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SiOX magnetic nanoparticles using atom transfer radical polymerization and their application for lipase immobilization
Materials Chemistry and Physics 125 (2011) 866–871
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Study on synthesis of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles using atom transfer radical polymerization and their application for lipase immobilization Lin Lei, Xiao Liu, Yanfeng Li ∗ , Yanjun Cui, Yong Yang, Guorui Qin State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering & Environmental Technology, Lanzhou University, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 24 January 2010 Received in revised form 8 September 2010 Accepted 19 September 2010 Keywords: Polymers Chemical synthesis Magnetic properties Surface properties
a b s t r a c t Functionalized superparamagnetic particles were prepared by atom transfer radical polymerization of glycidyl methacrylate onto the surface of modified Fe3 O4 /SiOX nanoparticles. The obtained particles were characterized by X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and vibrating sample magnetometer (VSM). Candida rugosa lipase was covalently immobilized on the magnetic particles in mild condition via covalent binding with a higher activity recovery. The resulting immobilized lipase had better resistance to pH and temperature inactivation in comparison to free lipase, the adaptive pH and temperature ranges of lipase were widened, and it exhibited good thermal stability and reusability. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction The immobilization of enzymes in biologically active states on solid support surfaces has attracted much attention, not only because of the interesting role of immobilization in pure surface sciences but also because of the important role of immobilization in the development of biocatalysts and their applications in areas such as biosensors [1], pharmaceutical [2] and organic synthesis [3]. Immobilization provides many advantages, such as use in continuous operations, product purification, catalyst recycling, enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth [4,5]. In recent years, magnetic nanoparticles became very popular when used in conjunction with biological materials such as proteins, peptides, enzymes, antibodies, and nucleic acids because of their unique properties [6–9]. This application is mainly based on the magnetic feature of the solid-phase that enables to achieve a rapid easy separation from the reaction medium in a magnetic field. In order to increase loading amount of the biomolecules immobilized on magnetic particles and improve the stability of immobilized biomolecules, the preparation of surface functionalized magnetic particles with water soluble, biocompatible and reactive groups is much desired. ATRP is a pseudo-living radical
∗ Corresponding author. Fax: +86 931 8912113. E-mail address: [email protected] (Y. Li).
polymerization, the polymers can be end-functionalized or block copolymerized upon the addition of other monomers [10]. This feature offers tailor ability of the polymer coating with a variety of compositions and functionalities. Therefore, it becomes the most attractive to obtain magnetic supports with reactive groups to immobilize biomolecules via ATRP. As a potential surface linker for biomolecules, poly-(glycidyl methacrylate) (GMA) has a promising application in advanced biotechnology. It has been successfully employed for the immobilization–stabilization of enzymes, resulting in a higher resistance to conformational changes induced by heat, organic solvents, or pH [11–13]. Immobilization is based on the reaction between epoxy groups and primary amino groups in enzymes, which are mostly -amino groups in lysine residues but also terminal amino groups. Many enzymes have been stabilized using this technique, including trypsin and chymotrypsin [14], alcalase [15], carboxypeptidase A [16], catalases [17], and lipases from different sources [18]. The objective of this work was to demonstrate the potential use of magnetic nanoparticals in the bioengineering application. Firstly, magnetic microspheres coated with SiOX were synthesized, the SiOX shell not only better providing a good degree of biocompatibility and a high specific area whose rich chemistry allows easy functionalization but also protecting iron oxides in magnetic microspheres from acid pH values. Secondly, organic molecules were introduced onto the Fe3 O4 /SiOX surface to prepare macromolecule initiator and poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles were obtained by atom transfer radical polymerization. The
0254-0584/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2010.09.031
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Scheme 1. Surface modification and atom transfer radical polymerization of poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles and the immobilization of lipase.
