Method for preparing immobilized lipase Janus particles by emulsion template one-step method and application of immobilized lipase Janus particles in Pickering emulsion catalysis

Abstract

The invention discloses a method for preparing immobilized lipase Janus particles by an emulsion template one-step method based on a zymoprotein directional adsorption strategy and application of the immobilized lipase Janus particles in a Pickering emulsion catalytic system. The natural hydrophilicity of lipase protein is utilized to drive the lipase protein to form directional adsorption on the surfaces of hydrophobic particles, and the Janus particles with hydrophilic and hydrophobic characteristics are constructed. Firstly, an organic phase containing hydrophobic particles and a water phase containing lipase are oscillated to form an emulsion, so that the lipase in the water phase is accurately adsorbed on the single side of the surface of the particles, and directional connection of zymoprotein and a hydrophobic matrix is realized. Afterwards, the Janus particles are used for constructing a Pickering emulsion system, and a microreactor is built, so that the catalytic efficiency of lipase in hydrolysis, ester synthesis and transesterification reaction is improved. The method is easy to operate, efficient and stable enzyme immobilization can be achieved, the obtained immobilized lipase Janus particles are remarkably superior to free enzyme in biological catalytic activity and cyclic utilization performance, and the application value of lipase in industrial large-scale production is remarkably improved.

Description

Technical Field

[0001] The present invention relates to a technology for the directional adsorption of enzyme proteins and hydrophobic particles, belonging to the fields of biocatalytic materials and interfacial chemistry. Specifically, it includes utilizing the natural hydrophilicity of enzyme proteins to regulate their distribution on the surface of hydrophobic particles, preparing amphiphilic Janus particles by a one-step emulsion templating method, and applying them to interfacial catalysis, emulsion stabilization, and industrial biocatalytic processes. Background Art

[0002] Due to their extremely high catalytic activity and selectivity, lipases are widely used in fields such as chemical engineering, food, medicine, energy, and the environment. However, free lipases are not easily recovered and reused during use, and their stability is poor, which greatly limits their large-scale application. For this reason, various immobilization techniques are usually adopted at home and abroad to maximize the catalytic activity and operational stability of enzymes and improve their catalytic efficiency under harsh conditions.

[0003] In many enzymatic reactions, the substrate needs to be dissolved in the organic phase, while the enzyme itself is water-soluble. Especially for lipases, which are typical interfacial catalytic enzymes and need to carry out efficient catalysis at the oil-water interface. Increasing the oil-water interface area is the key method to improve the enzymatic efficiency. The conventional method is to add surfactants to form emulsions, thereby providing a larger reaction interface. However, the additional addition of surfactants will not only introduce new complex components, increasing the difficulty of product purification and enzyme recovery, but may also interfere with the reaction system.

[0004] To overcome the above disadvantages, Pickering emulsions stabilized by solid particles have received extensive attention. Similar to traditional surfactant-stabilized emulsions, they disperse one phase into micron-sized droplets in another immiscible liquid, greatly increasing the interface area, and at the same time having more advantages in terms of stability, product separation, and catalyst recovery. In the prior art, various particles have been used to construct Pickering emulsion catalytic systems. For example, in Patent CN202411369289.4, spherical lignin nanoparticles are used as emulsifiers to form O / W type Pickering emulsions at a stirring speed of 800 - 2000 rpm; in Patent CN202411415404.7, hydrophilic cations are mixed with shellac nanoparticles to prepare emulsifiers, and O / W type Pickering emulsions are prepared by high-speed shear homogenization.

