Appl Biochem Biotechnol DOI 10.1007/s12010-014-1033-9
Solvothermal Synthesis of Functional Magnetic Nanoparticles for Biocatalyst Huaqing Shen & Jianxuan Hou & Zexin Gao & Biqiang Chen & Tianwei Tan
Received: 8 March 2014 / Accepted: 19 June 2014 # Springer Science+Business Media New York 2014
Abstract A facile and simple one-step solvothermal method has been developed to synthesize polyethyleneimine (PEI)-modified magnetic nanoparticles. Characterization of morphology, surface charges, crystal structure, and magnetic property confirmed the efficiency of this facile synthesis route. Lipase immobilized on the PEI-modified magnetic nanoparticles was used to synthesize vitamin A palmitate from vitamin A acetate and palmitic acid. The reuse of immobilized lipase can be extended to eight times by removing water during esterification with a conversion rate above 80 % for 12 h. Keywords Solvothermal method . Magnetic nanoparticles . Biocatalysis . Vitamin A palmitate
Introduction Vitamin A (retinol), indispensable for human health, has been used in cosmetics and pharmaceuticals [1, 2], but retinol is unstable and easily decomposed by light, heat, oxygen, and irritating to skin [3]. To solve the problems above, vitamin A is usually converted into vitamin A esters, and the long-chain esters of vitamin A are more stable. At present, commercially available vitamin A esters are produced by chemical methods. However, the disadvantage is obviously the hightemperature process that easily results in retinol degradation and the formation of by-products [4]. As an alternative, biological process has been studied in recent years [5–7]. Lipase has been used to catalyze the production of vitamin esters in organic solvent. Various acyl donors have been studied; the yield of retinol with methyl oleate as acyl donor reached 90 %, but the reaction process took a very long time (50 h) and higher temperature (55 °C) [5] that would influence the stability of retinol and the activity of lipase. Vitamin A acetate and palmitic acid are used to produce vitamin A palmitate and have a better conversion [7]. The main challenge for the synthesis of vitamin A palmitate by lipase is the recycle and the cost of enzyme, so enzymes are often immobilized on support to solve the problem of catalyst recycle. Various materials as support for immobilization including magnetic nanoparticles (MNPs) have H. Shen : J. Hou : Z. Gao : B. Chen (*) : T. Tan (*) Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China e-mail: [email protected] e-mail: [email protected]
Appl Biochem Biotechnol
been studied in recent decades [8]. MNPs with well-defined nanoscale structures and unique properties have attracted tremendous research efforts because of the potential applications in many fields [9, 10], such as magnetic resonance imaging [11, 12], drug delivery [13], chemical sensor [14], bioseparation [15], and enzyme immobilization [16, 17]. The magnetic property of MNPs can make the recycle of enzyme much more convenient and controllable. In this article, we report the synthesis of polyethyleneimine-modified MNPs (PMNPs) through a facile and simple one-step solvothermal method and its application in the synthesis of vitamin A palmitate from vitamin A acetate and palmitic acid. It turned out that PMNPs loaded with lipase had a good performance on the biocatalyst with a high yield and good stability.
