Preparation of light core/shell magnetic composite microspheres and their application for lipase immobilization

Abstract

Fe₃O₄@P(GMA-DVB-MAA) magnetic composite microspheres were prepared by facile one-pot distillation–precipitation polymerization. The composite microspheres were formed using a P(GMA-DVB-MAA) copolymer as the shell and hollow magnetic Fe₃O₄ nanoparticles as the core. During the distillation–precipitation polymerization, non-modified Fe₃O₄ nanoparticles can be encapsulated and the thickness of the polymer shell layer can be adjusted by the amount of monomer used. The as-prepared particles showed excellent magnetic responsibility, well-defined core/shell structure and monodispersity. Lipase is one of the best biocatalysts which can catalyze a variety of reactions, such as alcoholysis, hydrolysis, aminolysis, esterification and transesterification. Fe₃O₄@P(GMA-DVB-MAA)–NH₂ composite microspheres were obtained by modification of hexamethylenediamine and they were used for Candida rugosa lipase (CRL) immobilization. The immobilization conditions were systematically studied and the hydrolysis of olive oil emulsion was used for the examination of lipase activity. The characterization of the immobilized lipase indicated that the immobilization amount was up to 131.9 mg g⁻¹ and that the relative activity remained at 65% after ten cycles. The immobilized lipase still held 41% relative activity after 140 h at 50 °C. All these results show that the immobilized lipase exhibited excellent thermal stability and reusability. This kind of immobilized lipase has great potential in industrial application because it is easy to separate from the system.

---

1. Introduction

Recently, magnetic nanoparticles have aroused considerable research interest owing to their excellent magnetic responsiveness and their applications in different areas, such as the separation and purification of proteins,¹ drug delivery,² cancer therapy,³ imaging,⁴ catalysis,^5,6 water treatment,⁷ enzyme immobilization,⁸ and so forth. Of all the magnetic nanoparticles, magnetic Fe₃O₄ microspheres are the most widely used due to their easy separation and non-toxicity. Several popular methods including co-precipitation, thermal decomposition, hydrothermal synthesis have been developed to prepare iron oxide nanoparticles.⁹ Particles prepared by co-precipitation tend to be rather polydisperse and the magnetic saturation value is in the range of 30–50 emu g⁻¹. Nearly monodisperse magnetic nanoparticles of smaller size (∼50 nm) can essentially be synthesized by the thermal decomposition method. Unfortunately, particles synthesized by these methods are usually hydrophobic, smaller and have lower magnetic responsiveness, which makes it difficult to quickly separate them from the solution. To overcome the disadvantages, hydrothermal synthesis has been widely adopted for the preparation of monodisperse magnetic nanoparticles with water-dispersible, superparamagnetic properties and tunable sizes in the range of 200–800 nm. Chen et al.¹⁰ reported the synthesis of porous Fe₃O₄ beads and their application in lithium ion batteries. Liu et al.¹¹ successfully prepared monodisperse water-dispersible hollow magnetic microspheres with excellent photonic crystal properties through a one-pot hydrothermal synthesis. However, magnetic Fe₃O₄ nanoparticles are sensitive to aggregation due to their high specific area and strong magneto-dipole interparticle interactions, and are prone to oxidation and corrosion in air or under acidic conditions.

Coating a layer of polymer shell onto the magnetic Fe₃O₄ microspheres is one of the most attractive and effective methods for solving these problems. Core/shell magnetic composite microspheres combine the easy separation properties of the core with the abundant functional groups of the polymer shell, and have been widely applied in many areas.^12–17 Chitosan (CS),¹⁸ N-isopropyl acrylamide (NIPAM),^19,20 2-hydroxyethyl methacrylate (HEMA),²¹ acrylamide (AM),²² ethylene glycol dimethacrylate (EGDMA),²³ methacrylic acid (MAA),²⁴ acrylic acid (AA)²⁵ and glycidyl methacrylate (GMA)²⁶ are often used as monomers for the preparation of magnetic polymer composite microspheres. Among these monomers, GMA is an excellent candidate due to its epoxide group which is easy to react with several functional groups via ring-opening reactions.

Although various approaches including miniemulsion polymerization,^27,28 microemulsion polymerization,²⁹ soap-free emulsion polymerization^30,31 and conventional emulsion polymerization³² have been used for preparing magnetic composite microspheres, these methods are relatively complex and time-consuming. In addition, magnetic composite microspheres prepared by the above methods have shown a lower magnetic content and lower encapsulation efficiency. Yang et al.³³ reported a direct and efficient distillation–precipitation polymerization (DPP) method for the preparation of polymer microspheres. Wang et al.^34–37 prepared various magnetic core/shell microspheres by DPP. Liu et al.²¹ synthesized Fe₃O₄@polymer microspheres by DPP and applied them as a support for the loading of metal nanoparticles.

