Accepted Manuscript Title: Removal of sulfonamide antibiotics by oriented immobilized laccase on Fe3 O4 nanoparticles with natural mediators Author: Lili Shi Fuying Ma Yuling Han Xiaoyu Zhang Hongbo Yu PII: DOI: Reference:

S0304-3894(14)00554-8 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.06.070 HAZMAT 16082

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

30-1-2014 7-6-2014 21-6-2014

Please cite this article as: L. Shi, F. Ma, Y. Han, X. Zhang, H. Yu, Removal of sulfonamide antibiotics by oriented immobilized laccase on Fe3 O4 nanoparticles with natural mediators, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.06.070 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.

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Removal of sulfonamide antibiotics by oriented immobilized laccase on Fe3O4 nanoparticles with natural mediators

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Lili Shi, Fuying Ma, Yuling Han, Xiaoyu Zhang, Hongbo Yu*

Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Luoyu

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Road 1037,Wuhan 430074, PR China.

*Corresponding author. Tel.: +86 27 87792108; fax: +86 27 87792128.

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E-mail addresses: yuhongbo@ hust.edu.cn

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Abstract

A novel strategy was applied in the oriented immobilization of laccase from Echinodontium taxodii on concanavalin A-activated Fe3O4 nanoparticles (GAMNs-Con A) based on laccase surface analysis. These nanoparticles showed higher enzyme loading and activity recovery compared with conventional covalent binding. Along with the improvement in thermal and operational stabilities, the oriented immobilized laccase (GAMNs-Con A-L) exhibited higher substrate affinity than free laccase. Free laccase and GAMNs-Con A-L were then applied in the removal of sulfonamide antibiotics (SAs). Although both free and immobilized laccase resulted in the rapid removal of SAs, GAMNs-Con A-L showed a higher removal rate of SAs compared with the free

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counterpart in the presence of S-type compounds present in lignin structure. Syringic acid mediated the fastest removal efficiency of SAs among S-type

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compounds and resulted in an almost complete removal of these substances after incubation for 5 min. The oxidation products of SAs were identified via LC-ESI+-MS. The results suggested the transformation of SAs and S-type compounds were catalyzed by laccase, resulting in the formation of

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cross-coupled products.

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Keywords:

Introduction

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magnetic nanoparticles; laccase; immobilization; sulfonamide antibiotics; natural mediator.

Antibiotics have been investigated as sources of emerging environmental contaminants. The presence of these substances in the environment changes the microbial ecology, increases the proliferation of antibiotic resistant pathogens, and provokes toxic effects on aquatic species and negative effects on

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human health [1]. As a major class of antibiotics, sulfonamide antibiotics (SAs) are widely used for the treatment of bacterial, protozoal, and fungal

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infections in human therapy, livestock production, and aquaculture [2]. These substances account for a high proportion of the total usage of antibiotics worldwide [3]. However, these substances are frequently overused and incompletely metabolized in human and animal bodies [4]. Several SA residues

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remain and are transferred into water bodies because of their low sorption rates in soils and sediments [5]. SAs cannot be effectively eliminated in conventional wastewater treatment plants because of their anionic characteristics [6]. Therefore, searching for effective and eco-friendly solutions to

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remove SAs in water bodies is necessary. The use of oxidative enzymes, such as laccases, for the treatment of antibiotics (e.g., fluoroquinolones,

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tetracyclines, and SAs) [7-9] have received considerable attention because of their rapid oxidative ability in mild and energy-saving conditions. Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) are multicopper oxidases that can catalyze the one-electron removal of various

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substrates coupled with four-electron reduction of molecular oxygen to water [10]. Studies have indicated that laccases have enormous potential in the remediation and treatment of contaminated water or soil, such as decolorization of dyes [11] and degradation of pesticides [12]. However, the low stability and high production costs of laccases have limited their industrial application [13]. Immobilization is an effective and the most straightforward way to overcome these limitations by improving enzyme properties, such as increasing their thermostability and resistance to extreme conditions [14]. Various carriers have been reported to immobilize laccases successfully, such as nanoparticles, chitosan, and nylon membrane [15]. Among various carriers, Fe3O4

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nanoparticles are highly suitable supports for enzyme immobilization because of their high specific surface areas, low mass transfer resistances, low

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operational costs, facility of reusability and modification on the surface with various active groups [16]. Conventional immobilization of laccases on magnetic nanoparticles relies on either non-specific physical adsorption or covalent binding. Physical

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adsorption is a mild and easy method of immobilizing laccase, but nonuniform adsorption leads to enzyme leakage from carriers. Covalent immobilization is usually more stable, and the enzyme is not released into the solution upon use. However, laccase activity may be hindered since amino acid residues of

