Article pubs.acs.org/IECR
Rapid Decolorization of Phenolic Azo Dyes by Immobilized Laccase with Fe3O4/SiO2 Nanoparticles as Support Hongxia Wang,*,† Wei Zhang,‡ Jingxiang Zhao,† Lulu Xu,† Chunyan Zhou,† Lin Chang,† and Liyan Wang§ †
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Key Laboratory for Photonic and Electronic Bandgap Materials, Ministry of Education, College of Chemistry and Chemical Engineering, Harbin Normal University, Harbin, China 150025 ‡ School of Materials Science and Engineering, Hebei University of Technology, Tianjin, China 300130 § School of Science, Harbin University, Harbin, China 150086 S Supporting Information *
ABSTRACT: Fe3O4/SiO2 nanoparticles with particle size below 30 nm were used as the support for laccase immobilization through glutaraldehyde coupling. Investigation of the immobilized laccase was carried out by X-ray diffractometry (XRD), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), vibrating sample magnetometry (VSM), UV−vis spectrophotometry, and cyclic voltammogram (CV) measurements. Two phenolic azo dyes, Procion Red MX-5B and azophloxine, were selected to investigate the enzyme activity of the immobilized laccase toward degradation of phenolic azo dyes. The immobilized laccase presents unusual performance for dye decolorization and easy separation with an external magnetic field. Finally, the possible mechanism for the unusual decolorization of phenolic azo dyes by the immobilized laccase is discussed.
1. INTRODUCTION As the largest group of synthetic dyes, azo dyes are widely used in textile dyeing but are highly recalcitrant to conventional wastewater treatment processes. Therefore, efficient removal of azo dyes from wastewater would accelerate the purification of dyeing wastewater without a doubt. With the development of diverse wastewater treatment processes, the recent focus for this has shifted toward enzymatic treatment,1−6 during which a laccase mediator system has proved to be highly efficient and eco-friendly,4,7−9 not only because of the rapid decolorization of azo dyes but also because of the concomitant reduction of oxygen to water. Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2), a multicopper oxidase produced by many plants and fungi, has the ability to catalyze a variety of phenolic substrates and aromatic amines. In the presence of some small molecular redox mediators, some nonsubstrate dyes of laccase could be degraded to a desired level.10−15 Moreover, with the development of immobilization of laccase on some support materials, such as alumina, activated carbon, chitosan, membranes, porous glass, mesoporous silica, polymers, and nanoporous gold, separation of laccase from reaction medium became much easier along with enhanced thermal, storage, and operation stability.16−27 As a result, laccase has attracted much attention in the treatment of wastewater from textile and paper industries. However, cost was a big problem when the laccase mediator system was used in continuous bioreactors, since periodic addition of redox mediator was necessary. If the redox mediator is absent, the reaction becomes time-consuming.10,18,23,28 For instance, Arica et al.23 reported the degradation of Reactive Red 120 by immobilized laccase with nonporous poly(glycidyl methacrylate/ethylene glycol dimethacrylate) beads as the © 2013 American Chemical Society
carrier, they found that realizing 90% color removal required 10 h. Bayramoğlu et al.28 used immobilized laccase with poly(4vinylpyridine) grafted and Cu(II) ions chelated magnetic beads as support to decolorize three industrial azo dyes, Reactive Green 19, Procion Red MX-5B, and Reactive Brown 10. The results demonstrated that the color removal of the three azo dyes in 6 h was 38%, 51% and 59%, respectively, and then reached 77%, 89% and 92% in 18 h. In another report,18 they investigated the degradation of Reactive Yellow 2 by laccase, and found that the total degradation of Reactive Yellow 2 by free and immobilized laccase in 18 h were about 71% and 82%, respectively. Ciullini et al.10 found that the decolorization degree of the azo