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Chinese Chemical Letters 23 (2012) 197–200 www.elsevier.com/locate/cclet
Immobilization of laccase by Cu2+ chelate affinity interaction on surface modified magnetic silica particles and its use for the removal of pentachlorophenol Ying Wang a,b,*, Di Zhang a, Fu Rong He c, Xiao Chun Chen c a
The Key Laboratory of Water and Sediment Sciences, Ministry of Education, School of Environment, Beijing Normal University, Beijing 100875, China b State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, China c Department of Chemical Engineering, Beijing University of Chemical Technology, Beijing 100029, China Received 27 May 2011 Available online 21 December 2011
Abstract Magnetic Cu2+-chelated silica particles using polyacrylamide as a metal-chelating ligand was developed and used for the immobilization of laccase by coordination. The effect of pH and temperature on the enzymatic property of immobilized laccase and its catalytic capacity for pentachlorophenol (PCP) degradation were evaluated systemically. Compared with free laccase, the immobilized laccase showed improved acid adaptability and thermal stability. The immobilized laccase prepared in this work exhibited a good catalytic capacity for PCP removal from aqueous solutions. # 2011 Ying Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Magnetic silica; Immobilization; Laccase; Pentachlorophenol
Chlorophenols, a kind of toxic and non-biodegradable organic compounds, have been listed by the Environmental Protection Agency as prior pollutants [1]. Currently, many physical and chemical methods, such as adsorption, ozonation and filtration, are available for decontamination of chlorinated phenols. However, more attention has been focused on biological degradation methods because they are often cheaper and more environmentally friendly [2]. Laccase, a multi-copper oxidase, is capable of catalyzing the degradation of many phenolic compounds. Presently, many researchers focused on the immobilization of laccase and found that the activity of the immobilized laccase is dependent largely upon the properties of the carrier and the immobilization procedure [3,4]. The magnetic silica particles have been regarded as excellent carriers because of many important properties such as easy recovery, large surface areas, tunable pore sizes and volumes. Furthermore, they have well-defined surface properties which make them easily modified. For the immobilization procedure, the metal-chelated method based on the interaction between metal ions and the histidine or cysteine exposed on the surface of enzyme by the covalent conjugation was believed to be a good option because of its strong adsorption for enzyme, simple operation and reusability.
* Corresponding author at: The Key Laboratory of Water and Sediment Sciences, Ministry of Education, School of Environment, Beijing Normal University, Beijing 100875, China. E-mail address:
[email protected] (Y. Wang). 1001-8417/$ – see front matter # 2011 Ying Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2011.10.011
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Y. Wang et al. / Chinese Chemical Letters 23 (2012) 197–200
The aim of our study is to develop a kind of novel magnetic metal-chelated silica particles for laccase immobilization. Cu(II) ion was used because of its affinity towards proteins. The effects of pH and temperature on the enzymatic property of immobilized laccase and the catalytic capacity of the immobilized laccase for pentachlorophenol (PCP) degradation were also evaluated. 1. Experimental Preparation of magnetic SiO2: Firstly, Fe3O4 magnetic particles were prepared by coprecipitation of Fe2+ and Fe3+ ions in alkaline solution. Then the above magnetic particles were coated with silica using a typical sol–gel method. Modification of magnetic SiO2: 1.5 g of magnetic SiO2 particles was dispersed in 37.5 mL of methanol and 2.0 mL of deionised water. Then 37.5 mL of glycerol and 50 mL of methanol solution containing 5 mL of (CH3O)3CH2CH2CH2Cl (CP) were added and stirred at 80 8C for 3 h. The resultant product was washed with methanol and deionised water six times respectively and subsequently dried at 50 8C in air. The sample was mentioned as magnetic SiO2/CP. Preparation of magnetic Cu2+-chelated silica particles: The prepared magnetic SiO2/CP particles (0.8 g) were added into 2 mg/L of polyacrylamide (PAM) solution (100 mL) and stirred at 50 8C for 3 h. Then the resultant product was washed with distilled water and dried at 50 8C in air. Subsequently, 0.5 g of above product was added into CuSO4 solution (0.2 mol/L Cu2+) and mixed at 30 8C for 10 h. Finally, the resultant product was washed thoroughly with distilled water and dried at 50 8C. The sample was mentioned as magnetic Cu2+-chelated silica. Laccase immobilization: 0.1 g of magnetic Cu2+-chelated silica support was added in 7 mL of phosphate buffer solution (PBS) containing 2 mg of laccase. The system was slowly stirred at 25 8C to immobilize laccase. Subsequently, the immobilized laccase was washed with 5 mL of PBS (pH 3.0) for five times and then dried in a vacuum oven at room temperature. The activity of immobilized laccase was determined by a UV-2450 spectrophotometer (Shimadzu, Japan) by using 1 mmol/L 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) in distilled water as a substrate. One unit of activity is defined as the amount of enzyme required to catalyze 1 mmol of substrate per minute. The catalytic degradation of PCP by immobilized laccase was carried out in a batch reactor with an effective volume of 50 mL. The reactor was placed in a dark shaker with rotating speed of 100 r/min at 25 8C. Samples were taken at different time intervals. The concentration of PCP was analyzed by HPLC (Waters, USA). 