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active center are involved in the covalent linkage to the functional groups of magnetic nanoparticles from the random cross-linking between proteins and

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supports. Bioaffinity-based enzyme immobilization is a potential strategy that is mild and only has slight negative effects on the immobilized enzymes. Moreover, this procedure confers oriented immobilization to enzymes, which facilitates good expression of activity and reusability [17, 18]. Enzymes can

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be immobilized via biospecific interactions, such as antibody-antigen, avidin-biotin, and lectin-sugar interactions [19]. Laccase is a glycoprotein and can be oriented immobilized on carriers based on the interaction between lectin concanavalin A (Con A) and glycosyl of glycoprotein. The oriented immobilization on Fe3O4 nanoparticles via interaction between Con A and glycosyl of laccase has not been reported. In

this

work,

Fe3O4

nanoparticles were

prepared

rapidly

via

chemical

co-precipitation,

and

were

then

functionalized

with

3-aminopropyltriethoxysilane (APTES), glutaraldehyde, and Con A. Subsequently, laccase was oriented immobilized on the functionalized Fe3O4

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nanoparticles by affinity adsorption and was used in the removal of SAs. The best types of natural mediators were screened, and the effects of the screened

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natural mediators on the removal of SAs were analyzed. The removal products of SAs were identified via LC-ESI+-MS, and the possible removal mechanism was proposed.

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2. Experimental

2.1 Preparation of the amino-functionalized Fe3O4 nanoparticles (AFMNs)

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Fe3O4 nanoparticles were prepared simply and rapidly via chemical co-precipitation and were then coated with poly(ethylene glycol) (PEG, Mn=4000)

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[20]. Ferrous chloride tetrahydrate (FeCl2·4H2O) and ferric chloride hexahydrate (FeCl3·6H2O) as iron sources (Fe2+/Fe3+=1/1.75), and PEG 4000 were

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dissolved in distilled water under nitrogen gas with vigorous stirring at 80 °C. Fe3O4 nanoparticles were obtained by adding aqueous ammonia (NH4OH, 30%) to adjust the pH value to 10 under vigorous stirring. After stirring for 30 min, the synthesized Fe3O4 nanoparticles were washed with deionized water and ethanol thrice and were coated directly with APTES. This process was performed as previously described [21]. The Fe3O4 nanoparticles and APTES with a molar ratio of 1:4 were dissolved in 150 mL of ethanol/water (1:1 volume ratio) solution. The solution was vigorously stirred under N2 at 40 °C for 2 h. The synthesized AFMNs were collected using a magnetic separation device, washed with deionized water and ethanol for three times, and stored at 4 °C.

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2.2 Preparation of laccase from Echinodontium taxodii

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Laccase was produced from the white-rot fungus E. taxodii, which was obtained from Shennongjia Scenic Area in Hubei of China. The production and purification steps of laccase were carried according to the reference[22]. The laccase was harvested at the peak of laccase activity and purified by three

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steps including salt-out with ammonium sulfate, hydrophobic interaction chromatography with Phenyl Sepharose 6 Fast Flow and ion-exchange chromatography with DEAE-Sepharose 6 Fast Flow (GE Healthcare, Uppsala, Sweden). The sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE) result showed that only one band was observed in the purified sample. The purified laccase solution (30.8 U mg-1, 0.3 mg mL-1) was used

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directly for subsequent immobilization.

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2.3 Oriented immobilization of laccase

The oriented immobilization of laccase on the surface of the Fe3O4 nanoparticles was performed via interaction between Con A and glycosyl of laccase. Random immobilization via covalent binding was used as the control process. Fig.S1 shows the scheme for conjugating laccase onto the surface of the nanoparticles via two different strategies. For random immobilization, 100 mg of AFMNs was dispersed in 10 mL of phosphate buffer (20 mmol·L-1, pH 8.0) and treated ultrasonically for 30 min. Subsequently, glutaraldehyde (4% final concentration) was added and shaken for 3 h at 30 °C. The nanoparticles were then separated via magnetic decantation and washed thrice to remove the unattached glutaraldehyde. The glutaraldehyde-activated

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Fe3O4 nanoparticles (GAMNs) were directly added to 10 mL of laccase solution (30.8 U·mg-1, 0.3 mg·mL-1), and the resulting mixture was shaken at 200

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rpm at 30 °C for 12 h. For oriented immobilization, the same amount of GAMNs were added into a pre-activated Con A solution (0.8 mg·mL-1, Con A was pre-activated in 0.1 mol·L-1 phosphate buffer at pH 7.0 containing 0.1 mol·L-1 KCl, 0.1 mmol·L-1 CaCl2, and 0.1 mmol·L-1 MnCl2 for 6 h) and shaken at

