A method for preparing immobilized lipase and its application in catalyzing synthesis of 1,3-diglyceride
By combining PPS nanosphere templates and metal-organic frameworks during lipase immobilization, the problems of reduced enzyme activity and limited mass transfer were solved, achieving highly efficient catalysis of 1,3-diglyceride generation, which is suitable for lipase immobilization applications in the food industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUNAN AMAZING GRACE BIOTECH
- Filing Date
- 2026-02-05
- Publication Date
- 2026-05-29
AI Technical Summary
In existing technologies, lipase immobilization methods lead to reduced or inactivated enzyme activity, and it is difficult to selectively catalyze the generation of 1,3-diglycerides with lipid-lowering effects, resulting in severe mass transfer limitations.
A PPS nanosphere template was formed by styrene polymerization, and Mg2+ and 2,5-dihydroxyterephthalic acid were combined to form a metal-organic framework to immobilize lipase. The lipase was also immobilized through specific group interactions, and a three-dimensional through-pore structure was constructed to ensure the stability of the enzyme active site and the mass transfer channel of macromolecular substrate.
It significantly improves the selectivity and production of 1,3-glycerol while maintaining enzyme activity and stability, making it suitable for industrial production.
Smart Images
Figure CN122104666A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of materials, specifically relating to a method for preparing immobilized lipase and its application in the catalytic synthesis of 1,3-glycerol diesters. Background Technology
[0002] Lipases are widely used in the food industry due to their green and efficient catalysis of fat hydrolysis and synthesis. However, free lipases in the reaction system are difficult to recover and reuse. Immobilizing lipases on a carrier can improve their recovery rate, but it can lead to a decrease or loss of their activity. Enzyme immobilization can be divided into three categories: post-synthesis adsorption (physical adsorption), covalent bonding (chemical cross-linking), and coprecipitation (in-situ synthesis). Adsorption relies on weak hydrophobic interactions, making it easy for the enzyme to detach from the carrier surface. Covalent bonding can modify some important functional groups of the enzyme, leading to its inactivation. Coprecipitation can cause shielding of the active site, resulting in a significant decrease in enzyme activity.
[0003] The conversion of triglycerides (TAG) to diglycerides (DAG) catalyzed by lipases has become a hot topic in the development of functional oils in recent years. However, DAG is divided into 1,2-DAG and 1,3-DAG due to differences in the position of the fatty chain. The former is degraded into 2-monoglyceride (MAG) in the gastrointestinal tract and can be resynthesized into TAG in the body after absorption, while the latter cannot. Therefore, only 1,3-DAG can truly play a lipid-lowering role (Mao Yilin, Zhou Jun, Chen Kai, et al. Research progress on the metabolic mechanism and nutritional function of diglycerides. China Oils and Fats, 2023, 48 (11): 80-89). However, 1,2-DAG is more easily obtained in the process of lipase catalyzing TAG to DAG (Zhu Yuxiao, Wang Yingying, Jin Qingzhe, et al. Research on the process of enzymatic synthesis of 1,2-diglycerides. China Oils and Fats, 2017, 42(1): 56-59), which leads to a serious reduction in the lipid-lowering effect of actual DAG products. Because the reaction process begins with esterification at the 1,3 positions of TAG to generate 1,2-DAG, further esterification yields 2-monoglycerate, and simultaneously, esterification of fatty acids with glycerol occurs to produce a small amount of 1,3-DAG and a larger amount of 1,2-DAG. Therefore, how to induce lipase to selectively catalyze the generation of 1,3-DAG remains a challenging problem to be solved. Summary of the Invention
[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a method for preparing immobilized lipase.
[0005] Another object of the present invention is to provide the application of the immobilized lipase prepared by the above method in the catalytic synthesis of 1,3-glycerol diester.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing immobilized lipase includes the following steps: (1) Template preparation: Styrene (PS) was washed with NaOH aqueous solution and then washed with deionized water until the washing solution was neutral; the washed PS was dispersed in deionized water, polyvinylpyrrolidone (PVP) was added first, and then potassium persulfate aqueous solution was added to react, and the polystyrene (PPS) nanospheres were obtained by filtration. (2) Lipase immobilization: magnesium acetate tetrahydrate and 2,5-dihydroxyterephthalic acid (DHTA) were dissolved in deionized water, and lipase was added to obtain a precursor solution; the PPS nanospheres obtained in step (1) were immersed in the precursor solution, then the PPS nanospheres were separated by filtration, and then the PPS nanospheres were immersed in ammonia solution. After that, the PPS nanospheres were taken out and immobilized to obtain an immobilized lipase template. (3) Template removal: The immobilized lipase template obtained in step (2) is immersed in N,N-dimethylformamide (DMF), the polystyrene is removed by sonication, and then the solid is collected by centrifugation and vacuum dried to obtain the immobilized lipase.
