Lipase Immobilized on Layer-by-Layer ... - ACS Publications

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Lipase Immobilized on Layer-by-Layer Polysaccharide-Coated Fe3O4@SiO2 Microspheres as a Reusable Biocatalyst for the Production of Structured Lipids Zhixiang Cai,† Yue Wei,† Min Wu,† Yalong Guo,† Yanping Xie,† Ran Tao,† Ruiqi Li,† Pengguang Wang,† Aiqin Ma,‡ and Hongbin Zhang*,† †

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Department of Polymer Science and Engineering, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China ‡ Affiliated Sixth People’s Hospital South Campus, Shanghai Jiao Tong University, 6600 Nanfeng Road, Fengxian District, Shanghai 201499, China S Supporting Information *

ABSTRACT: A promising strategy for lipase immobilization based on the natural polymers of polysaccharides (hyaluronan (HA) and chitosan (CHI)) functionalized magnetic microspheres (Fe3O4@SiO2@{CHI/HA}3) was developed. First, Fe3O4 magnetic microspheres and Fe3O4@SiO2 core/shell microspheres were synthesized by hydrothermal reaction and sol−gel method, respectively. Owing to the abundant carboxyl groups in HA chains, the lipase was covalently bonded on the surface of the polysaccharide-functionalized magnetic supports by ultilizing 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide and N-hydroxysuccinimide chemistry to produce robust biocatalysts of Fe3O4@SiO2@{CHI/HA}3@lipase. The morphology, core−shell structure, and magnetic property of the supports and immobilized lipase were investigated through various analytical techniques, including FT-IR analysis, transmission electron microscopy, scanning electron microscopy equipped with energy dispersive spectrometer, elemental analysis, vibrating sample magnetometer, thermogravimetric analysis, and X-ray diffraction. Consequently, the magnetic Fe3O4@SiO2@{CHI/HA}3 microspheres exhibited a superior performance in terms of immobilizing lipase. The magnetic immobilized lipase showed good thermal and long-term stability, reusability, and catalytic activity for the synthesis of the structured lipid of 1,3-dioleoyl-2-palmitoylglycerol (OPO), which has recently received much interest as a healthy component of food, oil, and pharmaceutical intermediates. The magnetic immobilized lipase could be considered a green and sustainable biocatalyst for the highly efficient synthesis of OPO. KEYWORDS: Magnetic microspheres, Layer-by-layer self-assembly, Polysaccharide, Structured lipids

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INTRODUCTION Enzymes are versatile biocatalysts and find various applications in many areas due to their high effectiveness, excellent regioand stereoselectivity, low cost, and mild reaction conditions.1−4 However, the practical applications of native enzymes are generally hampered by their unsatisfactory operational and storage stability, difficulties in their recovery and reusability of enzyme, high cost, and high sensitivity to environmental conditions.5,6 The immobilization of enzyme molecules on substrates has been a promising and economical method to improve the stability to overcome the preceding drawbacks and facilitate the separation and reusability of enzyme from reaction systems.7 Several enzyme immobilization techniques have been employed for the enzyme immobilization, such as physical,8 covalent bonding,9 and affinity interactions of enzymes.10 Among the various immobilization methods, covalent bonding method has been widely used to bind enzyme onto the supports, which can prevent enzyme leaching from the support surface.11 To date, various carriers and © XXXX American Chemical Society

scaffolds have been utilized for enzyme immobilization, including silica dioxide,12 polysaccharides,13 carbon nanomaterials,14,15 mesoporous materials,16 and magnetic particles.17 Specifically, magnetic particles have attracted special attention for enzyme immobilization because of their easy separation by external magnetic treatment.18,19 Iron oxide (Fe3O4) particles have attracted considerable attention in the field of enzyme immobilization because of their advantages, including easy isolation from sample solutions and effective recycling under an external magnetic field.20 However, bare Fe3O4 particles have a tendency to aggregate in solutions because of the strong magnetic dipole− dipole attractions between particles.21 Fe3O4 particles are also very susceptible to oxidation and acid conditions, which markedly limits their applications. In addition, active groups on Received: November 7, 2018 Revised: February 24, 2019 Published: March 5, 2019 A