[0005] Among the particles that can stabilize Pickering emulsions, Janus particles with "amphiphilic" characteristics have attracted much attention. Such particles are lipophilic on one side and hydrophilic on the other side, and can be more effectively anchored to the oil-water interface, showing outstanding advantages in stabilizing emulsions. However, the existing preparation methods of Janus particles (such as interface modification method, paraffin emulsion method, phase separation method, microfluidic method, etc.) are often cumbersome in steps and harsh in conditions, making it difficult to meet the requirements of large-scale industrial applications. For example, in Patent CN201711065032.X, amphiphilic SiO 2 nano-particles were prepared by the paraffin protection modification method, and then assembled into a single-wall stable Pickering emulsion at the oil-water interface, and the enzyme was dispersed in the aqueous phase of the emulsion to measure the stability and catalytic performance of the Pickering emulsion; in Patent 202411468168.5, a magnetic living Janus microgel sphere was obtained by injecting a mixture of acid silicone oil, Chlorella vulgaris and iron tetroxide into a microfluidic chip through the microfluidic method. However, these methods are limited in their promotion in industrial-scale applications either due to complex operations and high device requirements, or due to the easy loss of enzyme activity during multi-step chemical modifications. At the same time, most methods for immobilizing lipase require multi-step chemical modification or functionalization on the surface of the carrier, with complex operations and limited types of materials, and often fail to integrate "lipase" and "Janus particles" on the same carrier. Therefore, starting from the natural properties of lipase, the present invention proposes a simple, efficient and emulsion template method without complex chemical modification. Through the hydrophilicity of the enzyme protein, its precise directional adsorption on the surface of hydrophobic particles is realized, and "lipase Janus microparticles" with both hydrophilic and hydrophobic characteristics are prepared in one step, which can play the dual functions of stabilizing emulsions and catalysis at the oil-water interface.

[0006] Based on the emulsion template method, the present invention precisely regulates the distribution of enzyme protein on the surface of hydrophobic particles by using the hydrophilicity of the enzyme protein, so as to prepare Janus particles with both hydrophilic and hydrophobic characteristics. In addition, by adjusting the droplet size in the emulsion template, the adsorption distribution and coverage ratio of the enzyme protein on the particle surface can be further controlled, so as to flexibly adjust the hydrophilic / hydrophobic characteristics of the Janus particles to meet the requirements of forming W / O or O / W type emulsions. The Janus particles can significantly improve the stability of the constructed emulsion and maintain the high catalytic activity and stability of the enzyme protein. The lipase Janus microparticles prepared by the one-step emulsion template method can be used to construct a Pickering emulsion catalytic system. In the environment of a microreactor, by optimizing the particle characteristics and the Pickering emulsion preparation process, the efficiency of catalytic reactions such as lipid hydrolysis, ester synthesis and transesterification can be greatly improved. The present invention has broad application prospects in the fields of chemical industry and green energy development. Summary of the Invention

[0007] The present invention aims to provide a simple method for preparing Janus particles by directly utilizing the interaction between enzyme proteins and hydrophobic particles without the need for complex chemical modification of the carrier. Through the directional adsorption between the natural hydrophilic property of the enzyme protein and the surface of the hydrophobic particles, Janus particles with both hydrophilic and hydrophobic functions are prepared and widely applied in the stabilization of Pickering emulsions and biocatalytic systems, especially suitable for the catalytic reaction of lipase at the oil-water interface.

[0008] To achieve the above object, the present invention adopts the following technical solutions: (1) Preparation of enzyme protein solution: Lipases from different sources are selected and dispersed in an appropriate buffer solution (pH = 6 - 8, 0.05 mol / L) to prepare a lipase solution with a concentration of 0.1 - 10 mg / mL.

[0009] In one embodiment of the present invention, the lipase can be derived from animals, plants or microorganisms, or a combination of different lipases can be used to meet the requirements of enzyme activity and selectivity in different application scenarios.

[0010] (2) Preparation of amphiphilic Janus particles: Prepare a certain amount of organic phase system containing hydrophobic particles (such as n-heptane, n-hexane, decane, octane, toluene, etc.). The hydrophobic particles can be selected from silica, organic polymers, metal oxides or other surface-hydrophobic non-porous / porous materials, and the particle size range can be 50 - 1000 nm.

[0011] Add the lipase solution described in (1) to the organic phase containing hydrophobic particles in proportion, and perform vortex oscillation for 3 minutes (stirring or ultrasonic treatment can also be used) to form an emulsion. Subsequently, place the obtained emulsion in a shaker at 20 °C and 200 rpm and oscillate for 0.5 - 4 h.

[0012] After the oscillation is completed, quickly demulsify by centrifugation (5000 - 10000 g), recover the hydrophobic particles containing the bound enzyme protein, and wash with a suitable organic phase to remove the residual oil phase to obtain amphiphilic Janus particles. Finally, freeze-dry the amphiphilic Janus particles and store them in a 4 °C refrigerator.

[0013] In one embodiment of the present invention, the addition amount of hydrophobic particles in the organic phase system can be 10 - 100 mg / mL, and the volume fraction of the oil phase can account for 10 - 90% (v / v) of the total volume.

