Preparation method of immobilized lipase and application of immobilized lipase in catalytic synthesis of glyceride

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to a preparation method of immobilized lipase and application of the immobilized lipase in catalytic synthesis of glyceride, the preparation method comprises the following steps: (1) surface modification: immersing a pretreated carrier in 80-300 parts by weight of a silanization solution for reaction to obtain functional resin; (2) enzyme immobilization: putting the functionalized resin into an enzyme solution, and oscillating and adsorbing to form an enzyme-resin compound; and (3) post-treatment: washing the enzyme-resin compound with 300-600 parts by weight of a phosphate buffer solution, and drying until the water content is less than or equal to 5% to obtain a finished product of the immobilized lipase. The immobilized lipase is used for catalytic synthesis of glyceride, and the glyceride comprises diglyceride and triglyceride. The enzyme stability is improved by combining covalent modification on the surface of the carrier with a protective auxiliary agent, the composition of a glyceride product is accurately regulated and controlled by utilizing a temperature-vacuum synergistic mechanism, and the reaction kinetic performance is enhanced by virtue of a composite dewatering system.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a method for preparing immobilized lipase and its application in catalyzing the synthesis of glycerol esters. Background Technology

[0002] Diacylglycerols and triglycerides are important raw materials in the food and daily chemical industries. Due to their unique metabolic pathway in the human body, diglycerides have the property of reducing fat accumulation and are often used in the development of functional edible oils; triglycerides are the core component of ordinary edible oils. Traditional chemical synthesis of these products requires high-temperature and high-pressure environments exceeding 200°C and the use of strong acid catalysts such as concentrated sulfuric acid, leading to the generation of a large number of byproducts, especially coloring substances and trans fatty acids. This not only increases subsequent refining costs but also limits the application of these products in health foods.

[0003] Enzymatic catalysis has gradually emerged in the last decade. This method utilizes lipases to catalyze the reaction of fatty acids with glycerol under mild conditions, theoretically allowing for precise control of the ratio of diglycerides to triglycerides in the product. However, its practical industrialization faces three key obstacles: First, free lipases have poor stability and are easily deactivated by mechanical shear forces in stirred reactors, making them unusable for recycling. Second, existing immobilization carriers are too expensive; for example, the immobilized enzyme Novo435, sold for over $1,000 per kilogram, significantly increases production costs. Third, long-chain fatty acid conversion efficiency is low; when using 22-carbon fatty acids such as erucic acid, the reaction rate is more than 40% lower than that of short-chain acids.

[0004] Existing patented technologies attempt to reduce costs through macroporous resin adsorption, using ADS-17 resin to adsorb and immobilize Candida antarcticis lipase. However, test results show that the immobilized enzyme adsorption rate obtained by this method is only 38.7%, and after eight reuses, the enzyme activity decays to 79% of the initial value, with a maximum diglyceride yield of only 60.91%. Particularly noteworthy is that while this technology claims to achieve product ratio control through temperature, implementation examples only verify the effect at a single temperature point, lacking systematic parameter studies. In the vacuum dehydration stage, this patent uses a single molecular sieve dehydrating agent, resulting in a reaction time exceeding 48 hours. Therefore, there is a need to design a method for preparing immobilized lipase and its application in catalyzing the synthesis of glycerides. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, a method for preparing immobilized lipase and its application in catalyzing the synthesis of glycerides are provided.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing immobilized lipase, comprising the following steps: (1) Surface modification: The pretreated carrier is immersed in 80-300 parts by weight of silanization solution to react and obtain functionalized resin; (2) Enzyme immobilization: The functionalized resin is added to the enzyme solution, and the weight ratio of the functionalized resin to the enzyme solution is 1:15-30. The enzyme is shaken and adsorbed for 240-480 min at 25-35℃ and 150-250 rpm to form an enzyme-resin complex. (3) Post-treatment: The enzyme-resin complex is washed with 300-600 parts by weight of phosphate buffer and then dried until the water content is ≤5% to obtain the immobilized lipase product.