In the current reports^{19,23,38–40} on the preparation of magnetic composite microspheres using precipitation polymerization, the magnetic Fe₃O₄ nanoparticles must be modified before being encapsulated. Unfortunately, the process is inconvenient and results in low magnetism composite microspheres. Hence, it is very necessary to increase the magnetism of the magnetic composite microspheres, but high magnetism needs an increase in the magnetic content, leading to heavier magnetic composite microspheres which is unfavorable for their further applications. Therefore, it is highly desirable to develop novel light-weight high-saturation magnetism composite microspheres featuring no easy sedimentation, and easy separation. Based on this view, hollow magnetic Fe₃O₄ nanoparticles were the best candidate. Compared to the solid magnetic Fe₃O₄ nanoparticles, hollow magnetic Fe₃O₄ nanoparticles have a preponderance of qualities, such as low density (light), high specific surface area and distinct optical performance.^41,42

Candida rugosa lipase (CRL) is one of the best biocatalysts which has been widely used in chemical and pharmaceutical industries. However, some drawbacks of free lipase have limited its application, including difficulties in recovery, low stability and poor reusability. Enzyme immobilization has been proven to be an effective way in overcoming the above shortcomings. Recently, various carriers have been used for enzyme immobilization, such as metal–organic frameworks (MOFs),^43–45 inorganic nanoflowers,^46,47 polymers⁴⁸ and magnetic polymer composite microspheres.⁴⁹ Magnetic polymer composite microspheres are the best candidates for enzyme carriers due to their stability and ease of separation.

In this study, firstly, monodisperse water dispersible hollow magnetic Fe₃O₄ nanoparticles were prepared, which had high saturation magnetism and were light weight. Secondly, magnetic composite microspheres with a well-defined core/shell structure were synthesized using facile efficient one-pot distillation–precipitation polymerization (DPP). Importantly, the as-prepared Fe₃O₄ nanoparticles can be directly encapsulated without modification. The effects of the crosslinking degree and the amount of monomer on the morphology of the composite microspheres were also investigated. Furthermore, the microspheres were used for Candida rugosa lipase immobilization and the catalytic performance was systematically studied. The novelty of this article includes two aspects: one is that the “light-weight” property lies in the preparation of hollow magnetic Fe₃O₄ nanoparticles, the other one is that non-modified Fe₃O₄ nanoparticles have been successfully coated and that the magnetic composite microspheres have a well-defined morphology. The synthetic route of the whole experiment is shown in Fig. 1.

Fig. 1 The synthetic route of the magnetic composite microspheres for the lipase immobilization.

2. Experimental

2.1 Materials

Glycidyl methacrylate (GMA, purchased from Sartomer Company). Divinylbenzene (DVB, 80% mixture of isomers, obtained from Aldrich Chemical Co., USA). Sodium citrate and methacrylic acid (MAA) were purchased from Tianjin Fuchen Chemical Reagent Factory. 2,2-Azobisisobutyronitrile (AIBN, Shanghai Shanpu Chemical Reagent Company), ferric chloride (FeCl₃·6H₂O, Tianjin Guangfu Chemical Engineering Institute). Sodium polyacrylate (PAAS, Tianjin Kemiou). Urea (Zhengzhou Paini Chemical Reagent Factory). Candida rugosa lipase (Sigma-Aldrich Co. LLC). Glutaraldehyde (25%, Tianjin Kemiou). All reagents are analytical grade.

2.2 Synthesis of Fe₃O₄ hollow nanoparticles

The magnetic Fe₃O₄ nanoparticles were synthesized through a solvothermal method according to the literature.¹¹ Briefly, 1.36 g of FeCl₃·6H₂O and 3.22 g of sodium citrate were dissolved in 80 g of distilled water to form a transparent solution, followed by the addition of urea (0.50 g) and sodium polyacrylate (0.68 g) under vigorous stirring. The mixture solution continued stirring for a time and then was transferred to a Teflon-lined stainless-steel autoclave (with a capacity of 100 mL) to react at 200 °C for 12 h. After that, the products were washed several times with distilled water and then dried.

2.3 Synthesis of Fe₃O₄@P(GMA-DVB-MAA) microspheres

Fe₃O₄@P(GMA-DVB-MAA) composite microspheres were prepared by one-pot distillation–precipitation polymerization. A typical procedure was as follows: 0.10 g of the Fe₃O₄ particles was dispersed in 50 mL of acetonitrile in a 100 mL three-necked flask. Then 10 mL of acetonitrile containing GMA (0.40 g), MAA (0.40 g), DVB (0.20 g) and AIBN (0.02 g, 2 wt% relative to all monomers) was added to the aforementioned mixture. The mixture was heated from ambient temperature to the boiling state within 15 min. The reaction ended after half of the acetonitrile was distilled out. The final products were collected by magnetic separation and washed several times with ethanol, then dried by vacuum freeze-drying.