[0014] This operation can utilize the hydrophilic end of the enzyme protein to form directional adsorption with the hydrophobic region of the particles without additional chemical modification of the particles, thereby obtaining a Janus structure with both hydrophilic and hydrophobic characteristics on the particle surface.

[0015] (3) Construction of Pickering emulsion catalytic system: Disperse the immobilized lipase Janus particles obtained in step (2) in a selected organic solvent (such as n - hexane, decane, octane, toluene, etc.). The volume fraction of the organic phase can account for 10 - 90% (v / v) of the total volume. According to specific requirements, the organic substrate to be reacted can be directly added at this stage, or added after the emulsion is formed.

[0016] If there is a water - soluble substrate, it can be dissolved in the aqueous phase, then mixed with the above - mentioned organic phase, and through simple oscillation (3 min) or stirring operation, the system can quickly form a Pickering emulsion.

[0017] The addition amount of the immobilized lipase Janus particles can be adjusted to 10 - 100 mg / mL. By selecting particles of different sizes or properties (see step (2)), W / O or O / W type emulsions can be stably formed respectively, and sufficient enzyme molecules can be ensured to be exposed at the oil - water interface for efficient catalysis.

[0018] The temperature of the entire reaction system can be selected in the range of 20 - 70 °C, and the reaction time can be flexibly set to 0.5 - 48 h according to the specific substrate and target yield.

[0019] Specific application examples are as follows: Esterification reaction: One or more acids among acetic acid, hexanoic acid, octanoic acid, oleic acid, palmitic acid, stearic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid react with one or more alcohols among methanol, ethanol, hexanol, octanol, isopropanol, glycerol or polyols; Transesterification reaction: One or more esters among methyl hexanoate, ethyl octanoate, fatty acid methyl ester, fatty acid ethyl ester or triglyceride react with one or more alcohols among methanol, ethanol, glycerol, or react with one or more esters among methyl ester, ethyl ester or triglyceride; Hydrolysis reaction: One or more among triglyceride, monoglyceride, fatty acid ester, methyl ester, ethyl ester.

[0020] (4) Recovery of particles Demulsification and product separation: After the reaction is completed, quickly demulsify by centrifugation or other methods, separate the upper organic phase to obtain the required reaction product; when purifying or detecting the product, gas chromatography (GC) analysis method can be used.

[0021] Recycling of particles: The separated immobilized lipase Janus particles can be resuspended with fresh organic solvent and added with substrate to carry out the next round of catalysis; due to the firm and oriented adsorption of the enzyme protein on the surface of the hydrophobic carrier, the particles can still maintain good catalytic activity and stability after being recycled many times.

[0022] Advantages of the present invention: 1) Simple process and no need for chemical modification: By using the emulsion template one-step method, lipase is directly "physically" combined with the hydrophobic carrier without complex chemical modification of the carrier or construction of a special template. The process is green and environmentally friendly and is easy to scale up industrially.

[0023] 2) High-efficiency interfacial catalysis and stability: Through the interaction between the natural hydrophilicity of the enzyme protein and the hydrophobic particles, the obtained Janus particles can simultaneously perform the functions of "emulsification" and "catalysis", significantly improving the emulsion stability and catalytic efficiency, and enhancing the durability of the enzyme.

[0024] 3) Wide applicability: ● It can be flexibly adapted to substrates and reaction systems for various types of hydrophobic particles and lipases from different sources; ● By selecting different particles, adjusting the oil-water ratio of the emulsion and the preparation conditions, W / O or O / W Pickering emulsions can be obtained to meet the requirements of different catalytic reactions; · It has good effects on various reactions such as esterification, transesterification and hydrolysis, and has important application values especially in the chemical industry and green energy development.

[0025] 4) Integration of enzyme immobilization and construction of Janus particles: Compared with the traditional multi-step chemical modification or the method of independently treating the enzyme / carrier, in the present invention, lipase is directly immobilized on the surface of the hydrophobic carrier, making one side hydrophilic and the other side lipophilic, to obtain Janus microparticles with both surface activity and biocatalytic ability, which are convenient for recycling, reuse and maintaining high enzyme activity. Description of the drawings

[0026] Figure 1 : Effects of different oil phase volumes and addition amounts of hydrophobic SiO 2 nanoparticles on the enzyme loading and immobilization yield of immobilized lipase Janus microparticles.