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200 rpm at 30 °C for 3 h. GAMNs-Con A complexes were then added to 10 mL of laccase solution (30.8 U·mg-1, 0.3 mg·mL-1) on a rotary shaker at 200 rpm at 30 °C for 2 h after repeated washing. The immobilized laccase was removed via magnetic decantation and washed three times with phosphate

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buffer (20 mmol L-1, pH 7.0) after completion of the immobilization to eliminate the unbound laccase. The laccase binding efficiency is defined as:

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E(%)=C1-C0/C1×100, where E is the laccase binding efficiency, and C1 and C0 are the amounts of the laccase protein that exist in the solution before and after immobilization, respectively. The activity recovery of the immobilized laccase is calculated as: R(%)=Ai/Af×100, where R is the activity recovery of

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the immobilized laccase, Ai is the activity of the immobilized laccase, and Af is the activity of the same amount of free laccase in solution as that immobilized on nanoparticles[23]. 2.4 Activity and protein assays

The activity of free and immobilized laccase was determined spectrophotometrically at 420 nm with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate [23]. The reaction mixture (2 mL) contained 100 μL of 10 mmol·L-1 ABTS, 1.8 mL of 50 mmol·L-1 sodium acetate buffer

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solution (pH 4.8) and 100 μL of a suitable amount of free or immobilized laccase sample. The amount of free and immobilized laccase was chosen to

measured for 3 min at 30 °C with the

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make the absorbance of the reaction product within the linear of the spectrophotometer used in the experiment. The oxidation of substrate to ABTS+ was molar extinction coefficient of 36,000 M-1·cm-1. One unit of enzyme activity was defined as the amount of enzyme

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that can oxidize 1 umol of substrate per minute. The amount of laccase protein was determined via the Bradford method by using bovine serum albumin (BSA) as the standard.

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2.5 Properties of free and immobilized laccase

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The thermal stability was measured after incubating the free and immobilized laccase at 60 °C for 2 h, and the residual activity was determined every 30 min by using ABTS as the substrate. The kinetic parameters of the free and immobilized laccase were determined using different substrates,

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whose concentration ranged from 0.1 mM to 1.0 mM. The protein concentration of free and immobilized laccase was equal. Km and Vmax were calculated based on Lineweaver-Burk plots at 30 °C and pH 4.8 in each case. The operational stability of the immobilized laccase was assessed by performing several consecutive operating cycles by using ABTS as the substrate. At the end of each cycle, the immobilized laccase was separated using a magnet, washed thrice with sodium acetate buffer (pH 4.8), and the process was repeated with a fresh aliquot of substrate. 2.6 Removal of sulfonamide antibiotics (SAs)

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Sulfadiazine (SDZ), sulfamethazine (SMZ), and sulfamethoxazole (SMX) were selected as representative SAs to study the effect of

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laccase-mediated removal. The concentration of 1000 mg L-1 of the stock solution was prepared by dissolving or dispersing each in methanol. The stock solution was then added into sodium acetate buffer (20 mmol·L-1, pH 5.0), and the final concentration of each constituent in the reaction solutions was 50

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mg·L-1. Three types of lignin-derived compounds were added into the reaction mixture as mediators with a final concentration of 1 mM, and the structure type with the best promotion effect on SAs removal was selected for further study. Control samples were run in parallel without the addition of mediators.

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The reaction was initiated by adding free or immobilized laccase at mild shaking conditions in the presence of mediator. The time of removal was

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determined by monitoring the residual concentrations of the SAs via high-performance liquid chromatography (HPLC). The immobilized laccase was separated using a magnet after its treatment. The solution was filtered with cellulose acetate filter (0.22 µm) prior to the HPLC analysis of residual SAs.

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The removal percentage was calculated as: removal percentage (%)=C0 – Ct/C0 × 100, where C0 is the initial concentrations of SAs and Ct is the residual concentrations of SAs after t minutes of treatment at experimental conditions. To ensure that the removal percentage was attributed only to laccase catalysis and not to adsorption on the carrier, a control process was carried out at the same operational conditions by using the same amount of heat-denatured immobilized laccase. The free laccase was also used as a control sample, and the sample treatment was slightly different from that of the immobilized laccase. After treatment of the same activity of free laccase, 1 N NaOH was added into the mixture solution, and the pH was adjusted to 7–8

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to stop the enzyme reaction prior to HPLC analysis. All reactions were performed in triplicate.