Materials and Methods Materials All the reagents used in this work were of analytical purity, purchased from Beijing Chemical Reagent Factory, and were used as received without further purification unless otherwise noted, including ferric nitrate (FeCl3 ·6H2O, 99 %), polyethyleneimine (PEI, MW=15,000, 30 % solution), anhydrous sodium acetate (NaAc), and ethylene glycol (EG). Yarrowia lipolytica lipase Lip2 was prepared in our laboratory. Vitamin A acetate (98 %) was obtained from Beijing Brilliance Biochemical Co., Ltd. Vitamin A palmitate (99 %) was obtained from SIGMA (USA). Synthesis of PMNPs A typical synthetic process of PMNPs was described as follows [18]: 1 g FeCl3 ·6H2O and 2 g NaAc were added into 20-mL EG under ultrasonication at room temperature to form a clear solution, then 2.5-g PEI was poured into the mixture with magnetic stirring to form an uniform suspension. Next, the above solution was transferred to into a 40-mL Teflon-sealed autoclave and maintained at 180 °C for 24 h. After the autoclave cooled down to room temperature, the black products were collected at the bottom of the container. The products were alternately washed by ethanol and distilled water with a permanent magnet for three times, followed by oven-drying at 60 °C for more than 4 h. For the synthesis of unmodified MNPs (UMNPs), a similar procedure was conducted without PEI. Immobilization of Lipase on MNPs The obtained MNPs were used as magnetic carrier for the immobilization of lipase. To enhance the physical immobilization of lipase on the rough surface of Fe3O4 carrier, 10-mL lipase solution with the concentration of 100 mg/mL in 0.1 mol/L pH 7.0 phosphate buffer solution was mixed with 100 mg MNPs and shaked at 160 rpm for 25 °C. Thirty-six hours later, the immobilized lipase was separated from the solution under external magnetic field and washed twice with 0.1 mol/L pH 7.0 phosphate buffer solution to remove the free lipase. After that, the immobilized enzymes on magnetic carriers were freeze-dried for the following experiments. Characterization The morphology and size of the synthesized samples were determined by using the scanning electron microscope (SEM, Zeiss SUPRA 55). Powder X-ray diffraction (XRD) data was
Appl Biochem Biotechnol
taken on a D/max-Ultima III (Rigaku) with a Cu Kα X-ray radiation source (λ=0.15418 nm). The samples were step-scanned in steps of 0.02° (2θ) in the range 5~90° with a speed of 10° per minute. The surface composition of the nanoparticles was studied by Fourier transform infrared (FTIR) spectroscopy with a Varian 3100 FTIR in 400–4,000 cm−1 spectral range and a resolution of 2 cm−1. Solid samples were ultrasonicated in deionized water for more than 10 min to form a transparent solution and measured by a Zeta sizer instrument (ZS90 Malvern Instruments) to define their surface charge properties. The magnetizations of the MNPs were tested on a JSM-13 vibrating sample magnetometer (VSM) at 298 K and under the applied magnetic field at ±20 kOe. Synthesis of Vitamin A Palmitate Vitamin A acetate was hydrolyzed into vitamin A. One-gram vitamin A acetate was dissolved in 15-mL methanol in a 100-mL flask, followed by adding 0.3 mL of 12.5 mol/L NaOH. The solution was hydrolyzed for 1 h under nitrogen protection. The hydrolysate was extracted by 60-mL hexane for three times to obtain vitamin A. The extraction was washed five times by 50-mL deionized water to remove the methanol. Finally, 0.25-g palmitic acid, 0.3-g 4-Å molecular sieves, 100-mg immobilized lipase, and 10-mL vitamin A solution were added into a 50-mL flask and shaked at 180 rpm and 30 °C for 12 h. HPLC Analysis The concentration of substrate was determined by high-performance liquid chromatography (HPLC) system (Shimadzu 20AVP, Japan) equipped a C18 column (250 mm×4.6 mm, 4.5 μm). The detector was SPD-20AVP UV/V operated at 327 nm. A 20-μl sample with proper dilution of the reaction mixture was injected. One hundred percent methanol was used as the mobile phase at 30 °C with a flow rate of 1 mL/min. The conversion of vitamin A palmitate was defined as the following: conversion% ¼ Mp=ðMa þ MpÞ 100% where Mp is the mole of vitamin A palmitate and Ma is the mole of vitamin A acetate in the reaction solution.
Results and Discussion Structure Characterization MNPs were prepared through high-temperature hydrolysis of Fe+3 with addition of NaAc in the presence of modificatory reagent. Due to the reductive power provided by EG at high temperature, the hydrolysis product, Fe(OH)3, partially transformed to Fe(OH)2 and finally formed magnetic iron oxide [19]. A typical positively charged organic molecule (PEI) was chosen to adjust the surface charge and type of functional groups. The crystalline structure and phase purity were determined by XRD, as shown in Fig. 1. The sharp, strong peaks confirmed that the products were well crystallized and consistent with the standard data of typical cubic iron oxide Fe3O4 (JCPDS, no. 65–3107), which is indexed to the (220), (311), (400), (422), (511), and (440) [18]. Furthermore, the peak shape and broadening in XRD patterns indicated that polymer affected the crystallinity of MNPs.