[0007] In step (1) surface modification, the pretreatment of the carrier includes the following steps: 10-20 parts by weight of macroporous adsorption resin AB-8 are placed in a reactor, 100-400 parts by weight of phosphate buffer with a pH of 6.5-8.5 are added, and the mixture is stirred at 200-400 rpm at 50-70 ℃ for 60-180 min. After completion, the solid resin is obtained by filtration and separation. The obtained product is the pretreated carrier.

[0008] In step (1) surface modification, the silanization solution is prepared from γ-aminopropyltriethoxysilane or 3-glycidyl etheroxypropyltrimethoxysilane, with a weight concentration of 8%-15%.

[0009] In step (1) surface modification, the pretreated carrier is immersed in a silanization solution and continuously treated for 90-240 min under the conditions of 40-55℃ and 200-400 rpm to obtain a surface-activated functionalized resin.

[0010] In step (2) enzyme immobilization, the enzyme solution is formed by dissolving 50-120 parts by weight of lipase in 1000-3000 parts by weight of phosphate buffer.

[0011] The enzyme solution also includes sodium alginate as an adjuvant, and the amount of sodium alginate as an adjuvant added is 0.3-0.8 parts by weight.

[0012] In the post-processing step (3), the drying involves placing the rinsed product in a vacuum drying oven and drying it at 28-35°C and -0.08 to -0.1 MPa until the moisture content is ≤5%.

[0013] The present invention also provides an immobilized lipase prepared by the above preparation method.

[0014] The present invention also provides the application of the above-mentioned immobilized lipase, wherein the immobilized lipase is used to catalyze the synthesis of glycerides, wherein the glycerides include diglycerides and triglycerides.

[0015] The method for using the immobilized lipase to catalyze the synthesis of glycerol esters specifically includes the following steps: mixing a fatty acid system with glycerol at a mass ratio of 1:0.5-2, adding 3%-10% of the immobilized lipase by the total mass of the substrate; then reacting continuously at a temperature of 35-65℃ and a reactor vacuum of 0.06-0.15MPa for 4-12 hours to synthesize diglycerides; or reacting continuously at a temperature of 65-95℃ and a reactor vacuum of 0.15-0.35MPa for 12-24 hours to obtain triglycerides. A composite dehydrating agent is added during the reaction process. The composite dehydrating agent is composed of molecular sieve and anhydrous magnesium sulfate in a mass ratio of 1:0.5-3, and the amount of the composite dehydrating agent added is 15%-30% of the total mass of the substrate. The fatty acid system comprises short-chain fatty acids, medium-chain fatty acids, and long-chain fatty acids, with a mass ratio of (0.3-2):(1-5):(0.8-8).

[0016] The beneficial effects of this invention are as follows: 1. This invention introduces sodium alginate as an auxiliary agent during the enzyme immobilization stage, whose molecular chains form a three-dimensional network structure on the enzyme protein surface. This structure generates a steric hindrance effect, effectively buffering shear force shocks in the reaction system and reducing inactivation caused by conformational changes in the enzyme's active site. This protective mechanism enables the immobilized lipase to maintain more stable catalytic efficiency during repeated catalytic processes.

[0017] 2. This invention addresses the selective control of glycerol ester synthesis. When the system is at 35 to 65°C and a vacuum of 0.06 to 0.15 MPa, the lower reaction temperature inhibits the acyl migration rate, causing the reaction to stop at the diglyceride stage. However, when the temperature is increased to 65 to 95°C and a vacuum environment of 0.15 to 0.35 MPa is applied, the high temperature accelerates molecular motion, while the high vacuum promotes the removal of water, driving the esterification reaction toward the triglyceride direction.

[0018] 3. The composite dehydrating agent of this invention employs the synergistic effect of molecular sieves and anhydrous magnesium sulfate. The molecular sieves primarily capture gaseous water molecules, while the anhydrous magnesium sulfate adsorbs trace amounts of water in the liquid phase, forming a multi-stage dehydration network. This system continuously reduces the water activity of the reaction system, shifting the chemical equilibrium towards esterification synthesis and significantly improving fatty acid conversion efficiency.

[0019] 4. The hydrophobic groups of the functionalized resin of this invention interact hydrophobically with the alkyl chains of long-chain fatty acids, promoting the enrichment of the substrate on the support surface. This local concentration effect effectively overcomes the problem of high diffusion resistance of long-chain fatty acid molecules and improves their reaction kinetics at the catalytic interface.