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2.7 Analytical procedures

The concentrations of SAs were determined via HPLC by using a system equipped with a UV detector at 270 nm. Ultrapure water containing 0.1%

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formic acid (v/v) and methanol were used as the mobile phases[24], and the ratio(v/v) was 60:40. A WondaSil-18 column (250 mm×4.6 mm, 5 μm) was used to separate SAs at room temperature with a flow rate of 1.0 mL·min−1 and an injection volume of 10 μL for each sample. The intermediates were

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analyzed via high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Chromatographic separation was

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performed using a WondaSil-18 column (250 mm×4.6 mm, 5 μm) at room temperature with a flow rate of 1.0 mL·min-1 and an injection volume of 50 μL for each sample. The detection wavelength was 270 nm, and a Hitachi L-7455 diode array detector was used. The mobile phase comprised 0.1% formic

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acid (eluent A) and methanol (eluent B). The elution started with 20% of eluent B up to a gradient linear increase to 80% for 30 min. LC-ESI-MS analyses were conducted in the positive ionization mode. The drying gas was operated at a flow rate of 10 mL min-1 at 280 °C. The nebulizer pressure and the capillary voltage were 40 psig and 3500 V, respectively. 2.8 Assay for antibacterial activity The well agar diffusion method [25] was used to determine the antibacterial activity of three SAs solutions in the laccase–mediator system (LMS)

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oxidation process. The sampling times for both SAs were 5 min in the LMS with syringic acid, 30 min in the LMS with syringaldehyde and

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acetosyringone. The antibacterial activity assay employs Staphylococcus aureus and Escherichia coli as the typical bacteria. The diameter of the inhibition zone was measured after 12 h of incubation at 37 °C. The antibacterial activity was evaluated by measuring inhibition diameter surrounding the wells. The

3. Results and discussion

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solution of SAs, the mixture solution of SAs and mediators act as the control. Each experiment was carried out in triplicate.

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3.1 Characterization of the amino-functionalized Fe3O4 nanoparticles (AFMNs)

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PEG4000 was used in the synthesis of Fe3O4 nanoparticles in this study because this compound can be coated around Fe3O4 nanoparticles and can prevent further aggregation, thereby enhancing the stability of magnetic fluids [26]. The successful coating of PEG and 3-APTES on these particles was

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confirmed via FT-IR and TGA thermogram analysis (Fig.S2). Fig.S2A shows the FTIR spectra of both unfunctionalized and amino-functionalized Fe3O4 nanoparticles. Compared with Fig.S2A(a), the significant features observed in Fig.S2A(b) are the appearance of peaks at1080 cm-1 (Si–O stretching) and at near 2900 cm-1 (–CH2 stretching).The peak at 3400 cm-1 (Fig.S2A (b)) is attributed to the free amino groups overlapped by the O–H stretching vibration. The results demonstrate the successful attachment of amino groups to the surface of PMNs. Fig. S2B shows the TGA thermograms of PMNs and AFMNs, in which AFMNs shows an increase in weight loss at above 350 °C compared with that of PMNs, which is attributed to the loss of the APTES layer. The

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weight loss of APTES coated on the PMNs is about 4.6%. The XRD pattern in Fig.S2C suggests that the nanoparticles were Fe3O4 with a comparatively

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high purity. The diameters of PMNs and AFMNs are 13.4 and 16.7 nm, respectively. 3.2 Immobilization of laccase

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3.2.1 Surface analysis of laccase

The laccase protein structure was analyzed to determine the interaction between the NH2 groups of amino acid residues and N-glycosylation sites of

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laccase with the functionalized groups of PMNs. The gene sequence of laccase (No. KF432088.1) from E. taxodii was obtained from NCBI service. The

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3D structure of the enzyme was obtained via de novo modeling by using the Rosetta software suite and analyzed using PyMoL software simulation. Fig. 1 shows that the laccase from E. taxodii contained six NH2 groups (one N-terminus and five Lys) on the surface, and three NH2 groups were located at the

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rear part of the substrate-binding pocket of the enzyme. However, the other NH2 groups formed an area near the substrate-binding pocket, which indicated that random covalent binding could cause partial loss of laccase activity because of multiple-point binding and steric hindrance of carriers. On the contrary, three N-glycosylation sites were dispersed on the laccase surface and far from the substrate-binding pocket of the enzyme, which indicates that the affinity absorption via lectin-sugar interaction could result in good activity expression. 3.2.2 Characterization of immobilized laccase

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3.2.2.1 Enzyme loading and activity recovery

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Purified laccase was immobilized on the GAMNs and the GAMNs-Con A complexes via covalent binding (designated as GAMNs-L) and affinity absorption (designated as GAMNs-Con A -L), respectively. Table 1 shows the laccase binding efficiency, enzyme loading and activity recovery for two