DOI: 10.1021/acssuschemeng.8b05786 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering Scheme 1. Synthesis of OPO by Free Lipase or Immobilized Lipase

high-quality HMFS in infant formulas. As one of the most versatile enzymes, sn-1,3 regiospecific lipase has been widely used for the synthesis of OPO-rich TAGs.47 However, the drawbacks of the free lipase severely restrict its use on the industrial scale. Thus, the immobilization of lipase has been a promising and economical method to overcome the preceding drawbacks mentioned. In this study, in order to provide environmentally friendly procedures for OPO synthesis, a novel PEM-coated Fe3O4@ SiO 2 was first prepared as a support for the lipase immobilization. The Fe3O4@SiO2 magnetic microspheres comprised a uniform Fe3O4 core and a thin SiO2 middle layer providing high magnetic response and charged surfaces, respectively. HA is a naturally unbranched polyanionic polysaccharide composed of repeating disaccharide units of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine.48,49 PEMs assembled from HA and CHI were then coated onto the surface of magnetic microspheres via the LbL assembly technique. HA-coated magnetic microspheres have received much interest due to the abundant functional groups on the HA chains, which were used for the lipase immobilization in this study. To the best knowledge of the authors, the utilization of PEM-coated magnetic microspheres from the LbL technique for lipase immobilization has not been reported. Finally, the catalytic efficiency of the immobilized lipase was tested for the synthesis of OPO. The pH, thermal, storage stabilities, and reusability of the immobilized lipase were also investigated to illustrate its broad applicability.

the surface of Fe3O4 particles are limited. Therefore, the functionalization modification on the surface of Fe3O4 is an effective strategy that not only improves the stability of magnetic particles but also provides layers for enzyme immobilization.9,22 Various approaches have been developed to modify the surface of magnetic particles, including polysaccharides,23 carbon,24 and silica.25 Despite the improvement of the enzyme stability and recyclability of many pioneering works, the development of efficient substrates and ways to immobilize enzymes with high performances has a substantial demand. Among the various techniques used to modify surfaces, the layer-by-layer (LbL) self-assembly technique introduced by Decher and Hong26,27 has been one of the most rapidly growing strategies to develop polyelectrolyte multilayers (PEMs) on solid substrates due to its easy handling, low cost, automation, and reproducibility.28−31 The technique is based on the alternating desorption and self-assembly of anionic and cationic polyelectrolytes onto solid surfaces.32 The existing electrostatic interaction between the two polyelectrolytes of opposite charges is the driving force for the construction of PEM with tunable properties.33 Over the past few decades, various PEMs constructed by the LbL assembly technique have garnered considerable interest because they have been shown to serve as promising candidates for various applications in biomedical areas, such as optical devices, biomaterials coatings, drug delivery carriers, and biosensors.34−38 In recent years, natural polyelectrolyte biopolymers (such as hyaluronan (HA), poly-L-lysine, chitosan (CHI), and alginate) have become key candidates for the preparation of biopolymer-based PEMs using the LbL assembly technique mostly due to their tunability through assembly conditions, hydrophilicity, biocompatibility, bioactivity, and biodegradability.39−43 In the human body, high-energy substances, such as fats and oils, mainly exist as triacylglycerols (TAGs > 95%). However, the nutritional value of TAGs is determined by the fatty acid (FA) composition and the positional distribution of FA along the glycerol backbone.44 Hence, increasing attention has been provided to improve the nutritional value of TAGs by modification of composition and positional distribution of FA. Structured lipids (SLs), a form of TAGs modified to introduce new FA or alter the distribution of FA in glycerol backbone, have been extensively investigated in the field of food because of their improved nutritional and physiochemical properties.45 1,3-Dioleoyl-2-palmitoylglycerol (OPO), a type of SLs, is widely used as human milk fat substitute (HMFS) in infant formula.46 Numerous investigations have been focused on the large-scale synthesis of OPO-rich TAGs to achieve

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EXPERIMENTAL SECTION

For details of the preparation and characterization of immobilized lipase and the determination of the loading amount of lipase on the support, please refer to the Supporting Information. Measurement of the Free and Immobilized Lipase Activity. The enzymatic activities of immobilized lipase were determined with the interesterification of tripalmitin (PPP) and oleic acid (OA). One activity unit (U) is defined as the amount of lipase required to produce 1 μmol of OPO per min under the standard assay condition (Scheme 1). In a typical experiment, the reaction mixture containing 10 mmol PPP, 30 mmol oleic acid (OA), immobilized lipase (5 wt %, the amount of lipase on the Fe3O4@SiO2@{CHI/HA}3 or the resin was the same as that of free lipase) or free lipase, and 4 mL of n-hexane was incubated in an Erlenmeyer shaker (50 mL) on a rotary shaker at 200 rpm for 6 h. The molecular sieves (4 Å) were added to the solution to remove the byproduct water. Over the time course of the reaction, 50 μL aliquot was withdrawn from the system at different time intervals for HPLC-ELSD analysis. After completing the reaction, the immobilized lipase was collected by the external magnetic field (Fe3O4@SiO2@{CHI/HA}3@lipase) or centrifugation (Lipozyme RM IM) and washed with n-hexane three times to remove B


