A method for preparing immobilized lipase and its application in pickering interfacial catalysis

Abstract

The application discloses a kind of immobilized lipase and its preparation method and application in Pickering interface catalysis.The preparation method uses one of genipin, glutaraldehyde or aqueous solution of vanillin as crosslinking agent, to prepare emulsion with shellac nanoparticles and chitosan respectively, then the emulsion droplet containing crosslinking agent and crosslinking material is fused, to realize the immobilization of lipase and the hydrophobic modification of enzyme-loaded particles.The enzyme activity of the immobilized lipase is 10.53±0.48U / mg, higher than 3.71±0.50U / mg of empty particles+lipase and 0.92±0.02U / mg of free lipase.After 10 cycles, the enzyme activity of the immobilized lipase can still remain 86.52±3.09%, indicating that it has good reusability.The application provides a feasible way for using natural green materials to construct more secure immobilized lipase, which helps the application of immobilized lipase in food and daily necessities industry.

Description

Technical Field

[0001] This invention belongs to the field of immobilized lipase preparation technology, specifically relating to an immobilized lipase, its preparation method, and its application in Pickering interface catalysis. Background Technology

[0002] Lipase (EC 3.1.1.3) possesses specific catalytic activity for ester hydrolysis, ester synthesis, and transesterification reactions. Its reaction conditions are mild, the process is simple, and the product purity is high, making it widely used in food, chemical, pharmaceutical, and bioenergy industries. However, using free lipases in processing has many limitations, such as low enzyme stability and difficulty in recovery after catalysis. Furthermore, because lipid substrates are insoluble in water, lipases can only catalyze reactions at the oil-water interface, limiting catalytic efficiency to interfacial area and mass transfer. Immobilized lipase technology can overcome these shortcomings and offer additional superior properties such as magnetic response, pH response, and light response. In discontinuous reaction systems, immobilized lipases can be easily separated from solution with minimal loss of catalytic activity, resulting in satisfactory reusability.

[0003] Immobilized lipase carrier materials mainly include various inorganic and organic porous particles, hollow microspheres, and Janus particles, such as SiO2, TiO2, calcium alginate, cellulose, and modified starch. These particles, after hydrophobic modification, can be used to construct Pickering emulsions, providing a stable emulsion reaction system for the substrate and lipase. Because these particles avoid the use of emulsifiers, reducing lipase activity loss and the adverse effects of emulsifiers, they are widely used in the chemical industry. Studies have shown that these particles themselves or their modifiers possess a certain degree of toxicity, which limits their application in the food and consumer goods industries. Immobilized lipase technology often requires the use of cross-linking agents (such as glutaraldehyde, hexamethylenediamine, and diazoxide benzidine), which can lead to reduced enzyme activity and introduce certain toxicity.

[0004] Natural and green materials are widely available and abundant, and possess high biocompatibility. Immobilized lipases constructed using these materials offer advantages such as low cost, high safety, and biodegradability. Current research has successfully constructed immobilized lipase particles using materials such as chitosan and cellulose, and applied them to the synthesis of structured lipids in the food industry, thereby improving the nutritional value of lipids. Summary of the Invention

[0005] To overcome the shortcomings and drawbacks of existing technologies, the primary objective of this invention is to provide a method for preparing immobilized lipase. This method uses genipin aqueous solution, glutaraldehyde aqueous solution, or vanillin aqueous solution as a crosslinking agent, prepares emulsions using shellac nanoparticles (SNPs) and chitosan respectively, and then fuses the emulsion droplets containing the crosslinking agent and the crosslinked substance to achieve lipase immobilization and hydrophobic modification of the enzyme-carrying particles. The enzyme-carrying particles are characterized by an outer layer covered by shellac nanoparticles and an inner layer of chitosan microgel crosslinked with the lipase.

