# ABTS-Modified Silica Nanoparticles as Laccase Mediators for Decolorization of Indigo Carmine Dye

Authors: Youxun  Liu; Mingyang Yan; Yuanyuan Geng; Juan Huang
Journal: Journal of Chemistry
 (2015)

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## Abstract

Efficient reuse and regeneration of spent mediators are highly desired for many of the laccases’ biotechnology applications. This investigation demonstrates that a redox mediator 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) covalently attached to silica nanoparticles (SNPs) effectively mediated dye decolorization catalyzed by laccase. Characteristics of ABTS-modified silica nanoparticles (ABTS-SNPs) were researched by scanning electron microscopy and Fourier-transformed infrared spectroscopy. When ABTS and ABTS-SNPs were used as laccase mediators, the decolorization yields of 96 and 95% were, respectively, obtained for indigo carmine dye. The results suggest that ABTS immobilized on SNPs can be used as laccase mediators as they retain almost the same efficiency as the free ABTS. The oxidized ABTS-SNPs were regenerated by their reduction reaction with ascorbic acid. Decolorization efficiency of regenerated ABTS-SNPs and their initial forms were found to be almost equivalent. Six reuse cycles for spent ABTS-SNPs were run for the treatment of indigo carmine, providing decolorization yields of 96–77%. Compared with free mediator, the immobilized mediators have the advantage of being easily recovered, regenerated, and reused making the whole process environmentally friendly.


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## Body

## 1. Introduction

It is known that laccase (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) belongs to the family of the multicopper oxidases [1, 2]. It can oxidize various organic substrates including phenols and arylamines, even some inorganic compounds. In catalytic reactions of laccase, oxygen acts as the terminal electron acceptor with water as the only by-product [3, 4]. Due to their broad substrate specificity, laccases play a major role in a number of biotechnological and industrial applications [5, 6]. In general, the potential of fugal laccase varies within 430–780 mV [2]. The laccase cannot oxidize these substrates whose redox potential exceeds 800 mV. Interestingly, when several chemicals called as mediators such as ABTS are added into catalytic reactions, laccase is able to oxidize those substrates with high redox potential like nonphenolics [7, 8]. Therefore, application range of laccase is further expanded by laccase-mediator systems (LMSs). It has been reported that for a number of industrial processes mediated by laccases, redox mediators, are required in order to oxide complex substrates [7]. LMSs are known to catalyze diverse reactions, and in wastewater treatment they are used to detoxify or remove xenobiotic compounds [9–12]. ABTS and 1-hydroxybenzotriazole have been found to be efficient laccase mediators in the decolorization of recalcitrant dyes [13, 14]. When ABTS is used as a mediator, redox interaction with laccase could producein situ two reactive intermediates from ABTS, namely, A
B
T
S
+
• or ABTS2+ [8]. However, it has been reported that their degradation by-products formedin situ by hydrolysis during the laccase-ABTS reactions may also be responsible for the observed oxidation of nonphenolics [8]. The redox potential of the laccase-ABTS system is around 885 mv which may be enough to oxidize some dyes [7], but the efficiency of substrate degradation will depend on the redox potential of used laccase, mediator, and substrate.Many mediators used in these applications are soluble in the reaction medium. There are, however, several disadvantages of using dissolved mediators including their interferences during product purification, their limited reusability, and their cost-intensive removal from wastewater. Numerous ways of laccase immobilization are presented in the literature [15–17], whereas the accounts of immobilization of mediators are scarce. Recently, the electrode modified with ABTS polymer exhibited electrocatalytic activity towards dioxygen when immersed in laccase solution [18]. The fungal laccase has previously been successfully entrapped in poly-o-phenylenediamine matrix, which can mediate the enzymatic reaction [19]. Covalently attached 2,2,6,6-tetramethylpiperidine1-oxyl to polyethylene glycol has also been successfully utilized as a laccase mediator for dye decolorization in a membrane reactor [20]. On the one hand, small molecule mediators noncovalently adsorbed to insoluble carriers lead to mediator leakage in use. On the other hand, polymers or films modified with molecule mediators ordinarily may result in formidable mass-transfer resistance to the laccase-catalyzed reactions. To circumvent these problems, mediators can be covalently attached to nanosized particles that significantly reduce the external resistance to mass transfer. Further, mediator-modified nanoparticles can be recovered easily for repeated use, after effectively mediating the reactions catalyzed by enzymes. Resulting from the ability of the dication ABTS2+ to undergo reversible oxidation to A
B
T
S
+
• radical, ABTS is considered as a one of the most efficient mediator of laccase [21–24]. It has been reported that laccase with ABTS is able to degrade various environmental contaminants [8, 25]. Indigo carmine (Figure 1) is the most representative indigoid dye, and it is widely used as textile coloring agent [26]. It has been proved that the dye is carcinogenic and can lead to reproductive, developmental, neuron, and acute toxicity [27]. Thus, considering the toxicity of this dye, numerous attempts have been made to remove indigo carmine from wastewater [26, 27]. Because indigo carmine is not a typical substrate of laccase, the complete decolorization of this dye by laccase needs the help from a mediator. Therefore it was chosen as the model recalcitrant compound for carrying out decolorization reaction in this study.Figure 1
Molecular structure of indigo carmine.In the present study, our goal was to utilize ABTS-modified silica nanoparticles (SNPs) as mediators for a laccase-catalyzed decolorization of indigo carmine dye. First, ABTS was covalently attached to SNPs to yield ABTS-immobilized SNPs (ABTS-SNPs), which were utilized as laccase mediators to decolorize indigo carmine dye. Secondly, these immobilized ABTS-SNPs were proved to be effective reusable mediators and easily recovered by centrifugation of the reaction medium.