nanoparticles were characterized by X-ray powder diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and vibrating sample magnetometer. Then lipase was covalently immobilized onto the magnetic nanoparticles via active epoxy groups. The properties of the immobilized lipase such as activity recovery, thermal stability, reusability and kinetic behavior were investigated. 2. Materials and methods 2.1. Materials Glycidyl methacrylate (GMA) were obtained from Ciba Specialty Chemicals (China) Ltd. Guangzhou; ␥-aminopropyltriethoxysilane (APTES, 98%) was purchased from Wuhan University Silicone New Material Co. Ltd. (China); Candida rugosa lipase (CRL, Type VII, 1180 units mg−1 solid) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co.; 2,2 -bipyridine (Bpy), copper(I) chloride, copper(II) chloride, tetraethoxysilane (TEOS, 98%) and dimethylformamide (DMF) were obtained from Tianjing Chemical Reagent Company (China); triethylamine (TEA), ferric chloride hexahydrate (FeCl3 ·6H2 O), ferrous chloride tetrahydrate (FeCl2 ·4H2 O), ammonium hydroxide (25 wt%) and other chemicals and solvents were analytical grade, obtained from Tianjing Chemical Reagent Company (China). 2.2. Synthesis of Fe3 O4 /SiOX -g-P(GMA) nanoparticles 2.2.1. Synthesis of Fe3 O4 nanoparticles The preparation of Fe3 O4 nanoparticles was followed by a chemical coprecipitation of Fe2+ and Fe3+ ions with some modifications. 50 ml of 1.0 M FeCl2 and 1.75 M FeCl3 solution were prepared with deionized water in separate beakers, and added to a 250 ml three-necked flask, stirred together under nitrogen. When
the solution was heated to 60 ◦ C, NH3 ·H2 O (25 wt%) was added drop-wise until pH = 10–11. Immediately after base addition solution became dark-brown indicating that iron oxide has been formed in the system. The solution was heated at 80 ◦ C for 1 h. The precipitates were isolated from the solvent by magnetic decantation and repeatedly washed with deionized water until neutral, and were then dried at room temperature under evacuated for 12 h. 2.2.2. Silica-coating of magnetic nanoparticles The silica-coating procedure was performed by the Deng et al. [19] method with minor modifications. Typically, 0.5 g of Fe3 O4 nanoparticles was added to 160 ml of ethanol and the dispersion was homogenized under ultrasonic vibration in water bath. After 10 min, 40 ml of water, 5 ml of aqueous ammonia, and 1 ml of tetraethyl orthosilicate (TEOS) were added into the dispersion, which was continuously mechanically stirred for another 24 h. The magnetite was magnetically separated with a permanent magnet. The obtained product was washed with distilled water several times. 2.2.3. Immobilization of initiator on Fe3 O4 /SiOX The preparation procedures of the initiator modified Fe3 O4 /SiOX , bromoacetyl modified Fe3 O4 /SiOX , could be schematically shown as Scheme 1. APTES-modified Fe3 O4 /SiOX nanoparticles were achieved by the reaction between APTES and the hydroxyl groups on the surface of magnetite. Typically, 1.0 g of Fe3 O4 /SiOX nanoparticles were dispersed in 60 ml of ethanol by sonication for about 1 h, then 6.0 ml of ammonium hydroxide was added and sonicated to homogenize for 10 min. Under continuous mechanical stirring, 4.0 ml of APTES was added to the reaction mixture. The reaction was allowed to proceed at 50 ◦ C for 8 h under continuous stirring. After that, the resultant products were obtained by magnetic separation with permanent magnet and were thoroughly washed with ethanol and deionized water until neutral, then were dried at room temperature under evacuated for 12 h. After 2.0 g of APTES-modified Fe3 O4 /SiOX was dispersed into 30 ml of toluene containing 4 ml of TEA under electromagnetic stirring in an ice bath. After the above
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mixture was cooled down to 0 ◦ C, a solution of chloroacetyl chloride (4 ml) and toluene (8 ml) was added drop by drop into the dispersoid and the mixture was stirred with an electromagnetic stirrer for 12 h at room temperature. Then, the nanoparticles were separated with a permanent magnet and washed with toluene and ethanol thoroughly and then dried in vacuum for the subsequent polymerization.
2.2.4. Surface-initiated ATRP of GMA For the preparation of GMA polymer on the Fe3 O4 /SiOX surface, the reaction was carried out by adding a [GMA]/[CuCl]/[CuCl2 ]/[Bpy] feed ratio of 100:1:0.2:2 in 20 ml of a mixed solvent [DMF/water (1:1, v/v)] at room temperature into a dry round-bottom flask containing 0.5 g of nanoparticles synthesized before. The mixture was irradiated with ultrasonic vibrations for 30 min, while bubbling with nitrogen (N2 ). The reaction was allowed to proceed for a predetermined period of time to give rise to the poly(GMA)-grafted Fe3 O4 /SiOX surface. After the reaction, the poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles was washed thoroughly by extraction with acetone for about 48 h to ensure the complete removal of the adhered and physically adsorbed polymer, and then, the sample was dried in vacuum at 40 ◦ C. The contents of epoxy group on the surface of the magnetic nanoparticles sample were measured according to Ref. [20].
2.3. Characterization Power X-ray diffraction (XRD) was used to investigate the crystal structure of the magnetic nanoparticles. The size and shape of the nanoparticles were determined by transmission electron microscope (TEM). The sample was dispersed in ethanol and spread a small drop onto a 400 mesh copper grid. The IR spectra were recorded by a Fourier transform infrared spectrophotometer (FT-IR), and the sample and KBr were pressed to form a tablet. The magnetization curves of samples were measured with a vibrating sample magnetometer (VSM) at room temperature.
Fig. 1. XRD patterns of (a) pure Fe3 O4 nanoparticles; (b) poly(GMA)-grafted Fe3 O4 /SiOX carrier.
3. Results and discussion 3.1. Properties of Fe3 O4 and poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles
2.4. Immobilization of lipase Due to the epoxy groups on the surface of nanoparticles, lipase immobilization was carried out by the treatment of the lipase solution with the particles directly. The particles (1.0 g) were added into 40 ml of phosphate buffer (0.1 M, pH 6.5) containing lipase (0.2 g). The mixture was placed in a shaking incubator at 30 ◦ C and 150 rpm, while continuously shook for 6 h to finish immobilization of lipase. The immobilized lipase was recovered by magnetic separation, and washed with phosphate buffer (0.1 M, pH 6.5) three times to remove excess lipase. The resulting immobilized lipase was held at 4 ◦ C prior to use. The enzymatic activities of free and immobilized lipase were measured by the titration of the fatty acid which comes from the hydrolysis of olive oil. One unit of lipase activity (U) is defined as the amount of enzyme that hydrolyzes olive oil liberating 1.0 mol fatty acid per minute under the assay conditions. The relative recovery (%) was the ratio between the activity of immobilized lipase and the activity of free lipase.