[0006] The second objective of this invention is to provide an immobilized lipase. Experiments revealed that the immobilized lipase (SNPs-CS@Lipase) prepared according to this invention has an enzyme activity of 10.53 ± 0.48 U / mg, which is higher than that of empty particles + lipase (3.71 ± 0.50 U / mg) and free lipase (0.92 ± 0.02 U / mg). After 10 cycles of use, the enzyme activity of SNPs-CS@Lipase still retained 86.52 ± 3.09%, indicating good reusability.

[0007] A third objective of this invention is to provide an application of immobilized lipase in Pickering interface catalysis. This invention offers a feasible approach to constructing safer immobilized lipases using natural, green materials, which will facilitate the application of immobilized lipases in the food and consumer goods industries.

[0008] The primary objective of this invention is achieved through the following technical solution:

[0009] A method for preparing immobilized lipase includes the following steps:

[0010] (1) Mix shellac nanoparticle aqueous dispersion with crosslinking agent solution to prepare mixture I, then add mixture I to liquid paraffin containing Span 80, and homogenize by high-speed shearing to obtain unstable emulsion A containing crosslinking agent;

[0011] (2) Mix the Candida Sp. lipase solution with the acidic chitosan solution to prepare mixture II. Add mixture II to liquid paraffin containing Span 80 and homogenize by high-speed shearing to obtain unstable emulsion B containing cross-linked substances.

[0012] (3) Mix emulsion A obtained in step (1) with emulsion B obtained in step (2) and incubate to perform droplet fusion cross-linking and dehydration shrinkage to obtain immobilized lipase (SNPs-CS@Lipase).

[0013] Preferably, the volume ratio of the shellac nanoparticle dispersion and the genipin aqueous solution in step (1) is (4-6):1; the mass concentration of the shellac nanoparticle dispersion is 0-2 wt.%, wherein the diameter of the shellac nanoparticles is 100-200 nm; the crosslinking agent is one of the following: a genipin aqueous solution with a mass concentration of 5-15 mg / mL, a glutaraldehyde aqueous solution with a volume concentration of 2-10% v / v, or a vanillin aqueous solution with a mass concentration of 1-8 mg / mL.

[0014] Preferably, the specific preparation method of the shellac nanoparticle dispersion in step (1) is as follows:

[0015] (a) Dissolve 1.2 g of shellac in 80 mL of anhydrous ethanol to obtain a 15 mg / mL shellac ethanol solution. Pass the shellac ethanol solution through a 0.22 μm filter to remove a small amount of insoluble matter.

[0016] (b) Using a stainless steel needle with a diameter of 0.43 mm, drop the filtered shellac ethanol solution into 100 mL of distilled water at a rate of 0.45 mL / min. To ensure that almost all of the shellac is converted into SNPs, stir the distilled water with a magnetic stirrer at a speed of 600 rpm until the shellac ethanol solution is completely dropped.

[0017] (c) Use filter paper to filter out a small amount of shellac aggregates that have not formed nanoparticles to obtain the original SNPs dispersion, and use a rotary evaporator to remove all ethanol from the original dispersion. Finally, add distilled water to the evaporated dispersion to adjust the total volume of the dispersion to 100 mL.

[0018] Preferably, the concentration of the liquid paraffin containing Span 80 in steps (1) and (2) is 0.04-0.08 g / mL, the ratio of the aqueous phase to the liquid paraffin is 1:(1-3), and the high-speed shear homogenization conditions are a rotation speed of 10000-14000 rpm and a time of 1-3 min.

[0019] Preferably, the volume ratio of the Candida lipase solution to the acidic chitosan solution in step (2) is 1:(1-3), wherein the lipase concentration of the lipase solution is 0.05-0.2 g / mL, the lipase solution is pre-diluted 4-6 times with a phosphate buffer solution with a phosphate concentration of 5-15 mM and a pH of 7.0-8.0, the chitosan concentration in the acidic chitosan solution is 1-3 wt.%, and the acetic acid volume concentration is 0.4-0.8%.

[0020] Preferably, the volume ratio of emulsion A and emulsion B in step (3) is (0.8-1.2):1; the conditions for fusion crosslinking and dehydration shrinkage are: open constant temperature water bath stirring, temperature is 30-45℃, rotation speed is 500-1500rpm, and time is 40-60h.