## 2. Materials and Methods

### 2.1. Chemicals

ABTS and laccase (catalogue number 38429) were purchased from Sigma-Aldrich, USA. Indigo carmine (purity≥
9
6%, MW 466.35) was obtained from Aladdin, China. The SNPs (catalogue number XF105, purity >
99%) used in this study were milky white dispersion and purchased from purchased from Nanjing XFNANO Materials Tech Co., Ltd., China. The diameter of SNPs ranges between 10 nm and 50 nm. All chemicals were used as received without further purification. Deionized water was used throughout the whole experiment.


### 2.2. Preparation and Characteristics of Silica Nanoparticles Modified with ABTS

The researchers have recently developed a procedure for the modification of the carbon nanotubes with residues of ABTS [28, 29]. By employing the same procedure, amino-functionalized SNPs were conjugated with ABTS. In brief, first, ABTS (ammonium salt) was converted into potassium salt by reaction with potassium tert-butoxide. Then, disulfonyl bromide was obtained by the reaction of potassium salt with triphenylphosphine/Br2. Finally, ABTS was covalently bonded to SNPs by the disulfonyl bromide reacted with amino-functionalized SNPs (Scheme 1). The resulting samples were centrifuged and washed thoroughly several times with dimethyl sulfoxide, methanol, and distilled water, respectively, until unbounded species were completely removed. ABTS covalently attached to SNPs were obtained and resuspended in distilled water. The functional groups of SNPs, ABTS-SNPs, and ABTS were studied using Fourier transform infrared (FT-IR) spectroscopy (Bruker Optics Tensor 37) in the range 4000–450 cm−1 at a resolution of 2 cm−1. These samples were initially dried in drying oven and then were ground with KBr, respectively. The mixture was molded into a disc, which was analyzed by FT-IR. According to elemental analysis, there were about 0.09% S in the ABTS-SNPs; this meant that the amount of ABTS attached to SNPs was about 7 μmol/g. The microstructure of ABTS-SNPs was characterized by a JEOL JSM-6700F field emission scanning electron microscope (SEM).Scheme 1
Structure of dominating SNP modifications obtained by the route for covalent attachment of ABTS.


