Micron-sized flower-like Fe₃O₄@GMA magnetic porous microspheres for lipase immobilization

Abstract

Flower-like Fe₃O₄ microspheres prepared by a fast solvothermal method were selected to fabricate micron-sized Fe₃O₄@glycidyl methacrylate (GMA) magnetic porous microspheres. The magnetic porous microspheres were endowed with appropriate specific surface area (39.54 m² g⁻¹) and appropriate pore diameter (16 nm), and could serve as a carrier for immobilized lipase. Meanwhile, the prepared magnetic microspheres with high saturation magnetization could realize rapid separations. After those microspheres were aminated and activated, the obtained immobilized lipase possessed high efficiency of immobilization and catalytic activity, with the optimum pH and temperature of 8.0 and 40 °C, respectively. The thermal stability of immobilized lipase was obviously exceeding free lipase.

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1. Introduction

For the past few years, more and more attention has been paid to biocatalysts, especially enzymes.^1–4 Compared with the issues such as low yield, severe pollution in the traditional pharmaceutical intermediates and manufacturing technology of fine chemical products, enzymatic synthesis has been widely used in medicine, food processing, biomass energy and other fields because of its simple process, high product purity, low cost, no poisonous reagents and being environmentally friendly.^5,6

Lipase (EC 3.1.1.3) is a significant biocatalyst which can be available for catalyzing a variety of reactions, such as esterification, transesterification, esterolysis, and it especially has an important role in the preparation of chiral drugs.^7–9 However, it is sensitive to the environment for the free lipase when directly used in the catalytic process. The free lipase would be unstable and easily deactivated in alkali, acid, high temperature and organic solvents, and contaminates the products. Immobilized lipase, which can be prepared by immobilizing free lipase on a carrier by certain physical and chemical methods, not only overcomes the deficiencies of the free lipase, but also maintains the inherent biological catalytic activity. As a result, immobilized lipase has been extensively researched and extensively used in biological medicine, food, etc.^2,10–12

Among the numerous carriers of immobilized lipase, magnetic composite microspheres with magnetic responsiveness are the most common choice. Firstly, magnetic microspheres are easy to modify (like the silane coupling agent) due to abundant functional groups on the surface, and good biocompatibility;^13–15 for another, because of the property of rapid separation, it improves the reusability of the immobilized enzyme and reduces production costs. Tan’s group¹⁶ prepared submicron hollow Fe₃O₄ particles via a solvothermal method for immobilized lipase after amino functionalization. Further, Zhu et al.⁶ utilized succinic anhydride modified co-precipitation Fe₃O₄ nanoparticles to immobilize porcine pancreatic lipase. Li’s group immobilized Candida rugosa lipase (CRL) using hollow Fe₃O₄ nanoparticles¹⁷ and amine or acid modified Fe₃O₄ nanoparticles;¹⁸ the immobilized lipase revealed excellent catalytic activity and reusability. Nevertheless, the particle size of the above-mentioned Fe₃O₄ particles is popularly in the nanometer or submicron range. Although a relatively high specific surface area is achieved, the direct contact area with lipase is low, and binding sites are limited by the steric hindrance. Therefore, it is crucial to fabricated micron-sized hierarchical Fe₃O₄ porous microspheres for immobilized lipase.

In this study, micron-sized flower-like Fe₃O₄ porous microspheres were produced by a solvothermal method with the aftertreatment of calcination under a nitrogen atmosphere. Then, the Fe₃O₄ porous microspheres modified with methacryloxy propyl trimethoxyl silane (MPS) were polymerized with GMA to fabricate flower-like Fe₃O₄@GMA magnetic porous microspheres (MPMs) for the immobilized lipase CRL. The structure and character of the MPMs were studied in detail, and the performance of the immobilized lipase was also investigated.

2. Materials and methods

2.1. Materials

Tetraethoxysilane (TEOS), methacryloxy propyl trimethoxyl silane (MPS), Candida rugosa lipase (CRL) were purchased from Sigma-Aldrich Co. LLC. Anhydrous FeCl₃, potassium persulfate (KPS), ethylene glycol dimethacrylate (EGDA), glutaraldehyde and ethylene glycol (EG) were provided by Tianjin Fuchen Chemical Reagent Factory. Glycidyl methacrylate (GMA) was obtained from Sartomer Company. Deionized water was ultrapure produced by an apparatus for pharmaceutical purified water (Aquapro Co. Ltd.).