[0021] The second objective of this invention is achieved through the following technical solution:

[0022] An immobilized lipase prepared by the above method.

[0023] The third objective of this invention is achieved through the following technical solution:

[0024] An application of an immobilized lipase in Pickering interface catalysis, the application specifically comprising: mixing an acid-alcohol solution with deionized water, adding the immobilized lipase, and stirring at a constant temperature to carry out the reaction.

[0025] Preferably, the acid-alcohol solution is a hexanoic acid and hexanol solution in n-hexane, and the concentration of hexanoic acid and hexanol in the n-hexane solution is 0.3-0.5 mol / L; the ratio of the acid-alcohol solution to deionized water is 1-2:1; and the mass concentration of the immobilized lipase in the reaction system is 20-50 mg / mL.

[0026] Preferably, the constant temperature stirring conditions are: temperature 35-45℃, rotation speed 300rpm, and reaction time 10-14h.

[0027] The technical solution of the present invention has the following advantages compared with the prior art:

[0028] (1) The immobilized lipase described in this invention is constructed entirely using natural and green materials, avoiding the toxicity caused by immobilized materials and the potential safety hazards in its application in the food and daily necessities industries.

[0029] (2) In the prepared immobilized lipase, the enzyme and the chitosan microgel are connected by covalent bonds, and the microgel is covered by shellac nanoparticles, which effectively improves the reusability of the lipase and reduces the loss of lipase catalytic activity.

[0030] (3) The hydrophilicity and hydrophobicity of the immobilized lipase particles described in this invention can be adjusted by the amount of shellac nanoparticles added. The adjusted particles can form a stable W / O type Pickering emulsion, which increases the contact area between the enzyme and the substrate and improves the catalytic activity of the lipase. Attached Figure Description

[0031] Figure 1 (a) Figure 1 (b) Figure 1 (c) and Figure 1 (d) The results of particle size, particle size distribution, PDI and potential of SNPs in Example 1 are respectively;

[0032] Figure 2 The infrared spectrum of the SNPs in Example 1;

[0033] Figure 3 The image shows a transmission electron microscope (TEM) image of the SNPs in Example 1.

[0034] Figure 4 The infrared spectra of genipin and chitosan before and after the cross-linking reaction in Example 2 are shown.

[0035] Figure 5 The changes in moisture content, moisture distribution, and appearance of SNPs-CS@Lipase during cultivation in Example 2;

[0036] Figure 6 The contact angle of particles at the hexane-water interface after hydrophobic modification of SNPs-CS@Lipase using different concentrations of SNPs in Example 3.

[0037] Figure 7 The results of laser confocal observation after marking SNPs-CS@Lipase and the microreactor formed therefrom with fluorescein isothiocyanate in Example 4;

[0038] Figure 8 This is a simplified diagram illustrating the structure and catalytic mechanism of the Pickering microreactor constructed from SNPs-CS@Lipase in Example 4;

[0039] Figure 9 The conversion rate of lipases from different sources in Example 4 after immobilization for 15 min catalyzing the esterification of hexanoic acid and hexanol;

[0040] Figure 10 The reaction kinetic curves for the esterification of hexanoic acid and hexanol catalyzed by SNPs-CS@Lipase, empty particles + lipase, and free lipase in Example 4 are shown.

[0041] Figure 11 The conditional enzyme activity was determined in Example 4 based on the conversion rate during the first 15 minutes of the reaction kinetic curve.

[0042] Figure 12 The results show the reusability of the SNPs-CS@Lipase-catalyzed hexanoic acid and hexanol esterification in Example 4. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. All materials used in the examples of the present invention are commercially available.

[0044] Example 1

[0045] SNPs were prepared by anti-solvent precipitation and their physicochemical properties were characterized.