### 2.3. Dye Decolorization

The effect of pH on decolorization of indigo carmine (20 mg/L dye) by 3000 U/L laccase and 10μM ABTS at different pH ranging from 2.5 to 6 (50 mM sodium acetate buffer) was studied. Results from the preliminary experiments indicated that the optimal pH value was about 4.5, so decolorization experiments were performed using 20 mg/L dye, 3000 U/L laccase, and mediator (10 g/L for ABTS-SNPs containing 70 μM ABTS or 70 μM ABTS) at pH 4.5. After the addition of these samples into 5 mL centrifuge tube, the reaction mixture was incubated in 50 mM sodium acetate buffer with gentle shaking at 150 rpm at room temperature. Controls reactions were also carried out to study the decolorization efficiency by laccase without a mediator or with ABTS or ABTS-SNPs alone. The data presented are the average values of measurements from triplicate technical repeats. The absorption spectra of ABTS (70 μM ABTS) and indigo carmine (20 mg/L) in 50 mM sodium acetate buffer at pH 4.5 were shown in Figure 2, which indicated that ABTS has a major ultraviolet light absorption peak at 340 nm and indigo carmine has the absorbance peak at around 615 nm. Decolorization was determined spectrophotometrically by monitoring the decrease in absorbance at the maximum wavelength of dye (around 615 nm) using Shimadzu UV-2550 spectrophotometer. The percentage decolorization of a dye was calculated based on the formula: conversion (
%
)
=
(
A
0
-
A
1
)
/
A
0, where A
0 was the initial absorbance of the dye at its adsorption maximum and A
1 was its absorbance of the reaction mixture at the different reaction time.Figure 2
The absorption spectra of ABTS (70μM ABTS) and indigo carmine (20 mg/L) in 50 mM sodium acetate buffer at pH 4.5.


### 2.4. Regeneration of the Oxidized ABTS-SNPs

After the decolorization reaction was complete, the oxidized ABTS-SNPs were separated and then transferred into a 5 mL centrifuge tube containing 3.5 mL distilled water. With mixing, 10μL of 1 M ascorbic acid was added. After shaking at 150 rpm for 5 min, the mixture was centrifuged for 2 min at 10000 ×g. The regenerated ABTS-SNPs were separated and washed thrice with 3 mL distilled water again.


### 2.5. Reusability of ABTS-SNPs

The recyclability of ABTS-SNPs was assessed in a discontinuous decolorization reaction. Decolorization of indigo carmine (20 mg/L) were performed in a 5 mL centrifuge tube containing 3000 U/L of laccase and 10 g/L ABTS-SNPs at pH 4.5, shaking at 150 rpm for 30 min at room temperature. At the end of every cycle, ABTS-SNPs were separated, regenerated, and washed thrice with 3 mL distilled water. Next, the ABTS-SNPs was added into a fresh reaction solution (20 mg/L indigo carmine, 3000 U/L laccase, pH 4.5) to start a fresh cycle. The data presented are the average values of measurements from triplicate technical repeats.


## 2.1. Chemicals

ABTS and laccase (catalogue number 38429) were purchased from Sigma-Aldrich, USA. Indigo carmine (purity≥
9
6%, MW 466.35) was obtained from Aladdin, China. The SNPs (catalogue number XF105, purity >
99%) used in this study were milky white dispersion and purchased from purchased from Nanjing XFNANO Materials Tech Co., Ltd., China. The diameter of SNPs ranges between 10 nm and 50 nm. All chemicals were used as received without further purification. Deionized water was used throughout the whole experiment.


## 2.2. Preparation and Characteristics of Silica Nanoparticles Modified with ABTS

The researchers have recently developed a procedure for the modification of the carbon nanotubes with residues of ABTS [28, 29]. By employing the same procedure, amino-functionalized SNPs were conjugated with ABTS. In brief, first, ABTS (ammonium salt) was converted into potassium salt by reaction with potassium tert-butoxide. Then, disulfonyl bromide was obtained by the reaction of potassium salt with triphenylphosphine/Br2. Finally, ABTS was covalently bonded to SNPs by the disulfonyl bromide reacted with amino-functionalized SNPs (Scheme 1). The resulting samples were centrifuged and washed thoroughly several times with dimethyl sulfoxide, methanol, and distilled water, respectively, until unbounded species were completely removed. ABTS covalently attached to SNPs were obtained and resuspended in distilled water. The functional groups of SNPs, ABTS-SNPs, and ABTS were studied using Fourier transform infrared (FT-IR) spectroscopy (Bruker Optics Tensor 37) in the range 4000–450 cm−1 at a resolution of 2 cm−1. These samples were initially dried in drying oven and then were ground with KBr, respectively. The mixture was molded into a disc, which was analyzed by FT-IR. According to elemental analysis, there were about 0.09% S in the ABTS-SNPs; this meant that the amount of ABTS attached to SNPs was about 7 μmol/g. The microstructure of ABTS-SNPs was characterized by a JEOL JSM-6700F field emission scanning electron microscope (SEM).Scheme 1
Structure of dominating SNP modifications obtained by the route for covalent attachment of ABTS.