[0027] Figure 2 : Effects of different oil phase volumes and addition amounts of hydrophobic SiO 2 nanoparticles on the enzyme activity and specific activity of immobilized lipase Janus microparticles.

[0028] Figure 3 : Construction of W / O and O / W Pickering emulsion catalytic systems based on different immobilized lipase microparticles.

[0029] Figure 4 : Effects of the catalytic esterification reaction efficiency of the W / O Pickering emulsion constructed by immobilized lipase Janus microparticles.

[0030] Figure 5 : Reaction efficiency of transesterification catalyzed by W / O Pickering emulsion constructed with immobilized lipase Janus particles.

[0031] Figure 6 : Reaction efficiency of ester hydrolysis catalyzed by W / O Pickering emulsion constructed with immobilized lipase Janus particles. Specific embodiments

[0032] In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, the following examples are only examples of the present invention and do not represent the scope of the claimed rights of the present invention. The scope of the claimed rights of the present invention shall be subject to the claims.

[0033] Candida antarctica lipase B (CalB) used in the following examples was purchased from Novozymes (China) Biotechnology Co., Ltd., and the enzyme activity was 5000 LU / g; the alkylsilylated modified SiO 2 nanoparticles (i.e., hydrophobic SiO 2 nanoparticles, ML-SiO 2 -WN500) were purchased from Zhejiang Manli Nano Technology Co., Ltd., and the particle size was 500 nm.

[0034] Example 1 Preparation of immobilized lipase Janus particles based on an emulsion template with 60% oil phase volume: Take a certain amount of the original CalB lipase solution and add it to a phosphate buffer solution (pH = 7, 0.05 mol / L) to dilute it into a lipase solution with a set concentration. Subsequently, take a certain amount of hydrophobic SiO 2 nanoparticles and add them to n-heptane to prepare a n-heptane solution containing hydrophobic SiO 2 nanoparticles. Take 4.8 mL of the above lipase solution and add it to 7.2 mL of the n-heptane solution containing hydrophobic SiO 2 nanoparticles, and vortex oscillate at room temperature for 3 min for emulsification to form a W / O emulsion with a total volume of 12 mL.

[0035] Place the formed W / O emulsion in a shaker at 200 rpm and 20 °C and oscillate for 0.5 h. After the oscillation is completed, demulsify by centrifugation (12000 g, 10 min), recover the aqueous phase and the precipitate respectively, wash the precipitate twice with n-heptane, and freeze-dry to obtain immobilized lipase Janus particles.

[0036] Among them, in the n - heptane solution containing hydrophobic SiO 2 nanoparticles, the addition amount of the hydrophobic SiO 2 nanoparticles is 10 - 50 mg / mL, and the concentration of the lipase solution is 0.25 - 1 mg / mL.

[0037] Example 2 Preparation of immobilized lipase Janus particles based on an emulsion template with 50% oil - phase volume: Take a certain amount of the original CalB lipase solution and add it to a phosphate - buffered solution (pH = 7, 0.05 mol / L) to dilute it into a lipase solution with a set concentration. Subsequently, take a certain amount of hydrophobic SiO 2 nanoparticles and add them to n - heptane to prepare an n - heptane solution containing hydrophobic SiO 2 nanoparticles. Take 6 mL of the above - mentioned lipase solution and add it to 6 mL of the n - heptane solution containing hydrophobic SiO 2 nanoparticles, and vortex - oscillate at room temperature for 3 min for emulsification to form a W / O - type emulsion with a total volume of 12 mL.

[0038] Place the formed W / O - type emulsion in a shaker at 200 rpm and 20 °C and oscillate for 0.5 h. After the oscillation ends, demulsify by centrifugation (12000 g, 10 min), recover the aqueous phase and the precipitate respectively, wash the precipitate twice with n - heptane, and freeze - dry to obtain the immobilized lipase Janus particles.

[0039] Among them, in the n - heptane solution containing hydrophobic SiO 2 nanoparticles, the addition amount of the hydrophobic SiO 2 nanoparticles is 10 - 50 mg / mL, and the concentration of the lipase solution is 0.25 - 1 mg / mL.