[0046] 1. Particle size, particle size distribution, PDI, and potential of SNPs

[0047] Figure 1 (a) Figure 1 (b) Figure 1 (c) and Figure 1 (d) The particle size, particle size distribution, PDI, and potential results of the SNPs are shown in Figure 1. SNPs were prepared by reverse-phase solvent precipitation, taking advantage of the difference in solubility of shellac in ethanol and water. The average particle size of the obtained SNPs was 143.56 ± 2.48 nm, exhibiting good uniformity. The PDI value of the SNPs was 0.79 ± 0.07, indicating minimal aggregation between nanoparticles and uniform dispersion in water. Due to the influence of residual groups on the nanoparticle surface from shellac molecules, the surface potential of the SNPs was -29.54 ± 0.79. This is because some cyclic terpene acids in the shellac molecules did not form ester bonds with their -COOH groups, and under hydrophilic interaction, they tended to distribute on the surface of the SNPs. After ionization in water, they generated -COO- groups, thus giving the SNPs a negative potential. The electrostatic repulsion between the SNPs helps resist particle aggregation, thereby improving the stability of the SNPs in the dispersion.

[0048] 2. Surface groups of SNPs

[0049] Figure 2 The image shows the infrared spectrum of the SNPs. Among them, 940 cm⁻¹... -1 1716cm -1 and 2929cm -1 The presence of the carboxyl group was confirmed by the corresponding OH bending vibration, C=O stretching vibration, and OH stretching vibration. (3450cm) -1 The broad peak at 1251 cm⁻¹ corresponds to the OH stretching vibration of the hydroxyl group, confirming the presence of the hydroxyl group. -1 and 1716cm -1 The presence of carboxylic acid esters was confirmed by the COC stretching vibration and C=O stretching vibration corresponding to the ester, respectively. The C=C stretching vibration at 1636 cm⁻¹ confirmed the presence of alkenes without trans substituents.

[0050] 3. Microscopic morphology of SNPs

[0051] Figure 3 These are transmission electron microscopy (TEM) images of the SNPs. The SNPs are observed to be spherical particles with good uniformity, consistent with particle size analysis results. All particles have smooth surfaces and show no obvious aggregation.

[0052] Example 2

[0053] A method for preparing SNPs-CS@Lipase and its property characterization, the preparation method comprising:

[0054] (1) Take 4 mL of the SNPs dispersion prepared in Example 1 and mix it with 1 mL of deionized water and 1 mL of genipin aqueous solution (10 mg / mL). Then add 12 mL of Span 80 liquid paraffin solution (0.06 g / mL) to the mixture. Homogenize the mixture at 12,000 rpm for 2 min using a high-speed shear press to form an unstable W / O type emulsion A.

[0055] (2) Mix 0.4 mL of Candida lipase solution with 1.6 mL of acidic chitosan solution (containing 2 wt.% chitosan and 0.6% acetic acid), add 12 mL of Span 80 liquid paraffin solution (0.06 g / mL), and homogenize by high-speed shearing (12000 rpm, 2 min) to obtain unstable W / O type emulsion B.

[0056] (3) Mix emulsion A obtained in step (1) with emulsion B obtained in step (2), first homogenize by high-speed shearing (12000 rpm, 2 min), and then incubate by stirring in a water bath (40℃, 500 rpm, 48 h) to obtain SNPs-CS@Lipase.

[0057] Performance testing

[0058] 1. Changes in functional groups before and after cross-linking chitosan with genipin

[0059] Figure 4 The images show the infrared spectra of genipin and chitosan before and after the cross-linking reaction. The image shows the 1074 cm⁻¹... -1 2847cm -1 and 2922cm -1 The absorption peak intensity decreases at 1623 cm⁻¹ -1 and 1681cm -1 The absorption peak intensity increases at this point. This change is related to the cross-linking reaction between genipin and chitosan. At the first reaction site, the ester bond on genipin reacts with the amino group of chitosan to form a new amide bond. At the second reaction site, the hemiacetal group of genipin reacts with the amino group of chitosan to form a tertiary amine. Both of these reactions also occur simultaneously at the amino group of lipase in SNPs-CS@Lipase, causing the lipase to covalently cross-link to the chitosan-formed gel network. Simultaneously, the cross-linking of chitosan itself or with the enzyme increases the strength of the gel network.