Appl Biochem Biotechnol
Intensity (a.u.)
PMNPs
MNPs
PDF#65-3107
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2 Theta Fig. 1 XRD patterns of the resulted samples and comparison with standard iron oxide
Surface Property Characterization To characterize morphology and size of the synthesized MNPs, SEM study was performed. The results indicated that the diameter and surface properties of UMNPs can be greatly changed by introducing modifier PEI. Figure 2a showed that the average diameter of UMNP was about 200 nm while Fig. 2b demonstrated that PMNPs had a much smaller size, indicating that PMNPs had acquired a much larger specific area. Both kinds of these MNPs have coarse surfaces and secondary structure consisting of primary crystal, making them superparamagnetic. The coarse surface and superparamagnetism may contribute to its application in adsorption and immobilization [18]. Surface charges were investigated by zeta potential measurement. Figure 3 showed zeta values of the two kinds of MNPs under different pH condition. It is demonstrated that the modification was quite efficient, and PEI did affected their surface charges with changing the isoelectric point from 4.02 to 10.0. This big change on surface property would benefit the immobilization process because lipase was negatively
Fig. 2 SEM images of UMNPs (a) and PMNPs (b)
Appl Biochem Biotechnol
Fig. 3 Zeta potential of two types of MNPs under different pH
charged while PMNPs was positively charged under adsorption condition so that they can combine via electrostatic attraction. The FTIR spectroscopy was performed to reveal evidence of the modifier present on the MNPs’ surfaces. Figure 4 showed a typical spectrum for PMNPs. Strong bands around 590 cm−1 corresponding to the Fe–O stretching modes of the magnetite lattice [20]. The intense band between 3,400 and 3,500 cm−1 indicates the stretching vibration of free N–H. Bands around 1,576 cm−1 correspond to N–H bending vibration. These results indicated that PEI was successfully bound to Fe3O4 MNPs. Magnetic Properties Better magnetic properties can make bioseparation process faster and easier. Figure 5 demonstrated the magnetization curves of two kinds of MNPs in which steeply increased magnetization with increasing field could be observed. Both of these two types of MNPs have very low remanence and small coercivity (
Solvothermal Synthesis of Functional Magnetic Nanoparticles for Biocatalyst Huaqing Shen & Jianxuan Hou & Zexin Gao & Biqiang Chen & Tianwei Tan
Received: 8 March 2014 / Accepted: 19 June 2014 # Springer Science+Business Media New York 2014
Abstract A facile and simple one-step solvothermal method has been developed to synthesize polyethyleneimine (PEI)-modified magnetic nanoparticles. Characterization of morphology, surface charges, crystal structure, and magnetic property confirmed the efficiency of this facile synthesis route. Lipase immobilized on the PEI-modified magnetic nanoparticles was used to synthesize vitamin A palmitate from vitamin A acetate and palmitic acid. The reuse of immobilized lipase can be extended to eight times by removing water during esterification with a conversion rate above 80 % for 12 h. Keywords Solvothermal method . Magnetic nanoparticles . Biocatalysis . Vitamin A palmitate
Introduction Vitamin A (retinol), indispensable for human health, has been used in cosmetics and pharmaceuticals [1, 2], but retinol is unstable and easily decomposed by light, heat, oxygen, and irritating to skin [3]. To solve the problems above, vitamin A is usually converted into vitamin A esters, and the long-chain esters of vitamin A are more stable. At present, commercially available vitamin A esters are produced by chemical methods. However, the disadvantage is obviously the hightemperature process that easily results in retinol degradation and the formation of by-products [4]. As an alternative, biological process has been studied in recent years [5–7]. Lipase has been used to catalyze the production of vitamin esters in organic solvent. Various acyl donors have been studied; the yield of retinol with methyl oleate as acyl donor reached 90 %, but the reaction process took a very long time (50 h) and higher temperature (55 °C) [5] that would influence the stability of retinol and the activity of lipase. Vitamin A acetate and palmitic acid are used to produce vitamin A palmitate and have a better conversion [7]. The main challenge for the synthesis of vitamin A palmitate by lipase is the recycle and the cost of enzyme, so enzymes are often immobilized on support to solve the problem of catalyst recycle. Various materials as support for immobilization including magnetic nanoparticles (MNPs) have H. Shen : J. Hou : Z. Gao : B. Chen (*) : T. Tan (*) Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China e-mail: [email protected] e-mail: [email protected]
Appl Biochem Biotechnol
been studied in recent decades [8]. MNPs with well-defined nanoscale structures and unique properties have attracted tremendous research efforts because of the potential applications in many fields [9, 10], such as magnetic resonance imaging [11, 12], drug delivery [13], chemical sensor [14], bioseparation [15], and enzyme immobilization [16, 17]. The magnetic property of MNPs can make the recycle of enzyme much more convenient and controllable. In this article, we report the synthesis of polyethyleneimine-modified MNPs (PMNPs) through a facile and simple one-step solvothermal method and its application in the synthesis of vitamin A palmitate from vitamin A acetate and palmitic acid. It turned out that PMNPs loaded with lipase had a good performance on the biocatalyst with a high yield and good stability.