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ACS Sustainable Chemistry & Engineering

Figure 1. Schematic illustration of the synthesis process for the preparation of lipase immobilized magnetic microspheres (Fe3O4@SiO2@{CHI/ HA}3@lipase). any remaining products or substrate species and dried under vacuum. All reactions were conducted at least in triplicate. OPO content was analyzed by Agilent 1200 series equipped with an evaporative light-scattering detector (ELSD) equipment. A reverse phase C18 column (250 mm × 4.6 mm, 5 μm particle size, Waters) was used for the analysis. Acetonitrile and dichloromethane (8:2) were used as the mobile phases of HPLC-ELSD. The chromatographic conditions were set as follows: injection volume, 5 μL; flow rate, 1 mL/min; nitrogen pressure, 320 kPa; column temperature, 40 °C; detector temperature, 70 °C. The standard calibration curve for standard OPO (in CH2Cl2) was constructed under the aforementioned conditions by plotting the peak area versus concentration. For details of the effect of pH and temperature on the synthesis of OPO catalyzed by immobilized lipase, and the stability and reusability test of immobilized lipase, please see Supporting Information.

SiO2 microspheres by the usual electrostatic attraction (Figure 1B). An initial “precursor” layer of CHI was adsorbed on the SiO2 surface, resulting in a positively charged surface, followed by HA. The alternate adsorption process at each step was followed by the surface electrical potentials (zeta potential) of the Fe3O4@SiO2 microspheres. The Fe3O4@SiO2 microspheres were coated with three bilayers of CHI and HA. The resulting CHI−HA shells on Fe3O4@SiO2 microspheres (Fe3O4@SiO2@{CHI/HA}3) are in multilayer form and contain numerous carboxylic acid groups. In this study, the carboxylic acid groups in HA chains were employed for the immobilization of lipase using EDC/NHS reaction between the carboxyl groups of HA and the amino groups of lipase. The carboxylic acid groups were first activated using EDC/NHS to form reactive succinimidyl esters, which subsequently reacted with the amino in lipase to form strong amide bonds (Figure 1C). Thus, lipase can be bound onto the CHI/HA-coated Fe3O4@SiO2 microspheres through amidation reaction. As shown in Figure 2, zeta potential alterations were plotted as a function of the number of CHI and HA layers to confirm the occurrence of LbL assembly on the surface of Fe3O4@SiO2

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RESULTS AND DISCUSSION Synthesis and Characterization of Lipase-Immobilized Microspheres. The immobilization of lipase onto suitable substrates has been considered to be a promising solution to improve the long-term stability, activity, and efficiency of the free lipase. Figure 1 outlines the general synthetic route for the preparation of the lipase-immobilized microspheres (Fe3O4@SiO2@{CHI/HA}3@lipase). The synthesis of the lipase-immobilized microspheres was accomplished by combining several procedures. First, the magnetic Fe3O4 microspheres were synthesized using a well-established solvothermal method. The obtained magnetic microspheres can be uniformly dispersed in alcohol and water. Subsequently, the obtained Fe3O4 microspheres were coated with an amorphous layer of silica by TEOS using typical sol−gel process to form core−shell structure Fe3O4@SiO2 microspheres (Figure 1A). Researchers have reported that the silica coating on Fe3O4 can prevent their aggregation in solution and increase their stability against heat and acidic medium.50 The thickness of SiO2 shells could be tuned through varying the concentrations of ammonium hydroxide and TEOS. In addition, another advantage of silica coating is that the surface of Fe3O4@SiO2 microspheres is negatively charged.51 Polymer layers can be directly assembled onto the negatively charged SiO2 surface using LbL assembly technique. In this study, CHI and HA bilayers were assembled on the surface of Fe3O4@