[0040] Example 3 Determination of the enzyme - protein loading amount of immobilized lipase Janus particles: Use the BCA (Bicinchoninic Acid) method to measure the initial protein concentration of the free enzyme and the remaining protein content recovered from the supernatant at different immobilization time periods. The specific operation is as follows: Take 20 μL of the protein solution and add it to 200 μL of the BCA working solution, oscillate in an enzyme - label instrument for 30 s, then incubate in an environment at 37 °C for 30 min, and then measure the absorbance value at 562 nm. Each sample is set with three parallels. According to formula (1) and formula (2), calculate the protein loading amount (enzyme - loading amount) per gram of the carrier and the protein immobilization yield.

[0041] In the formula: q e : enzyme loading amount (mg / g); C 0 —protein concentration of the initial lipase solution (mg / mL); C t : protein concentration in the lipase solution at time t (mg / mL); m: weight of the empty carrier (wet) (g); V: total volume of the lipase solution (mL); V 0 : initial volume of the protein solution (mL); η protein (%) : immobilization yield (%).

[0042] The aqueous phase recovered after preparing the immobilized lipase Janus particles in Example 1 was measured for protein content using the above method, and the protein loading amount (enzyme loading amount) per gram of carrier and the protein immobilization yield were calculated. The results are as Figure 1 shown. Among them, H, M, and L respectively represent that the lipase solution concentrations are 1.0, 0.5, and 0.25 mg / mL.

[0043] It can be seen from Figure 1 that when the addition amount of hydrophobic SiO 2 nanoparticles increases from 10 mg / mL to 50 mg / mL, the enzyme loading amount of the immobilized lipase Janus particles decreases significantly. This is because after the particles increase, the emulsion droplets decrease, the emulsion interface area increases, and the particles at the interface increase, resulting in a decrease in the amount of enzyme adsorbed by each particle, leading to a decrease in the enzyme loading amount; however, with the increase of the particles, the total amount of enzyme adsorbed increases, and the immobilization yield increases. Continuing to increase the particles, the immobilization yield no longer increases, indicating that the addition amount of the particles reaches saturation.

[0044] Example 4 Determination of the enzyme activity of the immobilized lipase Janus particles: The enzyme activity was determined by the high-throughput phenol method. According to the keto-enol tautomerism between vinyl acetate and n-butanol catalyzed by lipase, the transesterification reaction that releases acetaldehyde and vinyl alcohol in proportion was used as the model reaction, and the released acetaldehyde was measured in an enzyme-linked immunosorbent assay (ELISA) by a colorimetric method derived from 3-methyl-2-benzothiazolinone (MBTH).

[0045] The transesterification reaction is as follows: 100 mmol / L vinyl acetate and 100 mmol / L n-butanol were dispersed in n-hexane (the total reaction volume was 1 mL), the enzyme addition amount was 5 mg, and the reaction was shaken at 30 °C and 1000 rpm for 5 min in a constant temperature shaking incubator. In the blank group experiment, no enzyme was added, and other conditions were the same as those in the experimental group. After the reaction, the supernatant was diluted with ultrapure water by a certain multiple as the sample to be measured, and the concentration of the diluted sample was 0.001 - 0.15 mmol / L.

[0046] Determination of acetaldehyde content by phenol reagent method: Acetaldehyde reacts with MBTH to form a hydrazine substance, which is oxidized by ferric ions under acidic conditions to form a blue-green compound (TAPMC). The content of acetaldehyde in the sample to be measured is determined by colorimetry according to the depth of color. The specific operation method is as follows: Add 100 μL of the sample to be measured and 100 μL of 0.1% (w / w) MBTH into a 96-well plate, mix well (shake and mix for 15 seconds with a microplate reader), and incubate at 30 °C in a molecular incubator for 10 min. Then immediately add 40 μL of 1% ammonium ferric sulfate, mix well and incubate at 30 °C in a molecular incubator for 30 min. After the reaction is completed, measure the absorbance value of the sample at 598 nm.

[0047] Preparation of the standard curve for the phenol reagent method: According to Table 1, using distilled water as the solvent, prepare acetaldehyde standard solutions with concentrations ranging from 0.001 to 0.15 mmol / L. After MBTH derivatization, measure the absorbance values at different concentrations at 598 nm. In the experiment, the acetaldehyde concentration is used as the abscissa, and the absorbance value at 598 nm is used as the ordinate to plot the detection standard curve. The equation of the standard curve obtained is y = 11.654x + 0.0973.

[0048] Definition of enzyme activity unit: The amount of enzyme required for 1 g of immobilized lipase Janus particles to catalyze the transesterification reaction of vinyl acetate and n-butanol to produce 1 μmol of acetaldehyde within 1 min is defined as one enzyme activity unit (U).