2.5. Protein determination The amount of protein in the lipase solution and in supernatant after immobilization was determined by the Bradford method [21], and the amount of protein [(C −C )V ]
(p) bound on the supports was calculated from the formula:p = i W f where, p is the amount of bound lipase onto supports (mg g−1 ), Ci and Cf are the concentrations of the lipase protein initial and final in the reaction medium (mg ml−1 ), V is the volume of the reaction medium (ml) and W is the weight of the supports (g). All data used in this formula are the average of triplicate of experiments.
2.6. Effect of pH and temperature on free and immobilized lipase activity The effect of temperature on free and immobilized lipase activities was determined after incubation in 20 ml phosphate buffer (0.1 M, pH 7.0) for 30 min in the range of 20–80 ◦ C. The effect of pH on the activities of free and immobilized CRL was investigated at 37 ◦ C under the variety of pH (0.1 M phosphate buffer for pH 6.0–9.0, boric acid–NaOH buffer solution pH range of 8.0–9.0) for 30 min.
2.7. Thermal stability and reusability Thermal stabilities of the free and immobilized lipase were studied by measuring the relative activities of the enzymes after incubation in phosphate buffer (0.1 M, pH 7.0) for 30 min in the range of 25–80 ◦ C with continuous shaking. In addition, the reusability of the immobilized lipase was determined by hydrolysis of olive oil by the recovered immobilized lipase with magnetic separation and compared with the first run (activity defined as 100%).
The process for synthesis of poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles and immobilization of lipase onto them is shown in Scheme 1. The XRD patterns of pure Fe3 O4 and poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles are shown in Fig. 1. It is apparent that the diffraction pattern of the Fe3 O4 nanoparticles is close to the standard pattern for crystalline magnetite (Fig. 1(a)). According to the XRD data, the standard Fe3 O4 crystal with spinel structure has six diffraction peaks: (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0). They could be well indexed to the inverse cubic spinel structure of Fe3 O4 (JCPDS Card No. 85-1436), were also observed for poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles (Fig. 1(b)). This revealed that modified and graft polymerized on the surface of Fe3 O4 nanoparticles did not lead to their phase change. In addition, from the data of XRD, the average crystallite size D was about 15 nm, obtained from Sherrer equation: D = K/(ˇ cos ), where K is constant, is X-ray wavelength, and ˇ is the peak width at halfmaximum. FT-IR spectroscopy was used to show the structure of Fe3 O4 /SiOX composite particles containing Initiator (Fig. 2(a)) and poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles (Fig. 2(b)). From the IR spectra presented in Fig. 2, the absorption peaks at 579 cm−1 belonged to the stretching vibration mode of FeO bonds in Fe3 O4 , the absorption peak presented at 1082 cm−1 most probably due to stretching vibration of framework and terminal Si–O-groups. The absorption peaks presented at 1650 cm−1 should be attached to the stretching vibrations of –C O from amide. Comparing with the IR spectrum (a), the IR spectrum (b) of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles holds a stronger absorption peak at 1729 cm−1 , it should belong to the stretching vibration mode of C O from GMA, i.e. an evidence that the GMA were successfully grafted to Fe3 O4 /SiOX composite nanoparticles. However, the identification of a peak attributable to the stretching vibrations of the epoxy groups from GMA link (normally at about 1250, 900 cm−1 ) was problematic due to the overlapping of the peaks of Si–O-groups. The TEM micrographs of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles (Fig. 3) is shown. Observing the photograph, the
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Fig. 2. FT-IR spectra of (a) Fe3 O4 /SiOX composite particles containing initiator; (b) poly(GMA)-grafted Fe3 O4 /SiOX carrier.
Fig. 4. Magnetic hysteresis loop of (a) Fe3 O4 /SiOX nanoparticles; (b) poly(GMA)grafted Fe3 O4 /SiOX carrier.