Figure 2. Zeta potential measurements of Fe3O4@SiO2 surface charge alteration during LbL self-assembly process. C


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were measured by DLS (for detail, see Figure S2 in the Supporting Information). The chemical composition of microspheres was further confirmed by energy dispersive spectrometer (EDS). The EDS spectrum of final lipase-immobilized microspheres showed that the elements Fe, Si, C, N, and O existed in Fe3O4@ SiO2@{CHI/HA}3@lipase microspheres (Figure 4). Si peak was observed in the EDS analysis, which indicated the successful silanization reaction on the Fe3O4 surface. The weak C and N signals may be originated from the surfacebound polysaccharides (CHI and HA) and the immobilized lipase. EDS mapping analysis was employed in this study to obtain the precise distribution of these elements. Figure 5 showed that the EDS mapping images of Fe 3 O 4 @ SiO2@{CHI/HA}3@lipase microspheres also indicated the presence of elements Fe, Si, and O. In the EDS mapping, the distribution of element Fe and O is located in the central part of the microspheres, while element Si is homogeneously distributed in the outer layer of the entire microspheres. The result demonstrated the successful silanization reaction and the homogeneous distribution of SiO2 on Fe3O4 surfaces. The EDS mapping images also confirmed the Fe 3 O 4 @ SiO2@{CHI/HA}3@lipase core−shell structure. In addition, the morphological characteristic of the synthesized magnetic microspheres was further evaluated by transmission electron microscopy (TEM). The TEM image showed that the naked Fe3O4 particles were spherical with an average size of 450 nm (Figure 6A). After SiO2 coating, core− shell Fe3O4@ SiO2 microspheres comprising Fe3O4 cores (black) with 450 nm and thin silica layer (gray) were observed (Figure 6B). Differentiating the Fe3O4 cores and SiO2 shells from TEM images is easy because of the distinct density contrast between the two components.52 The average thickness of SiO2 shells was approximately 50 nm (inset of Figure 6B). Furthermore, the surface of the Fe3O4@SiO2 was smoother than that of naked Fe3O4. After surface modification with CHI and HA, the morphological characteristic of these magnetic microspheres (Fe3O4@SiO2@{CHI/HA}3) showed no change compared with that of Fe3O4@SiO2 (Figure 6C) but the average shell thickness increased to 60 nm (inset of Figure 6C). Figure 6D showed that the morphology and size of lipase-immobilized Fe3O4@SiO2@{CHI/HA}3 microspheres remained unchanged compared with those of Fe3O4@ SiO2@{CHI/HA}3 microspheres. Notably, the lipase immobilized on the magnetic microspheres could be hardly observed from the TEM image (inset of Figure 6D) because lipase immobilization only occurred on the surface of magnetic microspheres and the molecules grafted were not sufficiently thick to be observed. These TEM results were consistent with those of SEM observation. Combustion elemental analysis was employed for systematical analysis of these microspheres to further determine the composition and the proportion of each component of magnetic microspheres clearly. Element content data of Fe 3 O 4, Fe 3 O 4 @SiO 2 , Fe 3 O 4 @SiO 2 @{CHI/HA} 3 , and Fe3O4@SiO2@{CHI/HA}3@lipase microspheres are shown in Table 1. Evidently, C, H, and N elements were absent in Fe3O4 and Fe3O4@SiO2 microspheres. The H element of Fe3O4@SiO2@{CHI/HA}3 was attributable to the HA and CHI, and the N element of Fe3O4@SiO2@{CHI/HA}3@lipase was mainly attributable to the lipase. Given the elemental analysis results, the lipase was successfully attached to the surface of Fe3O4@SiO2@{CHI/HA}3 microspheres.

microspheres. The synthesized Fe3O4@SiO2 microspheres presented a zeta potential of approximately −37 mV. When CHI was coated on Fe3O4@SiO2, the surface charge changed to +39 mV due to the positive charge of amine pending groups in CHI chains. The surface potential was tuned to be −46 mV after further coating HA onto the CHI-coated Fe3O4@SiO2. The change of surface charge from negative to positive values observed with each coating step indicated that Fe3O4@SiO2 microspheres were successfully coated by HA and CHI polymers via the LbL assembly technique. The Fe3O4@SiO2 microspheres with outermost layers of HA exhibited zeta potentials of −44 mV. As the number of bilayers (>3) on Fe3O4@SiO2 microspheres was increased, the CHI/HA-coated Fe3O4@SiO2 microspheres demonstrated the tendency to aggregate in aqueous solution. Therefore, the Fe3O4@SiO2 microspheres in this study were coated with three bilayers of CHI and HA. In addition, FT-IR was also used to characterize the changes of groups over the course of LbL assembly and immobilization (Figure S1). The FT-IR results confirmed the successful preparation of magnetic supports for lipase immobilization (see the detailed discussion in the Supporting Information). The morphological characteristics and size of these magnetic microspheres were observed by scanning electron microscopy (SEM) (Figure 3). As displayed in Figure 3A, the naked Fe3O4