[0049] Definition of the specific activity of the immobilized enzyme: The enzyme activity possessed by the protein loading amount per unit mass of immobilized lipase Janus particles, expressed as (U / mg).

[0050] Table 1 Preparation of acetaldehyde standard solutions The enzyme activities of the immobilized lipase Janus particles prepared in Example 1 and Example 2 were measured respectively, and the results are as Figure 2 shown. Figure 2 In it, A and C respectively represent the transesterification enzyme activities of the immobilized lipase Janus particles prepared by shaking under the conditions of 60% and 50% oil phase volumes. Among them, H, M, and L respectively represent the lipase solution concentrations of 1.0, 0.5, and 0.25 mg / mL. The results show that under the condition of adding a high enzyme concentration (H), the enzyme activity of the particles is higher than that at medium and low enzyme concentrations. Among them, when the addition amount of hydrophobic SiO 2 particles is 10 mg / mL, the enzyme activities of the prepared immobilized lipase Janus particles reach the highest, which are 147437.5 U / g (60% oil phase) and 77870.34 U / g (50% oil phase) respectively. Figure 2 In it, B and D respectively represent the transesterification specific activities of the immobilized lipase Janus particles. With the increase of hydrophobic SiO2 With the increase in the addition amount of nanoparticles, the enzyme loading gradually decreases, resulting in a decrease in both enzyme activity and specific activity. In addition, the specific activity of the immobilized lipase Janus particles prepared under the condition of 60% oil phase volume is higher than that of the immobilized lipase Janus particles under the condition of 50% oil phase volume. Among them, under the condition of low-hydrophobic SiO 2 nanoparticle addition amount (60% oil phase volume), the specific activity of the obtained immobilized lipase Janus particles is the highest, reaching 16294.59 U / mg.

[0051] Example 5 Preparation of immobilized lipase particles based on pure aqueous phase: Take a certain amount of the original CalB lipase solution and add it to a phosphate buffer solution (pH = 7, 0.05 mol / L) to dilute it into a lipase solution with a set concentration. Subsequently, take a certain amount of hydrophobic SiO 2 nanoparticles and wet them with 0.2 mL of 90% ethanol to obtain a SiO 2 nanoparticle dispersion. Take 4.8 mL of the above lipase solution and mix it with 0.2 mL of the SiO 2 nanoparticle dispersion, and place it in a shaker at 200 rpm and 20 °C for 0.5 h. After the oscillation, recover the precipitate by centrifugation (12000 g, 10 min), wash the precipitate twice with deionized water, and freeze-dry it to obtain the immobilized lipase particles prepared by adsorption based on the pure aqueous phase.

[0052] Among them, the addition amount of the hydrophobic SiO 2 nanoparticles in the final system is 10 - 100 mg / mL, and the concentration of the lipase solution is 0.25 - 1 mg / mL.

[0053] Example 6 Construct a W / O or O / W type Pickering emulsion catalytic system based on the immobilized lipase Janus particles prepared differently: First, prepare the Nile red staining solution. Dissolve 10 mg of Nile red solid particles in 10 mL of isopropanone, and then add it to n - heptane at a volume ratio of 1:10 to obtain the stained n - heptane solution. Then, disperse the immobilized lipase Janus particles prepared in Example 1 in a certain amount of the stained n - heptane. After ultrasonic treatment for 10 min, mix it with pure water as the aqueous phase at different volume ratios to form a total system of 1 mL. Disperse the immobilized lipase particles prepared by the pure - water - phase adsorption method in Example 5 in pure water. After ultrasonic treatment for 10 min, mix it with n - heptane as the oil phase at different volume ratios to form a total system of 1 mL. Oscillate at room temperature for 3 min to obtain Pickering emulsions. Take 20 μL of the Pickering emulsion, disperse it in 100 μL of n - heptane, and then observe the emulsion state using a fluorescence microscope.

[0054] Figure 3 Figure 4 shows the fluorescence images of Pickering emulsions formed by different immobilized lipase particles. Since Nile red is used to stain n - heptane, the oil phase appears red and the aqueous phase appears black. Figure 3 Figure A shows the Pickering emulsion stabilized by the immobilized lipase Janus particles prepared in Example 1. It can be seen from the figure that the continuous phase appears orange - red, which is the n - heptane solution. Therefore, the type of emulsion formed by these particles is the W / O - type Pickering emulsion. Figure 3 Figure B shows the Pickering emulsion stabilized by the immobilized lipase particles prepared by the pure - water - phase adsorption method in Example 5. It can be observed from the figure that the dispersed phase appears orange - red. Then, the type of emulsion formed by these particles is the O / W - type Pickering emulsion.

