Preparation method of Janus magnetic nanoparticles with asymmetric structure and application thereof in immobilization of lipase

Abstract

The application relates to a Janus magnetic nanoparticle with an asymmetric structure, in particular to a preparation method of a Janus magnetic nanoparticle with an asymmetric structure and application of the Janus magnetic nanoparticle in immobilized lipase, and belongs to the field of biological materials, enzyme engineering and nanotechnology. The method provided by the application can orientally anchor a lipase hydrophobic cover through a phospholipid layer, maintain a natural conformation, improve activity compared with a traditional covalent fixation method, cooperate with CO2 demulsification through a pulse magnetic field (0.5 T, 1 Hz), shorten a particle recovery time to 2 minutes, and has high recovery rate; the product adsorption is removed through in-situ regeneration by N2 / CO2 mixed gas, the enzyme activity is reserved to be high after multiple cycles, the carrier loss is low, and the comprehensive catalytic efficiency is improved. The application provides a high-activity, centrifuge-free and recyclable solution for an emulsion catalytic system such as biodiesel.

Description

Technical Field

[0001] This invention relates to Janus magnetic nanoparticles with an asymmetric structure, specifically to a method for preparing Janus magnetic nanoparticles with an asymmetric structure and their application in immobilized lipases, belonging to the fields of biomaterials, enzyme engineering and nanotechnology. Background Technology

[0002] Existing lipase immobilization technologies mostly employ single-carrier methods, resulting in low enzyme loading, poor stability, and difficulty in achieving targeted catalysis. While Chinese patent CN202510298656.4 describes a three-step method combining amino grafting, aldehyde crosslinking, and embedding with a magnetic carrier, its interfacial catalytic effect is insufficient, failing to fully utilize the catalytic function of the lipase. Chinese patent CN202510254699.2 discloses an immobilized lipase using modified nano-SiO2 as a carrier and its preparation method, modified with amino and epoxy silanes; however, due to the small size of nano-SiO2, it is also difficult to recover, thus not fully solving the lipase recovery problem. Patent CN202510191935.0 discloses a pancreatic lipase immobilization composite magnetic nanobead, its preparation method, and its application; however, the lipase is uniformly distributed on the surface of the magnetic nanobeads, also failing to fully utilize the catalytic activity of pancreatic lipase.

[0003] Janus particles, due to their asymmetric structure, can achieve bifunctional interfaces. However, existing methods for immobilizing lipases using Janus particles often suffer from drawbacks such as demulsification and difficulties in recovery. Although CN202510188663.9 discloses a one-step method for preparing immobilized lipase Janus microparticles using an emulsion template based on an enzyme-protein directional adsorption strategy and its application in a Pickering emulsion catalytic system, this emulsion template method relies on the hydrophobic structure of the lipase, resulting in insufficient adsorption capacity. Furthermore, the construction of the Pickering emulsion requires homogenization, leading to lipase detachment and affecting catalytic efficiency. In addition, the strong stability of the Pickering emulsion makes it difficult to separate the lipase from the emulsion, reducing the lipase recovery rate. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a method for preparing Janus magnetic nanoparticles with an asymmetric structure.

[0005] Another objective of this invention is to provide the application of the aforementioned Janus magnetic nanoparticles in immobilized lipases. By optimizing the core-shell structure, Pickering emulsion masking technology, and double-crosslinked enzyme immobilization, a high-performance method for preparing Janus lipases is developed. Janus magnetic particles with both hydrophilic and hydrophobic sides are prepared, achieving directional enzyme immobilization. Furthermore, through the synergistic effect of CO2-triggered demulsification and pulsed magnetic field, efficient recovery and regeneration of the enzyme carrier are achieved, solving the problems of large activity loss, low recovery efficiency, and limited lifespan inherent in traditional immobilized enzymes.