The synthesis of the composite microspheres with different crosslinking degrees (5%, 10%, 15%, 20%) was similar to the aforementioned process only with an adjustment of the proportion of crossing agent while other reagent consumptions remained the same. For the preparation of composite microspheres with different shell thicknesses, the amounts of monomer used were 0.25 g, 0.50 g, 0.75 g and 1.00 g, while keeping the amount of acetonitrile (60 mL), Fe₃O₄ nanoparticles (0.10 g) and the degree of crosslinking (20%) constant.

2.4 Preparation of amino-functionalized magnetic composite microspheres

The aforementioned magnetic composite microspheres were modified by hexamethylenediamine to afford amino groups on the surface. Typically, 0.10 g of composite microspheres were dispersed in 50 mL of ethanol, and 2.5 mL of hexamethylenediamine was added to the mixture. After 24 h of reaction at 80 °C, the amino-functionalized microspheres were collected by magnetic separation and washed several times with ethanol. Then, the products were dried using vacuum freeze-drying.

2.5 Lipase immobilization

2.5.1 Activated particles for lipase immobilization. 0.05 g of the amino-functionalized particles was dispersed in 15 mL of phosphate buffer solution (pH = 7.4) and 2 mL of glutaraldehyde was added to the above solution. The mixture was placed in a shaking incubator for 4 h at 25 °C. The activated particles were separated using a magnet and washed several times with PBS and stored at 4 °C to reserve.

2.5.2 Lipase immobilization. The above activated particles were swollen in 20 mL of PBS for 12 h. Then, 9 mL of lipase solution (1 mg mL⁻¹) was added to the above solution. The mixture was placed in a shaking incubator for 4 h at 25 °C. After immobilization, the residual lipase was washed and the supernatant was collected to assay the immobilization amount. The activity of lipase was measured by the hydrolysis of olive oil emulsion according to the process of Yamada et al.⁵⁰ Briefly, 5 mL of olive oil emulsion, 4 mL of phosphate buffer solution (pH = 7.5) and 1 mL CaCl₂ were mixed in a 50 mL centrifuge tube. After 30 min of incubation at 37 °C, 1 mL of the immobilization enzyme solution was added to the mixture. Then, the blend solution was placed in a shaking incubator for 15 min. Immediately after incubation, the hydrolysis was terminated by the addition of 20 mL of ethanol and the free fatty acid was titrated with 0.01 mol L⁻¹ NaOH. The relative activity was defined as the ratio of the activity of every sample to the maximum activity of the sample.

2.6 Characterization

Transmission electron microscopy (TEM) was performed on a JEM-3010 transmission electron microscope at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) imaging was measured by a JEOL JSM-6700F SEM. The crystal structure of the magnetic Fe₃O₄ nanoparticles was characterized by X-ray diffraction (Shimadzu XRD-7000, Cu Kα radiation, λ = 1.5406 Å). The shell contents of the magnetic composite microspheres were obtained by thermogravimetric analysis (TGA, Q50 TA instruments) in the range from room temperature to 800 °C with a heating rate of 10 °C min⁻¹. Fourier transform infrared spectroscopy (FTIR) was carried out with a TEN-SOR27 FTIR spectrometer (Bruker). The magnetic properties of the microspheres were assessed using a vibrating sample magnetometer (VSM, LakeShore 7307). The immobilization amount of lipase can be detected using an UV-visible spectrophotometer (Lab Tech).