2. Results and discussion The activities of laccase immobilized on magnetic SiO2 particles, magnetic SiO2/CP with grafted PAM particles and magnetic Cu2+-chelated silica particles was compared in Table 1. The activity of laccase immobilized on magnetic Cu2+-chelated silica particles was 234.42 U/mg, which was 1.50 and 5.81 times as high as those on magnetic SiO2/CP with grafted PAM particles and magnetic SiO2 particles, respectively. Because laccase was immobilized on the magnetic SiO2 particles only through physical adsorption, laccase could easily be desorbed from the carrier, which led to the decrease of laccase activity. After magnetic SiO2 was grafted with PAM, strong adsorption occurred between the protonated amino group of PAM and laccase with negative charge besides physical adsorption. So the immobilized laccase was not easily desorbed from the carrier and thus the activity of laccase increased. When laccase was immobilized on magnetic Cu2+-chelated silica particles, most laccase was immobilized by metal-chelated adsorption, which made the immobilization efficiency of laccase greatly improved. So the magnetic Cu2+-chelated silica particles are the best carrier for laccase immobilized in our study. Fig. 1 shows the effects of pH and temperature on the activity of the immobilized and free laccase. The activity observed in optimal conditions was taken as 100%. Higher activity of immobilized laccase was observed at the pH range of 2.0–3.5. For free laccase, the maximum activity was observed at pH 3.5, while it was shifted to pH 3.0 after Table 1 Effect of different carriers on the activity of immobilized laccase. Carriers
Magnetic SiO2
Magnetic SiO2/CP with grafted PAM
Magnetic Cu2+-chelated silica
Activity (U/mg)
40.37
156.62
234.42
Y. Wang et al. / Chinese Chemical Letters 23 (2012) 197–200
Relative activity (%)
(a) 100
100
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80
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Immonilized laccase Free laccase
0 5
199
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Temperature (ºC )
pH
Fig. 1. (a) Effect of pH on the activities of immobilized and free laccase (temperature: 25 8C); (b) effect of temperature on the activities of immobilized and free laccase (pH 3.0).
immobilized. The microenvironment of the immobilized laccase and the bulk solution usually has unequal partitioning of H+ and OH concentrations due to electrostatic interactions with the matrix, which often leads to the displacements in the pH activity profile [5]. So the optimal pH of the immobilized enzyme was displaced towards more acidic values as predicted from the cationic PAM. The same results have been also observed by other investigators [6,7]. Additionally, it is necessary to further study the modification of the immobilized laccase in order to increase its activity in neutral condition and then use it for actual wastewater. 100
4
support immobilized laccase
60
Cl concentration (mg/L)
PCP removal efficiency (%)
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40
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2
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0
1 0
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t (h)
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8
0
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Fig. 2. (a) The removal of PCP by adsorption of support and degradation of immobilized laccase (immobilized laccase: 8 g/L; solution volume: 50 mL; pH: 5.0; temperature: 25 8C); (b) the concentration of Cl ion during the degradation of PCP by immobilized laccase (immobilized laccase: 8 g/L; solution volume: 50 mL; pH: 5.0; temperature: 25 8C).
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The temperature value has an obvious effect on the activities of immobilized and free laccase. Higher activity of immobilized laccase was observed at the temperature of 25–40 8C. The optimum temperature for the immobilized laccase was 40 8C, which was obviously higher than that of the free laccase. A similar case has been reported by Sarı et al. [8]. This result was attributed to multipoint chelate interactions, which consequently leads to an increase in the activation energy for reorganization of the enzyme to an optimum conformation for binding to its substrate [8]. Meanwhile, compared with free laccase, the immobilized laccase exhibited higher relative activity at higher temperature, indicating that the immobilized laccase was more stable. The degradation of PCP by immobilized laccase was assessed in Fig. 2. When no carrier and laccase was added in PCP solution, the concentration of PCP rarely changed. In order to distinguish the removal of PCP by laccase catalysis with carrier adsorption, PCP removal by carrier adsorption was also evaluated using blank carrier without immobilized laccase. When using blank carrier, PCP concentration decreased and the removal efficiency reached 38.31% at the initial 2 h adsorption. Then no further decrease was observed, which indicated the adsorption of PCP reached equilibrium. Comparably, when using the immobilized laccase, PCP concentration decreased rapidly at the initial 0.5 h and the removal efficiency was 82.89%. After 5 h reaction, the concentration of Cl reached 3.90 mg/L. This result demonstrated that the immobilized laccase showed obvious catalytic capacity for PCP degradation. Above experiment was repeated for four times. The removal efficiency of PCP was still above 70% after 4 consecutive operations, indicating that the immobilized laccase exhibited excellent stability. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21177013 and 50708007), the National Basic Research Program of China (973 Project, No. 2010CB429003), the Fundamental Research Funds for the Central Universities and special fund of State Key Laboratory of Water Environment Simulation (No. 11K04ESPCN). References [1] [2] [3] [4] [5] [6] [7] [8]
T. Essam, M.A. Amin, O. El Tayeb, Chemosphere 66 (2007) 2201. J.B. Zhang, X.P. Liu, Z.Q. Xu, Int. Biodeterior. Biodegrad. 61 (2008) 351. S.A. Cetinus, E. Sahin, D. Saraydin, Food Chem. 114 (2009) 962. S. Akgol, N. Ozturk, A.A. Karagozler, J. Mol. Catal. B: Enzym. 51 (2008) 36. D.L. Jiang, S.Y. Long, J. Huang, Biochem. Eng. J. 25 (2005) 15. A. Rekuc, J. Bryjak, K.S. Ska, Proc. Biochem. 44 (2009) 191. J.B. Zhang, Z.Q. Xua, H. Chen, Biochem. Eng. J. 45 (2009) 54. M. Sarı, S. Akgol, M. Karatas, Ind. Eng. Chem. Res. 45 (2006) 3036.