dyes treated by laccase was significantly dependent on the dye structures; moreover, all the tested azo dyes needed more than 24 h to be decolorized efficiently. All these results demonstrate that rapid decolorization of azo dyes by immobilized laccase in the absence of mediators is difficult to achieve. So, something must be done to improve the rapidity of the process. In the present study, magnetic Fe3O4/SiO2 nanoparticles with particle size below 30 nm were selected as support to immobilize laccase, and the enzyme activity toward degradation of phenolic azo dyes was tested. It was not the first time to immobilize laccase on magnetic Fe3O4/SiO2 support;22,29,30 however, it is the first time such small Fe3O4/SiO2 nanoparticles (below 30 nm) have been used as support. As support for laccase immobilization, nonporous Fe3O4/SiO2 nanoparticles not only could abate the diffusion limitation of dyes Received: Revised: Accepted: Published: 4401
September 26, 2012 March 3, 2013 March 9, 2013 March 9, 2013 dx.doi.org/10.1021/ie302627c | Ind. Eng. Chem. Res. 2013, 52, 4401−4407
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(pH 7.0) for 12 h at room temperature. Then the resulting composites (denoted as IM-laccase, or immobilized laccase) were obtained by repeated washing and magnetic separation and stored at 4 °C under refrigeration for later use. The diffuse reflectance UV−vis spectra of samples are shown in Figure S2 in Supporting Information, and the amount of laccase attached on the Fe3O4/SiO2 support was determined by the Coomassie Brilliant Blue method with BSA as a standard.32 2.4. Activity Assay of Free and Immobilized Laccase. Laccase activity was determined on a UV−vis spectrophotometer with ABTS as substrate.33 One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 micromole substrate per minute. The activity recovery R (percent) of IM-laccase is calculated according to the following equation:28
or intermediates but also could avoid excessive laccase adsorption that is inevitable on porous Fe3O4/SiO2 support. Moreover, the specific surface area of small Fe3O4/SiO2 nanoparticles will be larger than that of bigger Fe3O4/SiO2 nanoparticles. Once multipoint attachment is realized on the support with larger specific surface area, high loading of laccase with enhanced stability will be obtained.
2. MATERIALS AND METHODS 2.1. Chemicals and Instrumentation. Laccase (EC 1.10.3.2: p-diphenol:dioxygen oxidoreductase; 23.1U/mg) from Trametes villosa, ABTS [2,2′-azinodi(ethylbenzothiazoline-6-sulfonic acid)], Procion Red MX-5B, azophloxine, APS (3-aminopropyltrimethoxysilane), and TEOS (tetraethoxysilane) were all purchased from Sigma−Aldrich. Coomassie Brilliant Blue, BSA (bovine serum albumin), FITC (fluorescein isothiocyanate), and Sephadex G-15 were purchased from Shanghai Yongye Chemical Ltd., China. All other reagents were analytical grade and used without further purification. The crystal structure of sample was measured by use of a Rigaku-12KW X-ray diffractometer (XRD). Diffuse reflectance UV−vis spectra were measured on a Shimadzu UV 2550 spectrophotometer with BaSO4 as a reference. UV−vis absorption spectra were recorded on a Perkin-Elmer Lambda 45 spectrophotometer. Magnetization curves at room temperature were recorded on a America Lake shore 7410 VSM. Transmission electron microscopy (TEM) measurement was performed on a JEOL JEM-2010 with accelerating voltage of 200 kV. Sample for TEM was prepared via sonication in deionized water and dropped on a carbon-coated copper grid, followed by drying for observation. FITC-labeled free laccase and IM-Laccase were observed on a TCS SP5 confocal laser scanning microscope (CLSM) (Germany). Electrochemical measurements were performed with a CHI 660D electrochemical workstation (Shanghai Huachen Co., China) that had a glassy carbon (GC) working electrode, a platinum wire auxiliary electrode, and a Ag/AgCl reference electrode. 