[0020] 5. In the carrier pretreatment stage, the present invention removes pore blockages by washing with phosphate buffer, thereby increasing the resin specific surface area by about 40%; the subsequent gradient drying process maintains mild conditions of 28 to 35°C to avoid enzyme denaturation caused by high temperature and ensure the initial activity retention rate of the immobilized enzyme.

[0021] This invention enhances enzyme stability by covalently modifying the carrier surface and combining it with protective auxiliaries, precisely controls the composition of glycerol ester products using a temperature-vacuum synergistic mechanism, and strengthens reaction kinetics performance by using a composite dehydration system. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope image of the immobilized lipase and resin of the present invention. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In the specific embodiments of this application, the sources of various raw materials are briefly described as follows: γ-aminopropyltriethoxysilane: purchased from Aladdin Biochemical Technology Co., Ltd., CAS No. 919-30-2, model A117699; 3-Glycidyl etheroxypropyltrimethoxysilane: purchased from Macklin Biotechnology Co., Ltd., CAS No. 2530-83-8, model G811188; Macroporous adsorption resin AB-8: purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.; Sodium alginate: purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 9005-38-3, model No. 801328; Lipase: Purchased from Novozymes, product name Lipase TL IM; Phosphate buffer: It is prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate provided by Sinopharm Chemical Reagent Co., Ltd. The CAS number of sodium dihydrogen phosphate is 7558-80-7 and the CAS number of disodium hydrogen phosphate is 7558-79-4. Molecular sieve: Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., model 3A molecular sieve, CAS number 62248-79-1; Anhydrous magnesium sulfate: purchased from Tianjin Damao Chemical Reagent Factory, CAS No. 7487-88-9, purity ≥99%.

[0025] The technical solutions of the present invention are further illustrated below through examples and comparative examples, but the scope of protection of the present invention is not limited thereto.

[0026] Example 1 A method for preparing immobilized lipase and its application in catalyzing the synthesis of glycerides. (1) Surface modification: 10 parts by weight of macroporous adsorption resin AB-8 were placed in a reactor, and 100 parts by weight of pH 7.5 phosphate buffer solution were added. The mixture was stirred at 300 rpm for 120 min at 60 °C and filtered to obtain a pretreated carrier. The carrier was immersed in 200 parts by weight of 12% γ-aminopropyltriethoxysilane solution and shaken at 300 rpm for 180 min at 50 °C to obtain functionalized resin. (2) Enzyme immobilization: Dissolve 100 parts by weight of lipase in 2000 parts by weight of phosphate buffer, add 0.6 parts by weight of sodium alginate as an auxiliary agent, and prepare enzyme solution; The functionalized resin and enzyme solution were mixed at a weight ratio of 1:22 and shaken at 30°C and 200 rpm for 360 min to form an enzyme-resin complex. (3) Post-treatment: The obtained enzyme-resin complex was washed three times with 450 parts by weight of buffer solution and dried under vacuum at 32℃ and -0.09 MPa to a water content of 4.5% to obtain immobilized lipase. (4) Catalytic application: Mixed oleic acid (long chain fatty acid) and glycerol (mass ratio 1:1), add 7% immobilized lipase, react at 45℃ and 0.10 MPa vacuum for 8h, and simultaneously add 25% composite dehydrating agent (molecular sieve: anhydrous magnesium sulfate = 1:1.5).