[0060] 2. Changes in moisture content, moisture distribution, and appearance of SNPs-CS@Lipase during water bath agitation culture.

[0061] Figure 5The changes in moisture content, moisture distribution, and appearance of SNPs-CS@Lipase during cultivation are shown. Moisture content was measured using a direct drying method, and moisture distribution was measured using low-field pulsed nuclear magnetic resonance (NMR). It was found that when the two unstable W / O emulsions in Example 2 were first fused, the moisture content of the emulsion was 30.55 ± 3.40%. Of this, free water accounted for 75.51 ± 0.72%, immobile water for 24.45 ± 0.18%, and bound water for 0.032 ± 0.00%. After 36 hours of cultivation, the moisture content decreased to 0.54 ± 0.04%, with 99.10 ± 1.01% of the remaining water being immobile water. During cultivation, free water was removed first, decreasing to 2.44 ± 0.25% by 12 hours. Immobile water was subsequently removed, and its absolute content at 36 hours of cultivation was below 0.53%, but due to the initial removal of free water, its relative content increased to 99.10 ± 1.01%. Throughout the entire cultivation process, the bound water remained largely unaffected. The new groups generated by the cross-linking of chitosan with genipin gave the system a dark green color, which gradually deepened as the reaction progressed and water was removed, changing from white at 0h to a deep dark green at 36h.

[0062] Example 3

[0063] A method for hydrophobic modification of SNPs-CS@Lipase, the method comprising:

[0064] In the preparation method of SNPs-CS@Lipase described in Example 2, the amounts of SNPs and deionized water were adjusted to 0 mL, 1 mL, 2 mL, 3 mL, 4 mL and 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, while the total volume remained constant at 5 mL. All other parameters and conditions were the same as in Example 2. Figure 6 As shown, after hydrophobic modification, the contact angle of SNPs-CS@Lipase at the water-n-hexane interface increased from 100.5° to 124.4°, making it possible to regulate the hydrophobicity of SNPs-CS@Lipase and form a stable W / O emulsion.

[0065] Example 4

[0066] A method for constructing a Pickering emulsion microreactor formed from SNPs-CS@Lipase and determination of its enzymatic properties, the construction method comprising:

[0067] Take 6 mL of the SNPs-CS@Lipase liquid paraffin dispersion prepared in Example 2 into a 10 mL centrifuge tube, centrifuge at 8000 rpm for 5 min to precipitate the particles and remove the oil phase. Add 5 mL of n-hexane and vortex for 3 min to redisperse the particles, then centrifuge at 5000 rpm for 3 min to remove the organic phase. Repeat the above steps 5 times to completely remove the liquid paraffin and unadsorbed Span 80. Redisperse the resulting precipitate in 3 mL of n-hexane, add 3 mL of deionized water, and vortex for 30 s to form a W / O type Pickering emulsion microreactor.

[0068] SNPs-CS@Lipase in the microreactor were labeled using fluorescein isothiocyanate, and their microstructure was observed using laser confocal microscopy. The results are as follows: Figure 7 It can be observed that SNPs-CS@Lipase is uniformly adsorbed on the surface of water droplets in n-hexane, forming a bright adsorption layer. The size of the resulting microreactor is approximately 20 μm. Individual labeling and observation of SNPs-CS@Lipase reveals that its size is approximately 2 μm, slightly larger than the thickness of the interfacial adsorption layer, possibly due to a small amount of aggregation of particles during individual labeling.