Materials and Methods Materials All the reagents used in this work were of analytical purity, purchased from Beijing Chemical Reagent Factory, and were used as received without further purification unless otherwise noted, including ferric nitrate (FeCl3 ·6H2O, 99 %), polyethyleneimine (PEI, MW=15,000, 30 % solution), anhydrous sodium acetate (NaAc), and ethylene glycol (EG). Yarrowia lipolytica lipase Lip2 was prepared in our laboratory. Vitamin A acetate (98 %) was obtained from Beijing Brilliance Biochemical Co., Ltd. Vitamin A palmitate (99 %) was obtained from SIGMA (USA). Synthesis of PMNPs A typical synthetic process of PMNPs was described as follows [18]: 1 g FeCl3 ·6H2O and 2 g NaAc were added into 20-mL EG under ultrasonication at room temperature to form a clear solution, then 2.5-g PEI was poured into the mixture with magnetic stirring to form an uniform suspension. Next, the above solution was transferred to into a 40-mL Teflon-sealed autoclave and maintained at 180 °C for 24 h. After the autoclave cooled down to room temperature, the black products were collected at the bottom of the container. The products were alternately washed by ethanol and distilled water with a permanent magnet for three times, followed by oven-drying at 60 °C for more than 4 h. For the synthesis of unmodified MNPs (UMNPs), a similar procedure was conducted without PEI. Immobilization of Lipase on MNPs The obtained MNPs were used as magnetic carrier for the immobilization of lipase. To enhance the physical immobilization of lipase on the rough surface of Fe3O4 carrier, 10-mL lipase solution with the concentration of 100 mg/mL in 0.1 mol/L pH 7.0 phosphate buffer solution was mixed with 100 mg MNPs and shaked at 160 rpm for 25 °C. Thirty-six hours later, the immobilized lipase was separated from the solution under external magnetic field and washed twice with 0.1 mol/L pH 7.0 phosphate buffer solution to remove the free lipase. After that, the immobilized enzymes on magnetic carriers were freeze-dried for the following experiments. Characterization The morphology and size of the synthesized samples were determined by using the scanning electron microscope (SEM, Zeiss SUPRA 55). Powder X-ray diffraction (XRD) data was
Appl Biochem Biotechnol
taken on a D/max-Ultima III (Rigaku) with a Cu Kα X-ray radiation source (λ=0.15418 nm). The samples were step-scanned in steps of 0.02° (2θ) in the range 5~90° with a speed of 10° per minute. The surface composition of the nanoparticles was studied by Fourier transform infrared (FTIR) spectroscopy with a Varian 3100 FTIR in 400–4,000 cm−1 spectral range and a resolution of 2 cm−1. Solid samples were ultrasonicated in deionized water for more than 10 min to form a transparent solution and measured by a Zeta sizer instrument (ZS90 Malvern Instruments) to define their surface charge properties. The magnetizations of the MNPs were tested on a JSM-13 vibrating sample magnetometer (VSM) at 298 K and under the applied magnetic field at ±20 kOe. Synthesis of Vitamin A Palmitate Vitamin A acetate was hydrolyzed into vitamin A. One-gram vitamin A acetate was dissolved in 15-mL methanol in a 100-mL flask, followed by adding 0.3 mL of 12.5 mol/L NaOH. The solution was hydrolyzed for 1 h under nitrogen protection. The hydrolysate was extracted by 60-mL hexane for three times to obtain vitamin A. The extraction was washed five times by 50-mL deionized water to remove the methanol. Finally, 0.25-g palmitic acid, 0.3-g 4-Å molecular sieves, 100-mg immobilized lipase, and 10-mL vitamin A solution were added into a 50-mL flask and shaked at 180 rpm and 30 °C for 12 h. HPLC Analysis The concentration of substrate was determined by high-performance liquid chromatography (HPLC) system (Shimadzu 20AVP, Japan) equipped a C18 column (250 mm×4.6 mm, 4.5 μm). The detector was SPD-20AVP UV/V operated at 327 nm. A 20-μl sample with proper dilution of the reaction mixture was injected. One hundred percent methanol was used as the mobile phase at 30 °C with a flow rate of 1 mL/min. The conversion of vitamin A palmitate was defined as the following: conversion% ¼ Mp=ðMa þ MpÞ 100% where Mp is the mole of vitamin A palmitate and Ma is the mole of vitamin A acetate in the reaction solution.