[0027] Example 2 A method for preparing immobilized lipase and its application in catalyzing the synthesis of glycerides. (1) Surface modification: 15 parts by weight of macroporous adsorption resin AB-8 were placed in a reactor, and 300 parts by weight of pH 8.5 phosphate buffer solution were added. The mixture was stirred at 400 rpm for 60 min at 70 °C and filtered to obtain a pretreated carrier. The carrier was immersed in 80 parts by weight of 15% 3-glycidyl ether oxypropyltrimethoxysilane solution and shaken at 200 rpm for 90 min at 55 °C to obtain functionalized resin. (2) Enzyme immobilization: Dissolve 50 parts by weight of lipase in 1000 parts by weight of phosphate buffer, add 0.3 parts by weight of sodium alginate as an auxiliary agent, and prepare enzyme solution; The functionalized resin and enzyme solution were mixed at a weight ratio of 1:30 and shaken at 35°C and 150 rpm for 480 min to form an enzyme-resin complex. (3) Post-treatment: The obtained enzyme-resin complex was washed three times with 500 parts by weight of buffer solution and dried at 28℃ and -0.10 MPa to a water content of 4.0% to obtain immobilized lipase; (4) Catalytic application: Mix octanoic acid (short-chain fatty acid), palmitic acid (medium-chain), and erucic acid (long-chain) (mass ratio 0.3:5:0.8), mix with glycerol at a ratio of 1:2, and add 3% immobilized enzyme. React at 90℃ and 0.35 MPa vacuum for 24 h, and add 15% composite dehydrating agent (molecular sieve: magnesium sulfate = 1:3).

[0028] Example 3 A method for preparing immobilized lipase and its application in catalyzing the synthesis of glycerides. (1) Surface modification: 20 parts by weight of macroporous adsorption resin AB-8 were placed in a reactor, and 400 parts by weight of pH 6.5 phosphate buffer solution were added. The mixture was stirred at 200 rpm for 180 min at 50 °C and filtered to obtain a pretreated carrier. The carrier was immersed in 300 parts by weight of 8% γ-aminopropyltriethoxysilane solution and shaken at 40 °C and 400 rpm for 240 min to obtain functionalized resin. (2) Enzyme immobilization: Dissolve 120 parts by weight of lipase in 3000 parts by weight of phosphate buffer, add 0.8 parts by weight of sodium alginate as an auxiliary agent, and prepare the enzyme solution. The functionalized resin and enzyme solution were mixed at a weight ratio of 1:15 and shaken at 25°C and 250 rpm for 240 min to form an enzyme-resin complex. (3) Post-treatment: The obtained enzyme-resin complex was washed three times with 450 parts by weight of buffer solution and dried at 35℃ and -0.08MPa to a water content of 3.8% to obtain immobilized lipase; (4) Catalytic application: Mix decanoic acid (short chain), lauric acid (medium chain), and dodecanoic acid (long chain) (mass ratio 2:1:8), and mix with glycerol at a ratio of 1:0.5, and add 10% immobilized enzyme. React at 65℃ and 0.25 MPa vacuum for 18h, and add 30% composite dehydrating agent (molecular sieve: magnesium sulfate = 1:0.5).

[0029] like Figure 1 As shown, the upper and lower images are electron microscope scans of the immobilized lipase and resin AB-8 of this invention, respectively. Compared with resin without enzyme loading, the surface of the immobilized lipase spheres of this invention is relatively smooth and dense, with only a few dents and fine textures. This indicates that the microstructure of the immobilized lipase loaded with enzyme in this invention has good uniformity, and the enzyme is densely distributed on the carrier surface with fewer defects. This morphological feature may mean a high degree of enzyme immobilization, which is beneficial for maintaining the enzyme's catalytic activity and stability.

[0030] Comparative Example 1 In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows: The surface modification step is omitted, and the unfunctionalized vector is used directly for enzyme immobilization.

[0031] Comparative Example 2 In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows: No sodium alginate was added to the enzyme solution.

[0032] Comparative Example 3 In this comparative example, the similarities with Example 3 will not be repeated, and the differences are as follows: The composite dehydrating agent was eliminated from the catalytic reaction and replaced with a single molecular sieve.

[0033] Comparative Example 4 In this comparative example, the similarities with Example 1 will not be repeated, and the differences are as follows: The diglyceride synthesis stage employs high temperature and high pressure conditions: the reaction is carried out at 80℃ and 0.30 MPa.

[0034] Comparative Example 5 In this comparative example, the similarities with Example 2 will not be repeated, and the differences are as follows: The fatty acid system uses only erucic acid (long-chain acid), without adding short-chain or medium-chain acids, and has a mass ratio of 1:1 with glycerol.

[0035] The performance test results and analysis are as follows: The performance of the obtained immobilized lipase was tested according to the parameters of the examples and comparative examples, and the test results are shown in Table 1.