[0007] Preferably, in step (1), the styrene (PS) is washed with NaOH aqueous solution 2 to 4 times, the concentration of NaOH aqueous solution is 0.5 to 1.5 mol / L, and the volume ratio of styrene to NaOH aqueous solution is controlled to be 3 to 4:1 each time.
[0008] Preferably, in step (1), the styrene is washed with deionized water 2 to 4 times, and the volume ratio of deionized water to styrene is controlled to be 0.2 to 0.3:1 each time.
[0009] Preferably, in step (1), the washed PS is dispersed in 5 to 10 times its volume of deionized water, the mass-to-volume ratio of PVP to styrene is controlled to be 30 to 50 g / L, the concentration of potassium persulfate aqueous solution is controlled to be 10 to 30 g / L, the amount of potassium persulfate aqueous solution is controlled to be 70 to 80% of the volume of styrene, the reaction temperature of potassium persulfate aqueous solution is controlled to be 65 to 75°C, and the reaction time is 18 to 24 h.
[0010] Preferably, in step (2), the lipase is Aspergillus niger lipase, and the amount added is 0.5 to 1 times the mass of magnesium acetate tetrahydrate; the mass ratio of magnesium acetate tetrahydrate to DHTA is 1.5 to 2.5: 1, the mass-volume ratio of magnesium acetate tetrahydrate to deionized water is 90 to 120 g / L, the mass-volume ratio of PPS nanospheres to precursor solution solvent is 40 to 60 g / L, and the time for PPS nanospheres to be soaked in the precursor solution is 1 to 3 h.
[0011] Preferably, in step (2), the concentration of the ammonia solution is 2% to 5%, the volume-to-mass ratio of the ammonia solution to the PPS nanospheres is 20 to 30:1 mL / g, and the polystyrene nanospheres are soaked in the ammonia solution for 1 to 3 hours.
[0012] Preferably, in step (2), the reaction temperature of the immobilization reaction is 40-45°C and the reaction time is 10-12h.
[0013] Preferably, in step (3), the volume-to-mass ratio of DMF to immobilized lipase template is 10-20:1 mL / g, the ultrasonic power is 50-150W, and the ultrasonic time is 6-8 h.
[0014] Preferably, in step (3), the centrifugation speed is 3000-5000 rpm and the centrifugation time is 10-30 min; the vacuum drying temperature is 30-40℃ and the drying time is 12-14 h.
[0015] The application of the immobilized lipase prepared by any of the above methods in the catalytic synthesis of 1,3-diglycerides includes the following steps: After mixing vegetable oil, glycerol and deionized water evenly, the immobilized lipase prepared above was added, the mixture was stirred and reacted, and then filtered. The filtrate was vegetable oil catalyzed by immobilized enzyme rich in 1,3-DAG.
[0016] Preferably, the mass ratio of vegetable oil to glycerol is 3-5:1; the amount of deionized water is 4%-6% of the total mass of vegetable oil and glycerol; the amount of immobilized lipase is 5%-10% of the total mass of the reaction system; the reaction temperature is 40-50℃, the stirring rate is 500-1500 rpm, and the reaction time is 16-24 h.
[0017] Preferably, the immobilized enzyme catalyzes 80% to 90% of the 1,3-diglycerides in the vegetable oil.