3. Results and discussion

3.1 Synthesis of Fe₃O₄ nanoparticles

Hollow magnetic Fe₃O₄ nanoparticles were prepared through a solvothermal method with water as the solvent. As can be seen from the SEM and TEM images in Fig. 2, the Fe₃O₄ nanoparticles exhibited sphere-shape morphologies and a narrow particle size. The SEM image shows that the nanoparticles had a coarse surface and that each particle was composed of many small nanocrystals with a size of approximately 10 nm. Moreover, a large number of cracks on the surface of the spheres can be observed from the SEM image, which provides them with a large surface area. The spheres have a pale center region in contrast to a dark edge in the TEM image, which suggests that the nanoparticles are hollow. It can clearly be seen from the images that the range of the particle size was from 300 nm to 400 nm and the average diameter was about 350 nm. In addition, Fig. 2(c) shows the XRD pattern of the Fe₃O₄ nanoparticles, and the eight characteristic peaks located at (111), (220), (311), (222), (400), (422), (511) and (440) agree with standard magnetic Fe₃O₄ nanoparticles (JCPDS 79-0419). Obviously, no other impurity peaks can be found, indicating that Fe₃O₄ nanoparticles with high purity had been successfully obtained. The strongest peak (311) calculated by Scherrer’s formula revealed that the average crystallite size of the sample was about 20.5 nm. The images of electron microscopy and the XRD data indicated that hollow Fe₃O₄ nanoparticles had been successfully obtained. The formation mechanism of Fe₃O₄ can be explained in that sodium citrate serves as a reductant due to its hydroxyl group and chelating ligand properties. At elevated temperatures, part of the Fe³⁺ present will be reduced to Fe²⁺, at the same time, sodium citrate will coordinate with the iron ions (Fe²⁺ or Fe³⁺) to form stable complexes to dramatically reduce the availability of free iron ions in aqueous solution. Urea acts as an alkaline resource which slowly decomposes into NH₃ and CO₂ to provide an alkaline atmosphere which is favourable for forming Fe(OH)₃ and Fe(OH)₂. Fe(OH)₃ and Fe(OH)₂ will be transformed into Fe₃O₄ after dehydration. Sodium polyacrylate in addition to stabilizing a charge can also increase the solution viscosity. A larger viscosity means that primary nanoparticles have enough time to aggregate into regular round spheres.⁵¹

Fig. 2 SEM (a) and TEM (b) images of Fe₃O₄ nanoparticles. (c) XRD pattern of magnetic Fe₃O₄ nanoparticles.

3.2 Synthesis of Fe₃O₄@P(GMA-DVB-MAA) microspheres

In this work, Fe₃O₄@P(GMA-DVB-MAA) core/shell magnetic composite microspheres were prepared by distillation–precipitation polymerization. In this polymerization system, the hollow non-modified Fe₃O₄ particles were used as the core, MAA and GMA served as functional monomers and DVB was the crosslinking agent. The corresponding SEM and TEM images of the composite microspheres are shown in Fig. 3. Compared to the SEM image of the Fe₃O₄ nanoparticles, the size of the composite microspheres has increased and the surface of the encapsulated particles has become smooth due to the fact that a large number of cracks have been filled with polymer shell. The TEM image of the encapsulated particles demonstrated that the composite microspheres showed a well-defined core/shell structure which consisted of an inside black core and a peripheral gray shell, which resulted from two kinds of contrast materials. From the analysis of the results, the core was the Fe₃O₄ nanoparticles and the shell was the polymer, moreover, the thickness of the shell was about 62 nm. All the results indicate that the encapsulation of hollow Fe₃O₄ nanoparticles without modification was successfully achieved. For the encapsulation mechanism of the nanoparticles without modification, there are two possible cases, the first case is: in the early stage of the reaction, due to the presence of abundant hydroxyl and carboxyl groups on the surface of Fe₃O₄, which can form hydrogen bonds with the carboxyl groups of MAA and the ester groups of GMA, the Fe₃O₄ particles capture GMA and MAA monomers or oligomers onto its surface to nucleate. Subsequently, oligomer radicals continue to capture the monomer to grow the chain. The second case is: as P(GMA-DVB-MAA) species are not soluble in acetonitrile, the generated P(GMA-DVB-MAA) chains would continuously precipitate from the solution, attaching to the surface of the Fe₃O₄ particles to form a robust shell with the simultaneous distillation of the solvent.

Fig. 3 SEM (a) and TEM (b) images of Fe₃O₄@P(GMA-DVB-MAA) microspheres.

In order to indicate the functional groups and composition of the as-synthesized materials, Fourier transform infrared spectroscopy (FTIR) and TGA spectra were determined. Fig. 4 presents the FTIR spectra of the Fe₃O₄ nanoparticles, Fe₃O₄@P(GMA-DVB-MAA) composite microspheres and Fe₃O₄@P(GMA-DVB-MAA)–NH₂ microspheres. The strong absorption peak at 587 cm⁻¹ corresponds to the Fe–O vibrations. The peak at 1633 cm⁻¹ of curve a implies the existence of –COOH on the surface of the Fe₃O₄ nanoparticles, which was favourable for the encapsulation process due to the formation of hydrogen bonds between carboxyl and ester groups. In contrast with curve a, curve b shows apparent absorption peaks at 840 cm⁻¹ and 906 cm⁻¹ which belong to the characteristic peaks of the epoxy groups. In addition, the peaks at 1510 cm⁻¹ and 1443 cm⁻¹ were attributed to the skeleton vibration absorption peaks of the benzene rings. These results imply that the composite microspheres contain GMA and DVB. Compared with curve b, the absorption peaks at 840 cm⁻¹ and 906 cm⁻¹ of curve c disappeared which suggests that the epoxy groups decreased and parts were aminated. Moreover, the absorption peak at 3442 cm⁻¹ became sharp, indicating the generation of amino groups. Infrared analysis showed that the magnetic Fe₃O₄ microspheres had been successfully encapsulated and amination of the epoxy groups was achieved.

