates — Bioremediation tool for decolorization of distinct classes of recalcitrant dyes, Sci. Total Environ., 487 (2014) 830-839. [21] S. Talekar, A. Joshi, G. Joshi, P. Kamat, R. Haripurkar, S. Kambale, Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs), RSC Adv., 3 (2013) 12485-12511. [22] V.V. Kumar, H. Cabana, Towards high potential magnetic biocatalysts for on-demand elimination of pharmaceuticals, Bioresour. Technol., 200 (2016) 81-89. [23] A. Arca-Ramos, V.V. Kumar, G. Eibes, M.T. Moreira, H. Cabana, Recyclable
cross-linked laccase aggregates coupled to magnetic silica microbeads for elimination of pharmaceuticals from municipal wastewater, Environ. Sci. Pollut. Res., 23 (2016) 8929-8939. [24] M. Llorca, S. Rodríguez-Mozaz, O. Couillerot, K. Panigoni, J. de Gunzburg, S. Bayer, R. Czaja, D. Barceló, Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap, Chemosphere, 119 (2015) 90-98. [25] T. Suda, T. Hata, S. Kawai, H. Okamura, T. Nishida, Treatment of tetracycline antibiotics by laccase in the presence of 1-hydroxybenzotriazole, Bioresour. Technol., 103 (2012) 498-501. [26] H. Ding, Y. Wu, B. Zou, Q. Lou, W. Zhang, J. Zhong, L. Lu, G. Dai, Simultaneous removal and degradation characteristics of sulfonamide, tetracycline, and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorption, J. Hazard. Mater., 307 (2016) 350-358. [27] G. Songulashvili, G.A. Jimenéz-Tobón, C. Jaspers, M.J. Penninckx, Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants, Fungal Biol., 116 (2012) 883-889. [28] J. Yang, Q. Lin, T.B. Ng, X. Ye, J. Lin, Purification and characterization of a novel laccase from Cerrena sp. HYB07 with dye decolorizing ability, PLoS ONE, 9 (2014) e110834. [29] S.-C. Chen, P.-H. Wu, Y.-C. Su, T.-N. Wen, Y.-S. Wei, N.-C. Wang, C.-A. Hsu, A.H.-J. Wang, L.-F. Shyur, Biochemical characterization of a novel laccase from the basidiomycete fungus Cerrena sp. WR1, Protein Eng. Des. Sel., 25 (2012) 761-769.
[30] A. Michniewicz, S. Ledakowicz, R. Ullrich, M. Hofrichter, Kinetics of the enzymatic decolorization of textile dyes by laccase from Cerrena unicolor, Dyes Pigm., 77 (2008) 295-302. [31] A. Jarosz-Wilkołazka, T. Ruzgas, L. Gorton, Use of laccase-modified electrode for amperometric detection of plant flavonoids, Enzyme Microb. Technol., 35 (2004) 238-241. [32] D.T. D'Souza, R. Tiwari, A.K. Sah, C. Raghukumar, Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes, Enzyme Microb. Technol., 38 (2006) 504-511. [33] R. Bourbonnais, M.G. Paice, Demethylation and delignification of kraft pulp by Trametes
versicolor
laccase
in
the
presence
of
2,2
′
-azinobis-(3-ethylbenzthiazoline-6-sulphonate), Appl. Microbiol. Biotechnol., 36 (1992) 823-827. [34] S. Dhawan, R. Lal, M. Hanspal, R.C. Kuhad, Effect of antibiotics on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus, Bioresour. Technol., 96 (2005) 1415-1418. [35] J.A. Mir-Tutusaus, M. Masis-Mora, C. Corcellas, E. Eljarrat, D. Barcelo, M. Sarra, G. Caminal, T. Vicent, C.E. Rodriguez-Rodriguez, Degradation of selected agrochemicals by the white rot fungus Trametes versicolor, Sci. Total Environ., 500-501 (2014) 235-242. [36] J. Yang, X. Yang, X. Ye, J. Lin, Optimal parameters for laccase-mediated destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels, Data Brief, 7 (2016) 1-7. [37] M.R. Hu, Y.P. Chao, G.Q. Zhang, Z.Q. Xue, S. Qian, Laccase-mediator system in
the decolorization of different types of recalcitrant dyes, J. Ind. Microbiol. Biotechnol., 36 (2009) 45-51. [38] J. Margot, P.-J. Copin, U. von Gunten, D.A. Barry, C. Holliger, Sulfamethoxazole and isoproturon degradation and detoxification by a laccase-mediator system: Influence of treatment conditions and mechanistic aspects, Biochem. Eng. J., 103 (2015) 47-59. [39] S. Kurniawati, J.A. Nicell, Efficacy of mediators for enhancing the laccase-catalyzed oxidation of aqueous phenol, Enzyme Microb. Technol., 41 (2007) 353-361. [40] U. Fillat, A. Prieto, S. Camarero, Á.T. Martínez, M.J. Martínez, Biodeinking of flexographic inks by fungal laccases using synthetic and natural mediators, Biochem. Eng. J., 67 (2012) 97-103. [41] A.I. Cañas, M. Alcalde, F. Plou, M.J. Martínez, Á.T. Martínez, S. Camarero, Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil, Environ. Sci. Technol., 41 (2007) 2964-2971.