[0018] The technical principle of this invention: This invention first forms a PPS nanosphere template through styrene polymerization and self-assembly, and then Mg... 2+ Lipase is immobilized by forming a metal-organic framework with 2,5-dihydroxyterephthalic acid (DHTA) in the template interstitial space. After template removal, the immobilized lipase is obtained. Since the lipase molecule consists of both hydrophilic and hydrophobic parts, and the active site (Asp-His-Ser triplet) is located near the hydrophobic end of the molecule, the immobilization method of this invention immobilizes the lipase by binding the hydroxyl and carboxyl groups in DHTA to the hydrophilic part of the lipase. PPS, being hydrophobic, can induce the hydrophobic end of the lipase to face outwards. Mg 2+It can coordinate with the enzyme's active site, stabilizing its three-dimensional conformation and ensuring the catalytic center is in an optimal active state. On the other hand, the specific nanochannels formed after template removal allow triglycerides (TAGs) to pass through smoothly. The fatty acid chains of TAGs are captured by the hydrophobic portion of the lipase, thus efficiently hydrolyzing the intermediate fatty acid chains to form fatty acids. Subsequently, the fatty acids undergo esterification with glycerol to yield 1,3-dAGs. By addressing the limitations of mass transfer in vegetable oils and insufficient accessibility to the enzyme's active site through template action and removal, the 1,3-DAG content in the product is significantly increased.
[0019] The present invention has the following advantages and effects compared with the prior art: (1) The present invention uses specific group interactions to immobilize lipase, which not only prevents enzyme detachment but also maintains enzyme activity. At the same time, a template method is used to construct a porous immobilized lipase with a three-dimensional interconnected mesoporous structure, which solves the problem of limited mass transfer of macromolecular substrates such as vegetable oil TAG. When used to catalyze the preparation of DAG from vegetable oil, it can significantly improve the selectivity of 1,3-DAG.
[0020] (2) The preparation method of the present invention is mild, the template removal is simple, the obtained material has a regular and controllable pore structure, good repeatability and operational stability, and is easy to scale up for industrial production. Attached Figure Description
[0021] Figure 1 The images are SEM images of PPS nanospheres, where a is the morphology of the polyethylene nanosphere stack template, and b, c, and d are magnified views of a.
[0022] Figure 2 The images show SEM images of immobilized lipase grown in the gaps between PPS nanospheres, where a is the immobilized lipase template and b is a magnified view of a.
[0023] Figure 3 The images show SEM images of the immobilized lipase after template removal, where a is the morphology of the immobilized lipase after template removal; b is a magnified view of a; and c and d are magnified views of different locations in b.
[0024] Figure 4 The images are SEM comparisons with those of the immobilized lipases prepared in Comparative Examples 1 and 2, where a1-a3 are products of Comparative Example 1; b1-b3 are products of Example 1; c1-c3 are products of Comparative Example 2; and 1, 2, and 3 are images magnified step by step. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Example 1
[0026] (1) Take 70 mL of PS and wash it three times with 20 mL of 1.3 mol / L NaOH solution to remove the polymerization inhibitor. Then wash it three times with 17 mL of deionized water until the aqueous phase is neutral. Add the washed PS to 500 mL of deionized water, add 2.8 g of PVP to the system, mix well, and then add 50 mL of potassium persulfate aqueous solution (concentration 20 g / L). React at 75℃ for 18 h. After the reaction is completed, filter and collect the PPS nanospheres.
[0027] (2) Take 10 g magnesium acetate tetrahydrate and 5 g DHTA, dissolve them in 100 mL deionized water, add 7 g Aspergillus niger lipase to the solution, mix well to obtain a precursor solution; take 6 g of PPS nanospheres obtained in step (1) and soak them in the precursor solution. After 1 h, take them out and soak them in 150 mL ammonia solution (concentration 3%). After 2 h, take them out and immobilize them at 45℃ for 10 h to obtain an immobilized lipase template.
[0028] (3) Take 10g of the immobilized lipase template obtained in step (2) and soak it in 100 mL of DMF solvent. Then, sonicate it at 100W for 7h to dissolve and remove PPS. Subsequently, collect the solid by centrifugation at 3000rpm for 30min and vacuum dry it at 40℃ for 12h to obtain the immobilized lipase.
[0029] (4) Mix 8 g of tea seed oil, 2.5 g of glycerol and 0.5 g of deionized water evenly, and then add 1.0 g of the prepared immobilized lipase. Stir at 1000 rpm for 20 h at a constant temperature of 50℃, and filter to obtain the immobilized lipase-catalyzed vegetable oil. Example 2
[0030] (1) Take 70 mL of PS and wash it twice with 23 mL of 1.0 mol / L NaOH solution to remove the polymerization inhibitor, then wash it four times with 14 mL of deionized water until the aqueous phase is neutral. Add the washed PS to 350 mL of deionized water, add 2.1 g of PVP to the system, mix well, add 50 mL of potassium persulfate aqueous solution (concentration 20 g / L), and react at 65℃ for 24 h. After the reaction is completed, filter and collect the PPS nanospheres.
