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Lipase Immobilization on Oleic Acid-Pluronic (L-64) Block Copolymer Coated Magnetic Nanoparticles, for Hydrolysis at the Oil/Water Interface Iram Mahmood,†,‡ Chen Guo,† Hansong Xia,†,‡ Junhe Ma,† Yangyang Jiang,†,‡ and Huizhou Liu*,† Laboratory of Separation Sciences and Engineering, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Zhongguancun N 1, St 2, P.O. Box 353, Beijing 100080, People’s Republic of China, and Graduate UniVersity of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
Here, we have reported a new approach for utilizing oleic acid-Pluronic L-64 block copolymer coated iron oxide nanoparticles as supports for enzyme immobilization. Iron oxide nanoparticles were prepared by a coprecipitation method and were coated with oleic acid and Pluronic to achieve higher stability and dispersibility. The surface morphology and size of the particle, as determined by transmission electron microscopy (TEM), was (10 nm. X-ray diffraction (XRD) patterns were taken over a range from 10° to 90° 2θ, using Cu KR radiation. Saturation magnetization values, measured at 300 K, varied from 34.6 emu/g to 60.8 emu/g. The possible interaction behavior of oleic acid and Pluronic was observed by Fourier transform infrared (FTIR) analysis and nuclear magnetic resonance (NMR) studies. Further potential of this material as a support for lipase immobilization and enzymatic hydrolysis at the oil/water interface was also investigated. The features of the surface-coated magnetic particles enable the adsorption of lipase from Candida cylindraces via strong hydrophobic interactions, which enhances the stability of the adsorbed enzyme molecules. The stability of the catalyst and, hence, its industrial applicability was tested by performing subsequent reaction cycles for the hydrolysis of olive oil. The activity of the immobilized lipase, pretreated with its substrate, was 510 U/g-matrix and was observed to be maintained at levels as high as 90% of its original activity for up to at least seven reuses. Introduction Nanoparticles provide an ideal remedy to the usually contradictory issues that are encountered in the optimization of immobilized enzymes: minimum diffusion limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis.1 The use of nanophase materials offers many advantages, because of their unique size and physical properties. Hybrid nanoscale materials are well-established in various bioprocesses, such as protein separation2 and immobilization of enzymes.3 An important area of interest is the immobilization of proteins and enzymes on magnetic particles. Several magnetic particles4 and magnetic supports, such as microspheres of various biomaterials that encapsulate the magnetic particles5,6 and copolymers with magnetic particles, have been used with good results. However, because of size constraints (usually 75-100 µm), these microparticles cannot be placed at specific locations that are relevant to bioprocesses. Preferably, such particles would possess a very low magnetic hysteresis and high stability. The fictionalized Fe3O4 magnetic nanoparticles used as a support in this communication possess all these traits. In addition, because of the inherent structure and size of these particles, they are superparamagnetic, which addresses aggregation and flocculation concerns. * To whom correspondence should be addressed. Tel.: +86 1062555005. Fax: +86 1062554264. E-mail address: hzliu@ home.ipe.ac.cn. † Laboratory of Separation Sciences and Engineering, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences. ‡ Graduate University of Chinese Academy of Sciences.
The use of magnetic nanoparticles as the support of immobilized enzymes has some advantages: (i) higher specific surface area obtained for the binding of a larger amount of enzymes, (ii) lower mass transfer resistance and less fouling, and (iii) the selective separation of immobilized enzymes from a reaction mixture via the application of a magnetic field. The demand for industrial enzymes, particularly of microbial origin, is ever increasing, because of their applications in a wide variety of processes. Enzyme-mediated reactions are attractive alternatives to tedious and expensive chemical methods. Enzymes find great use in a large number of fields, such as food, dairy, pharmaceutical, detergent, textile, and cosmetic industries. In the aforementioned scenario, enzymes such as lipases have dominated the world market, because of their hydrolytic reactions. Lipases are frequently used enzymes, because they are commonly used for the hydrolysis of fats and oils,7 enantioselective ester hydrolysis,8 synthesis of enantioenriched monomers, macromers and for polymerization reactions.9 However, with the realization of the catalytic potential of microbial lipases in both aqueous and nonaqueous media in the last one and a half-decades, industrial fronts have shifted toward utilizing this enzyme for a variety of reactions of immense importance. Enzymatic hydrolysis perhaps offers the greatest hope for successful fat splitting without a substantial investment in expensive equipment or the expenditure of large amounts of thermal energy. The scope for application of lipases in the oleochemical industry is enormous, because it saves energy and minimizes thermal degradation during hydrolysis, glycerolysis, and alcoholysis.10 The Miyoshi Oil and Fat Co. (Japan) reported the commercial use of Candida cylindracea lipase in the production of soap.11 In this communication, lipase C. cylindracea (E.C.3.1.3) was used to hydrolyze olive oil.