Figure 3. SEM images of pristine Fe3O4 particles (A), Fe3O4@SiO2 microspheres (B), Fe3O4@SiO2@{CHI/HA}3 microspheres (C), and Fe3O4@SiO2@{CHI/HA}3@lipase microspheres (D).

particles were typical spheres and had an average diameter of approximately 450 nm. After silanization with TEOS onto the surface of Fe3O4 particles, the Fe3O4@SiO2 microspheres were all well-dispersed and had an average diameter of around 500 nm with a spherical shape (Figure 3B). Furthermore, the morphology and structure of Fe3O4@SiO2@{CHI/HA}3 and Fe3O4@SiO2@{CHI/HA}3@lipase were similar to those of Fe3O4@SiO2 (Figure 3C,D), which indicated that the size and morphology of the Fe3O4@SiO2 microspheres remained unchanged after LbL self-assembly and lipase immobilization. Therefore, the two modifications did not considerably affect the dispersibility and average diameter of microspheres, indicating that these modifications occur only on the surface of Fe3O4@SiO2 magnetic microspheres. In addition, the particle size distributions of these magnetic microspheres D


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Figure 4. SEM image and corresponding EDS result of Fe3O4@SiO2@{CHI/HA}3@lipase microspheres.

Figure 5. EDS mapping images of Fe3O4@SiO2@{CHI/HA}3@lipase for elements Fe, O, and Si.

The magnetic properties of the Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@{CHI/HA}3, and the immobilized lipase at room temperature were studied by measuring the magnetization curves using vibrating sample magnetometer (VSM) (Figure 7A). The saturation magnetization of Fe3O4 particles was 62.5 emu g−1. Also, the saturation magnetization of Fe3O4@SiO2 microspheres were decreased to 42.2 emu g−1. Furthermore, after CHI and HA coating and lipase immobilization their saturation magnetization further decreased to 39.7 and 37.3 emu g−1, respectively. Clearly, the saturation magnetization of the magnetic microspheres were decreased after SiO2 coating, polysaccharide (CHI and HA) coating, and lipase immobilization. This reduction of saturation magnetization can be explained by the presence of diamagnetic silica shell around the magnetic cores. Nevertheless, only a small difference of saturation magnetization was observed between Fe3O4@SiO2@{CHI/HA}3 and Fe3O4@ SiO 2 @{CHI/HA} 3 @lipase. Therefore, the Fe 3 O 4 @ SiO2@{CHI/HA}3@lipase microspheres can be separated by external magnetic field from the reaction mixtures. Figure 7B depicted that the Fe3O4@SiO2@{CHI/HA}3@lipase microspheres can be rapidly collected from the solution using an external usual magnet and forming aggregates in only 3 s.

Figure 6. TEM images of pristine Fe3O4 (A), Fe3O4@SiO2 (B), Fe3O4@SiO2@{CHI/HA}3 (C), and Fe3O4@SiO2@{CHI/HA}3@ lipase microspheres (D).

Table 1. Elemental Content Data (Atomic %) of Different Magnetic Microspheres microspheres Fe3O4 Fe3O4@SiO2 Fe3O4@SiO2@{CHI/HA}3 Fe3O4@SiO2@{CHI/HA}3@lipase a

C (%)a

H (%)a

N (%)a

n.d. n.d. 2.08 2.28

n.d. n.d. 0.90 0.98

n.d. n.d. n.d. 0.46

n.d. = not detectable

When the magnetic field disappeared, the magnetic microspheres were rapidly redispersed under slight shaking (Figure 7B). Thus, the excellent magnetic responsivity and redispersibility of lipase supports are the advantages for biocatalysts. These results revealed that the magnetic biocatalyst possessed suitable properties for magnetic manipulation. The Fe3O4 particles with different modifications were also characterized by TGA analysis. Weight loss (%) was used in E