Results and Discussion Structure Characterization MNPs were prepared through high-temperature hydrolysis of Fe+3 with addition of NaAc in the presence of modificatory reagent. Due to the reductive power provided by EG at high temperature, the hydrolysis product, Fe(OH)3, partially transformed to Fe(OH)2 and finally formed magnetic iron oxide [19]. A typical positively charged organic molecule (PEI) was chosen to adjust the surface charge and type of functional groups. The crystalline structure and phase purity were determined by XRD, as shown in Fig. 1. The sharp, strong peaks confirmed that the products were well crystallized and consistent with the standard data of typical cubic iron oxide Fe3O4 (JCPDS, no. 65–3107), which is indexed to the (220), (311), (400), (422), (511), and (440) [18]. Furthermore, the peak shape and broadening in XRD patterns indicated that polymer affected the crystallinity of MNPs.
Appl Biochem Biotechnol
Intensity (a.u.)
PMNPs
MNPs
PDF#65-3107
10
20
30
40
50
60
70
2 Theta Fig. 1 XRD patterns of the resulted samples and comparison with standard iron oxide
Surface Property Characterization To characterize morphology and size of the synthesized MNPs, SEM study was performed. The results indicated that the diameter and surface properties of UMNPs can be greatly changed by introducing modifier PEI. Figure 2a showed that the average diameter of UMNP was about 200 nm while Fig. 2b demonstrated that PMNPs had a much smaller size, indicating that PMNPs had acquired a much larger specific area. Both kinds of these MNPs have coarse surfaces and secondary structure consisting of primary crystal, making them superparamagnetic. The coarse surface and superparamagnetism may contribute to its application in adsorption and immobilization [18]. Surface charges were investigated by zeta potential measurement. Figure 3 showed zeta values of the two kinds of MNPs under different pH condition. It is demonstrated that the modification was quite efficient, and PEI did affected their surface charges with changing the isoelectric point from 4.02 to 10.0. This big change on surface property would benefit the immobilization process because lipase was negatively
Fig. 2 SEM images of UMNPs (a) and PMNPs (b)
Appl Biochem Biotechnol
Fig. 3 Zeta potential of two types of MNPs under different pH
charged while PMNPs was positively charged under adsorption condition so that they can combine via electrostatic attraction. The FTIR spectroscopy was performed to reveal evidence of the modifier present on the MNPs’ surfaces. Figure 4 showed a typical spectrum for PMNPs. Strong bands around 590 cm−1 corresponding to the Fe–O stretching modes of the magnetite lattice [20]. The intense band between 3,400 and 3,500 cm−1 indicates the stretching vibration of free N–H. Bands around 1,576 cm−1 correspond to N–H bending vibration. These results indicated that PEI was successfully bound to Fe3O4 MNPs. Magnetic Properties Better magnetic properties can make bioseparation process faster and easier. Figure 5 demonstrated the magnetization curves of two kinds of MNPs in which steeply increased magnetization with increasing field could be observed. Both of these two types of MNPs have very low remanence and small coercivity (