10.1021/ie701788x CCC: $40.75 2008 American Chemical Society Published on Web 06/10/2008
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Given the current high costs of lipases, the possibility of regenerating and reusing the enzyme would be an attractive feature of biocatalysis. Immobilized enzymes are preferred for most large-scale applications, largely because of the ease in catalyst recycling, continuous operation, and product purification. Often, the catalytic efficiency limits the development of large-scale bioprocessing to compete with traditional chemical processes.12,13 However, although the ability to stabilize and recover the enzyme is achieved via chemical immobilization on magnetic nanoparticles, some enzymatic activity may be lost by chemical bonding, because the active site is hidden or restricted from assuming the conformation needed to initiate the catalysis.14 One important approach to improve the efficiency of immobilized enzymes is to manipulate the structure of carrier materials. Researchers have found some problems in performing lipolysis in aqeous media.16 First, the hydrolysis rates are low, because of the limitations of interfacial area. Second, the hydrolysis of lipids subjects its substrate to inhibition by more than 3%-5% of emulsion. There are at least three main reasons why enzyme hydrolysis may be influenced by the availability of an interface. The substrate concentration at the interface is significantly higher (mass-transfer limitation). Second, the enzyme concentration is higher at the interface. Interfacial absorption can be increased or decreased by decreasing interface than in bulk solution “Interfacial Adsorption”. In addition, the intrinsic specific activity of enzyme is significantly higher at the interface then in the bulk solution “Interfacial Activation”.18 Because this enzyme works best at the oil/water interface, it can be used as a catalyst for the preparation of industrially important compounds.15,17 Despite the obvious advantages of lipase in the oil/water interface system, deactivation of enzyme at interface and the difficulty to separate products from enzyme hinders its wide application. To date, extensive research ande development efforts have been conducted to optimize the carrier materials’ structure to make more-efficient biocatalysts.19–21 The supports should allow the effective utilization of the enzyme by having the enzyme molecule accessible to the substrates. Since the loss of activity is a certainty over time, the support material should be easy to be regenerated with active enzymes. In this regard, nanostructured materials will provide the upper limits in terms of balancing the contradictory issues including surface area, mass transfer resistance, and effective enzyme loading.22 Oleic acid (OA) is a surfactant that is commonly used to stabilize the magnetic nanoparticles synthesized by the traditional coprecipitation method, and some studies have proved that a strong chemical bond formed between the carboxylic acid and the amorphous iron and amorphous iron oxide nanoparticles.23 It has been widely used for steric stabilization in nonpolar solutions; however, OA has a low hydrophilic-lipophilic balance (HLB) value, which makes stabilization difficult.24 The addition of Pluronic block copolymers as a second layer enhances the stability.25,26 Currently, there is much research interest in poly(ethylene oxide)-poly(propyleneoxide)-poly(ethyleneoxide)(PEO-PPOPEO) triblock copolymers (commercially available under the trade names Poloxamers or Pluronics), as they are widely used for various applications in the nanotechnology, pharmaceutical, bioprocessing, and detergent industries.27,28 Block copolymers consisting of a central PPO block flanked by two PEO blocks display interesting amphiphilic properties. Their structure, properties and application in drug delivery have been widely
studied.29 Morales and his co-workers25 have used Pluronic F-127 as coating material for magnetic particles stability and to improve their dispensability. Based on the surface properties and High HLB,26 such polymers can be used as a material of interest for stabilized iron oxide coated nanoparticles at oil-water interface to perform hydrolysis more efficiently. Here, we report the stability and enzymatic activity of C. cylindraceslipase(E.C.3.1.1.3),immobilizedonoleicAcid-Pluronic L-64 functionalized magnetite nanoparticles, and furthers their application for olive oil hydrolysis at the oil/water interface. For the immobilized enzyme in the present case, separation is facilitated by the use of a magnet, where either the substrate solution is removed while the immobilized enzyme is held in place with a magnetic field or vice versa. It is shown here that these enzymes, when immobilized on magnetic nanoparticles, can be easily separated from the reaction medium, stored, and reused with consistent results. This system offers a relatively simple technique for separating and reusing enzymes. Materials and Methods Chemicals. Lipase (E.C. 3.1.1.3) type VII Candida cylindraces was purchased from Sigma Aldrich (containing 943 units/ mg solids and 1310 units/mg protein (Biuret)). Pluronic L-64 was kindly provided by BASF Chemicals Co., Ltd. D2O (g99.9 at. % 2H) was purchased from CIL Corp. All the other chemicals were analytical grade and were of the highest purity available (Beijing Chemical Reagent Co.), including