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Figure 7. (A) Magnetic hysteresis loops of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@{CHI/HA}3, and Fe3O4@SiO2@{CHI/HA}3@lipase; (B) magnetic separation and redispersion process of Fe3O4@SiO2@{CHI/HA}3@lipase.

imately 0.8% (8 mg polysaccharides/g Fe3O4). Furthermore, from the TGA result of the amount of Fe3O4@SiO2@{CHI/ HA}3@lipase microspheres (Figure 8D), the amount of the immobilized lipase in Fe3O4@SiO2@{CHI/HA}3@lipase microspheres was calculated to be 3.7% (38.4 mg lipase/g support), which was consistent with the results of the Bradford assay. Enzyme Loading Determination. Fe3O4@SiO2@{CHI/ HA}3 were employed to immobilize lipase for biocatalyst construction to demonstrate the feasibility of PEM-modified magnetic microspheres as support for lipase immobilization. The classical Coomassie blue-binding assay was used in this study to investigate the lipase-loading capacity on Fe3O4@ SiO2@{CHI/HA}3 microspheres. In enzyme immobilization, the immobilization capacity was affected by many factors, including polysaccharide layers, incubation time, pH, temperature, and feeding concentration of lipase. Therefore, the influence of these factors on the loading capacity and efficiency was studied and these results (Figure S4) and discussion are shown in Supporting Information. The loading amount and immobilization technique of this immobilized lipase were compared with those of other immobilized lipase in published reports, and these parameters were summarized in Table 2. The relatively higher loading capacity of lipase on substrates can be achieved by crosslinking with glutaraldehyde. However, glutaraldehyde, despite having good cross-linking performance, possesses a noticeable problem in cytotoxicity. The lipase immobilized by crosslinking method may have toxicity, which severely limit their applications in food and biomedical fields. In the present work, the lipase was immobilized on the prepared magnetic microspheres via the formation of amide bonds between

this study to define the coating and immobilization amount of silica, polysaccharides, and lipase compared with those of naked Fe3O4. As shown in Figure 8A, Fe3O4 microspheres

Figure 8. TGA of magnetic microspheres: Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@{CHI/HA}3, and Fe3O4@SiO2@{CHI/HA}3@lipase.

showed a weight loss of 6.4% when temperature rose to 300 °C, which was attributed to the release of absorbed water. For Fe3O4@SiO2 microspheres (Figure 8B), the weight loss below 800 °C was approximately 11.1%, which caused by the release of water and the decomposition of silica. The CHI and HA in Fe3O4@SiO2@{CHI/HA}3 microspheres decomposed from 200 °C,53 and when the temperature rose to 800 °C the overall weight loss was 11.9% (Figure 8C). The amount of CHI and HA in Fe3O4@SiO2@{CHI/HA}3 microspheres was approx-

Table 2. Supports, Lipase Loading Amount, and Immobilizing Technique of the Current Work and Other Reports support

lipase loading amount

immobilization technique

references

poly(acrylonitrile-co-maleic acid) chitosan/poly(vinyl alcohol) (PVA) hollow fiber membrane γ-Fe2O3 magnetic nanoparticles silica-coated modified magnetite nanoparticles amine-functionalized Fe3O4@C nanoparticles PEM-coated Fe3O4@SiO2

∼20 mg/g 22.6−72.8 mg/g 48.0−74.2 mg/m2 5.8 mg/g 16 mg/g 115.6 mg/g 48.6 mg/g

covalent binding cross-linker (glutaraldehyde) cross-linker (glutaraldehyde) cross-linker (glutaraldehyde) covalent binding cross-linker (glutaraldehyde) covalent

54 13 55 56 57 24 this work

F


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Figure 9. Relative activity of the free and the immobilized lipases (Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM) at different temperature (A) and different pH (B).