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different immobilization methods. The laccase binding efficiency and enzyme loadings were 60.7% and 18.2 mg protein g-1support for covalent binding, 98.0% and 29.4 mg protein g-1support for affinity adsorption. Compared with covalent binding, more laccase proteins were conjugated onto the

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GAMNs-Con A complexes and the loadings of laccase were enhanced 1.6-fold. This difference may be due to more binding sites on GAMNs-Con A

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complexes. At neutral pH, Con A provided four glycosyl binding sites, which is more than that of GAMNs, and resulted in more laccase proteins being captured. It may also attribute to the fact that too little amount of NH2 groups caused lower enzyme loading. A similar result was also observed in laccase

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and horseradish peroxidase immobilization on single-walled carbon nanotubes [27]. Activity recovery is an index that reflects the effectiveness of an immobilization method [27]. Only 6.2% decline in the activity of GAMNs-Con A-L was observed compared with that in free laccase, whereas the activity of GAMNs-L declined by 17.6% (Table 1). This result was consistent with that of the laccase surface analysis. The covalent binding of enzymes on carriers inevitably altered their conformations and thus decreased their activity. By contrast, affinity adsorption strategy minimized the conformational

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groups of laccase were far from the active center (Fig. 1).

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change in the enzymes during immobilization [27]. In addition, affinity adsorption strategy might not result in any steric hindrance because the glycosyl

Laccase immobilizations on different magnetic carriers, such as magnetic chitosan [28], magnetic PVA-DVB-g-GMA-IDA-Cu2+ particles [29],

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and Fe3O4/SiO2 composite particles [30], have been reported (Table 1). Compared with these carriers, GAMNs-Con A complexes exhibited higher enzyme activity and activity recovery, which suggested that oriented immobilization based on affinity adsorption between Con A and glycosyl is an efficient

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strategy for laccase immobilization.

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3.2.2.2 Thermal and operational stability

Fig. 2A shows the comparison of thermal stability of the free and immobilized laccase. GAMNs-Con A-L and GAMNs-L still both retained over

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40% of its initial activity when treated for 2 h at 60 °C, but the free laccase was almost deactivated at similar experimental conditions. These results revealed that the thermal stability of GAMNs-Con A-L was significantly enhanced compared with that of the free counterpart. This phenomenon was attributed to the interactions between laccase and carrier, which enhanced the rigidity of the laccase molecular structure of. Moreover, the immobilized laccase became more resistant to heat inactivation [31]. The operating stability of the immobilized laccase was also studied because of the importance of application potential in reducing processing costs. Fig. 2B shows that the immobilized laccase retained 60.1% of the original activity after 10 consecutive

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operations when directly immobilized on GAMNs. However, 82.8% of the original activity was retained on the GAMNs-Con A complexes under the same

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condition. The results of this study indicated that GAMNs-Con A-L had a better reusability. 3.2.2.3 Kinetic parameters

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The Vmax and Km values for free and immobilized laccase were calculated based on Lineweaver-Burk plots. Table 2 lists the kinetic parameters of the free and immobilized laccase. An increase in the apparent Km for GAMNs-L was observed, which was consistent with other studies where an affinity

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decrease for covalent immobilization has been reported. The decreased affinity of immobilized enzyme to its substrate was probably caused by mass

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transport constraint and by structural changes of the proteins during covalent binding [30]. By contrast, the apparent Km for GAMNs-Con A-L was lower than that of the free counterpart, which indicated GAMNs-Con A-L had a higher affinity for ABTS. A similar phenomenon was observed in the laccase

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immobilization on Con A-attached super macroporous cryogel [32]. Because the protein concentration of free laccase is the same as that of immobilized laccase, Vmax/Km value can be used to compare the catalytic efficiency between free and immobilized laccase. As shown in Table 2, the Vmax/Km of free laccase was 0.143 min-1, whereas those of GAMNs-L and GAMNs-Con A-L were 0.1 and 0.2 min-1. GAMNs-Con A-L exhibited the highest catalytic efficiency for ABTS compared with free laccase and GAMNs-L. 3.3 Removal of SAs

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3.3.1 Natural mediator screening

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Studies have indicated that the removal of SAs by laccase alone was limited because the redox potential of laccase was not high enough to oxidize SAs [7, 33]. A similar result was observed using free laccase and GAMNs-Con A-L as biocatalysts, which both showed no removal ability towards the

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tested SAs. However, the SAs were oxidized in the presence of natural mediators (Fig. 3). Nine lignin-derived compounds mediated the removal of SAs. These compounds contain H-, G-, and S-type subunits present in lignin and have been verified to mediate the decolorization of dyes and degradation of

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organic pollutants [15]. The effects on SAs removal by free laccase in the presence of mediators were similar to those by GAMNs-Con A-L. All the tested