Fig. 4 FTIR spectra of (a) Fe₃O₄ nanoparticles, (b) Fe₃O₄@P(GMA-DVB-MAA) composite microspheres and (c) amino-functionalized Fe₃O₄@P(GMA-DVB-MAA)–NH₂ microspheres.

Fig. 5(a)–(c) illustrate the TGA curves of the Fe₃O₄ nanoparticles, Fe₃O₄@P(GMA-DVB-MAA) composite microspheres and Fe₃O₄@P(GMA-DVB-MAA)–NH₂ functional microspheres. It was obvious that there was a little weight loss of about 2% for the Fe₃O₄ nanoparticles between 200 °C and 300 °C which was attributed to the adsorbed organics and the modified functional groups (–COOH). However, both curves b and c had a significant weight loss in the range from 200 °C to 450 °C. Analysis showed that the weight loss before 330 °C belonged to the chain breaking of PGMA and PMAA, while the weight loss between 330 °C and 450 °C was attributed to the loss of DVB polymer. It can be seen from the curves that the weight losses for the composite microspheres and amine-functionalized microspheres were 46% and 49%, respectively. Obviously, after being aminated, the composite microsphere increased weight loss is probably due to a tiny weight increase on the shell resulting from the modification with hexamethylenediamine.

Fig. 5 TGA curves of (a) Fe₃O₄ nanoparticles, (b) Fe₃O₄@P(GMA-DVB-MAA) composite microspheres and (c) Fe₃O₄@P(GMA-DVB-MAA)–NH₂ microspheres.

The magnetic responsivity of the magnetic particles, which is usually of great importance for practical applications, was determined using a vibrating sample magnetometer (VSM). The magnetic hysteresis curves in Fig. 6 demonstrate that all the microspheres had no obvious remanence or coercivity. The saturation magnetization (M_s) value of Fe₃O₄ nanoparticles was 68.9 emu g⁻¹. After being coated with a shell of polymer, the M_s value of the microspheres in curve b dramatically decreased to 32.5 emu g⁻¹. The M_s value of curve c was 31.5 emu g⁻¹. The M_s value of the amino-functionalized composite microspheres had dropped a little compared with the Fe₃O₄@P(GMA-DVB-MAA) composite microspheres. The reason for this was probably the little weight increase after being aminated.

Fig. 6 Magnetization curves of (a) Fe₃O₄ nanoparticles, (b) Fe₃O₄@P(GMA-DVB-MAA) composite microspheres and (c) Fe₃O₄@P(GMA-DVB-MAA)–NH₂ microspheres.

3.3 Controlled synthesis of Fe₃O₄@P(GMA-DVB-MAA) composite microspheres

The crosslinking degree of the polymer shell was an important factor influencing the properties and application of the core/shell particles, which can be easily tailored by changing the dosage of DVB while fixing other parameters. Fig. 7 shows the TEM images of different crosslinking degrees. As can be seen from Fig. 7(a), when the dosage of crosslinking agent was 5%, the core/shell structure composite microspheres were not apparent. However, the polymer tended to grow on the surface of the magnetic nanoparticles when the dosage of crosslinking agent was higher than 10%, and monodisperse core/shell Fe₃O₄@P(GMA-DVB-MAA) composite microspheres were obtained with good spherical morphology. Meanwhile, as shown in Fig. 7(b)–(d), with an increase of crosslinking agent, the polymer shells were around 35.72 nm and there were no significant changes in the thickness.

Fig. 7 TEM images of Fe₃O₄@P(GMA-DVB-MAA) microspheres with different crosslinking degrees; (a) crosslinking degree of 5%; (b) crosslinking degree of 10%; (c) crosslinking degree of 15%; and (d) crosslinking degree of 20%.

3.4 Synthesis of Fe₃O₄@P(GMA-DVB-MAA) microspheres with different shell thickness

For the synthesis of any core/shell structure, the shell thickness should be readily controlled to meet different requirements. As shown in the TEM graphs of Fig. 8, all the composite microspheres with different shell thicknesses exhibited an excellent spherical morphology, well-defined core/shell structure, uniform particle size and almost no secondary nucleation. With an increase of the amount of monomer, the shell thickness became thicker. When the amount of total monomer was increased from 0.25 g to 0.50 g, the average shell thickness increased from 28.06 nm to 38.50 nm. On further increasing the amount of monomer to 0.75 g, the shell thickness increased to 43.04 nm and the composite microspheres retained good spherical morphology. When the amount of monomer was 1.00 g, the shell thickness was up to 58.15 nm.