ferric chloride hexahydrate (FeCl3 · 6H2O), ferrous chloride tetrahydrate (FeCl2 · 4H2O), ammonium hydroxide (25% [w/w]), oleic acid, olive oil, glutaraldehyde, pyridine, cupric acetate, iso-octane, glycerol, etc. Synthesis of Oleic Acid-Pluronic-Coated Magnetic Nanoparticles. The preparation of oleic acid-coated magnetic Fe3O4 nanoparticles was performed using a coprecipitation method.30 The scheme used to synthesize these nanoparticles is depcited in Figure 1. Approximately 23.5 g of FeCl3 · 6H2O and 8.6 g of FeCl2 · 4H2O were dissolved in 800 mL of deionized water under nitrogen. When the solution was heated to 90 °C, 30 mL of NH3 · H2O was added rapidly. Later, 20 mL of oleic acid was added dropwise within 20 min. The reaction was kept at 90 °C for 1 h. The black lumpy Fe3O4 gel was cooled to room temperature and washed several times with deionized water. Pluronic L-64 was added after the solution cooled to room temperature. Iron oxide nanoparticles were recovered by ultracentrifugation, washed twice with deionized water, and redispersed in deionized water by sonication. The relative concentration (wt % basis) of iron oxide-OA and Pluronic was 40:60. Lyophilized powder samples were used for further characterization. Activated Particles for Lipase Immobilization. Particles were suspended in 20 mL of a 1 mM phosphate buffer solution (pH 7). Glutaraldehyde (2 mL of a 25% (w/v) solution) was added to the solution and incubated at 20 °C for 2 h to activate the nanoparticles. The activated particles were then washed with water and dried at 60 °C for 2 h. Lipase Preparation and Immobilization. Lipase (1 g) was suspended in 100 mL of 1 mM phosphate buffer (pH 7) and centrifuged at 4 °C and 4000g for 15 min. The supernatant was stored at 4 °C for further immobilization. Olive oil was added to 20 mL of a lipase solution. The mixture was incubated at 37 °C for 30 min with constant stirring. Subsequently, 10 mL of this solution was used for immobilization. The activated particles were mixed with 10 mL of lipase solution and incubated at 20 °C. The immobilized lipase was
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Figure 1. Representative scheme for the synthesis of oleic acid-Pluronic-coated magnetic nanoparticles.
Figure 2. Representative TEM images of (a) oleic acid (OA)-coated Fe3O4 particles; (b) OA-Pluronic L-64 coated Fe3O4, and (c) lipase-immobilized activated nanoparticles.
recovered by filtration, washed with water, and then dried overnight at room temperature. Characterization. The particle size and morphology was observed via transmission electron microscopy (TEM) (model techai 20, Philips, Eindhoven, The Netherlands). Vibrating sample magnetometry (VSM) (Digital Measurement System, Inc.) was performed to obtain the magnetization loop for the samples that were prepared at 300 K. The composition of the polymer particles was recorded by Fourier transform infrared spectroscopy (FTIR) (Bruker, model Vector 22). The existence of the oleic acid-Pluronic compound on the surface of magnetic particles was confirmed by FTIR and 1H nuclear magnetic resonance (NMR) (Bruker, model Avance 600 M spectrometer). The crystal structure of the samples was measured with X-ray diffraction (XRD), using an X-ray diffractometer (X’pert PRO MPD, PANaytical, B.V., The Netherlands). The XRD patterns were reocrded over a range from 10° to 90° (2θ), using Cu KR radiation. Assay of Immobilized Lipase Activity. Iso-octane (10 mL) that contained 10% (w/v) oOlive oil was added to 10 mL of a 50 mM phosphate buffer (pH 7) that contained 200 mg of immobilized lipase. The reaction mixture was incubated in a shaking water bath at 37 °C and 150 rpm for 30 min. A quantity of 2 mL of the upper layer was then transferred to a test tube and cupric acetate pyridine reagent (0.5 mL) was added. The activity of the lipase for olive oil hydrolysis was measured by ultraviolet/visible light (UV/vis) spectrophotometry (Perkin-Elmer, model Lambda Bio 40) at an absorbance of 715 nm.31 One unit of lipase activity was defined as the amount of the enzyme required to liberate 1 µmol free fatty acid per minute. Samples were taken at different time intervals and
analyzed. All experiments were repeated five times, and their relative values were recorded. Results and Discussions Magnetite nanoparticles were prepared via a chemical coprecipitation method. It was reported that magnetic nanoparticles prepared by the coprecipitation method have many hydroxyl groups on the surface of precipitates, from the contact with the aqueous phase.30 Because of dipole attraction, the uncoated magnetite particles are easy to agglomerate. To avoid this, oleic acid and Pluronic L-64 was coated on the particle surface. Figure 2 shows a representative TEM micrograph of the magnetic particles. It can be observed that the nanoparticles are spherical in shape, with an average size of (10 nm. The XRD spectra in Figure 3 reveal the nanocrystalline nature of the two samples. The position and relative intensity of all peaks match well with standard Fe3O4 diffraction data, indicating that each sample is crystalline Fe3O4. Magnetization studies (MS (H)) were performed at 300 K in a sample that contained 40 wt % iron oxide-oleic acid-coated nanoparticles, and 60 wt % Pluronic (see Figure 4). At 300 K, the loops showed magnetization fields in the range of 34.6-60.8 emu/g (average ) 41.72 emu/g). Magnetic Fe3O4 particles