average size of the magnetic nanoparticles were 100 nm, and the dispersion of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles was fine, which can be explained by the electrostatic repulsion force and steric hindrance between the polymer chains on the surface of Fe3 O4 /SiOX nanoparticles. As clearly seen here, the spheres have a spherical form. The magnetic properties of the microspheres are significant for all further applications. The magnetic properties of poly(GMA)grafted Fe3 O4 /SiOX magnetic nanoparticles were analyzed by VSM at room temperature. Fig. 4 shows the hysteresis loops of the samples. The saturation magnetization is found to be 23.6 kA m−1 and 8.3 kA m−1 for Fe3 O4 /SiOX composite nanoparticles and poly(GMA)-grafted Fe3 O4 /SiOX , respectively. The amounts of polymers coated on the surface of Fe3 O4 /SiOX nanoparticles were calculated to be about 64% from the data. The magnetization curve exhibits zero remanence and coercivity, which proves that these magnetic microsphere have superparamagnetic properties. With the large saturation magnetization, the magnetic microspheres can
be separated from the reaction medium rapidly and easily in a magnetic field. 3.2. Immobilization of lipase Poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles with active epoxy groups were prepared and used for immobilizing lipase by covalent reaction via the amino groups of the lipase and the epoxy groups of the magnetic nanoparticles under mild condition. The content of epoxy groups on the surface of the magnetic nanoparticles sample was determined to be 1.62 ± 0.05 mmol g−1 . The activity recovery of these particles immobilized lipase was assayed through hydrolysis olive oil, and the maximum activity recovery was up to 69.1%. The enzyme loading amount on the particles was also determined, it was 98.4 mg protein g−1 magnetic nanoparticles. Such immobilization capacity is considered to be high, when compared with other covalent immobilization methods reported in the literature (immobilization capacities are in the range of 0.2–63.0 mg g−1 of support) [22–25]. 3.3. Effect of pH and temperature on free and immobilized CRL activity
Fig. 3. TEM micrographs of poly(GMA)-grafted Fe3 O4 /SiOX carrier.
The effect of pH values of the solutions on the hydrolysis activity of free and immobilized lipase for olive oil was determined within 6.0–9.0 pH range at 37 ◦ C, and the results are shown in Fig. 5. The maximum activity of the free and immobilized lipase was both at pH 7.0, but the activity of immobilized lipase was observed to be higher than that of free enzyme at alkaline. Furthermore, the pH profiles of the immobilized lipase preparations are broader than that of the free enzyme (Fig. 5). This phenomenon is similar to those enzymes being immobilized using conventional methods [26–28]. The effect of temperature of the solutions on the hydrolysis activity of free and immobilized lipase for olive oil was determined, and the results are shown in Fig. 6. The optimum reaction temperature of free enzyme and the immobilized lipase was both at 40 ◦ C, but compared with free lipase, the relative activity of the immobilized lipase is about 50% within 70–80 ◦ C, still exhibiting a wider endurance for reaction temperature. The result from Fig. 6 shows that the immobilized lipase holds much more excellent heat resistance than that of the free enzyme.
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Fig. 5. Effect of pH value on the activity of free and immobilized lipase. Incubated in phosphate buffer at 37 ◦ C for 30 min in the pH range 6.0–9.0.
Fig. 7. Thermal stability of the free and the immobilized lipase. Incubated in phosphate buffer (0.1 M, pH 7.0) at 50 ◦ C for different time.
3.4. Thermal stability and reusability Thermal stability of the free and immobilized lipase was determined by using olive oil as substrate at 50 ◦ C in phosphate buffer (0.1 M, pH 7.0) as shown in Fig. 7. Both preparations exhibited a similar trend, however, the immobilized lipase is more stable than the free one. The immobilized lipase retained their initial activity of about 60% after 210 min, but the free lipase lost its activity within around 180 min. These results indicated that the immobilized lipase had better thermal resistance than the free one. The enhancement of thermal stability is probably attributed to the covalent bonding between lipase and PGMA, thus restricting the conformational change of lipase during heating [29,30]. The reusability of immobilized lipase is an important aspect from an application standpoint, especially in industrial applications. To investigate the reusability, the immobilized lipase was washed with phosphate buffer (0.1 M, pH 7.0) after one catalysis run and reintroduced into a fresh olive oil solution for another hydrolysis at 37 ◦ C. Fig. 8 shows the variation of activity of the immobilized lipase after reuses. It was observed that the immobilized lipase had still retained 83% of their original activities after the 6th reuse. It could be concluded that immobilized lipase has a good durability and reusability.
Fig. 8. Reuse of the immobilized lipase for hydrolyzing olive oil.
4. Conclusion In the present study, the poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles were prepared by atom transfer radical polymerization and utilized for lipase immobilization. The obtained magnetic nanoparticles had smaller diameter (100 nm), higher saturation magnetization (8.3 kA m−1 ), and showed superior magnetic responsibility. The activity recovery of immobilization lipase reached 69.1% with the protein loaded 98.4 mg g−1 carriers. The optimal pH and temperature of the immobilized lipase were 7.0 and 40 ◦ C, respectively. The residual activity of immobilized lipase decreased a little as it was kept at 50 ◦ C for 150 min, and the residual activity of the immobilized lipase was still over 83% when it was used repeatedly 6 times. It can be concluded that these magnetic nanoparticles provides an economical, efficient and selective system for enzyme immobilization. Acknowledgements
Fig. 6. Effect of temperature on the activity of free and immobilized lipase. Incubated in phosphate buffer (0.1 M, pH 7.0) for 30 min in the temperature range 20–80 ◦ C.
The authors thank the financial supports from the National Natural Science Foundation of China (No. 21074049), the financial supports from the Committee of Natural Science Foundation in China on National Training Fund for Person with Ability of Basic Subjects (J0730425) and Key Research Program of Gansu Province (2GS064-A52-036-02).