Fig. 8 TEM images of Fe₃O₄@P(GMA-DVB-MAA) microspheres with different shell thicknesses, (a) 0.25 g of monomer, (b) 0.50 g of monomer, (c) 0.75 g of monomer, and (d) 1.00 g of monomer; other conditions were the same: AN: 60 ml, crosslinking degree: 20%, AIBN: 2% of monomer, Fe₃O₄ nanoparticles: 0.05 g.

3.5 Application of as-prepared immobilization lipase

The magnetic composite microspheres featured with monodispersity and light weight properties as well as high saturation magnetism were the best candidate for the carrier of the immobilized enzyme. Observation and analysis by TEM images indicated that when the amount of monomer was 0.50 g, the composite microspheres had excellent monodispersity and a moderate shell layer. Thereby, this kind of composite microsphere was chosen as the carrier for immobilizing lipase.

In the process of enzyme immobilization, glutaraldehyde (GA) was usually used to connect the enzyme and the carrier, which was due to the formation of a Schiff base between the aldehyde of glutaraldehyde and the terminal amino of lipase. In order to find the optimal immobilization conditions, lipase concentration, immobilization time and immobilization temperature were systematically studied. The hydrolysis of olive oil emulsion was used for the examination of lipase activity. Furthermore, the storage stability and reusability of the as-prepared immobilized lipase were also tested.

3.5.1 Effect of lipase concentration on immobilization amount. Fig. 9 shows the effect of the lipase concentration on the immobilization amount. It can be seen that the maximum immobilization amount was up to 131.9 mg g⁻¹. The curve shows a trend of increase before the lipase amount reaches 9 mg and essentially keeps constant after reaching the maximum. The reason is that the lipase is more disperse at low lipase amounts, which increases the contact surface area between the lipase and the carrier. Thereby, at low lipase amounts, with an increase in the amount of lipase, the immobilization amount increases. However, the immobilization amount didn’t increase with an increasing dosage of lipase, which was due to the fact that the carriers were limited for the enzyme.

Fig. 9 Effect of addition amount of lipase on immobilization amount.

3.5.2 Effect of reaction time on immobilization amount. In addition to lipase concentration, the reaction time is also an important factor for the immobilization amount. Fig. 10 illustrates the immobilization amount at different immobilization times. The immobilization amount reached a maximum at 4 h and only had a slight change after that. It was considered that the coupling reaction was rapid at the beginning. Herein, the optimal reaction time of the lipase was 4 h.

Fig. 10 Effect of reaction time on immobilization amount.

3.5.3 Effect of reaction temperature on immobilization amount. The effect of reaction temperature on the immobilization amount was investigated from 20 °C to 70 °C and the result is shown in Fig. 11. It was found that the curve essentially kept constant in the range from 20 °C to 40 °C and had a large decline when the temperature was higher than 40 °C. It was inferred from this phenomenon that the change of temperature made the lipase conformation change and the functional groups were eventually embedded, which caused a greater decrease for the immobilization amount. Based on the temperature effect on enzyme activity, low temperatures benefited enzyme activity retention. So the optimal reaction temperature of the lipase was 20 °C.

Fig. 11 Effect of reaction temperature on immobilization amount.

3.5.4 Effect of hydrolysis temperature on enzyme activity. The temperature dependencies of both free lipase and immobilized lipase were investigated from 20 °C to 70 °C using olive oil as the substrate and the results are given in Fig. 12. Free lipase showed good activity in a low temperature range while immobilized lipase showed good activity at higher temperatures. The optimum hydrolysis temperature of the free lipase appeared at 40 °C, while the immobilized lipase had the highest hydrolytic activity at 50 °C, which was higher than the free lipase and indicated that the immobilized lipase had a higher heat resistance. The reason for it may be that the immobilized lipase was a combination of enzyme molecules and the carrier so that the necessary conformation for the it to be highly active required a larger entropy to form. While there was no limitation for the free enzyme molecules which can induce the conformation to catalyze the reaction by contacting with the substrate at low temperature.⁵²

Fig. 12 The relative activity of free lipase and the immobilized lipase.