[0036] In this application, the following general methods are used for testing: Enzyme loading: The difference in enzyme protein content before and after fixation was determined by the BCA method; Diglyceride selectivity: HPLC-ELSD detection of the proportion of DAG in the product; Long-chain acid conversion rate: erucic acid residue was determined by gas chromatography; Water removal efficiency: The condensate from the reactor is collected and weighed. Stability after repeated use: Residual enzyme activity was measured after 10 consecutive catalytic cycles; Resin specific surface area: BET nitrogen adsorption method.

[0037] As shown in Table 1, comparing the results of Example 1 and Comparative Example 1, surface modification increased the enzyme loading from 41.3 mg / g to 98.7 mg / g, an increase of 139%. The active groups formed by silanization anchor the enzyme molecule through covalent bonds, significantly reducing enzyme shedding. Comparative Example 1, lacking covalent bonding, showed only 63.4% enzyme activity after repeated use, confirming insufficient physical adsorption stability.

[0038] Table 1 Analysis of Test Results

[0039] Note: * indicates the proportion of TAG in the triglyceride synthesis mode.

[0040] Sodium alginate forms a protective layer on the enzyme surface, reducing the reusability stability of the unadded group in Comparative Example 2 by 16.4%. This adjuvant buffers mechanical shear forces through steric hindrance, reducing enzyme conformational changes. Notably, the erucic acid conversion rate in Comparative Example 2 was significantly reduced, indicating that the lack of a protective layer hindered the diffusion of long-chain acids. Utilizing the high molecular weight properties of sodium alginate to assist enzyme immobilization at the molecular level may form a protective layer or enhance binding, thereby synergistically improving the thermal stability, operational stability, and efficiency of the immobilized enzyme in high-temperature catalytic synthesis of glycerides while maintaining the excellent structure of macroporous resin, resulting in technical effects superior to those of a single carrier.

[0041] Compared with Comparative Example 3, the composite dehydrating agent achieved a dehydration rate of 11.7 g / h, which is 121% higher than that of the single molecular sieve (5.3 g / h). The synergistic effect of the molecular sieve capturing gaseous water and magnesium sulfate adsorbing liquid water shifts the esterification equilibrium towards the forward reaction direction, increasing the conversion rate of long-chain acids by 14.3 percentage points.

[0042] In Example 1, 73.5% DAG selectivity was achieved under a low temperature range of 35-65℃ and a low pressure of 0.06-0.15 MPa. In Comparative Example 4, the DAG content dropped sharply to 38.7% under high temperature and high pressure, confirming that high temperature accelerates acyl migration and promotes the conversion of diglycerides to triglycerides. This phenomenon is consistent with the kinetics of esterification reactions.

[0043] The mixed fatty acid system achieved an erucic acid conversion rate of 97.8%, compared to only 79.5% in the pure erucic acid system of Comparative Example 5. Short-chain acids enhanced the system's fluidity, while medium-chain acids promoted emulsification, synergistically reducing the diffusion resistance of long-chain acids. The interaction between the hydrophobic groups of the resin and the alkyl chains was more pronounced in the mixed system.

[0044] Phosphate buffer washing increased the resin's specific surface area to 580 m² / g, a 38% increase compared to untreated resin (approximately 420 m² / g), confirming that removing pore blockages effectively expands the enzyme-carrying space. Gradient drying (28-35℃) ensured enzyme activity retention >88%, avoiding the risk of high-temperature denaturation.

[0045] Test results show that this invention significantly improves the immobilization efficiency and binding stability of lipase by modifying the carrier surface with silanizing agents and constructing covalent anchoring sites on the resin surface. The introduction of sodium alginate forms a three-dimensional protective network, effectively buffering the mechanical shear force that damages the enzyme structure and extending the lifespan of the immobilized enzyme. A synergistic temperature and vacuum control mechanism is established, utilizing low-temperature conditions to inhibit acyl migration for the directional synthesis of diglycerides, and high-temperature environments to accelerate molecular motion and promote water molecule removal for the primary production of triglycerides. A composite dehydration system using molecular sieves and anhydrous magnesium sulfate is employed to reduce the system's water activity through multi-stage adsorption, promoting efficient esterification. The carrier pretreatment process is optimized to expand the resin's specific surface area and maintain mild, dry conditions, ensuring the initial activity of the enzyme. This invention enhances enzyme stability through covalent surface modification of the carrier combined with protective auxiliaries, precisely controls the composition of glycerol ester products using a temperature-vacuum synergistic mechanism, and strengthens reaction kinetics through a composite dehydration system.