L. Lei et al. / Materials Chemistry and Physics 125 (2011) 866–871
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Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Study on synthesis of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles using atom transfer radical polymerization and their application for lipase immobilization Lin Lei, Xiao Liu, Yanfeng Li ∗ , Yanjun Cui, Yong Yang, Guorui Qin State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering & Environmental Technology, Lanzhou University, Lanzhou 730000, China
a r t i c l e
i n f o
Article history: Received 24 January 2010 Received in revised form 8 September 2010 Accepted 19 September 2010 Keywords: Polymers Chemical synthesis Magnetic properties Surface properties
a b s t r a c t Functionalized superparamagnetic particles were prepared by atom transfer radical polymerization of glycidyl methacrylate onto the surface of modified Fe3 O4 /SiOX nanoparticles. The obtained particles were characterized by X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and vibrating sample magnetometer (VSM). Candida rugosa lipase was covalently immobilized on the magnetic particles in mild condition via covalent binding with a higher activity recovery. The resulting immobilized lipase had better resistance to pH and temperature inactivation in comparison to free lipase, the adaptive pH and temperature ranges of lipase were widened, and it exhibited good thermal stability and reusability. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction The immobilization of enzymes in biologically active states on solid support surfaces has attracted much attention, not only because of the interesting role of immobilization in pure surface sciences but also because of the important role of immobilization in the development of biocatalysts and their applications in areas such as biosensors [1], pharmaceutical [2] and organic synthesis [3]. Immobilization provides many advantages, such as use in continuous operations, product purification, catalyst recycling, enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth [4,5]. In recent years, magnetic nanoparticles became very popular when used in conjunction with biological materials such as proteins, peptides, enzymes, antibodies, and nucleic acids because of their unique properties [6–9]. This application is mainly based on the magnetic feature of the solid-phase that enables to achieve a rapid easy separation from the reaction medium in a magnetic field. In order to increase loading amount of the biomolecules immobilized on magnetic particles and improve the stability of immobilized biomolecules, the preparation of surface functionalized magnetic particles with water soluble, biocompatible and reactive groups is much desired. ATRP is a pseudo-living radical
∗ Corresponding author. Fax: +86 931 8912113. E-mail address: [email protected] (Y. Li).
polymerization, the polymers can be end-functionalized or block copolymerized upon the addition of other monomers [10]. This feature offers tailor ability of the polymer coating with a variety of compositions and functionalities. Therefore, it becomes the most attractive to obtain magnetic supports with reactive groups to immobilize biomolecules via ATRP. As a potential surface linker for biomolecules, poly-(glycidyl methacrylate) (GMA) has a promising application in advanced biotechnology. It has been successfully employed for the immobilization–stabilization of enzymes, resulting in a higher resistance to conformational changes induced by heat, organic solvents, or pH [11–13]. Immobilization is based on the reaction between epoxy groups and primary amino groups in enzymes, which are mostly -amino groups in lysine residues but also terminal amino groups. Many enzymes have been stabilized using this technique, including trypsin and chymotrypsin [14], alcalase [15], carboxypeptidase A [16], catalases [17], and lipases from different sources [18]. The objective of this work was to demonstrate the potential use of magnetic nanoparticals in the bioengineering application. Firstly, magnetic microspheres coated with SiOX were synthesized, the SiOX shell not only better providing a good degree of biocompatibility and a high specific area whose rich chemistry allows easy functionalization but also protecting iron oxides in magnetic microspheres from acid pH values. Secondly, organic molecules were introduced onto the Fe3 O4 /SiOX surface to prepare macromolecule initiator and poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles were obtained by atom transfer radical polymerization. The
0254-0584/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2010.09.031
L. Lei et al. / Materials Chemistry and Physics 125 (2011) 866–871
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Scheme 1. Surface modification and atom transfer radical polymerization of poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles and the immobilization of lipase.