[0006] This invention aims to solve the above-mentioned problems, specifically involving the preparation of efficient, stable, and magnetically separable Janus lipases through multi-level structural design and asymmetric surface modification, for use in catalytic reactions, biosensing, drug delivery and other fields.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for preparing Janus magnetic nanoparticles with an asymmetric structure, comprising the following steps: 1) Preparation of Fe3O4 magnetic nuclei: FeCl3·6H2O, NaAc, and PVP (polyvinylpyrrolidone) were dissolved in deionized water, and the mixed solution was ultrasonically dispersed evenly and subjected to hydrothermal reaction; the product was magnetically separated, washed and dried to obtain Fe3O4 magnetic nuclei; 2) SiO2 shell coating: Fe3O4 magnetic cores were dispersed in an ethanol-water mixed solution, sonicated, and the pH was adjusted; TEOS was added dropwise, and the reaction was carried out after the addition was completed; magnetic separation and washing were performed to obtain Fe3O4@SiO2 core-shell particles; 3) Pickering emulsion semi-masking: Fe3O4@SiO2 core-shell particles are ultrasonically dispersed in CTAB aqueous solution, melted paraffin is added, homogenized emulsification is carried out, and rapid cooling is performed to form semi-coated paraffin spheres; 4) Hydrophobic side modification: Immerse the semi-coated paraffin balls in HCl solution, shake and wash; immerse the exposed side in DMA ethanol solution, react, and obtain the modified paraffin balls; 5) Hydrophilic side modification: After wax dissolution, the particles are magnetically separated and washed; the exposed hydrophilic surface is immersed in a phospholipid-dopamine solution and shaken; after magnetic separation and washing, phospholipid-modified Janus nanomagnetic particles are obtained.

[0008] Furthermore, in step 1), the mass ratio of FeCl3·6H2O, NaAc, and PVP is 3-4:6-6.5:0.5; the hydrothermal reaction is carried out at 200℃ for 10 hours.

[0009] Further, in step 2), the concentration of the Fe3O4 magnetic nucleus in the ethanol-water mixed solution is 3-3.5 mg / mL; the volume ratio of ethanol to water in the ethanol-water mixed solution is 2:1; the pH is adjusted to 9.5 using 28% ammonia; the ratio of the Fe3O4 magnetic nucleus to TEOS is 0.5 g: 0.8 mL; TEOS is added dropwise in three portions, with an interval of 30 min between each addition; the volume ratio of the three TEOS additions is 3:3:2; the reaction is carried out at 60°C with stirring for 3 h.

[0010] Further, in step 3), the concentration of the Fe3O4@SiO2 core-shell particles in the CTAB aqueous solution is 0.01 g / mL; the concentration of the CTAB aqueous solution is 0.003 g / L; the mass ratio of the Fe3O4@SiO2 core-shell particles to the molten paraffin is 1:20-25; and the homogenization emulsification is performed at 10000 rpm for 5 min.

[0011] Furthermore, in step 4), the DMA:SiO2 ratio is 1:2, w / w; the mass fraction of the HCl solution is 10%; the oscillation is performed at 25°C for 1 hour; and the reaction is performed at 60°C under nitrogen protection for 24 hours.

[0012] Furthermore, in step 5), the phospholipid-dopamine solution is composed of 10 mg dioleoylphosphatidyl acid, 5 mg dopamine, and 50 mL of Tris-HCl buffer at pH 8.5; the shaking is performed at 25°C in the dark for 12 hours.

[0013] This invention also provides an application of Janus magnetic nanoparticles prepared by the above method in immobilized lipase and its regeneration, comprising the following steps: (1) Lipase immobilization: Janus magnetic nanoparticles were dispersed in PBS, lipase solution was added, and the mixture was gently shaken; the mixture was then separated, washed, and freeze-dried to obtain an emulsion system of Janus magnetic nanoparticles with hydrophobic cap anchored to lipase. (2) Introduce CO2 into the emulsion system to break the emulsion; simultaneously apply a pulsed magnetic field to achieve particle sedimentation and recovery; (3) Carrier regeneration process: The recovered particles are flushed with mixed gas to remove product adsorption and restore the hydrophobicity of the particles.

[0014] Preferably, in step (1), the mass ratio of the Janus nanomagnetic particles to the lipase is 1:2–1:0.5; the concentration of the lipase solution is 5 mg / mL; the oscillation is gentle oscillation at 4°C for 8 hours; and the field separation conditions are: 0.5 T, treatment for 5 minutes.

[0015] Preferably, in step (2), the flow rate of CO2 is 0.2 L / min; the conditions of the pulsed magnetic field are 0.5 T, frequency 1 Hz, and pulse width 0.5 s.