2.2. Preparation of Fe3O4/SiO2 Nanoparticles. Twenty milliliters of homemade Fe3O4 ferrofluid (9.7 mg/mL)31 was first dispersed in 50 mL of deionized water and mixed with freshly prepared APS solution. After 40 min, 100 mL of 2propanol and 2 mL of TEOS were successively added in the above solution and stirred for 12 h, during which the pH was controlled at 11 by the addition of NH4OH (28%). After that, the dark-brown precipitate was washed, separated with an external magnetic field several times, and denoted as Fe3O4/ SiO2. The XRD pattern of the as-prepared Fe3O4/SiO2 nanocomposites is shown in Figure S1 in Supporting Information. Then the Fe3O 4/SiO 2 nanoparticles were modified with amino groups. Fe3O4/SiO2 nanoparticles (100 mg) were dispersed into 20 mL of absolute ethanol under sonication, and then 0.2 mL of APS was added and the mixture stood for 30 min. After being stirred at room temperature for 12 h and repeated washing, Fe3O4/SiO2 nanocomposites modified with amino groups were obtained. 2.3. Laccase Immobilization. Fe3O4/SiO2 nanocomposites modified with amino groups (in 20 mL of phosphate buffer solution, pH 7.0) was first mixed with 2.5% glutaraldehyde solution (1.5 mL) and stirred for 3 h at room temperature. After repeated washing and magnetic separation, the resulting supports were slowly stirred with a certain amount of laccase (4−20 mg) in 20 mL of phosphate buffer solution
R = A i /A f × 100 where Ai is the activity (units) of IM-laccase and Af is the activity (units) of the same amount of free laccase as that immobilized on Fe3O4/SiO2 support. 2.5. Leaching Test. At the end of each dye decolorization, the immobilized laccase was separated from the dye solution with an external magnetic field and washed with phosphate buffer solution. Then both the washing solution and the remaining dye solutuion were mixed with 100 μL of 10 mM ABTS, respectively. The adsorption at 420 nm was recorded for 2 min to characterize the leached amount of laccase from the Fe3O4/SiO2 nanocomposites. 2.6. Preparation of Fluorescence-Labeled Free Laccase and Immobilized Laccase. The fluorescence-labeled laccase was prepared according to a similar procedure described by Dai et al.24 IM-laccase (50 mg) or free laccase (5 mg) in 1 mL of phosphate buffer solution (pH 7.0) was mixed with 1.5 mL of 0.5 mM carbonate buffer solution (pH 9.0) under vigorous stirring. The resulting solution was then mixed with freshly prepared FITC solution (0.5 mM carbonate buffer solution as solvent, pH 9.0) and stirred for 2 h at room temperature. After that, fluorescence-labeled IM-laccase was separated by use of an external magnetic field and fluorescencelabeled free laccase was collected by centrifugation. 2.7. Preparation of GC Electrodes Modified with Laccase. To modify GC electrode with laccase, the bare GC electrode was first polished with 0.05 μm α-alumina on a microcloth polishing pad, ultrasonically cleaned in water for about 5 min, and left to dry at room temperature. Then the polished GC electrode was activated in 0.5 M H2SO4 solution through measuring cyclic voltammograms (CV) at a scan rate of 100 mV/s in the range of −0.2 to 1.0 V until a stable CV curve is reached. After that, the modifications of the GC electrodes with free laccase and IM-laccase starts. Four microliters of 1% Nafion solution was first spread on the activated GC electrode and left to dry at room temperature. Then a mixture containing a fixed amount of free laccase or IMlaccase, 1% Nafion solution, absolute ethanol, and tripledistilled water was sonicated for 5 min to become a homogeneous slurry. Finally, the obtained slurry was spread on GC electrode to get modified GC electrode and dried at room temperature, where same amount of laccase on both modified GC electrodes was controlled. 2.8. Decolorization of Phenolic Azo Dyes by Laccase. Two azo dyes were selected as model dyes to test the activity of IM-laccase toward dye degradation. Chemical structures and some properties of the azo dyes are presented in Figure 1 and 4402
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Figure 1. Chemical structures of (A) Procion Red MX-5B and (B) azophloxine. Figure 2. Effect of initial laccase concentration on the laccase loading and retained laccase activity on Fe3O4/SiO2 support.