3.5.5 Storage stability of the immobilized lipase. The storage stability of the immobilized lipase was assessed by the measurement of enzyme activity at 50 °C. As can be seen from Fig. 13, with the extension of saving time, the relative activity of free lipase had a sharp decrease while the relative activity of the immobilized lipase had a moderate change. The relative activity of the free lipase decreased to 20% while the relative activity of the immobilized lipase only decreased to 52% after saving for 100 h at 50 °C, which indicated that the lipase became more stable after immobilization. When the preservation time was 140 h at 50 °C, the relative activity of the free lipase disappeared, while the immobilized lipase still held 41% relative activity. It can be explained that the increase of thermal stability may result from the stabilization of the weak intramolecular forces and the prevention of autolysis of the lipase.⁵³

Fig. 13 Storage stabilities of free lipase and immobilized lipase.

3.5.6 Reusability of the immobilized lipase. The reusability of immobilized lipase is one of the most important criteria in practical applications. The results of the relative activity after multiple reuses is shown in Fig. 14. Obviously, the relative activity of the immobilized lipase remained at 65% after ten cycles. The reason for the decline of the relative activity is the deactivation of the lipase and the leakage of lipase from the carriers upon use. The results suggest that the as-immobilized lipase has excellent reusability.

Fig. 14 Reuse of the immobilized lipase for hydrolyzing olive oil.

4. Conclusions

In this study, first, the magnetic composite microspheres Fe₃O₄@P(GMA-DVB-MAA) with different shell thicknesses, high saturation magnetization, a well-defined core/shell structure and excellent dispersibility were prepared by a facile one-pot distillation–precipitation polymerization process. Then the well-defined core/shell amino-functionalised magnetic composite microspheres Fe₃O₄@P(GMA-DVB-MAA)–NH₂ were employed in the immobilization of lipase. Meanwhile, various immobilization factors and the thermal stability of the immobilized lipase as well as the reusability were investigated. The characterization of the immobilized lipase indicated that the immobilization amount was up to 131.9 mg g⁻¹ and the relative activity remained at 65% after ten cycles. Besides, when the preservation time was 140 h at 50 °C, the relative activity of free lipase disappeared, while the immobilized lipase still held 41% relative activity. The combination of magnetic composite microspheres and lipase could significantly improve the stability of lipase and expand the applications of magnetic composite microspheres. It is believed that this magnetic immobilized lipase has the advantages of simple preparation, easy separation, excellent thermal stability and recyclability.

Acknowledgements

The work was financially supported by the State Key Program of National Natural Science of China (Grant No. 51433008), the Natural Science Foundation of Shaanxi Province (Grant No. 2015JQ2055, 2015JM2050) and the Basic Research Fund of Northwestern Polytechnical University (Grant No. 3102014JCQ01094, 3102014ZD).