[0069] Based on the preparation process of SNPs-CS@Lipase and the microstructure of the microreactor, the structure and catalytic mechanism of the Pickering microreactor constructed from SNPs-CS@Lipase can be simplified as follows: Figure 8 As shown, the lipase is covalently cross-linked in a chitosan gel network, with the outer layer of the gel covered by SNPs. The SNPs enhance interfacial strength and regulate particle hydrophobicity. SNPs-CS@Lipase adsorbs onto the interface of a W / O emulsion formed by n-hexane and water, stabilizing the emulsion by forming a mechanical barrier. During the catalytic reaction, the substrate is added to the outer phase (n-hexane) and diffuses into the SNPs-CS@Lipase to contact and react with the lipase. The product diffuses back into the hexane, while the water generated enters the inner phase of the emulsion. This method provides strong lipase immobilization and reduces environmental damage to the lipase, improving its reusability. The microreactor provides a mild hydrophobic environment for the lipase, inducing the opening of its active sites and increasing the contact area between the enzyme and substrate, thus enhancing the catalytic activity of the lipase.

[0070] Performance testing

[0071] 1. Activity assay of immobilized lipases from different sources catalyzing the esterification of hexanoic acid and hexanol

[0072] The preparation method of SNPs-CS@Lipase in Example 2 was adjusted so that the lipase used was either 0.4 mL of Candida lipase solution or 0.4 mL of PBS solution (0.1 g / mL, pH = 7.4, 10 mM) containing lipase powder from different sources. The conversion rates of lipases from different sources after immobilization for 15 min catalyzing the esterification of hexanoic acid and hexanol were as follows: Figure 9 As shown, the lipases derived from *Candida* showed significantly higher catalytic activity in the esterification of hexanoic acid and hexanol than the other five lipases, with a 15-minute conversion rate of 73.27 ± 1.31%. The 15-minute conversion rates of the five lipases from different sources (including *Candida pumilus* lipase, *Aspergillus niger* lipase, *Aspergillus oryzae* lipase, *Mucor javanica* lipase, and porcine pancreatic lipase) ranged from 2.96 to 3.62, with no significant difference in the 15-minute conversion rates among the other four lipases except for *Aspergillus oryzae* lipase.

[0073] 2. Activity determination of SNPs-CS@Lipase in the esterification of hexanoic acid and hexanol

[0074] The reaction kinetic curves for the esterification of hexanoic acid and hexanol catalyzed by lipase-loaded particles, empty particles with lipase, and free lipase are shown below. Figure 10 As shown in the inset, the small figure represents the kinetic curve within the first hour of the reaction. It can be observed that lipase catalyzes the esterification of hexanoic acid and hexanol in all three reaction systems, and the conversion rate of the free lipase system at the reaction endpoint is approximately 4.15% lower than the other two groups. SNPs-CS@Lipase exhibits the fastest reaction rate, achieving a conversion rate of 91.22±0.15% within 30 minutes, higher than the 53.08±10.95% for empty particles + lipase and the 12.50±0.46% for free lipase. The conditional enzyme activity (based on the mass of lipase contained) calculated from the conversion rate in the first 15 minutes of the reaction is shown below. Figure 11 As shown, the enzyme activity of SNPs-CS@Lipase was 10.53±0.48 U / mg, which was higher than that of empty particle + lipase (3.71±0.50 U / mg) and free lipase (0.92±0.02 U / mg).

[0075] 2. Reusability of SNPs-CS@Lipase in catalyzing the esterification of hexanoic acid and hexanol

[0076] The reusability results of SNPs-CS@Lipase-catalyzed hexanoic acid and hexanol esterification are as follows: Figure 12 As shown, after 10 cycles, the enzyme activity of SNPs-CS@Lipase remained at 86.52±3.09%, indicating good reusability. However, after 20 cycles, irreversible aggregation of particles led to a decrease in emulsifying performance, and SNPs-CS@Lipase could not effectively stabilize W / O Pickering emulsions, with the enzyme activity retention rate decreasing to 40.53±5.00%.