2.2. The preparation of flower-like Fe₃O₄ porous microspheres

The preparation of flower-like Fe₃O₄ porous microspheres was done according to the literature^19–21 with a certain improvement. A typical procedure was as follows: first, 0.1 g FeCl₃ and 3 g urea were dissolved in 40 ml EG. Then, the mixture was moved to a Teflon-lined stainless-steel autoclave at 200 °C for a period of time after stirring for 30 min at 50 °C. The product was washed with ethanol and water for three times. The flower-like Fe₃O₄ porous microspheres were produced after calcination under a nitrogen atmosphere for 1 h.

2.3. Synthesis of flower-like Fe₃O₄@GMA magnetic porous microspheres

0.5 g of prepared Fe₃O₄ porous microspheres were dispersed in 320 ml ethanol/100 ml water. Subsequently, 5 ml MPS was added. The mixture was immersed in a water bath at 40 °C for 12 h. The product was washed with ethanol and water three times, marked as Fe₃O₄–MPS. Then, 0.2 g of the above-mentioned Fe₃O₄–MPS microspheres were dispersed into the aqueous solution contained the right amount of GMA and 5% EGDA. When the temperature of the mixed solution reached 70 °C, 10 ml 0.5% KPS solution was added. The polymerization reaction lasted 6 h. The flower-like Fe₃O₄@GMA MPMs were formed after washing and drying.

2.4. Lipase immobilization

The lipase solution was prepared by PBS buffer solution with a certain concentration. 0.2 g of carrier microspheres were dispersed in the solution, then incubated for 4 h at room temperature. Finally, the residual lipase was washed, and the supernatant was collected to determine the efficiency of immobilized lipase using the Bradford method.²² Bovine serum albumin (BSA) was used as the standard.

The activity of immobilized lipase was monitored by titrating the fatty acid hydrolysed from olive oil according to the literature.²³ The relative activity (%) was calculated as the ratio of the activity of every sample and the maximum activity of the sample.

3. Results and discussion

3.1. Synthesis and characterization of flower-like Fe₃O₄@GMA magnetic porous microspheres

To identify the composition of the products, the typical FTIR spectra of the Fe₃O₄@GMA MPMs were firstly determined as shown in Fig. 1. In Fig. 1A, the absorption peak at 3433 cm⁻¹ was attributed to the stretching vibration absorption peak of hydroxyl on the surface of Fe₃O₄ microspheres. 557 cm⁻¹ is the characteristic absorption peak of Fe–O. It revealed the flower-like magnetic porous microspheres were Fe₃O₄. The absorption peak at 1627 cm⁻¹ in Fig. 1B was assigned to stretching vibration of C=C. The in-plane deformation vibration of C–H was at 1390 cm⁻¹, while 1178 cm⁻¹ and 1060 cm⁻¹ were attributed to the Si–O–Si bond. It illustrated MPS has been modified on the Fe₃O₄@GMA MPMs. 1730 cm⁻¹ belonged to the absorption peak of the carbonyl group (C=O) as shown in Fig. 1C and D. The peaks at 1149 cm⁻¹ and 906 cm⁻¹ were attributed to C–O–C and the epoxy group, respectively. Therefore, we could confirm Fe₃O₄@GMA MPMs were formed with the polymerization of GMA.

Fig. 1 FTIR spectra of flower-like Fe₃O₄ microspheres (A), Fe₃O₄–MPS microspheres (B), Fe₃O₄@GMA magnetic microspheres (C) and PGMA (D).

The crystal structure of the obtained magnetic porous microspheres was determined, and the XRD pattern is shown in Fig. 2. From the different intensities of the crystal diffraction peaks, it indicated the inorganic particles in the prepared Fe₃O₄@GMA MPMs have fine crystallinity. Compared to the standard Fe₃O₄ XRD card, its peak positions were basically the same, which showed the inorganic particles were Fe₃O₄. Therefore, combined with the FTIR analysis results, the produced magnetic porous microspheres were Fe₃O₄@GMA MPMs. The magnetic responsiveness of the Fe₃O₄@GMA MPMs is shown as the VSM curves in Fig. 3. In Fig. 3A, the maximum saturated magnetization of flower-like Fe₃O₄ microsphere was 61 emu g⁻¹. The fairly low residual magnetization, coercivity, and the coincidence of the hysteresis loop reflected that the prepared flower-like Fe₃O₄ microspheres possessed superparamagnetic properties. After polymerization with GMA, the saturation magnetization of Fe₃O₄@GMA MPM was reduce to 58 emu g⁻¹ (as shown in Fig. 3B). It revealed the prepared Fe₃O₄@GMA MPMs still had strong magnetic responsiveness which could play a significant role in rapid separation and reutilization.