## 2.3. Dye Decolorization

The effect of pH on decolorization of indigo carmine (20 mg/L dye) by 3000 U/L laccase and 10μM ABTS at different pH ranging from 2.5 to 6 (50 mM sodium acetate buffer) was studied. Results from the preliminary experiments indicated that the optimal pH value was about 4.5, so decolorization experiments were performed using 20 mg/L dye, 3000 U/L laccase, and mediator (10 g/L for ABTS-SNPs containing 70 μM ABTS or 70 μM ABTS) at pH 4.5. After the addition of these samples into 5 mL centrifuge tube, the reaction mixture was incubated in 50 mM sodium acetate buffer with gentle shaking at 150 rpm at room temperature. Controls reactions were also carried out to study the decolorization efficiency by laccase without a mediator or with ABTS or ABTS-SNPs alone. The data presented are the average values of measurements from triplicate technical repeats. The absorption spectra of ABTS (70 μM ABTS) and indigo carmine (20 mg/L) in 50 mM sodium acetate buffer at pH 4.5 were shown in Figure 2, which indicated that ABTS has a major ultraviolet light absorption peak at 340 nm and indigo carmine has the absorbance peak at around 615 nm. Decolorization was determined spectrophotometrically by monitoring the decrease in absorbance at the maximum wavelength of dye (around 615 nm) using Shimadzu UV-2550 spectrophotometer. The percentage decolorization of a dye was calculated based on the formula: conversion (
%
)
=
(
A
0
-
A
1
)
/
A
0, where A
0 was the initial absorbance of the dye at its adsorption maximum and A
1 was its absorbance of the reaction mixture at the different reaction time.Figure 2
The absorption spectra of ABTS (70μM ABTS) and indigo carmine (20 mg/L) in 50 mM sodium acetate buffer at pH 4.5.


## 2.4. Regeneration of the Oxidized ABTS-SNPs

After the decolorization reaction was complete, the oxidized ABTS-SNPs were separated and then transferred into a 5 mL centrifuge tube containing 3.5 mL distilled water. With mixing, 10μL of 1 M ascorbic acid was added. After shaking at 150 rpm for 5 min, the mixture was centrifuged for 2 min at 10000 ×g. The regenerated ABTS-SNPs were separated and washed thrice with 3 mL distilled water again.


## 2.5. Reusability of ABTS-SNPs

The recyclability of ABTS-SNPs was assessed in a discontinuous decolorization reaction. Decolorization of indigo carmine (20 mg/L) were performed in a 5 mL centrifuge tube containing 3000 U/L of laccase and 10 g/L ABTS-SNPs at pH 4.5, shaking at 150 rpm for 30 min at room temperature. At the end of every cycle, ABTS-SNPs were separated, regenerated, and washed thrice with 3 mL distilled water. Next, the ABTS-SNPs was added into a fresh reaction solution (20 mg/L indigo carmine, 3000 U/L laccase, pH 4.5) to start a fresh cycle. The data presented are the average values of measurements from triplicate technical repeats.