[0031] (2) Take 10 g magnesium acetate tetrahydrate and 4 g DHTA, dissolve them in 90 mL deionized water, add 5 g Aspergillus niger lipase to the solution, mix well to obtain a precursor solution; take 6.5 g of PPS nanospheres obtained in step (1) and soak them in the precursor solution. After 3 h, take them out and soak them in 130 mL ammonia solution (concentration 5%). After 1 h, take them out and immobilize them at 43℃ for 11 h to obtain an immobilized lipase template.
[0032] (3) Take 10g of the immobilized lipase template obtained in step (2) and soak it in 150 mL of DMF solvent. Soak it at 50W for 8 h to dissolve and remove PPS. Then, collect the solid by centrifugation at 4000rpm for 20 min and vacuum dry it at 30℃ for 14 h to obtain immobilized lipase.
[0033] (4) Mix 8 g soybean oil, 2.0 g glycerol and 0.6 g deionized water evenly, then add 0.8 g of the prepared immobilized lipase. Stir at 500 rpm for 24 h at a constant temperature of 45℃, and filter to obtain the immobilized lipase-catalyzed vegetable oil. Example 3
[0034] (1) Take 70 mL of PS and wash it four times with 18 mL of 1.5 mol / L NaOH solution to remove the polymerization inhibitor, then wash it twice with 20 mL of deionized water until the aqueous phase is neutral. Add the washed PS to 700 mL of deionized water, add 3.5 g of PVP to the system, mix well, add 50 mL of potassium persulfate aqueous solution (concentration 20 g / L), and react at 70℃ for 2 h. After the reaction is completed, filter and collect the PPS nanospheres.
[0035] (2) Take 10 g magnesium acetate tetrahydrate and 6.5 g DHTA, dissolve them in 120 mL deionized water, add 10 g Aspergillus niger lipase to the solution, mix well to obtain a precursor solution; take 5 g of PPS nanospheres obtained in step (1) and soak them in the precursor solution. After 2 h, take them out and soak them in 150 mL ammonia solution (concentration 2%). After 3 h, take them out and immobilize them at 40℃ for 12 h to obtain an immobilized lipase template.
[0036] (3) Take 10g of the immobilized lipase template obtained in step (2) and soak it in 200 mL of DMF solvent. Soak it at 150W for 6 h to dissolve and remove PPS. Then, collect the solid by centrifugation at 5000 rpm for 10 min and vacuum dry it at 35℃ for 13 h to obtain immobilized lipase.
[0037] (4) Mix 10 g peanut oil, 2.0 g glycerol and 0.5 g deionized water evenly, and then add 0.7 g of the prepared immobilized lipase. Stir at 1500 rpm for 16 h at a constant temperature of 40℃, and filter to obtain the immobilized lipase-catalyzed vegetable oil.
[0038] Comparative Example 1
[0039] The process is essentially the same as in Example 1, except that in step (2), 10 g of magnesium acetate tetrahydrate and 3.5 g of DHTA are used, with a mass ratio of magnesium acetate tetrahydrate to DHTA of 2.86:1. The resulting immobilized lipase does not form interconnected channels, as shown in Example 1. Figure 4 As shown in a. Comparative Example 2
[0040] The process is essentially the same as in Example 1, except that in step (2), 10 g of magnesium acetate tetrahydrate and 8 g of DHTA are used, with a mass ratio of magnesium acetate tetrahydrate to DHTA of 1.25:1. The resulting immobilized lipase exhibits a sheet-like accumulation, which is detrimental to substrate transport. Figure 4 As shown in c. Comparative Example 3
[0041] The experiment was basically the same as step (4) in Example 1, but the product was obtained by using free lipase instead of immobilized lipase, and its DAG content was only 15%. Comparative Example 4
[0042] The process is basically the same as step (4) in Example 1, but the product obtained by using commercially available immobilized lipase instead of the immobilized lipase of the present invention has a relative content of only 35% of 1,3-DAG.
[0043] Test Example 1
[0044] Observation of the structure and morphology of the immobilized lipase template products prepared in Example 1 and Comparative Examples 1 and 2.
[0045] Method: The prepared sample was ground into powder, then coated onto a copper film and sputtered with gold at a current of 20 mA for 200 s for SEM testing (SU8220 ultra-high resolution field emission electron microscope).