Table 1, respectively. The enzymatic degradation reaction of each dye was carried out at 20 °C, controlled by a DC-2015
about 80 mg/g. This behavior was ascribed to an intermolecular space inhibition of laccase for substrate dispersion.21 Figure 3 shows the TEM image of the as-prepared Fe3O4/ SiO2 nanoparticles and CLSM images of free laccase and IMlaccase. As shown in Figure 3a, a narrow size distribution of the as-prepared Fe3O4/SiO2 nanoparticles can be observed with particle sizes in the range 25−30 nm. In the meantime, the morphology of free laccase is identified from the fluorescence emitted from the FITC marker as shown in Figure 3b. Apart from long laccase in the image, those bright green spots should be the conjunction of FITC with laccase, that is, the location of FITC marker. In Figure 3c, the morphology of IM-laccase is presented. Since the fluorescence labeling of IM-laccase is performed after immobilization, the amount of amino groups left on laccase used for reacting with FITC is obviously lower than for free laccase. Therefore, the fluorescence emitted from laccase immobilized on Fe3O4/SiO2 nanoparticles was less than that from free laccase because there are less amino groups left on immobilized laccase. But it is still easy to distinguish laccase from Fe3O4/SiO2 support in Figure 3c. Figure 4 shows the magnetization curves of Fe3O4/SiO2 nanoparticles and IM-laccase measured at room temperature and a photograph of IM-laccase separated by an external magnetic field. The absence of a hysteresis loop in both curves indicates both the as-prepared Fe3O4/SiO2 nanoparticles and the IM-laccase show superparamagnetic behavior. Moreover, the IM-laccase has a magnetization saturation (Ms) value of 39.7 emu/g that is high enough for rapid separation under an external magnetic field (shown in Figure 4B). 3.2. Decolorization of Phenolic Azo Dyes by Immobilized Laccase. As azo dyes, Procion Red MX-5B and azophloxine are not substrates of laccase and therefore cannot be decolorized easily by laccase in the absence of redox medium. As expected, we did not observe any color removal of Procion Red MX-5B or azophloxine by free laccase in hours by measuring the absorbance of dye at λmax. However, rapid color removal of each dye was observed in 20 min when IM-laccase was used, and the decolorization percentage of each dye exceeds 80% at the end of 1 h (Figure 5). Figure 5 also presents the performance of dye decolorization by Fe3O4/SiO2 nanocomposites, in which the absorption−desorption equilibrium of dye on the support was observed. This indicates that dye decolorization in the IM-laccase system was not completely due to absorption by the support but to the combined effects of
Table 1. Characteristics of the Two Tested Azo Dyes dye
synonym
mol wt
λmax(nm)
color index
azophloxine Procion Red MX-5B
Acid Red 1 Reactive Red 2
509.42 615.55
531 535
18 050 18 200
low-temperature thermostat bath for 120 min under stirring. Each reaction medium comprised dye solution (15 mL, 10 mg/ L), phosphate buffer solution (pH 7.0, 15 mL), and IM-laccase (50 mg). Under the same conditions, 50 mg of Fe3O4/SiO2 nanocomposites was added instead of IM-laccase to establish the absorption−desorption equilibrium of the dye on support. At different time intervals, the IM-laccase or support was separated from the medium by an external magnetic field, and the percentage of dye decolorization was analyzed via PerkinElmer Lambda 45 spectrophotometer and expressed as the following formula: A − At decolorization (%) = 0 × 100 A0 where A0 is the initial absorbance at λmax and At is the absorbance at λmax at various time intervals. To test the reusability of IM-laccase for dye decolorization, the above decolorization reaction of each dye with a 60-min cycle was run for 10 cycles. At the end of each cycle, IM-laccase was separated and washed three times with phosphate buffer solution (pH 7.0), and then a second cycle was repeated. In this section, all the decolorization experiments were performed in triplicate.