[0016] Preferably, in step (3), the mixed gas is composed of N2 and CO2 in a volume ratio of 4:1; the flow rate of the mixed gas is 0.3 L / min, and the rinsing time is 10 min.

[0017] The semi-coating modification method provided by this invention allows particles to be directionally adsorbed at the oil / water interface. Due to surface energy differences and the hydrophilic / hydrophobic distribution regulated by CTAB, a portion of the particle surface is embedded in the paraffin phase, while the other portion is exposed in the aqueous phase. This results in particle janus formation, where one side is in contact with paraffin and the other side is exposed to the aqueous phase, allowing for subsequent modification. This invention utilizes the masking effect of paraffin to modify only the exposed side with silane (DMA).

[0018] The DMA modification used in this invention involves reacting the exposed SiO2 surface of semi-coated paraffin microspheres with a DMA ethanol solution. The amount of DMA added is determined by the mass ratio of DMA to SiO2. DMA is soluble in ethanol, and the target of modification is the exposed SiO2 surface. After emulsification and cooling, the Fe3O4@SiO2 particles in the prepared semi-coated paraffin microspheres are directionally embedded on the surface of the paraffin microspheres, resulting in one side of the particle being covered by paraffin while the other side is exposed. In this way, only the exposed side can contact the DMA solution and undergo modification, thereby achieving asymmetric modification. After removing the paraffin, individual Janus particles are obtained.

[0019] Based on the construction of Janus particles with dual functions of CO2-responsive demulsification and magnetic field recovery, this invention utilizes a biomimetic phospholipid anchoring method to immobilize lipases, resulting in the following beneficial effects: (1) The method provided by the present invention can anchor the hydrophobic cap of lipase in a phospholipid layer to maintain the natural conformation, which can greatly improve the activity. Furthermore, the particle recovery time is shortened and the recovery rate is improved by pulsed magnetic field and CO2 demulsification treatment. (2) Product adsorption is relieved by in-situ regeneration with N2 / CO2 mixed gas. After 15 cycles, the enzyme activity is 93% retained, the carrier loss is small, and the catalytic efficiency is improved. The method provided by this invention offers a highly active, centrifugation-free, and recyclable solution for emulsion catalytic systems such as biodiesel. Attached Figure Description

[0020] Figure 1 This is a SEM image of the Janus nanoparticles prepared in Example 1. Detailed Implementation

[0021] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0022] The DMA used in this invention is dimethylacetamide.

[0023] Example 1 1) Preparation of Fe3O4 magnetic cores: FeCl3·6H2O (3.24 g), NaAc (6.15 g), and PVP (0.5 g) (polyvinylpyrrolidone) were dissolved in deionized water (80 mL). The mixed solution was ultrasonically dispersed for 20 min and hydrothermally reacted at 200℃ for 10 h. The product was magnetically separated, washed three times with alternating ethanol and water, and dried under vacuum at 60℃ to obtain Fe3O4 magnetic cores.

[0024] 2) SiO2 shell coating: Fe3O4 (0.5 g) was dispersed in an ethanol-water mixed solution (160 mL, 2:1 v / v), sonicated for 30 min, and 28% ammonia was added to adjust the pH to 9.5; TEOS (0.3 + 0.3 + 0.2 mL) was added dropwise in three portions, with an interval of 30 min each time, and the reaction was stirred at 60℃ for 3 h; magnetic separation and washing with ethanol were performed to obtain Fe3O4@SiO2 core-shell particles.

[0025] 3) Pickering emulsion masking and asymmetric modification: Fe3O4@SiO2 (0.5 g) was ultrasonically dispersed in an aqueous solution containing CTAB (0.003 g / L, 50 mL), and molten paraffin (10 g, 60-62℃) was added. The mixture was homogenized and emulsified at 10,000 rpm for 5 min, and then rapidly cooled to 20℃ in an ice bath to form semi-coated paraffin spheres (with the SiO2 exposed side facing outward).

[0026] 4) Hydrophobic side modification: The semi-coated particles were immersed in 10% HCl, shaken at 25℃ for 1 h, and washed with ethanol; the exposed surface was immersed in DMA ethanol solution (DMA:SiO2=1:2, w / w), and reacted at 60℃ under nitrogen protection for 24 h, and DMA was grafted by silane condensation.