[0046] result: Figure 1 The images show SEM images of PPS nanospheres, where a represents the morphology of the polyethylene nanosphere stacking template, and b, c, and d are magnified views of a portion thereof, demonstrating the successful preparation of PPS nanospheres. Figure 2 The images show SEM images of immobilized lipase grown in the gaps between PPS nanospheres, where a is the immobilized lipase template and b is a magnified view of a, showing the successful preparation of the immobilized lipase template. Figure 3 The images show SEM images of the immobilized lipase after template removal, where a is the morphology of the immobilized lipase after template removal; b is a magnified view of a; and c and d are magnified views of different locations in b, showing its uniform pore size distribution. Figure 4SEM images comparing the immobilized lipases prepared with those of Comparative Examples 1 and 2 are shown, where a1-a3 are the products of Comparative Example 1; b1-b3 are the products of Example 1; c1-c3 are the products of Comparative Example 2; and 1, 2, and 3 are images magnified step by step. The results show that the product of Comparative Example 1 retains the circular pits left after template removal on its surface, but the channels are not effectively interconnected, and the porous structure exists only on the surface of the material, which is not conducive to substrate entry. The product of Comparative Example 2 has a sheet-like morphology with protruding surfaces and irregular channels, which is not conducive to enzyme catalysis. In contrast, the product of Example 1 has a uniformly distributed porous structure on its surface, forming a connected network, which is beneficial for substrate transport and enzyme activity. This indicates that the immobilized enzyme preparation conditions of the present invention are the result of optimization.
[0047] Test Example 2
[0048] The relative content of 1,3-DAG in vegetable oils catalyzed by immobilized lipase was determined in Examples 1 to 3.
[0049] Method: The immobilized lipases prepared in Examples 1 to 3 were reacted in step (4) and then filtered. The filtrate was the immobilized lipase-catalyzed vegetable oil, and the filter residue was the recovered immobilized lipase. The recovered immobilized lipase was recycled and reused 10 times in step (4). The filtrate collected in the last cycle was the immobilized lipase-catalyzed vegetable oil obtained after 10 cycles.
[0050] Take 30 µL of the immobilized lipase-catalyzed vegetable oil prepared above and dissolve it in 1 mL of acetone. After mixing thoroughly, filter through a 0.45 µm pore size filter membrane. Inject 50 µL of the filtrate using a syringe for analysis. Analyze the product components using HPLC-ELSD (APS80-16T-APS80-16E high-performance liquid chromatograph), and calculate the content of each component using the area normalization method. The instrument conditions are set as follows: (1) Liquid chromatography detection parameters: The chromatographic column is a C18 column, and the mobile phase is a binary mobile phase gradient elution (mobile phase A is 0.15% acetic acid-acetonitrile (v / v) solution, mobile phase B is isopropanol, and the gradient elution scheme of the mobile phase is set as follows: 0 min~4 min, A is 100%; 12 min~25 min, A is 90%, B is 10%; 30 min~35 min, A is 70%, B is 30%; 45 min, A is 20%, B is 80%; 48 min~55 min, A is 100%), the flow rate is 1.2 mL / min, and the chromatographic column temperature is 45℃; (2) Air generator parameters: The pressure is 1.05 bar; (3) Evaporative light detector parameters: The nebulizer temperature is 70℃, and the drift tube temperature is 50℃.
[0051]
[0052]
[0053] In the formula: The peak area of each 1,2-DAG in the sample is denoted as 1. The peak area of each 1,3-DAG in the sample is denoted as 1. This represents the peak area of each component in the sample.
[0054] Results: The DAG contents in the products of Examples 1-3 were 48%, 45%, and 47%, respectively; the relative contents of 1,3-DAG were 85%, 83%, and 90%, respectively; after 10 cycles, the DAG contents were 44%, 42%, and 45%, respectively; the relative contents of 1,3-DAG were 82%, 81%, and 85%, respectively. This indicates that the immobilized lipase prepared by this invention exhibits high selectivity in catalyzing the formation of 1,3-DAG from vegetable oil TAG, is reusable, and has good stability. In contrast, free lipase is prone to aggregation, resulting in a product with a DAG content of only 15%, and it cannot be recovered. The commercially available immobilized lipase produces a product with a DAG content of 40%, but only 35% 1,3-DAG. After 10 cycles, the DAG content of the product is 25%, with only 30% 1,3-DAG. This demonstrates the superior performance of the immobilized lipase prepared by this invention.