Figure legends Fig. 1 Preparation of M-CLEAs from Cerrena laccase. (A) Effect of enzyme loading on M-CLEA activity recovery. Fermentation broth and functionalized were mixed in different w:w ratios. Laccase was precipitated with ammonium sulfate and cross-linked with glutaraldehyde. (B) Effect of glutaraldehyde concentration and cross-linking time on M-CLEA activity recovery.
Fig. 2 SEM image of M-CLEAs of Cerrena laccase.
Fig. 3 Degradation of antibiotics by laccase. Individual antibiotics at 100 mg/L were treated with 20 U/mL immobilized laccase in the presence or absence of 5 μM ABTS at pH 7.0 and 25 ºC for 48 h. Residual antibiotic concentrations were determined by LC-MS/MS. Error bars represent standard deviation.
Fig. 4 Effect of pH (A) and enzyme activity (B) on TC (100 mg/L) degradation by laccase. Residual antibiotic concentrations were determined by LC-MS/MS.
Fig. 5 Mass spectra of TC transformation products uponafter laccase treatment. TC (100 mg/L) treatment was performed at pH 6 and 25 ºC with 40 U/mL laccase. TC (A, m/z 445.16) and TC transformation products (B-D, m/z 459.13, 431.11 and 396.07) were identified with LC-TOF MS.
Fig. 6 Formation of detected transformation products during TC degradation. TP, transformation product.
Fig. 7 Proposed degradation pathway of TC by laccase.
Tables Table 1 Optimized mass parameters and MRM transitions for antibiotics. Analytes
Tetracycline
Oxytetracycline
Ampicillin
Electrospray
MRM
Dwell
Declustering
Collision
mode
transition
time
potential
energy
(ms)
(V)
(V)
445>410
10
50
28
445>427
10
50
20
461>443
10
50
20
461>426
10
50
30
350>160
10
80
24
350>192
10
80
20
254>156
10
65
23
254>108
10
65
35
734>158
10
80
41
734>576
10
80
43
291>230
10
110
32
291>261
10
110
33
Negative
321>152
10
-85
-25
ions
321>257
10
-85
-16
Positive ions
Positive ions
Positive ions
Sulfamethoxazole Positive ions
Erythromycin
Trimethoprim
Chloramphenicol
Positive ions
Positive ions
The major MRM transition for quantification is indicated in boldface.
Table 2 IC50 values of TC and OTC for E. coli and B. licheniformis. IC50 (mg/L) TC
OTC
E. coli
2.2
2.5
B. licheniformis
1.4
1.2
Table 2 Antimicrobial activity of TC and OTC before and after laccase treatment.
TC
OTC
Growth inhibition (%)
Treatment time (h)
E.coli
B. licheniformis
0
99.5 ± 1.1
100.0 ± 0.4
24
27.8 ± 6.2
78.8 ± 3.5
48
5.8 ± 6.1
8.6 ± 4.6
0
98.4 ± 1.8
99.7 ± 0.4
24
34.9 ± 7.4
72.2 ± 4.7
48
7.3 ± 5.8
7.4 ± 6.5
Values represent means ± standard deviations (n=3).
Relative activity (%)
(A) 50 40 30 20 10 0 0.4:1
0.6:1
0.8:1
1.0:1
1.2:1
1.4:1
Laccase:MNPs (w/w) 60 Relative activity (%)
(B)
50 40 30 10 mM 20 mM 30 mM 40 mM
20 10 0 0
1
2
3
Time (h) Fig. 1
4
5
6
Fig. 2
Degradation (%)
80 Without ABTS
70 60 50 40 30 20
With ABTS
10 0
Antibiotics Fig. 3
(A) Degradation (%)
100 80 60 40 20 0 1
2
3
4
5
6
7
8
9 10
pH (B) Degradation (%)
120 100 80 60 40 20 0 0
10
20
30
Laccase (U/mL) Fig. 4
40
50
Fig. 5
Tetracycline TP 459 TP 431 TP 396
Relative abundance (%)
120 100 80 60 40 20 0 0
6
12
18
24
30
Time (h) Fig. 6
36
42
48
Fig. 7