nanoparticles were characterized by X-ray powder diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and vibrating sample magnetometer. Then lipase was covalently immobilized onto the magnetic nanoparticles via active epoxy groups. The properties of the immobilized lipase such as activity recovery, thermal stability, reusability and kinetic behavior were investigated. 2. Materials and methods 2.1. Materials Glycidyl methacrylate (GMA) were obtained from Ciba Specialty Chemicals (China) Ltd. Guangzhou; ␥-aminopropyltriethoxysilane (APTES, 98%) was purchased from Wuhan University Silicone New Material Co. Ltd. (China); Candida rugosa lipase (CRL, Type VII, 1180 units mg−1 solid) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co.; 2,2 -bipyridine (Bpy), copper(I) chloride, copper(II) chloride, tetraethoxysilane (TEOS, 98%) and dimethylformamide (DMF) were obtained from Tianjing Chemical Reagent Company (China); triethylamine (TEA), ferric chloride hexahydrate (FeCl3 ·6H2 O), ferrous chloride tetrahydrate (FeCl2 ·4H2 O), ammonium hydroxide (25 wt%) and other chemicals and solvents were analytical grade, obtained from Tianjing Chemical Reagent Company (China). 2.2. Synthesis of Fe3 O4 /SiOX -g-P(GMA) nanoparticles 2.2.1. Synthesis of Fe3 O4 nanoparticles The preparation of Fe3 O4 nanoparticles was followed by a chemical coprecipitation of Fe2+ and Fe3+ ions with some modifications. 50 ml of 1.0 M FeCl2 and 1.75 M FeCl3 solution were prepared with deionized water in separate beakers, and added to a 250 ml three-necked flask, stirred together under nitrogen. When
the solution was heated to 60 ◦ C, NH3 ·H2 O (25 wt%) was added drop-wise until pH = 10–11. Immediately after base addition solution became dark-brown indicating that iron oxide has been formed in the system. The solution was heated at 80 ◦ C for 1 h. The precipitates were isolated from the solvent by magnetic decantation and repeatedly washed with deionized water until neutral, and were then dried at room temperature under evacuated for 12 h. 2.2.2. Silica-coating of magnetic nanoparticles The silica-coating procedure was performed by the Deng et al. [19] method with minor modifications. Typically, 0.5 g of Fe3 O4 nanoparticles was added to 160 ml of ethanol and the dispersion was homogenized under ultrasonic vibration in water bath. After 10 min, 40 ml of water, 5 ml of aqueous ammonia, and 1 ml of tetraethyl orthosilicate (TEOS) were added into the dispersion, which was continuously mechanically stirred for another 24 h. The magnetite was magnetically separated with a permanent magnet. The obtained product was washed with distilled water several times. 2.2.3. Immobilization of initiator on Fe3 O4 /SiOX The preparation procedures of the initiator modified Fe3 O4 /SiOX , bromoacetyl modified Fe3 O4 /SiOX , could be schematically shown as Scheme 1. APTES-modified Fe3 O4 /SiOX nanoparticles were achieved by the reaction between APTES and the hydroxyl groups on the surface of magnetite. Typically, 1.0 g of Fe3 O4 /SiOX nanoparticles were dispersed in 60 ml of ethanol by sonication for about 1 h, then 6.0 ml of ammonium hydroxide was added and sonicated to homogenize for 10 min. Under continuous mechanical stirring, 4.0 ml of APTES was added to the reaction mixture. The reaction was allowed to proceed at 50 ◦ C for 8 h under continuous stirring. After that, the resultant products were obtained by magnetic separation with permanent magnet and were thoroughly washed with ethanol and deionized water until neutral, then were dried at room temperature under evacuated for 12 h. After 2.0 g of APTES-modified Fe3 O4 /SiOX was dispersed into 30 ml of toluene containing 4 ml of TEA under electromagnetic stirring in an ice bath. After the above
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mixture was cooled down to 0 ◦ C, a solution of chloroacetyl chloride (4 ml) and toluene (8 ml) was added drop by drop into the dispersoid and the mixture was stirred with an electromagnetic stirrer for 12 h at room temperature. Then, the nanoparticles were separated with a permanent magnet and washed with toluene and ethanol thoroughly and then dried in vacuum for the subsequent polymerization.
2.2.4. Surface-initiated ATRP of GMA For the preparation of GMA polymer on the Fe3 O4 /SiOX surface, the reaction was carried out by adding a [GMA]/[CuCl]/[CuCl2 ]/[Bpy] feed ratio of 100:1:0.2:2 in 20 ml of a mixed solvent [DMF/water (1:1, v/v)] at room temperature into a dry round-bottom flask containing 0.5 g of nanoparticles synthesized before. The mixture was irradiated with ultrasonic vibrations for 30 min, while bubbling with nitrogen (N2 ). The reaction was allowed to proceed for a predetermined period of time to give rise to the poly(GMA)-grafted Fe3 O4 /SiOX surface. After the reaction, the poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles was washed thoroughly by extraction with acetone for about 48 h to ensure the complete removal of the adhered and physically adsorbed polymer, and then, the sample was dried in vacuum at 40 ◦ C. The contents of epoxy group on the surface of the magnetic nanoparticles sample were measured according to Ref. [20].
2.3. Characterization Power X-ray diffraction (XRD) was used to investigate the crystal structure of the magnetic nanoparticles. The size and shape of the nanoparticles were determined by transmission electron microscope (TEM). The sample was dispersed in ethanol and spread a small drop onto a 400 mesh copper grid. The IR spectra were recorded by a Fourier transform infrared spectrophotometer (FT-IR), and the sample and KBr were pressed to form a tablet. The magnetization curves of samples were measured with a vibrating sample magnetometer (VSM) at room temperature.
Fig. 1. XRD patterns of (a) pure Fe3 O4 nanoparticles; (b) poly(GMA)-grafted Fe3 O4 /SiOX carrier.
3. Results and discussion 3.1. Properties of Fe3 O4 and poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles
2.4. Immobilization of lipase Due to the epoxy groups on the surface of nanoparticles, lipase immobilization was carried out by the treatment of the lipase solution with the particles directly. The particles (1.0 g) were added into 40 ml of phosphate buffer (0.1 M, pH 6.5) containing lipase (0.2 g). The mixture was placed in a shaking incubator at 30 ◦ C and 150 rpm, while continuously shook for 6 h to finish immobilization of lipase. The immobilized lipase was recovered by magnetic separation, and washed with phosphate buffer (0.1 M, pH 6.5) three times to remove excess lipase. The resulting immobilized lipase was held at 4 ◦ C prior to use. The enzymatic activities of free and immobilized lipase were measured by the titration of the fatty acid which comes from the hydrolysis of olive oil. One unit of lipase activity (U) is defined as the amount of enzyme that hydrolyzes olive oil liberating 1.0 mol fatty acid per minute under the assay conditions. The relative recovery (%) was the ratio between the activity of immobilized lipase and the activity of free lipase.