[0055] Example 7 Catalytic efficiency of the esterification reaction based on the constructed W / O - type Pickering emulsion catalytic system: Disperse the immobilized lipase Janus particles prepared in Example 1 in n - heptane containing 0.8 mol / L octanoic acid as the oil phase, and mix it with pure water containing 2.0 mol / L methanol at different volume ratios to form a total system of 1 mL. After obtaining the W / O - type Pickering emulsion by oscillating at room temperature for 3 min, carry out the esterification reaction at 30 °C for 7 - 180 min. After the reaction is completed, centrifuge at 12000 g for 3 min to quickly demulsify, take the upper organic phase, and determine the product by gas chromatography. Among them, the addition amount of the immobilized lipase Janus particles in the oil phase of the reaction system is 30 mg / mL.

[0056] After the esterification reaction, the upper organic phase was taken, filtered through an organic microporous membrane, placed in a 2 mL chromatographic injection vial, and detected using an Agilent 7890A gas chromatograph. The chromatographic conditions were as follows: HP-INNOWax capillary column (30.0 m × 0.25 mm × 0.25 μm, polyethylene glycol); flame ionization detector (FID); the inlet and detector temperatures were both 250 °C. Temperature programming: hold at 55 °C for 1 min, increase to 210 °C at a rate of 10 °C / min, hold for 10 min, then increase to 250 °C at a rate of 20 °C / min, and hold for 1 min. The carrier gas was pure nitrogen, with a pressure of 0.14 MPa; the hydrogen flow rate was 30 mL / min, the air flow rate was 350 mL / min, and the make-up gas nitrogen flow rate was 42 mL / min; split injection was used, with a split ratio of 50:1 and an injection volume of 1 μL. External standard method was used for quantitative analysis, and the conversion rate was calculated according to the following formula: Conversion rate (%) = (W 2 / W 1 ) × 100 Where: W 1 : the amount of substrate in the system before the catalytic reaction; W 2 : the amount of product in the system after the catalytic reaction.

[0057] A Pickering emulsion catalytic system was constructed at 30% oil phase volume. As Figure 4 shown, as the reaction time increased, the conversion rate of methyl octanoate gradually increased. After 60 min, the reaction rate decreased significantly, and the conversion rate reached 90.44% after 2 h.

[0058] Example 8 Catalytic efficiency of the transesterification reaction based on the constructed W / O Pickering emulsion catalytic system: The immobilized lipase Janus particles prepared in Example 1 were dispersed in a certain amount of n-heptane. After ultrasonic treatment for 10 min, a quantitative amount of octyl octanoate (final concentration 0.8 mol / L) was added as the oil phase, and it was mixed with pure water containing 2.0 mol / L methanol in different volume ratios to form a total system of 1 mL. After preparing the W / O Pickering emulsion by shaking at room temperature for 3 min, the transesterification reaction was carried out at 40 °C for a reaction time of 7 - 180 min. After the reaction ended, it was centrifuged at 12000 g for 3 min to quickly demulsify. The upper organic phase was taken and the content of methyl octanoate was determined by gas chromatography. The addition amount of the immobilized lipase Janus particles in the oil phase of the reaction system was 30 mg / mL.

[0059] After the transesterification reaction, the upper organic phase was taken, filtered through an organic microporous membrane, placed in a 2 mL chromatographic injection vial, and detected using an Agilent 7890A gas chromatograph. The chromatographic conditions were the same as in Example 7.

[0060] Construct a Pickering emulsion catalytic system at 30% oil phase volume, as Figure 5 shown, as the reaction time increases, the conversion rate of methyl octanoate also increases. After the reaction reaches 30 min, the reaction rate significantly decreases, and the conversion rate reaches 22.52% after 1 h.