amino of lipase and carboxyl groups of HA. This method provided a strategy to fabricate immobilized lipase with high loading capacity and high biocompatibility. Activity of the Immobilized Lipase. Reaction Condition Optimization and Activity Assay. Several process parameters, such as pH and temperature, can affect the enzyme activity. In addition, compared with free enzyme, immobilized enzyme usually exhibited reduced activity due to the mass transfer limitations.58,59 Thus, a comparative investigation between the free lipase and immobilized lipase (Fe3O4@SiO2@{CHI/ HA}3@lipase and Lipozyme RM IM) for the synthesis of OPO was provided in terms of pH and temperature. The influence of reaction temperature on the activity of the free and immobilized lipase ranging from 25 to 70 °C was investigated by measuring the amount of OPO. Figure 9A showed that the three temperature profiles for the free and immobilized lipase exhibited similar patterns. The maximum activity was at 55 °C for the Fe3O4@SiO2@{CHI/HA}3@lipase, which was the same for the free lipase and Lipozyme RM IM. The activity of the Fe3O4@SiO2@{CHI/HA}3@lipase was generally higher than most of the temperatures compared with that of the free lipase and Lipozyme RM IM. The effect of pH on the activity of the free and immobilized lipase (Fe3O4@SiO2@{CHI/ HA}3@lipase and Lipozyme RM IM) was investigated in the pH range of 6−11 at 55 °C. According to Figure 9B, the optimal pH of free and immobilized lipase was observed at pH values of 8.0. Compared with the free lipase and Lipozyme RM IM, the immobilized lipase maintained relatively high activity over a broad pH range of 6−11. Kinetic Parameters. Michaelis−Menten kinetics of interesterification was measured for the free and immobilized lipase using a series of initial concentrations (0.25−2 mg/mL) of PPP as a substrate to understand the lipase activity. The Michaelis−Menten constant (Km) and the maximum reaction velocity (Vmax) values for the free and immobilized lipase (Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM) were calculated by the Lineweaver−Burk plots (Figure 10). The kinetic parameters of the free and immobilized lipase were obtained and presented in Table 3. The results affirmed that the Km value for the free lipase was 0.82 mg/mL whereas those of the Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM were 1.40 and 1.16 mg/mL, respectively, which means that the affinity between enzyme and its substrate was slightly

Figure 10. Lineweaver−Burk plot for (A) free lipase, (B) Fe3O4@ SiO2@{CHI/HA}3@lipase, and (C) Lipozyme RM IM.

Table 3. Activity and Kinetic Parameters (Vmax, Km) for the Free Lipase, Fe3O4@SiO2@{CHI/HA}3@lipase, and Lipozyme RM IM under Optimum Conditions sample

Vmax (U/mg)

Km (mg/mL)

free lipase Fe3O4@SiO2@{CHI/HA}3@lipase lipozyme RM IM

35.7 20.0 18.3

0.82 1.40 1.16

decreased when lipase was immobilized on magnetic microspheres and resins. In addition, the Vmax value of the free lipase (35.7 U/mg lipase) was higher than that of the immobilized lipase (20.0 U/mg Fe3O4@SiO2@{CHI/HA}3@lipase and 18.3 U/mg Lipozyme RM IM). This phenomenon might be due to the strong interaction between lipase and support, leading to either structural change into the less active conformation of the enzyme or hindrance in substrate transfer toward active sites.19 Previously, an increase in Km and a decrease in Vmax were observed after the immobilization of enzyme on solid support materials with high activity.19,60−62 Thermal and Storage Stability of the Free and Immobilized Lipase. The thermal, pH, and storage stability properties are key factors in practical applications for immobilized enzyme. The thermal stability of the immobilized G


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Figure 11. Stability of free lipase and immobilized lipase (Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM): (A) thermal stability at 60 °C, (B) storage stability at 4 °C.