[0027] 5) Hydrophilic side modification: After dissolving paraffin in chloroform, the particles were magnetically separated and washed with ethanol; the exposed hydrophilic side was immersed in phospholipid-dopamine solution (10 mg dioleoylphosphatidyl acid, 5 mg dopamine and 50 mL Tris-HCl buffer, pH 8.5) and shaken at 25°C in the dark for 12 h; after magnetic separation and washing with PBS, phospholipid-modified Janus particles were obtained.

[0028] 6) Lipase fixation: 50g Janus particles were dispersed in 10mL PBS buffer (pH 7.0), 10mL lipase solution (5 mg / mL) was added, and the mixture was gently shaken at 4℃ for 8 h; then field separation was performed (0.5 T, 5 min), washed 3 times with PBS, and lyophilized to obtain Janus particles with hydrophobic cap anchored to lipase.

[0029] 7) Dual-response operation: After the Janus particles immobilized with lipase catalyze the reaction, CO2 is introduced into the emulsion system (flow rate 0.2 L / min), and demulsification is completed within 2 min; at the same time, a pulsed magnetic field (0.5 T, frequency 1 Hz, pulse width 0.5 s) is applied to achieve sedimentation and recovery of particles within 2 min.

[0030] 8) Carrier regeneration process: The recovered particles are flushed with a N2 / CO2 mixed gas (4:1 v / v, flow rate 0.3 L / min) for 10 min, which can remove the adsorption of the product and restore the hydrophobicity of the particles.

[0031] Scanning electron microscope image of Janus particles as follows Figure 1 As shown.

[0032] Comparative Example 1 The preparation steps for Fe3O4@SiO2 were the same as above. Without Pickering emulsion masking and asymmetric modification, Fe3O4@SiO2 particles (10 mg / mL) were directly dispersed in PBS. Lipase solution (5 mg / mL) was added, and the mixture was incubated at 4°C for 8 h, followed by static adsorption. The immobilized particles were collected using a static magnetic field separation method (0.5 T, continuous magnetic field). Emulsion demulsification was achieved solely through CO2 introduction (0.2 L / min), without pulsed magnetic field assistance.

[0033] Example 1 The methods provided in the examples and comparative examples were used to detect enzyme activity recovery, demulsification time, particle recovery, and cycling stability. The specific detection methods are as follows: Enzyme activity recovery rate: The enzyme activity was detected by UV-Vis spectrophotometer. p-nitrophenyl palmitate (p-NPP) was used as a substrate. Lipase hydrolyzed it to generate p-nitrophenol, which has a characteristic absorption at 410 nm. The enzyme activity was calculated by the change in absorbance.

[0034] Enzyme activity recovery rate = (Total activity of immobilized enzyme / Total activity of free enzyme) × 100%; Demulsification time: heptane and aqueous phase containing immobilized enzyme were ultrasonically emulsified at a volume ratio of 1:1 at 200W for 2 min to form an O / W type emulsion. The emulsion was placed in a graduated tube and allowed to stand at room temperature. The time for complete separation of oil and water was recorded.

[0035] Particle recovery rate: Utilizing the magnetic responsiveness of magnetic particles, the amount of residual particles in the supernatant is quantitatively detected after magnetic separation, and the recovery rate is calculated.

[0036] Particle recovery rate = (Initial dosage - Supernatant residue / Initial dosage) × 100% Cyclic stability: The enzyme activity was measured after each cycle of reaction, magnetic separation, washing and reuse.

[0037] The specific results are shown in Table 1.

[0038] Table 1 As can be seen from Table 1, the Janus magnetic nanoparticles prepared by this invention have a high recovery rate, short demulsification time, and good particle recovery rate and cycle stability.