3. RESULTS AND DISCUSSION 3.1. Laccase Immobilization and Separation. The effect of initial laccase concentration on the amount of laccase anchored on Fe3O4/SiO2 nanoparticles and the activity recovery of IM-laccase are shown in Figure 2. It can be seen that the amount of laccase anchored on support increases with the laccase concentration in the range 0.2−1.0 mg/mL, whereas the activity recovery of IM-laccase reached a maximum at an initial laccase concentration of 0.6 mg/mL. The maximum activity recovery of IM-laccase reached 55.2%, where the amount of laccase loading on Fe3O4/SiO2 support reached 4403
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Figure 4. (A) Magnetization curves of (a) Fe3O4 nanoparticles and (b) IM-laccase measured at room temperature and (B) photograph of IM-laccase separated under an external magnetic field.
Figure 3. (a) TEM image of Fe3O4/SiO2 nanoparticles. (b, c) CLSM images of fluorescence-labeled (b) free laccase and (c) immobilized laccase.
degradation by IM-laccase and adsorption by the Fe3O4/SiO2 support. The degradation of dye by IM-laccase was also confirmed by the UV−vis spectra of each dye before and after decolorization by the immobilized laccase, as shown in Figure S5 in Supporting Information. To test the operational stability of IM-laccase for dye decolorization, the IM-laccase was used repeatedly. Figure 6 shows the performance up to 10 cycles; during each cycle the reaction runs for 60 min. It can be seen that the percentage of decolorization of each dye decreases slightly as the cycle number increases. In the 10th cycle, the decolorization percentage of azophloxine remained 88% of that
Figure 5. Decolorization of dyes by immobilized laccase on Fe3O4/ SiO2 support.
in the first cycle, and Procion Red MX-5B remained about 79% of that in the first cycle. The slight decrease in decolorization percentage in 10 cycles also suggests a decrease in operational stability of the immobilized laccase for dye decolorization. However, what should be responsible for this? Laccase leaching or gradual deactivation of the immobilized laccase are possible 4404
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ABTS. After the GC electrode was modified with free laccase, the ABTS+·/ABTS2+ redox cycle with anodic potential at 930 mV and cathodic potential at 850 mV disappeared (Figure 7, curve b). Instead, the anodic peak corresponding to the oxidation of ABTS to ABTS+ • remained, and the cathodic peak shifted to ∼220 mV, demonstrating the electrocatalytic reduction of oxygen by laccase.34−36 The disappearance of ABTS+·/ABTS2+ redox cycle on GC electrode modified with laccase has also been ascribed to the slow oxidation of ABTS+· to ABTS2+ by laccase that cannot be measured.33 The same phenomenon was observed at the GC electrode modified with IM-laccase (Figure 7, curve c); however, it is interesting to notice that both the anodic and cathodic current increase rapidly, and the cathodic current is 10-fold more than that at GC electrode modified with free laccase. This seems to suggest that the oxidation of ABTS to ABTS+ • and the reduction of oxygen by laccase were both promoted after laccase was immobilized on Fe3O4/SiO2 nanocomposites. Similar results were reported by Qiu et al.,25 who found that the GC electrode modified with nanoporous gold-supported laccase has a much higher responsive current than that of GC electrode modified with gold sheet-supported laccase; they attributed this to the difference in the surface properties of carrier materials. Immobilization of laccase on nanoporous gold led to the effective direct electron transfer between GC electrode and laccase.25 Here, the rapid decolorization of phenolic azo dyes by laccase with Fe3O4/SiO2 nanoparticles as support was reported and the particle size of Fe3O4/SiO2 nanoparticles is below 30 nm. Based on the CV results in the present study and report from Qiu et al.,25 we learn that the GC electrode modified with Fe3O4/SiO2 nanocomposite-supported laccase is more helpful for direct electron transfer compared with GC electrode modified with free laccase. Moreover, this difference is mainly from the difference in the microenvironment between immobilized and free laccase. It is obvious that the microenvironment from the laccase immobilized on Fe3O4/SiO2 nanocomposites is different from that of free laccase. At least, multipoint attachment on the carrier could improve the rigidification of the protein and protect it from denaturation.37 Quan et al.38 have demonstrated that direct electron transfer between Pt electrode and laccase was not feasible when Pt electrode surface was modified with free laccase. Compared with the weak peak current at GC electrode modified with free laccase (Figure 7, curve b), the sharp increase in current at GC electrode modified with the immobilized laccase (Figure 7, curve c) exactly indicates that direct electron transfer between GC electrode and laccase was improved after the laccase was immobilized on Fe3O4/SiO2 nanocomposites. And then, it is easy to understand the better performance of IM-laccase toward decolorization of phenolic azo dyes in comparison with free laccase: since the laccase activity toward dye decolorization is closely related to the reduction of oxygen by laccase,9,39 the increase in cathodic peak current means enhancement of the ability of laccase to reduce oxygen.