lipase (Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM) was compared with that of the free lipase at pH 8 and 60 °C for 48 h (Figure 11A). In this study, the relative activity is the ratio of the specific activity measured at each time to the initial specific activity. The results verified that the immobilized lipase has relatively better thermal stability. After 48 h of heat treatment, the Fe3O4@SiO2@{CHI/HA}3@lipase, Lipozyme RM IM, and free lipase retained 53.9%, 50.1%, and 47.7% of their initial activity, respectively. This method of covalent bonding not only maintained the optimum temperature but also improved the thermal stability. The good performance in thermal stability of the Fe3O4@SiO2@{CHI/HA}3@lipase can be ascribed to the prevention of the conformation transition of lipase at high temperature by the HA and support, making it stable against the change in temperature.63 This Fe3O4@ SiO2@{CHI/HA}3@lipase with better stability at high temperature broadens its further practical applications. The activities of enzymes often decreased during the storage. Another advantage of immobilized enzyme is good storage stability without losing substantial activity. This advantage is especially crucial for the final assessment and selection of industrially important enzyme over other available enzymes in the similar domain. The activity of lipase stored at 4 °C for 30 days was investigated to assess the storage stability of the free and immobilized lipase, and these results are shown in Figure 11B. As the time was increased, the activity of the immobilized lipase (Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM) and free lipase reduced, whereas the immobilized lipase slowly decreased and became lesser than the free lipase. After storage for 30 days, the free lipase retained 45% of its initial activity, whereas the Fe3O4@SiO2@{CHI/HA}3@lipase and Lipozyme RM IM retained 78% and 58% of their initial activities, respectively (Figure 11B). Therefore, Fe3O4@ SiO2@{CHI/HA}3 played an important role in improving the storage stability of the immobilized lipase. Reusability of the Immobilized Lipase and Leaching Experiment. Further investigation was focused on the reusability of Fe3O4@SiO2@{CHI/HA}3@lipase. Compared with the free lipase and Lipozyme RM IM, the easy and rapid reusability of Fe3O4@SiO2@{CHI/HA}3@lipase is its most important advantage. The Fe3O4@SiO2@{CHI/HA}3@lipase can be separated from the reaction system using the external magnetic field. Figure 12 exhibited that Fe3O4@SiO2@{CHI/

Figure 12. Reusability of Fe3O4@SiO2@{CHI/HA}3@lipase for the synthesis of OPO.

HA}3@lipase showed good stability and retained 85% of its initial activity after nine consecutive cycles for OPO synthesis. This finding inferred that the Fe3O4@SiO2@{CHI/HA}3@ lipase had good stability in the enzymatic reaction, which can be attributed to the rigidification of lipase due to the formation of amide bonds between the lipase and HA on the surface of Fe3O4@SiO2@{CHI/HA}3 microspheres. The small decrease in enzyme activity after multiple reuses was commonly observed for the immobilized biocatalyst, which can be caused by many factors, including loss of constituents of the lipase complex, weak autodigestion, and gradual enzyme denaturation.64 In the present study, the small decrease in the activity of Fe3 O4 @SiO 2@{CHI/HA}3@lipase was ascribed to the denaturation of lipase after repeated usage. The Fe3O4@ SiO2@{CHI/HA}3@lipase microspheres exhibited better operational stability and reusability, which are crucial and attractive for practical applications. In addition, a leaching experiment was performed for the Fe3O4@SiO2@{CHI/HA}3@lipase. In a typical experiment, the Fe3O4@SiO2@{CHI/HA}3@lipase was separated from the reaction mixture using a magnet after 3 h of OPO synthesis, and the transesterification was allowed to continue for another H


Research Article

ACS Sustainable Chemistry & Engineering 3 h with the remaining filtrate. The result deduced that no additional OPO was observed in the reaction mixture. Furthermore, no leaching of the enzyme into the solution was observed, which indicated that the immobilization of lipase on this support only occurs via covalent bonds.

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CONCLUSIONS Overall, PEMs (HA and CHI) coated with Fe3O4@SiO2 core− shell microspheres via LbL assembly were successfully prepared and developed as an effective magnetic support for enzyme immobilization. Lipase was used as a model enzyme and was successfully immobilized onto the surface of Fe3O4@ SiO2@{CHI/HA}3. The Fe3O4@SiO2@{CHI/HA}3 microspheres exhibited good immobilization capacity and efficiency. Compared with the free lipase, the magnetic-immobilized lipase exhibited good thermal and long-term stability and catalytic activity for the synthesis of OPO. Moreover, the prepared immobilized lipase presented favorable reusability by magnetic treatment. The preceding results validate that the polysaccharide-coated magnetic microspheres are promising platforms for highly efficient lipase immobilization, and the immobilized lipase can be considered a robust and effective biocatalyst for the production of OPO.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b05786. The procedures and instruments used for the preparation and characterization of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2@{CHI/HA}3, and Fe3O4@SiO2@{CHI/ HA}3@lipase magnetic microspheres; determination of the lipase loading amount on the support; procedures for the determination of optimal pH and temperature on the synthesis of OPO catalyzed by immobilized lipase; stability and reusability test of the immobilized lipase; detailed results and discussions of FT-IR, DLS, and XRD of magnetic microspheres; results and discussion of lipase loading capacity onto support (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hongbin Zhang: 0000-0002-4419-4818 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Plan (2016YFD0400200) and the National Natural Science Foundation of China (Grants 21774075, 21074071).

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