2.5. Protein determination The amount of protein in the lipase solution and in supernatant after immobilization was determined by the Bradford method [21], and the amount of protein [(C −C )V ]
(p) bound on the supports was calculated from the formula:p = i W f where, p is the amount of bound lipase onto supports (mg g−1 ), Ci and Cf are the concentrations of the lipase protein initial and final in the reaction medium (mg ml−1 ), V is the volume of the reaction medium (ml) and W is the weight of the supports (g). All data used in this formula are the average of triplicate of experiments.
2.6. Effect of pH and temperature on free and immobilized lipase activity The effect of temperature on free and immobilized lipase activities was determined after incubation in 20 ml phosphate buffer (0.1 M, pH 7.0) for 30 min in the range of 20–80 ◦ C. The effect of pH on the activities of free and immobilized CRL was investigated at 37 ◦ C under the variety of pH (0.1 M phosphate buffer for pH 6.0–9.0, boric acid–NaOH buffer solution pH range of 8.0–9.0) for 30 min.
2.7. Thermal stability and reusability Thermal stabilities of the free and immobilized lipase were studied by measuring the relative activities of the enzymes after incubation in phosphate buffer (0.1 M, pH 7.0) for 30 min in the range of 25–80 ◦ C with continuous shaking. In addition, the reusability of the immobilized lipase was determined by hydrolysis of olive oil by the recovered immobilized lipase with magnetic separation and compared with the first run (activity defined as 100%).
The process for synthesis of poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles and immobilization of lipase onto them is shown in Scheme 1. The XRD patterns of pure Fe3 O4 and poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles are shown in Fig. 1. It is apparent that the diffraction pattern of the Fe3 O4 nanoparticles is close to the standard pattern for crystalline magnetite (Fig. 1(a)). According to the XRD data, the standard Fe3 O4 crystal with spinel structure has six diffraction peaks: (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0). They could be well indexed to the inverse cubic spinel structure of Fe3 O4 (JCPDS Card No. 85-1436), were also observed for poly(GMA)-grafted Fe3 O4 /SiOX nanoparticles (Fig. 1(b)). This revealed that modified and graft polymerized on the surface of Fe3 O4 nanoparticles did not lead to their phase change. In addition, from the data of XRD, the average crystallite size D was about 15 nm, obtained from Sherrer equation: D = K/(ˇ cos ), where K is constant, is X-ray wavelength, and ˇ is the peak width at halfmaximum. FT-IR spectroscopy was used to show the structure of Fe3 O4 /SiOX composite particles containing Initiator (Fig. 2(a)) and poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles (Fig. 2(b)). From the IR spectra presented in Fig. 2, the absorption peaks at 579 cm−1 belonged to the stretching vibration mode of FeO bonds in Fe3 O4 , the absorption peak presented at 1082 cm−1 most probably due to stretching vibration of framework and terminal Si–O-groups. The absorption peaks presented at 1650 cm−1 should be attached to the stretching vibrations of –C O from amide. Comparing with the IR spectrum (a), the IR spectrum (b) of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles holds a stronger absorption peak at 1729 cm−1 , it should belong to the stretching vibration mode of C O from GMA, i.e. an evidence that the GMA were successfully grafted to Fe3 O4 /SiOX composite nanoparticles. However, the identification of a peak attributable to the stretching vibrations of the epoxy groups from GMA link (normally at about 1250, 900 cm−1 ) was problematic due to the overlapping of the peaks of Si–O-groups. The TEM micrographs of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles (Fig. 3) is shown. Observing the photograph, the
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Fig. 2. FT-IR spectra of (a) Fe3 O4 /SiOX composite particles containing initiator; (b) poly(GMA)-grafted Fe3 O4 /SiOX carrier.
Fig. 4. Magnetic hysteresis loop of (a) Fe3 O4 /SiOX nanoparticles; (b) poly(GMA)grafted Fe3 O4 /SiOX carrier.