Claims

1. A method for preparing Janus magnetic nanoparticles with an asymmetric structure, characterized in that, Includes the following steps: 1) Dissolve FeCl3·6H2O, NaAc, and PVP (polyvinylpyrrolidone) in deionized water, ultrasonically disperse the mixed solution evenly, and carry out hydrothermal reaction; magnetically separate the product, wash and dry it to obtain Fe3O4 magnetic cores; 2) Disperse Fe3O4 magnetic nuclei in an ethanol-water mixed solution, sonicate, and adjust the pH; add TEOS dropwise, and proceed with the reaction after the addition is complete; Magnetic separation and washing yielded Fe3O4@SiO2 core-shell particles; 3) Pickering emulsion semi-masking: Fe3O4@SiO2 core-shell particles are ultrasonically dispersed in CTAB aqueous solution, melted paraffin is added, homogenized emulsification is carried out, and rapid cooling is performed to form semi-coated paraffin spheres; 4) Immerse the partially coated paraffin balls in HCl solution, shake and wash; immerse the exposed side in DMA ethanol solution, react, and obtain the modified paraffin balls; 5) After melting the wax, the particles are magnetically separated and washed; the exposed hydrophilic surface is immersed in a phospholipid-dopamine solution and shaken; after magnetic separation and washing, phospholipid-modified Janus nanomagnetic particles are obtained.

2. The preparation method according to claim 1, characterized in that, In step 1), the mass ratio of FeCl3·6H2O, NaAc, and PVP is 3-4:6-6.5:0.5; the hydrothermal reaction is carried out at 200℃ for 10 hours.

3. The preparation method according to claim 1, characterized in that, In step 2), the concentration of the Fe3O4 magnetic nucleus in the ethanol-water mixed solution is 3-3.5 mg / mL; the volume ratio of ethanol to water in the ethanol-water mixed solution is 2:1; the pH is adjusted to 9.5 using 28% ammonia; the ratio of the Fe3O4 magnetic nucleus to TEOS is 0.5 g: 0.8 mL; TEOS is added dropwise in three portions, with an interval of 30 min between each addition; the volume ratio of the three TEOS additions is 3:3:2; the reaction is carried out at 60°C with stirring for 3 h.

4. The preparation method according to any one of claims 1-3, characterized in that, In step 3), the concentration of the Fe3O4@SiO2 core-shell particles in the CTAB aqueous solution is 0.01 g / mL; the concentration of the CTAB aqueous solution is 0.003 g / L; the mass ratio of the Fe3O4@SiO2 core-shell particles to the molten paraffin is 1:20-25; and the homogenization emulsification is performed at 10,000 rpm for 5 min.

5. The preparation method according to claim 1 or 4, characterized in that, In step 4), the DMA:SiO2 ratio is 1:2, w / w; the mass fraction of the HCl solution is 10%; the oscillation is performed at 25°C for 1 hour; and the reaction is performed at 60°C under nitrogen protection for 24 hours.

6. The preparation method according to claim 1 or 5, characterized in that, In step 5), the phospholipid-dopamine solution is composed of 10 mg dioleoylphosphatidic acid, 5 mg dopamine and 50 mL of Tris-HCl buffer at pH 8.5; the shaking is performed at 25°C in the dark for 12 hours.

7. The application of Janus magnetic nanoparticles prepared by the preparation method according to any one of claims 1-6 in the immobilization and regeneration of lipases, characterized in that, Includes the following steps: (1) Disperse Janus nanomagnetic particles in PBS, add lipase solution, and gently shake; An emulsion system of Janus nanomagnetic particles with hydrophobic caps anchoring lipase was obtained by field separation, washing, and freeze-drying. (2) Apply a pulsed magnetic field simultaneously to achieve particle sedimentation and recovery; (3) The recovered particles are flushed with mixed gas to remove product adsorption and restore the hydrophobicity of the particles.

8. The application according to claim 7, characterized in that, In step (1), the mass ratio of Janus nanomagnetic particles to lipase is 1:2-1:0.5; the concentration of the lipase solution is 5 mg / mL; the oscillation is gentle oscillation at 4℃ for 8 hours; and the field separation conditions are: 0.5 T, treatment for 5 min.

9. The application according to claim 7, characterized in that, In step (2), the flow rate of CO2 is 0.2 L / min; the conditions of the pulsed magnetic field are 0.5 T, frequency 1 Hz, and pulse width 0.5 s.

10. The application according to claim 7, characterized in that, In step (3), the mixed gas is composed of N2 and CO2 in a volume ratio of 4:1; the flow rate of the mixed gas is 0.3 L / min, and the flushing time is 10 min.

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