[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. 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) Template preparation: Styrene was washed with NaOH aqueous solution and then washed with deionized water until the washing solution was neutral; the washed styrene was dispersed in deionized water, polyvinylpyrrolidone was added first, and then potassium persulfate aqueous solution was added to react. After filtration, polystyrene nanospheres were obtained. (2) Lipase immobilization: magnesium acetate tetrahydrate and 2,5-dihydroxyterephthalic acid were dissolved in deionized water, and lipase was added to obtain a precursor solution; the polystyrene nanospheres obtained in step (1) were immersed in the precursor solution, then the polystyrene nanospheres were separated by filtration, and then the polystyrene nanospheres were immersed in ammonia solution. Finally, the polystyrene nanospheres were taken out for immobilization reaction to obtain the immobilized lipase template. (3) Template removal: The immobilized lipase template obtained in step (2) is immersed in N,N-dimethylformamide, the polystyrene is removed by ultrasonication, and then the solid is collected by centrifugation and vacuum dried to obtain the immobilized lipase.
2. The preparation method according to claim 1, characterized in that, The styrene is washed with NaOH aqueous solution 2 to 4 times, the concentration of NaOH aqueous solution is 0.5 to 1.5 mol / L, and the volume ratio of styrene to NaOH aqueous solution is controlled at 3 to 4:1 during each washing; the styrene is washed with deionized water 2 to 4 times, and the volume ratio of deionized water to styrene is controlled at 0.2 to 0.3:1 during each washing.
3. The preparation method according to claim 1, characterized in that, In step (1), the washed PS is dispersed in 5 to 10 times its volume of deionized water, the mass-to-volume ratio of polyvinylpyrrolidone to styrene is controlled at 30 to 50 g / L, the concentration of potassium persulfate aqueous solution is controlled at 10 to 30 g / L, the amount of potassium persulfate aqueous solution is controlled at 70 to 80% of the volume of styrene, the reaction temperature of potassium persulfate aqueous solution is controlled at 65 to 75℃, and the reaction time is controlled at 18 to 24 h.
4. The preparation method according to claim 1, characterized in that, In step (2), the lipase is Aspergillus niger lipase, and the amount added is 0.5 to 1 times the mass of magnesium acetate tetrahydrate; the mass ratio of magnesium acetate tetrahydrate to 2,5-dihydroxyterephthalic acid is 1.5 to 2.5:1, the mass-volume ratio of magnesium acetate tetrahydrate to deionized water is 90 to 120 g / L; the mass-volume ratio of polystyrene nanospheres to precursor solution solvent is 40 to 60 g / L, and the polystyrene nanospheres are soaked in the precursor solution for 1 to 3 hours.
5. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the ammonia solution is 2% to 5%, the volume-to-mass ratio of the ammonia solution to the polystyrene nanospheres is 20 to 30:1 mL / g, and the polystyrene nanospheres are soaked in the ammonia solution for 1 to 3 hours.
6. The preparation method according to claim 1, characterized in that, In step (3), the volume-to-mass ratio of N,N-dimethylformamide to immobilized lipase template is 10-20:1 mL / g; the ultrasonic power is 50-150W and the ultrasonic time is 6-8 h; the centrifugation speed is 3000-5000 rpm and the centrifugation time is 10-30 min; the vacuum drying temperature is 30-40℃ and the drying time is 12-14 h.
7. The use of the immobilized lipase prepared by any one of claims 1-6 in the catalytic synthesis of 1,3-glycerol diester.
8. The application according to claim 7, characterized in that, The application method includes the following steps: after mixing vegetable oil, glycerol and deionized water evenly, the immobilized lipase prepared by any one of claims 1-6 is added, and then the mixture is stirred and reacted, filtered, and the filtrate is the immobilized enzyme-catalyzed vegetable oil rich in 1,3-diglycerides.
9. The application according to claim 8, characterized in that, The mass ratio of vegetable oil to glycerol is 3–5:1; the amount of deionized water is 4%–6% of the total mass of vegetable oil and glycerol; the amount of immobilized lipase is 5%–10% of the total mass of the reaction system; the reaction temperature is 40–50℃, the stirring rate is 500–1500 rpm, and the reaction time is 16–24 h.
10. The application according to claim 8, characterized in that, The immobilized enzyme catalyzes the proportion of 1,3-diglycerides in vegetable oil to 80%–90% of the total diglycerides.