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing immobilized lipase, characterized in that, Includes the following steps: (1) Mix shellac nanoparticle aqueous dispersion with crosslinking agent solution to prepare mixture I, then add mixture I to liquid paraffin containing Span 80, and homogenize by high-speed shearing to obtain unstable emulsion A containing crosslinking agent; (2) Mix Candida lipase solution with acidic chitosan solution to prepare mixture II. Add mixture II to liquid paraffin containing Span 80 and homogenize by high-speed shearing to obtain unstable emulsion B containing cross-linked substances. (3) Mix emulsion A obtained in step (1) with emulsion B obtained in step (2) and incubate to perform droplet fusion cross-linking and dehydration shrinkage, thereby obtaining immobilized lipase; The volume ratio of the shellac nanoparticle aqueous dispersion to the genipin aqueous solution in step (1) is 4-6:1; the mass concentration of the shellac nanoparticle aqueous dispersion is ≤2wt.%, wherein the diameter of the shellac nanoparticles is 100-200 nm; the crosslinking agent is one of the following: a genipin aqueous solution with a mass concentration of 5-15 mg / mL, a glutaraldehyde aqueous solution with a volume concentration of 2-10% v / v, or a vanillin aqueous solution with a mass concentration of 1-8 mg / mL. In step (2), the volume ratio of Candida lipase solution to acidic chitosan solution is 1:1-3, wherein the lipase concentration of the lipase solution is 0.05-0.2 g / mL, the lipase solution is pre-diluted 4-6 times with phosphate buffer solution with a phosphate concentration of 5-15 mM and a pH of 7.0-8.0, the chitosan concentration in the acidic chitosan solution is 1-3 wt.%, and the acetic acid volume concentration is 0.4-0.8%. The concentration of the liquid paraffin containing Span 80 in steps (1) and (2) is 0.04-0.08 g / mL, the ratio of aqueous phase to liquid paraffin is 1:1-3, and the high-speed shear homogenization conditions are 10000-14000 rpm and 1-3 min. The volume ratio of emulsion A and emulsion B in step (3) is 0.8-1.2:1; the conditions for fusion crosslinking and dehydration shrinkage are: open constant temperature water bath stirring, temperature is 30-45℃, rotation speed is 500-1500 rpm, and time is 40-60 h.

2. The method for preparing immobilized lipase according to claim 1, characterized in that, The specific preparation method of the shellac nanoparticle aqueous dispersion in step (1) is as follows: (a) Dissolve 1.2 g shellac in 80 mL of anhydrous ethanol to obtain a 15 mg / mL shellac ethanol solution. Pass the shellac ethanol solution through a 0.22 μm filter to remove a small amount of insoluble matter. (b) Using a stainless steel needle with a diameter of 0.43 mm, drop the filtered shellac ethanol solution into 100 mL of distilled water at a rate of 0.45 mL / min. To ensure that almost all of the shellac is converted into SNPs, stir the distilled water with a magnetic stirrer at a speed of 600 rpm until the shellac ethanol solution is completely dropped. (c) Use filter paper to filter out a small amount of shellac aggregates that have not formed nanoparticles to obtain the original SNPs dispersion, and use a rotary evaporator to remove all ethanol from the original dispersion. Finally, add distilled water to the evaporated dispersion to adjust the total volume of the dispersion to 100 mL.

3. An immobilized lipase, characterized in that, It is prepared according to the preparation method according to any one of claims 1 to 2.

4. An application of the immobilized lipase according to claim 3 in Pickering interface catalysis, characterized in that, The application includes: mixing an acid-alcohol solution with deionized water, adding immobilized lipase, and stirring at a constant temperature to carry out the reaction.

5. The application of the immobilized lipase according to claim 4 in Pickering interface catalysis, characterized in that, The acid-alcohol solution is a hexanoic acid and hexanol solution in n-hexane, with the concentration of hexanoic acid and hexanol in the n-hexane solution being 0.3-0.5 mol / L; the ratio of the acid-alcohol solution to deionized water is 1-2:1; and the mass concentration of the immobilized lipase in the reaction system is 20-50 mg / mL.

6. The application of the immobilized lipase according to claim 4 in Pickering interface catalysis, characterized in that, The constant temperature stirring conditions are: temperature 35-45℃, rotation speed 300 rpm, and reaction time 10-14 h.

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