[0061] Example 9 Catalytic efficiency of the ester hydrolysis reaction based on the constructed W / O Pickering emulsion catalytic system: Disperse the immobilized lipase Janus particles prepared in Example 1 in a certain amount of n-heptane. After ultrasonic treatment for 10 min, add a quantitative amount of methyl octanoate (final concentration 0.8 mol / L) as the oil phase, and mix it with pure water containing 2.0 mol / L methanol in different volume ratios to form a total system of 1 mL. After preparing the W / O Pickering emulsion by shaking at room temperature for 3 min, carry out the hydrolysis reaction at 40 °C for a reaction time of 7 - 180 min. After the reaction, centrifuge at 12000 g for 3 min to quickly demulsify, take the upper organic phase, and determine the content of octanoic acid by gas chromatography. The addition amount of the immobilized lipase Janus particles in the oil phase of the reaction system is 30 mg / mL.

[0062] After the hydrolysis reaction, take the upper organic phase, filter it through an organic microporous membrane and place it in a 2 mL chromatographic injection vial, and detect it using an Agilent 7890A gas chromatograph. The chromatographic conditions are the same as in Example 7.

[0063] Construct a system for the Pickering emulsion catalytic hydrolysis reaction at 30% oil phase volume, as Figure 6 shown, as the reaction time increases, the content of octanoic acid generated by hydrolysis gradually increases. After the reaction reaches 30 min, the reaction rate significantly decreases, and the conversion rate reaches 67.79% after 1 h.

[0064] The above are only some embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. A method for preparing immobilized lipase Janus microparticles using an emulsion template in one step, characterized in that: The following steps are involved: (1) dissolving lipase in a buffer solution to obtain a lipase solution; (2) dispersing the hydrophobic micro / nano particles in an organic solvent to obtain an organic phase dispersion system containing the hydrophobic micro / nano particles; (3) mixing the lipase solution obtained in step (1) with the organic phase dispersion system obtained in step (2) and emulsifying them to form an emulsion; (4) The emulsion obtained in step (3) is shaken at a preset temperature and speed, and then demulsified by centrifugation, and the precipitate is collected, washed and freeze-dried to obtain immobilized lipase Janus microparticles.

2. The method according to claim 1, characterized in that: The source of the lipase is selected from one or more of microorganisms, plants, and animals; the buffer solution is a phosphate buffer solution or other suitable buffer solution, with a pH value of 6-8 and a concentration of 0.01-0.1 mol / L; the concentration of the lipase solution is 0.1-10 mg / mL.

3. The method according to claim 1, characterized in that: The hydrophobic micro / nanoparticles are selected from one or more of silica particles, organic polymer particles or metal oxide particles, and have a particle size of 50-1000 nm; the organic solvent is selected from one or more of n-heptane, n-hexane, decane, octane and toluene; and the content of the hydrophobic micro / nanoparticles in the organic phase dispersion system containing the hydrophobic micro / nanoparticles is 10-100 mg / mL.

4. The method according to claim 1, characterized in that: The volume ratio of the lipase solution to the organic phase dispersion system containing hydrophobic micro / nano particles is 1:9-9:

1.

5. The method according to claim 1, characterized in that: The emulsification method is selected from one or more of vortex oscillation, stirring or ultrasonic treatment.

6. The method according to claim 1, characterized in that: The oscillation time is 0.5-4h, the temperature is 10-30°C, and the rotation speed is 50-300rpm.

7. The method according to claim 1, characterized in that: The centrifugal speed is 5000-13000×g, preferably 8000-10000×g, and the centrifugal time is 1-10 min.

8. An immobilized lipase Janus particle, characterized in that: The method is prepared by the method according to any one of claims 1 to 7.

9. Use of the immobilized lipase Janus microparticles according to claim 8 in esterification reaction, transesterification reaction or hydrolysis reaction.

10. The use according to claim 9, characterized in that: The esterification reaction is a reaction of one or more acids selected from acetic acid, caproic acid, caprylic acid, oleic acid, palmitic acid, stearic acid, eicosapentaenoic acid, docosahexaenoic acid, and linoleic acid with one or more alcohols selected from methanol, ethanol, hexanol, octanol, isopropanol, glycerol, or polyols; the transesterification reaction is a reaction of one or more esters selected from methyl caproate, ethyl caprylate, fatty acid methyl ester, fatty acid ethyl ester, or triglycerides with one or more alcohols selected from methanol, ethanol, and glycerol, or with one or more esters selected from methyl ester, ethyl ester, or triglycerides; the substrate of the hydrolysis reaction is one or more selected from triglycerides, monoglycerides, fatty acid esters, methyl esters, and ethyl esters.

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