[0046] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the preparation of an immobilized lipase, characterized in that, The method comprises the following steps: (1) surface modification: the pretreated carrier is immersed in 80-300 parts by weight of silanization solution to obtain a functionalized resin; (2) enzyme immobilization: the functionalized resin is put into an enzyme solution, the weight ratio of the functionalized resin to the enzyme solution is 1:15-30, and the enzyme-resin complex is formed by oscillation adsorption at 25-35°C and 150-250 rpm for 240-480 min; (3) post-treatment: the enzyme-resin complex is washed with 300-600 parts by weight of phosphate buffer and dried to a water content of ≤5% to obtain the finished product of immobilized lipase.

2. A process for the preparation of an immobilized lipase according to claim 1, characterized in that, In (1) surface modification, the pretreatment of the carrier comprises the following steps: 10-20 parts by weight of macroporous adsorption resin AB-8 is placed in a reactor, 100-400 parts by weight of phosphate buffer with a pH value of 6.5-8.5 is added, and stirring treatment is carried out at 50-70°C and a stirring speed of 200-400 rpm for 60-180 min. After completion, the solid resin is separated by filtration to obtain the pretreated carrier.

3. The method of claim 1, wherein the immobilized lipase is prepared by the steps of: In (1) surface modification, the silanization solution is prepared from γ-aminopropyl triethoxysilane or 3-glycidyl ether propyl trimethoxysilane with a concentration of 8%-15% by weight.

4. The method of claim 1, wherein the immobilized lipase is prepared by the steps of: In (1) surface modification, the pretreated carrier is immersed in the silanization solution, and the surface-activated functionalized resin is obtained by continuous treatment at a temperature of 40-55°C and an oscillation speed of 200-400 rpm for 90-240 min.

5. The method of claim 1, wherein the immobilized lipase is prepared by the steps of: In (2) enzyme immobilization, the enzyme solution is formed by dissolving 50-120 parts by weight of lipase in 1000-3000 parts by weight of phosphate buffer.

6. The method of claim 5, wherein the immobilized lipase is prepared by the steps of: The enzyme solution further comprises a sodium alginate auxiliary agent, and the addition amount of the sodium alginate auxiliary agent is 0.3-0.8 parts by weight.

7. The method of claim 1, wherein the immobilized lipase is prepared by the steps of: In (3) post-treatment, the drying is carried out by placing the washed product in a vacuum drying oven at 28-35°C and a pressure of -0.08 to -0.1 MPa until the water content is ≤5%.

8. An immobilized lipase obtainable by the process according to any one of claims 1 to 7, characterized in that, The immobilized lipase is used for catalytic synthesis of glycerides, including diglycerides and triglycerides.

9. Use of the immobilized lipase according to claim 8, characterized in that, The method for using the immobilized lipase to catalytically synthesize glycerides comprises the following steps: mixing a fatty acid system with glycerol at a mass ratio of 1:0.5-2, adding 3%-10% of the total mass of the substrate as immobilized lipase; then continuously reacting at a temperature of 35-65°C and a reactor vacuum degree of 0.06-0.15 MPa for 4-12 h to synthesize diglycerides; or continuously reacting at a temperature of 65-95°C and a reactor vacuum degree of 0.15-0.35 MPa for 12-24 h to obtain triglycerides; A composite water-removing agent is further added during the reaction, the composite water-removing agent is compounded from molecular sieves and anhydrous magnesium sulfate at a mass ratio of 1:0.5-3, and the addition amount of the composite water-removing agent is 15%-30% of the total mass of the substrate.

10. Use of immobilized lipase according to claim 9, characterized in that, The fatty acid system comprises short-chain fatty acids, medium-chain fatty acids, and long-chain fatty acids, and the mass ratio of the three is (0.3-2):(1-5):(0.8-8).

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