## 3. Results and Discussion

### 3.1. Structure, Biochemical, and Physicochemical Characteristics of ABTS-SNPs

The chemical structure of ABTS-SNPs is presented in Scheme1. Morphology of SNPs and ABTS-SNPs, as obtained by SEM, is shown in Figures 3(a) and 3(b). The particles obtained after covalent attachment of ABTS on SNPs were found to be nanosized (<50 nm). Original SNPs were white, but the SNPs covalently attached to ABTS exhibited light yellow color (Figure 3(c)). After ABTS-SNPs were mixed with laccase for a few minutes, the mixture turned from light yellow to green. The color change in the reaction was attributed to the fact that ABTS molecules attached on the nanoparticle surface were oxidized to green A
B
T
S
+
• by laccase.Figure 3
(a) SEM image of SNPs; (b) SEM image of ABTS-SNPs; (c) Tube 1, SNPs, Tube 2, SNPs covalently attached to ABTS, Tube 3, after reaction of ABTS-SNPs with laccase, Tube 4, regenerated ABTS-SNPs, Tube 5, after reaction of regenerated ABTS-SNPs with laccase, and (d) FT-IR spectra of ABTS, ABTS-SNPs, and SNPs.
(a)
(b)
(c)
(d)The comparison of transmission FT-IR spectral data for ABTS and ABTS-SNPs with those for the original SNPs is presented in Figure3(d). In FT-IR spectra of ABTS-SNPs, the obvious new peak observed at ~3400 cm−1 was due to the N-H group stretching vibrations [30]. Other new peaks at about 1550 and 825 cm−1 in the FT-IR spectra of ABTS-SNPs were characteristic bands due to the aryl group [31]. Additionally, new strong peaks at 1360 and 1150 cm−1 were attributed to the characteristic absorptions of SO2-N [30]. These results clearly indicate that ABTS was covalently attached to the SNPs.


### 3.2. Dye Decolorization

The ABTS-SNPs were evaluated as laccase mediators for dye decolorization. The comparison of decolorization efficiency of laccase/ABTS-SNPs system with those for ABTS-SNPs, laccase, and laccase/ABTS system is shown in Figure4. For ABTS-SNPs, only ~4% of dye decolorization, respectively, was achieved in 15 min. This decolorization was caused by the adsorption of ABTS-SNPs. For laccase alone, a dismal ~3% decolorization was reached. The low decolorization rate was due to the fact that indigo carmine is not a typical phenol substrate of laccase. These results showed that removal of dye by nanoparticles or laccase was negligible. Almost complete decolorization (96%) was achieved by laccase using ABTS as the mediator within 5 min, while about 95% decolorization was achieved within 15 min using ABTS-SNPs as mediators. These results suggest that ABTS immobilized on SNPs can be used as an efficient mediator of laccase for decolorization in a manner similar to free ABTS. Even though the decolorization rate is slower for ABTS-SNPs than for the free mediator, the immobilized mediator has the advantage of being easily recovered from the reaction system.Figure 4
Decolorization of indigo carmine (20 mg/L) by ABTS-SNPs (10 g/L containing 70μM ABTS), laccase (3000 U/L), laccase/ABTS-SNPs, and laccase/ABTS (70 μM ABTS) in 50 mM sodium acetate buffer at pH 4.5 with shaking at 150 rpm at room temperature.


### 3.3. Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs

In the case of laccase, the role of mediators in an enzymatic oxidation has been already described by several authors [7]. First, ABTS covalently attached to SNPs is converted into A
B
T
S
+
• cation radical by the enzymatic reaction. Subsequently, the A
B
T
S
+
• cation radical takes part in nonenzymatic reactions with dyes which are not oxidizable by laccases alone. Simultaneously, the A
B
T
S
+
• is reduced to its original form (ABTS) by the dye to be oxidized, and thus one catalytic cycle is completed. At the end of all catalytic cycle, the dye molecules are completely decolorized by oxidation and ABTS molecules are oxidized to A
B
T
S
+
• by laccase again. Moreover, A
B
T
S
+
• can be reduced by some strong reducing regents [23]. In our case, A
B
T
S
+
•-SNPs were reduced with ascorbic acid resulting in regeneration of ABTS-SNPs as shown in Scheme 2.Scheme 2
The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation.As shown in Figure3(c), after the regeneration process, the oxidized ABTS-SNPs turned from green to light yellow—the original color of the initial ABTS-SNPs. Further, the regenerated ABTS-SNPs can be oxidized by laccase again. Decolorization efficiency of regenerated ABTS-SNPs as mediators was 93.5%, which was almost the same as that of initial ABTS-SNPs (~95% decolorization). The results show that ABTS-SNPs after use can be recovered and reused as efficient mediators for decolorization similar to original ABTS-SNPs.