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mediators enhanced the removal of SAs, and the largest reductions in SAs concentration were observed in incubations with S-type compounds (syringaldehyde, syringic acid, and acetosyringone). On the contrary, G-type compounds (vanillin, acetovanillone, vanillic acid, and ferulic acid) and

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H-type compounds (p-coumaric acid and p-hydroxybenzoic acid) were less effective in enhancing the removal of SAs, although several were used as substrates for laccase (Fig. 3). These results could be attributed to the reason that more methoxy groups of S-type compounds decreased redox potentials and increased electron densities at the phenoxy group, which allowed these compounds to be easily oxidized by laccase [34]. Meanwhile, more stable radicals exist since the substitutions S-type compounds imposed steric hindrances for the polymerization via radical fusion [12]. Among three different structural types of mediators, all S-type compounds resulted in the significant removal of SAs among three structural types of mediators, and more than

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95% of the SAs were oxidized after incubation for 30 min with a laccase concentration of 0.2 U·mL-1. The following oxidization experiments were thus

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performed using S-type compounds as mediators. 3.3.2 Laccase-mediated removal of SAs

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As effective natural mediators, S-type compounds were used to mediate the removal of SAs with free laccase and GAMNs-Con A-L, both resulted in the quick removal of the SAs, and the reduction in SAs was consistent with the first-order kinetic model with correlation coefficient (R2) values ranging

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from 0.92 to 0.99 (Fig. 4A). The addition of syringaldehyde resulted in the rapid reductions of SAs, only 0.7% to 2.5% of residual SAs was detected via

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HPLC after incubation for 30 min with a laccase concentration of 0.4 U·mL-1. This result is better than Weng et al. [7], in which laccase from the Perenniporia strain TFRI 707 with syringaldehyde resulted in 83% to 90% removal of sulfadimethoxine and sulfamonomethoxine with 6 U·mL−1 laccase

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and the respective half-lives (t1/2s) or times required for 50% dissipation of the initial concentration were 19.0 and 32 min. This might be due to the difference in catalytic activities of laccases from different sources. A previous study has indicated that laccase from E. taxodii had a high redox potential and significantly decolorized dyes in the absence of any redox mediator. The rapid decline in SAs was also observed when acetosyringone was used as the mediator, in which 1.2% to 5.1% of the residual SAs remained after incubation for 30 min with a laccase concentration of 0.4 U·mL-1. Compared with syringaldehyde and acetosyringone, syringic acid demonstrated the best promotion effect on the removal of SAs, thereby resulting in an almost complete

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removal of these substances after incubation for 5 min at the same experimental conditions. The different affinities of laccase to syringic acid,

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syringaldehyde, and acetosyringone might cause the variation in the catalytic efficiency of SAs. This study indicated that laccase from E. taxodii exhibited a higher affinity for syringic acid than for syringaldehyde and acetosyringone (Table 2).

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Similar results were observed using GAMNs-Con A-L as the catalyst under the same experimental conditions. GAMNs-Con A-L exhibited the higher catalytic rate in the existence of S-type compounds compared with free laccase. For example, the respective t1/2s of SDZ, SMZ, and SMX were 1.41, 0.79,

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and 1.57 min with syringic acid by using free laccase as the catalyst, whereas those of SDZ, SMZ, and SMX were reduced to 0.99, 0.52, and 1.02 min (Fig.

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4B). Laccase usually oxidizes the mediator to free radicals, and the radicals consequently oxidize the non-specific substrates by resorting to removal mechanisms, which result in the effective removal of a wide spectrum of compounds. Thus, the affinity between laccase and mediator has a direct impact

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on the catalytic rate of substrates. Laccase was immobilized on the GAMNs-Con A complexes, which changed the affinity of syringic acid and caused the higher catalytic rates of SAs (Table 2). 3.3.3 Removal mechanism of SAs The intermediates were analyzed via LC-ESI+-MS to determine the possible removal mechanism. SAs were oxidized using syringaldehyde and syringic acid as mediators. The color of solution turned yellow, new peaks at m/z of 401, 404, and 429 were observed, which were attributed to SDZ,

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SMX, and SMZ, respectively (Table 3), and continually increased with the reduction in SAs. These results were not observed in the control process

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without laccase, which indicated that syringaldehyde and syringic acid were coupled with SAs in the presence of laccase. Moreover, the difference in the molecular weight of the new peaks was the same as that of SAs, which suggests that SAs shared a common reaction pathway in the LMS with