Fig. 2 XRD of flower-like Fe₃O₄@GMA magnetic microspheres.

Fig. 3 The magnetization curves of flower-like Fe₃O₄ microspheres (A) and Fe₃O₄@GMA magnetic microspheres (B).

Fig. 4 shows the flower-like Fe₃O₄ microspheres fabricated with different reaction times. From Fig. 4A, we can see that the flower-like Fe₃O₄ microspheres were assembled by multitudinous crystal plates. When the reaction time was 3 h, the petals of the prepared magnetic microspheres were small and dense. Then, flower morphology preliminarily revealed. With the further reaction to 4 h, the petals gradually grew up, and stretched to form the flower-like Fe₃O₄ microspheres. After reacting 6 h, the flower morphology disappeared. Spherical Fe₃O₄ particles were obtained, and their particle size was significantly reduced. So, the flower-like Fe₃O₄ microspheres prepared at 4 h were chose to be the carriers of immobilized lipase. The TEM image in Fig. 4D further proved the flower morphology of Fe₃O₄ microspheres which was attributed to petal aggregation.

Fig. 4 SEM images of flower-like porous Fe₃O₄ nanoparticles prepared at 3 h (A), 4 h (B) and 6 h (C), and TEM image prepared at 4 h (D).

Fig. 5 is the SEM and TEM images of prepared flower-like Fe₃O₄@GMA MPMs. From Fig. 5A, when the flower-like Fe₃O₄ microspheres were polymerized with GMA, Fe₃O₄@GMA MPMs still have the fine flower morphology, just the surface was more cluttered and rough. It illustrates that the prepared magnetic porous microspheres possessed shape retention, and were suitable for immobilized lipase. In Fig. 5B, Fe₃O₄ microspheres were successfully coated by GMA. Poly(GMA) was a thin layer which did not destroy the pore performance of the flower-like Fe₃O₄ microspheres.

Fig. 5 SEM (A) and TEM (B) images of Fe₃O₄@GMA magnetic microspheres.

To analyze the pore performance and specific surface area of the magnetic microspheres, Fig. 6 shows the N₂ adsorption–desorption curves of the flower-like Fe₃O₄ microspheres before and after polymerization. From Fig. 6A and B, both flower-like Fe₃O₄ and Fe₃O₄@GMA presented a hysteresis loop which belonged to the H3 type. It revealed that the pores of prepared microspheres were slit pores piled up by plate-like particles, which were consistent with the results observed in the SEM images. Fig. 6C shows the pore size distributions of the two kinds of microspheres were relatively wide. This was beneficial to degrade the mass transfer resistance of the substrate after immobilization of lipase. From the adsorption–desorption isotherms, the BET specific surface areas of prepared flower-like Fe₃O₄ and Fe₃O₄@GMA MPMs were 45.17 m² g⁻¹ and 39.54 m² g⁻¹, respectively. And average pore sizes were 16.08 nm and 15.99 nm. The above results indicated flower-like Fe₃O₄@GMA MPMs not only possessed an appropriate specific surface area, but also appropriate pore diameter, which were an excellent carrier for immobilized lipase.

Fig. 6 N₂ adsorption–desorption isotherm curves of Fe₃O₄ (A), Fe₃O₄@GMA magnetic microspheres (B) and their pore size distributions (C).

3.2. Lipase immobilization

GMA monomer endowed flower-like Fe₃O₄ microspheres with a large number of epoxide groups which were available to immobilize biomacromolecules using glutaraldehyde as a linker. The immobilized lipase was achieved via covalent immobilization of carriers and lipase CRL. So, the dosage of GMA is an important factor to influence the number of epoxy groups on prepared carriers. Table 1 contains the corresponding relationship between the dosage of GMA and immobilization efficiency of the immobilized lipase. From Table 1, although the immobilization efficiency was the highest when the amount of GMA used was 1.45 g, a white emulsion appeared after washing the product under an external magnetic field, which revealed the amount of GMA was excessive. Thus, 1 g of GMA was the optimal dosage.