### 3.4. Reusability

From the economic point of view, reusability of immobilized mediator plays a key role when LMSs will be used on a large scale for industrial applications [32]. The reusability of ABTS-SNPs was investigated for up to 6 cycles (Figure 5). When the reaction time was set to 30 min, which proved to be sufficient for almost complete decolorization, comparably high decolorization yields were obtained in 4 subsequent runs using the same recovered and regenerated ABTS-SNPs. Approximately 77% decolorization could be maintained after 6 cycles. A slight decrease in decolorization efficiency was observed, which was attributed to partial destruction of ABTS-SNPs during the reaction and/or the loss in quantity of ABTS-SNPs during supernatant discarding in the centrifugal recovery process. It has been reported that the radical cation form of ABTS was the main product observed after reacting with laccase, but a degradation product from ABTS was also formed [33]. The decomposition of ABTS during the laccase-ABTS reactions will be a drawback for the application of laccase/ABTS-SNPs system.Figure 5
Reusability of ABTS-SNPs (10 g/L) in six successive cycles using 3000 U/L of laccase, and 20 mg/L of indigo carmine at pH 4.5 with shaking at 150 rpm for 30 min at room temperature.


## 3.1. Structure, Biochemical, and Physicochemical Characteristics of ABTS-SNPs

The chemical structure of ABTS-SNPs is presented in Scheme1. Morphology of SNPs and ABTS-SNPs, as obtained by SEM, is shown in Figures 3(a) and 3(b). The particles obtained after covalent attachment of ABTS on SNPs were found to be nanosized (<50 nm). Original SNPs were white, but the SNPs covalently attached to ABTS exhibited light yellow color (Figure 3(c)). After ABTS-SNPs were mixed with laccase for a few minutes, the mixture turned from light yellow to green. The color change in the reaction was attributed to the fact that ABTS molecules attached on the nanoparticle surface were oxidized to green A
B
T
S
+
• by laccase.Figure 3
(a) SEM image of SNPs; (b) SEM image of ABTS-SNPs; (c) Tube 1, SNPs, Tube 2, SNPs covalently attached to ABTS, Tube 3, after reaction of ABTS-SNPs with laccase, Tube 4, regenerated ABTS-SNPs, Tube 5, after reaction of regenerated ABTS-SNPs with laccase, and (d) FT-IR spectra of ABTS, ABTS-SNPs, and SNPs.
(a)
(b)
(c)
(d)The comparison of transmission FT-IR spectral data for ABTS and ABTS-SNPs with those for the original SNPs is presented in Figure3(d). In FT-IR spectra of ABTS-SNPs, the obvious new peak observed at ~3400 cm−1 was due to the N-H group stretching vibrations [30]. Other new peaks at about 1550 and 825 cm−1 in the FT-IR spectra of ABTS-SNPs were characteristic bands due to the aryl group [31]. Additionally, new strong peaks at 1360 and 1150 cm−1 were attributed to the characteristic absorptions of SO2-N [30]. These results clearly indicate that ABTS was covalently attached to the SNPs.


## 3.2. Dye Decolorization

The ABTS-SNPs were evaluated as laccase mediators for dye decolorization. The comparison of decolorization efficiency of laccase/ABTS-SNPs system with those for ABTS-SNPs, laccase, and laccase/ABTS system is shown in Figure4. For ABTS-SNPs, only ~4% of dye decolorization, respectively, was achieved in 15 min. This decolorization was caused by the adsorption of ABTS-SNPs. For laccase alone, a dismal ~3% decolorization was reached. The low decolorization rate was due to the fact that indigo carmine is not a typical phenol substrate of laccase. These results showed that removal of dye by nanoparticles or laccase was negligible. Almost complete decolorization (96%) was achieved by laccase using ABTS as the mediator within 5 min, while about 95% decolorization was achieved within 15 min using ABTS-SNPs as mediators. These results suggest that ABTS immobilized on SNPs can be used as an efficient mediator of laccase for decolorization in a manner similar to free ABTS. Even though the decolorization rate is slower for ABTS-SNPs than for the free mediator, the immobilized mediator has the advantage of being easily recovered from the reaction system.Figure 4
Decolorization of indigo carmine (20 mg/L) by ABTS-SNPs (10 g/L containing 70μM ABTS), laccase (3000 U/L), laccase/ABTS-SNPs, and laccase/ABTS (70 μM ABTS) in 50 mM sodium acetate buffer at pH 4.5 with shaking at 150 rpm at room temperature.