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syringaldehyde and syringic acid. Both two mediators can be oxidized to 2,6-dimethoxybenzoquinone (2,6-DMBQ) [35]. The present study assumed that these compounds were first oxidized by laccase to an intermediate of 2,6-DMBQ and subsequently coupled with SAs to form cross-coupled products with

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molecular weights higher than those of SAs and 2,6-dimethoxyphenols (Fig. 5). The result was similar with a previous study that phenoloxidase mediated

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covalent cross-coupling of SAs and model humic constituents [33]. It turned to be purple during the removal of SAs with acetosyringone as the mediator. Besides the peaks at m/z of 401, 404, and 429, those at m/z of

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429, 432 and 457 also appeared and were attributed to SDZ, SMX, and SMZ, respectively (Table 3), which indicated that the oxidization of SAs may have another coupling route using acetosyringone as the mediator in the presence of laccase. The assumed coupling route started with the deprotonation of acetosyringone to generate the phenoxy radical [36], and this radical was coupled with SAs to form the cross-coupled products. The other route might be that acetosyringone could be oxidized to syringaldehyde [35] and then coupled with SAs in the presence of laccase (Fig. 5). A higher molecular weight of 28 g·mol-1 was attributed to the acetyl groups of the chemical structure in acetosyringone.

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3.4 Assay for antibacterial activity

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SAs can inhibit both gram-positive and gram-negative bacteria. After 12 h of incubation, three SAs all showed distinct inhibition zones against S. aureus and E. coli, and the diameter of the inhibition zone ranged from 9.4 mm to 12.2 mm. Addition of mediators didn’t affect the antibacterial activity of

ed

the three SAs. Interestingly, the inhibition zone disappeared when the solutions of three SAs oxidized by LMS were added into the wells, which indicated the antibacterial activity of SAs was destroyed after the LMS treatment. In general, the vacant para amino group is essential for the antibacterial activity of

pt

SAs[33], laccase mediated cross-coupling of SAs and S-type compounds, the covalent linkage formed via the anilinic nitrogen eliminated their

ce

antibacterial activities. However, it is reported that some natural mediators such as syringaldehyde can be oxidized to the intermediates, resulting in higher toxicity[37]. So the toxic effect of the solutions of SAs oxidized by LMS need be noticed, although these solutions don’t exhibit the antibacterial activity.

Conclusion

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Further studies need to be performed to screen the mediator with the lowest toxicity.

The laccase from E. taxodii was oriented immobilized on Fe3O4 nanoparticles based on the interaction between Con A and glycoprotein. Compared with conventional covalent binding method, the oriented immobilization showed an increase in enzyme loading and retained the activity. When syringic acid was used as the mediator, the oriented immobilized laccase resulted in an almost complete removal of SAs after incubation for 5 min and showed a

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higher removal rate compared with the free counterpart. The results indicated that this enzyme is a novel biocatalyst for application in bioremediation.

ed

Acknowledgement

pt

This work was supported by the National High Technology Research and Development Program of China (No. 2012AA101805), the National Natural Science Foundation of China (No. 31170104) and the Fundamental Research Funds for the Central Universities, HUST (No. 2013QN092). The authors

Ac

HPLC-ESI+-MS.

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also thank the Analytical and Testing Center of Huazhong University of Science and Technology for the analysis of FTIR, TGA, X-ray and

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[24] X.T. Zhao, Q.B. Lin, H. Song, Y.L. Pan, X. Wang, Development of an immunoaffinity chromatography purification and ultra performance liquid chromatography tandem mass spectrometry method for determination of 12 sulfonamides in beef and milk, J. Agric. Food Chem. 59 (2011) 9800-9805. [25] R.L. Thakur, U. Roy, Antibacterial activity of Leuconostoc lactis isolated from raw cattle milk and its preliminary optimization for the bacteriocin production, Res. J. Microbiol. 4 (2009) 122-131. [26] B. Hu, J. Pan, H.L. Yu, J.W. Liu, J.H. Xu, Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for

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[31] Y. Zhu, S. Kaskel, J. Shi, T. Wage, K.-H. van Pée, Immobilization of Trametes versicolor laccase on magnetically separable mesoporous silica spheres, Chem. Mat. 19 (2007) 6408-6413. [32] C. Altunbaş, M. Uygun, D.A. Uygun, S. Akgöl, A. Denizli, Immobilization of Inulinase on Concanavalin A-Attached Super Macroporous Cryogel for Production of High-Fructose Syrup, Appl. Biochem. Biotechnol. 170 (2013) 1-13.

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[33] H.M. Bialk, A.J. Simpson, J.A. Pedersen, Cross-coupling of sulfonamide antimicrobial agents with model humic constituents, Environ. Sci. Technol.

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Mol. Catal. B-Enzym. 97 (2013) 45-53.