Table 1 The immobilization efficiency of flower-like Fe₃O₄@GMA immobilized lipase synthesized with different amounts of GMA

Dosage of GMA	0.5 g	1.0 g	1.45 g
Immobilization efficiency	27.99 mg g^−1	133.32 mg g^−1	157.52 mg g^−1

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Considering the immobilized lipase required a suitable environment to develop its optimal catalytic performance, we examined the influence of the pH and temperature of the system on the activity of immobilized lipase, as shown in Fig. 7 and 8. The pH value directly decided the surface charge distribution of lipase which affected the activity.²⁴ Fig. 7 is the relative activity of immobilized lipase at various pH conditions compared to that at a pH of 6.0. From Fig. 7, the optimal pH was 8.0, which had far higher activity than that at other pH values.

Fig. 7 Relative activity of immobilized lipase at various pH conditions compared to that at pH 6.0.

Fig. 8 Relative activity of different lipases at various temperature conditions compared to the activity of free enzyme at 60 °C (A). Thermal stability of free and immobilized lipase (B).

Fig. 8A shows the relative activity of different lipases at various temperatures. From Fig. 8A, the optimal temperature for the immobilized lipase was 40 °C, with about 65% of the free lipase activity. In addition, the activity was higher than that of Novozym-435 in the determination of temperature. Fig. 8B shows the thermal inactivation analysis of the free lipase and immobilized lipase at 50 °C. From Fig. 8B, the activity of both free lipase and immobilized lipase declined. However, the decline extent of the free lipase was greater when compared at each test point. When incubated for 180 min, the activity of free lipase dropped to 6.3%, almost deactivated. From the thermal inactivation fitting curve, it was in accord with the two-step series-type enzyme deactivation model: . The residual enzyme activity could be expressed as:

Residual activity (%) = α exp(−k₁t) + β exp(−k₂t)

The fitting results are shown in Table 2. From Table 2, for the immobilized lipase, k₁ < k₂. It illustrated similarly that the stability of the immobilized lipase was better than the free lipase.

Table 2 Deactivation kinetic parameters of free lipase and immobilized lipase at 50 °C

	α	β	k1/min^−1	k2/min^−1	R^2
Free enzyme	19.2918	0.5385	0.3581	0.0273	0.999
Immobilized lipase	0.2188	1.4325	0.0097	0.0727	0.998

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The kinetics of free lipase and immobilized lipase were explored via hydrolyzing olive oil with different initial concentrations. The Lineweaver–Burk plots are shown in Fig. 9. From Fig. 9, it is shown that the K_m of free lipase and immobilized lipase were 15.4 and 3.1, respectively. And the V_max of immobilized lipase was 2.4 U mg⁻¹, lower than that of free lipase (16.3 U mg⁻¹). The low K_m and V_max of immobilized lipase revealed that the affinity of immobilized lipase for the substrate was higher than free lipase. It could be attributed to the formation of oil–water by the exposure of Fe₃O₄ and poly(GMA). The mass transfer limitation caused by the carriers was insignificant.

Fig. 9 Lineweaver–Burk plots for the immobilized lipase.

4. Conclusions

In conclusion, micron-sized flower-like Fe₃O₄@GMA magnetic porous microspheres were fabricated by a one-step emulsion polymerization of flower-like Fe₃O₄ microspheres. The magnetic porous microspheres possessed high specific surface area (39.54 m² g⁻¹) and appropriate pore diameter (16 nm). In addition, the magnetic responsiveness of the prepared Fe₃O₄@GMA magnetic porous microspheres was still up to 58 emu g⁻¹, which can be achieved rapid separation. It is doubtless an excellent carrier for immobilized lipase. The optimum pH and temperature for immobilized lipase were 8.0 and 40 °C, respectively. Through the analysis of the thermal inactivation, the thermal stability of immobilized lipase was obviously higher than free lipase. The micron-sized flower-like Fe₃O₄@GMA magnetic porous microspheres greatly enriched the magnetic carrier of immobilized lipase, and expanded the application of magnetic composite microspheres.

Acknowledgements

The authors are grateful for the financial support provided by the National High-Tech Research and Development Program of China (863 Program) (No. 2012AA02A404), the National Natural Science Foundation of China (No. 51433008), basic research fund of Northwestern Polytechnical University (3102014JCQ01094, 3102014ZD) and the problem plan of Xi’an (No. CX12164).

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