## 3.3. Regeneration and Catalytic Cycle Mechanism of ABTS-SNPs

In the case of laccase, the role of mediators in an enzymatic oxidation has been already described by several authors [7]. First, ABTS covalently attached to SNPs is converted into A
B
T
S
+
• cation radical by the enzymatic reaction. Subsequently, the A
B
T
S
+
• cation radical takes part in nonenzymatic reactions with dyes which are not oxidizable by laccases alone. Simultaneously, the A
B
T
S
+
• is reduced to its original form (ABTS) by the dye to be oxidized, and thus one catalytic cycle is completed. At the end of all catalytic cycle, the dye molecules are completely decolorized by oxidation and ABTS molecules are oxidized to A
B
T
S
+
• by laccase again. Moreover, A
B
T
S
+
• can be reduced by some strong reducing regents [23]. In our case, A
B
T
S
+
•-SNPs were reduced with ascorbic acid resulting in regeneration of ABTS-SNPs as shown in Scheme 2.Scheme 2
The proposed catalytic cycle of decolorizing the dye by ABTS-SNPs mediated laccase oxidation.As shown in Figure3(c), after the regeneration process, the oxidized ABTS-SNPs turned from green to light yellow—the original color of the initial ABTS-SNPs. Further, the regenerated ABTS-SNPs can be oxidized by laccase again. Decolorization efficiency of regenerated ABTS-SNPs as mediators was 93.5%, which was almost the same as that of initial ABTS-SNPs (~95% decolorization). The results show that ABTS-SNPs after use can be recovered and reused as efficient mediators for decolorization similar to original ABTS-SNPs.


## 3.4. Reusability

From the economic point of view, reusability of immobilized mediator plays a key role when LMSs will be used on a large scale for industrial applications [32]. The reusability of ABTS-SNPs was investigated for up to 6 cycles (Figure 5). When the reaction time was set to 30 min, which proved to be sufficient for almost complete decolorization, comparably high decolorization yields were obtained in 4 subsequent runs using the same recovered and regenerated ABTS-SNPs. Approximately 77% decolorization could be maintained after 6 cycles. A slight decrease in decolorization efficiency was observed, which was attributed to partial destruction of ABTS-SNPs during the reaction and/or the loss in quantity of ABTS-SNPs during supernatant discarding in the centrifugal recovery process. It has been reported that the radical cation form of ABTS was the main product observed after reacting with laccase, but a degradation product from ABTS was also formed [33]. The decomposition of ABTS during the laccase-ABTS reactions will be a drawback for the application of laccase/ABTS-SNPs system.Figure 5
Reusability of ABTS-SNPs (10 g/L) in six successive cycles using 3000 U/L of laccase, and 20 mg/L of indigo carmine at pH 4.5 with shaking at 150 rpm for 30 min at room temperature.


## 4. Conclusion

This investigation has demonstrated that ABTS covalently linked to SNPs can be utilized to mediate dye decolorization by laccase. Even though the decolorization efficiency of ABTS-SNPs as mediators is slower than that of the free mediators, the ABTS-SNPs can easily be recovered, regenerated, and reused, therefore improving the efficiency of mediator utilization in LMSs for biotechnological and industrial applications. This process has the potential to alleviate environmental problems caused by the release of free mediators in the wastewater system. Moreover, a large scale application of reusable and immobilized mediators will lower the cost of dye treatment using the laccase mediator systems.



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*Source: 670194-2015-12-29.xml*