Figure 6. Operation stability of IM-laccase for decolorizing (a) azophloxine and (b) Procion Red MX-5B at pH 7.0 and 20 °C (60 min per cycle).
reasons. In leaching tests, ABTS was selected as substrate to detect laccase in wash solution and the remaining dye solution, however, almost no increase of absorbance at 420 nm was observed. This indicates that the laccase on Fe3O4/SiO2 nanocomposites was stable engough to prevent leaching. Therefore, the decrease of decolorization by laccase leaching can be ruled out. Gradual deactivation of IM-laccase during repeated use might be the true reason, and further investigation will be performed. In order to well understand the activity of IM-laccase toward substrate decolorization, cyclic voltammogramms (CVs) at bare glassy carbon (GC) electrode and modified GC electrodes in phosphate buffer solution (pH 7.0) containing 1.0 mM ABTS were investigated, and the results are shown in Figure 7. Two pairs of redox peaks with anodic potentials at 530 and 930 mV and cathodic potentials at 450 and 850 mV were observed at bare GC electrode (Figure 7, curve a). Bourbonnais et al.33 attributed this to direct electrochemical oxidation of ABTS in two steps to ABTS2+ and reverse reduction from ABTS2+ to
4. CONCLUSIONS In summary, immobilization of laccase on Fe 3 O 4 /SiO 2 nanoparticles with particle size below 30 nm was performed in this study by the glutaraldehyde coupling method. Laccase activity toward decolorization of phenolic azo dyes Procion Red MX-5B and azophloxine, as well as operational stability for dye decolorization, are improved remarkably after immobilization
Figure 7. Cyclic voltammograms of 1 mM ABTS at GC electrode, (a) bare or modified with (b) free laccase and (c) IM-laccase in 0.1 M phosphate buffer solution. Scan rate: 10 mV/s. 4405
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for the decolorization of different classes of textile dyes. Bioresour. Technol. 2008, 99, 7003. (11) Moya, R.; Hernández, M.; García-Martín, A. B.; Ball, A. S.; Arias, M. E. Contributions to a better comprehension of redox-mediated decolouration and detoxification of azo dyes by a laccase produced by Streptomyces cyaneus CECT 3335. Bioresour. Technol. 2010, 101, 2224. (12) Bourbonnais, R.; Paice, M. G. Oxidation of non-phenolic substrates: An expanded role for laccase in lignin biodegradation. FEBS Lett. 1990, 267, 99. (13) Wong, Y. X.; Yu, J. Laccase-catalyzed decolorization of synthetic dyes. Water Res. 1999, 33, 3512. (14) Claus, H.; Faber, G.; König, H. Redox-mediated decolorization of synthetic dyes by fungal laccases. Appl. Microbiol. Biotechnol. 2002, 59, 672. (15) Campos, R.; Kandelbauer, A.; Robra, K. H.; Cavaco-Paulo, A.; Gübitz, G. M. Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii. J. Biotechnol. 2001, 89, 131. (16) Durán, N.; Rosa, M. A.; D’Annibale, A.; Gianfreda, L. Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb. Technol. 2002, 31, 907. (17) Osma, J. F.; Toca-Herrera, J. L.; Rodríguez-Couto, S. Biodegradation of a simulated textile effluent by immobilised-coated laccase in laboratory-scale reactors. Appl. Catal., A 2010, 373, 147. (18) Bayramoğlu, G.; Yilmaz, M.; Arica, M. Y. Reversible immobilization of laccase to poly(4-vinylpyridine) grafted and Cu(II) chelated magnetic beads: Biodegradation of reactive dyes. Bioresour. Technol. 2010, 101, 6615. (19) Jiang, D. S.; Long, S. Y; Huang, J.; Xiao, H. Y.; Zhou, J. Y. Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem. Eng. J. 2005, 25, 15. (20) Lu, L.; Zhao, M.; Wang, Y. Immobilization of laccase by alginate−chitosan microcapsules and its use in dye decolorization. World J. Microbiol. Biotechnol. 2007, 23, 159. (21) Wang, F.; Guo, C.; Yang, L. R.; Liu, C. Z. Magnetic mesoporous silica nanoparticles: Fabrication and their laccase immobilization performance. Bioresour. Technol. 2010, 101, 8931. (22) Zhu, Y. F.; Kaskel, S.; Shi, J. L.; Wage, T.; van Pée, K. H. Immobilization of Trametes wersicolor laccase on magnetically separable mesoporous silica spheres. Chem. Mater. 2007, 19, 6408. (23) Arica, M. Y.; Altintas, B.; Bayramoğlu, G. Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: Application for textile dye degradation. Bioresour. Technol. 2009, 100, 665. (24) Dai, Y. R.; Niu, J. F.; Liu, J.; Yin, L. F.; Xu, J. J. In situ encapsulation of laccase in microfibers by emulsion electrospinning: Preparation, characterization, and application. Bioresour. Technol. 2010, 101, 8942. (25) Qiu, H. J.; Xu, C. X.; Huang, X. R.; Ding, Y.; Qu, Y. B.; Gao, P. J. Adsorption of laccase on the surface of nanoporous gold and the direct electron transfer between them. J. Phys. Chem. C 2008, 112, 14781. (26) Qiu, H .J.; Xu, C. X.; Huang, X. R.; Ding, Y.; Qu, Y. B.; Gao, P. J. Immobilization of laccase on nanoporous gold: Comparative studies on the immobilization strategies and the particle size effects. J. Phys. Chem. C 2009, 113, 2521. (27) Liu, Y.; Zeng, Z.; Zeng, G.; Tang, L.; Pang, Y.; Li, Z.; Liu, C.; Lei, X.; Wu, M.; Ren, P.; Liu, Z.; Chen, M.; Xie, G. Immobilization of laccase on magnetic bimodal mesoporous carbon and the application in the removal of phenolic compounds. Bioresour. Technol. 2012, 115, 21. (28) Bayramoğlu, G.; Yilmaz, M.; Arica, M. Y. Preparation and characterization of epoxy-functionalized magnetic chitosan beads: Laccase immobilized for degradation of reactive dyes. Bioprocess. Biosyst. Eng. 2010, 33, 439. (29) Zheng, X. B.; Wang, Q.; Jiang, Y. J.; Gao, J. Biomimetic synthesis of magnetic composite particles for laccase immobilization. Ind. Eng. Chem. Res. 2012, 51, 10140.
on Fe3O4/SiO2 nanoparticles. The color removal of each dye by the immobilized laccase exceeded 80% in 1 h. A series of measurements suggest that the improvement of laccase activity toward decolorization of phenolic azo dyes after immobilization is possibly the result of the change in microenvironment of the laccase formed by immobilization on Fe3O4/SiO2 nanoparticles, which led to fast effective direct electron transfer and thus the improved reduction of oxygen by laccase.
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ASSOCIATED CONTENT
S Supporting Information *
Five figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org/.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +86-451-88060570. E-mail: hsdwanghx@yahoo. com. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful for financial support from the Program for Scientific and Technological Innovation Team Construction in Universities of Heilongjiang (2011TD010), the Key Project of Chinese Ministry of Education (210060), the National Natural Science Foundation of China (21203048), and the Natural Science Foundation of Heilongjiang Province of China (B201011). Dr. Gang Hu (Exponent, Inc.) is acknowledged for helpful discussions.
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REFERENCES
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