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[37] A. Fillat, J.F. Colom, T. Vidal, A new approach to the biobleaching of flax pulp with laccase using natural mediators, Bioresour. Technol. 101 (2010)

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Tables

GAMNs-Con A

Echinodontium taxodi Echinodontium taxodi Trametes versicolor Trametes versicolor

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Fe3O4/SiO2 composite particles

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GAMNs

Magnetic chitosan

Immobilization method

Laccase source

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Immobilization support

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Table 1 Performance of laccase immobilization on various supports

ECa (mg g-1 carrier)

ACb (U g-1 carrier)

Rc(%)

References

covalent binding

60.7%

18.2

462

82.4%

This work

affinity adsorption

98.0%

29.4

849

93.8%

This work

covalent binding

31.3%

62.6

224

83.9%

covalent binding

--

16.3

260

79.6%

62.7%

94.1

7

68.0%

Magnetic Pycnoporus affinity adsorption PVA-DVB-g-GMA-IDA-Cu2+ sanguineus particles a EC: Enzyme content per gram particles. b AC: Activity of immobilized laccase. c R (%) is the activity recovery of immobilized enzyme.

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Binding efficiency (%)

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Zheng et al., (2012) Bayramoglu et al., (2010) Wang et al., (2008)

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Km(umol L-1)

Vmax(μmol·L-1·min-1)

Vmax/ Kma(min-1)

Free laccase GAMNs-L GAMNs-Con A-L Free laccase GAMNs-Con A-L Free laccase GAMNs-Con A-L Free laccase GAMNs-Con A-L

41.4 42.7 35.2 74.0 44.2 985.2 907.4 668.6 652.2

5.9 4.3 7.0 56.1 77.0 160.1 179.3 120.7 135.3

0.14 0.10 0.20 0.76 1.74 0.16 0.19 0.18 0.21

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Acetosyringone

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Syringaldehyde

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Syringic acid

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Form of enzyme

Substrate ABTS

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Table 2 Kinetic constants of free and immobilized laccase

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a Catalytic efficiency was defined as the ratio Vmax/Km.

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Table 3 Molecular ions detected by LC-ESI+-MS for the main removal products of sulfonamide antibiotics in laccase mediator systems.

17.8 19.5 19.1 21.4 22.5 23.9 19.5 21.4 23.9 19.5 21.4 23.9

429 401 457 429 432 404 401 429 404 401 429 404

SDZ

acetosyringone

SDM SMX

syringaldehyde

syringic acid

SDZ SDM SMX SDZ SDM SMX

(a) (d) (b) (e) (c) (f) (d) (e) (f) (d) (e) (f)

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te

d

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a New peak: Not sulfonamide and mediator peaks. b RT: Retention time. c Proposed structure are shown on Fig 5.

Proposed structurec

ip t

M/Z(+)

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RT(min)b

cr

New peaka

SAs

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2,6-dimethoxyphenols

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Figure Captions Figure 1 Distribution of NH2 groups (N-terminus and Lys residues) and N-glycosylation sites on the surface of laccase from E. taxodii. NH2 groups are shown

ip t

in blue, N-glycosylation sites are shown in green and the active site is shown in red. (A) front view;(B) rear view.

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Figure 2 Properties of immobilized laccase. ( ■ )free laccase, ( ●)GAMNs-L, and

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(▲)GAMNs-Con A-L. (A) thermal stability at 60°C; (B) operational stability.

Figure 3 Screening for natural mediators based on the removal of SAs after 30 min

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treatment with GAMNs-Con A-L at a concentration of 0.2 U mL-1.

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Figure 4 Mediated removal of SAs in the presence of natural mediators at a concentration of 0.4 U mL-1.

d

(A) Comparison of the mediated removal of SAs with free laccase: (a) SDZ; (b) SMZ;

te

(c) SMX. The mediators are syringic acid(▲), syringaldehyde(■) and

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acetosyringone(●) at concentration of 1 mM. Control(◇)is treated with free laccase without the mediator.

(B) Comparison of syringic acid- mediated removal of SAs with free laccase and GAMNs-Con A-L: (a) SDZ; (b) SMZ; (c) SMX. (■) free laccase; (●) GAMNs-Con A-L.

Figure 5 The proposed removal pathways of the SAs in laccase-mediator systems using syringic acid, syringaldehyde and acetosyringone as mediators.

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ip t cr Ac c

ep te

d

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Fig. 1

(A)

(B)

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ip t cr Ac c

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d

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Fig. 2

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Ac

ce pt

ed

M

an

us

cr

ip t

Fig. 3

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ip t cr Ac c

ep te

d

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Fig.4

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ip t cr Ac c

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d

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Fig.5

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