References

1. X. Li, B. Zhang, W. Li, X. Fan and L. Tian, Biosens. Bioelectron., 2014, 51, 261.
2. B. Zhang, H. Zhang and L. Tian, Chem. Eng. J., 2015, 275, 235.
3. P. R. Gil and W. J. Parak, ACS Nano, 2008, 2, 2200.
4. M. A. McAteer, A. M. Akhtar, C. Muhlen and R. P. Choudhury, Atherosclerosis, 2010, 209, 18.
5. J. Ge, Q. Zhang, T. Zhang and Y. Yin, Angew. Chem., Int. Ed., 2008, 47, 8924.
6. M. Zhu and G. Diao, J. Phys. Chem. C, 2011, 115, 18923.
7. M. Kumari, C. U. Pittman and D. Mohan, J. Colloid Interface Sci., 2015, 442, 120.
8. Z. Ali, L. Tian, P. Zhao, B. Zhang and N. Ali, RSC Adv., 2015, 5, 92449.
9. A. H. Lu, E. L. Salabas and F. Schuth, Angew. Chem., Int. Ed., 2007, 46, 1222.
10. Y. Chen, H. Xia, L. Li and J. Xue, J. Mater. Chem., 2012, 22, 5006.
11. Y. Liu, C. Li, H. Zhang and X. Fan, Chem. Eng. J., 2015, 259, 779.
12. S. Pan, H. Shen, L. Zhou, X. Chen and Y. Zhao, J. Mater. Chem. A, 2014, 2, 15345.
13. L. Fan, M. Li, Z. Lv, M. Sun and C. Luo, Colloids Surf., B, 2012, 95, 42.
14. J. Zhang, N. Gan, S. Chen and M. Pan, J. Chromatogr. A, 2015, 1401, 24.
15. R. Chalasani and S. Vasudevan, ACS Nano, 2013, 7, 4093.
16. H. Li, H. Mohammad, E. L. Dakdouki and C. David, Chem. Commun., 2012, 48, 3385.
17. B. Zhang, H. Zhang, X. Li and X. Lei, Mater. Sci. Eng., C, 2013, 33, 4401.
18. L. Fan, Y. Zhang, C. Luo and F. Lu, Int. J. Biol. Macromol., 2012, 50, 444.
19. G. Liu, D. Wang, F. Zhou and W. Liu, Small, 2015, 11, 2807.
20. B. Zhang, H. Zhang, X. Fan and X. Li, J. Colloid Interface Sci., 2013, 398, 51.
21. B. Liu, W. Zhang, F. Yang, H. Feng and X. Yang, J. Phys. Chem. C, 2011, 115, 15875.
22. R. Tong, Y. Wang, G. Yang, A. Ma and K. Sun, S. Afr. J. Chem., 2015, 68, 99.
23. X. Jia, X. Fan, Y. Liu, W. Li and L. Tian, RSC Adv., 2015, 5, 60691.
24. F. Zhang, S. Yu, G. Hou, N. Xu, Z. Wu and L. Yue, Colloid Polym. Sci., 2015, 293, 1893.
25. H. Shang, W. S. Chang, H. Shi, S. A. Majetich and L. U. Gil, Langmuir, 2006, 22, 2516.
26. M. Ma, Q. Zhang, J. Dou, H. Zhang and D. Yin, J. Colloid Interface Sci., 2012, 374, 339.
27. F. Yang, J. Li, J. Zhang, F. Liu and W. Yang, J. Nanopart. Res., 2009, 11, 289.
28. H. Xu, L. Cui, N. Tong and H. Gu, J. Am. Chem. Soc., 2006, 128, 15582.
29. N. Sahiner and P. Ilgin, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, 5239.
30. S. Gu, J. Colloid Interface Sci., 2005, 289, 419.
31. X. Fan, X. Jia, H. Zhang, B. Zhang, C. Li and Q. Zhang, Langmuir, 2013, 29, 11730.
32. L. Cui, H. Xu, P. He, K. Sumitomo and Y. Yamaguchi, J. Polym. Sci., Part A: Polym. Chem., 2007, 45, 5285.
33. G. Liu, H. Wang and X. Yang, Polymer, 2009, 50, 2578.
34. W. Ma, S. Xu, J. Li, J. Guo, Y. Lin and C. Wang, J. Polym. Sci., Part A: Polym. Chem., 2011, 49, 2725.
35. Y. Zhang, Y. Yang, W. Ma, J. Guo, Y. Lin and C. Wang, ACS Appl. Mater. Interfaces, 2013, 5, 2626.
36. L. Liu, M. Yu, Y. Zhang, C. Wang and H. Lu, ACS Appl. Mater. Interfaces, 2014, 6, 7823.
37. S. Xu, Z. Weng, J. Tan, J. Guo and C. Wang, Polym. Chem., 2015, 6, 2892.
38. L. Wang, J. Bai, Y. Li and Y. Huang, Angew. Chem., Int. Ed., 2008, 47, 2439.
39. K. Cai, J. Li, Z. Luo, Y. Hu, Y. Hou and X. Ding, Chem. Commun., 2011, 47, 7719.
40. X. Yan, J. Kong, C. Yang and G. Fu, J. Colloid Interface Sci., 2015, 445, 9.
41. X. Lou, L. A. Archer and Z. Yang, Adv. Mater., 2008, 20, 3987.
42. L. Zhu, H. Xiao, W. Zhang, G. Yang and S. Fu, Cryst. Growth Des., 2008, 8, 957.
43. X. Wu, C. Yang, J. Ge and Z. Liu, Nanoscale, 2015, 7, 18883.
44. X. Wu, J. Ge, C. Yang, M. Hou and Z. Liu, Chem. Commun., 2015, 51, 13408.
45. F. Lyu, Y. Zhang, R. N. Zare, J. Ge and Z. Liu, Nano Lett., 2014, 14, 5761.
46. X. Wu, M. Hou and J. Ge, Catal. Sci. Technol., 2015, 5, 5077.
47. J. Ge, J. Lei and R. N. Zare, Nat. Nanotechnol., 2012, 7, 428.
48. Y. Zhang, Y. Dai, M. Hou, T. Li, J. Ge and Z. Liu, RSC Adv., 2013, 3, 22963.
49. J. Ge, J. Lei and R. N. Zare, Nano Lett., 2011, 11, 2551.
50. N. Watanabe, Y. Ota, Y. Minoda and K. Yamada, Agric. Biol. Chem., 1977, 41, 1353.
51. W. Cheng, K. Tang, Y. Qi, J. Sheng and Z. Liu, J. Mater. Chem., 2010, 20, 1799.
52. B. Zhang, P. Li, H. Zhang, H. Wang and X. Li, Chem. Eng. J., 2016, 291, 287.
53. L. Lei, Y. Bai, Y. Li, L. Yi, Y. Yang and C. Xia, J. Magn. Magn. Mater., 2009, 321, 252.

---