average size of the magnetic nanoparticles were 100 nm, and the dispersion of poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles was fine, which can be explained by the electrostatic repulsion force and steric hindrance between the polymer chains on the surface of Fe3 O4 /SiOX nanoparticles. As clearly seen here, the spheres have a spherical form. The magnetic properties of the microspheres are significant for all further applications. The magnetic properties of poly(GMA)grafted Fe3 O4 /SiOX magnetic nanoparticles were analyzed by VSM at room temperature. Fig. 4 shows the hysteresis loops of the samples. The saturation magnetization is found to be 23.6 kA m−1 and 8.3 kA m−1 for Fe3 O4 /SiOX composite nanoparticles and poly(GMA)-grafted Fe3 O4 /SiOX , respectively. The amounts of polymers coated on the surface of Fe3 O4 /SiOX nanoparticles were calculated to be about 64% from the data. The magnetization curve exhibits zero remanence and coercivity, which proves that these magnetic microsphere have superparamagnetic properties. With the large saturation magnetization, the magnetic microspheres can
be separated from the reaction medium rapidly and easily in a magnetic field. 3.2. Immobilization of lipase Poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles with active epoxy groups were prepared and used for immobilizing lipase by covalent reaction via the amino groups of the lipase and the epoxy groups of the magnetic nanoparticles under mild condition. The content of epoxy groups on the surface of the magnetic nanoparticles sample was determined to be 1.62 ± 0.05 mmol g−1 . The activity recovery of these particles immobilized lipase was assayed through hydrolysis olive oil, and the maximum activity recovery was up to 69.1%. The enzyme loading amount on the particles was also determined, it was 98.4 mg protein g−1 magnetic nanoparticles. Such immobilization capacity is considered to be high, when compared with other covalent immobilization methods reported in the literature (immobilization capacities are in the range of 0.2–63.0 mg g−1 of support) [22–25]. 3.3. Effect of pH and temperature on free and immobilized CRL activity
Fig. 3. TEM micrographs of poly(GMA)-grafted Fe3 O4 /SiOX carrier.
The effect of pH values of the solutions on the hydrolysis activity of free and immobilized lipase for olive oil was determined within 6.0–9.0 pH range at 37 ◦ C, and the results are shown in Fig. 5. The maximum activity of the free and immobilized lipase was both at pH 7.0, but the activity of immobilized lipase was observed to be higher than that of free enzyme at alkaline. Furthermore, the pH profiles of the immobilized lipase preparations are broader than that of the free enzyme (Fig. 5). This phenomenon is similar to those enzymes being immobilized using conventional methods [26–28]. The effect of temperature of the solutions on the hydrolysis activity of free and immobilized lipase for olive oil was determined, and the results are shown in Fig. 6. The optimum reaction temperature of free enzyme and the immobilized lipase was both at 40 ◦ C, but compared with free lipase, the relative activity of the immobilized lipase is about 50% within 70–80 ◦ C, still exhibiting a wider endurance for reaction temperature. The result from Fig. 6 shows that the immobilized lipase holds much more excellent heat resistance than that of the free enzyme.
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Fig. 5. Effect of pH value on the activity of free and immobilized lipase. Incubated in phosphate buffer at 37 ◦ C for 30 min in the pH range 6.0–9.0.
Fig. 7. Thermal stability of the free and the immobilized lipase. Incubated in phosphate buffer (0.1 M, pH 7.0) at 50 ◦ C for different time.
3.4. Thermal stability and reusability Thermal stability of the free and immobilized lipase was determined by using olive oil as substrate at 50 ◦ C in phosphate buffer (0.1 M, pH 7.0) as shown in Fig. 7. Both preparations exhibited a similar trend, however, the immobilized lipase is more stable than the free one. The immobilized lipase retained their initial activity of about 60% after 210 min, but the free lipase lost its activity within around 180 min. These results indicated that the immobilized lipase had better thermal resistance than the free one. The enhancement of thermal stability is probably attributed to the covalent bonding between lipase and PGMA, thus restricting the conformational change of lipase during heating [29,30]. The reusability of immobilized lipase is an important aspect from an application standpoint, especially in industrial applications. To investigate the reusability, the immobilized lipase was washed with phosphate buffer (0.1 M, pH 7.0) after one catalysis run and reintroduced into a fresh olive oil solution for another hydrolysis at 37 ◦ C. Fig. 8 shows the variation of activity of the immobilized lipase after reuses. It was observed that the immobilized lipase had still retained 83% of their original activities after the 6th reuse. It could be concluded that immobilized lipase has a good durability and reusability.
Fig. 8. Reuse of the immobilized lipase for hydrolyzing olive oil.
4. Conclusion In the present study, the poly(GMA)-grafted Fe3 O4 /SiOX magnetic nanoparticles were prepared by atom transfer radical polymerization and utilized for lipase immobilization. The obtained magnetic nanoparticles had smaller diameter (100 nm), higher saturation magnetization (8.3 kA m−1 ), and showed superior magnetic responsibility. The activity recovery of immobilization lipase reached 69.1% with the protein loaded 98.4 mg g−1 carriers. The optimal pH and temperature of the immobilized lipase were 7.0 and 40 ◦ C, respectively. The residual activity of immobilized lipase decreased a little as it was kept at 50 ◦ C for 150 min, and the residual activity of the immobilized lipase was still over 83% when it was used repeatedly 6 times. It can be concluded that these magnetic nanoparticles provides an economical, efficient and selective system for enzyme immobilization. Acknowledgements
Fig. 6. Effect of temperature on the activity of free and immobilized lipase. Incubated in phosphate buffer (0.1 M, pH 7.0) for 30 min in the temperature range 20–80 ◦ C.
The authors thank the financial supports from the National Natural Science Foundation of China (No. 21074049), the financial supports from the Committee of Natural Science Foundation in China on National Training Fund for Person with Ability of Basic Subjects (J0730425) and Key Research Program of Gansu Province (2GS064-A52-036-02).
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