Process for the enzymatic synthesis of isosorbide esters

Abstract

The present application relates to a method for synthesizing isosorbide ester by enzyme, and belongs to the technical field of organic synthesis. In a reaction kettle, isosorbide, fatty acid, immobilized lipase and solvent are added, and after stirring reaction, the immobilized lipase is separated from the reaction mixture by filtering through a Buchner funnel, and then is washed with solvent and used for the next batch of reaction; the filtrate is subjected to vacuum distillation to remove the solvent to obtain a crude product, which is purified by silica gel column chromatography, and then concentrated under reduced pressure to obtain high-purity isosorbide ester; the immobilized lipase is prepared by using an epoxy carrier, a crosslinking agent, a buffer and a lipase; and the present application can efficiently and greenly prepare high-purity isosorbide ester, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a method for the enzymatic synthesis of isosorbide esters. Background Technology

[0002] Isosorbide dimethyl esters are an important organic compound with wide applications in plastics, cosmetics, pharmaceuticals and many other fields.

[0003] Traditional methods for synthesizing isosorbide esters mostly employ chemical catalysis. However, chemical catalysis processes often require harsh reaction conditions such as high temperature and high pressure, and the catalysts are difficult to recover, easily generating large amounts of waste liquid and putting significant pressure on the environment. With the continuous development of green chemistry concepts, developing a mild, efficient, and environmentally friendly method for synthesizing isosorbide esters has become a key research direction.

[0004] The patent with authorization announcement number CN112724154B discloses an isosorbide ester plasticizer and its preparation method. The method involves dissolving isosorbide in an ionic liquid, adding a monocarboxylic acid and a catalyst, stirring while purging with nitrogen, reacting in the presence of microwaves at 40-95℃, cooling after the reaction is completed, and obtaining the isosorbide ester plasticizer by vacuum distillation.

[0005] Patent application CN106632369A discloses a method for preparing isosorbide dicarboxylate. The method uses sorbitol as a raw material, and a dehydration reaction is carried out at 100-180°C under a N2 atmosphere with the aid of a dehydration catalyst for 0.5-48 hours. Then, a monobasic acid and an esterification catalyst are added, and the reaction temperature is increased to 185-250°C for esterification for 0.5-48 hours. The isosorbide dicarboxylate product is obtained by vacuum distillation.

[0006] Patent application CN119306734A discloses a bio-based isosorbide carbonate plasticizer, its preparation method, and its application. The preparation method of the bio-based isosorbide carbonate plasticizer includes: carrying out an ester exchange reaction with carbonate diester, isosorbide, and catalyst under a protective gas atmosphere; after the reaction is completed, post-treatment is performed to obtain the isosorbide carbonate plasticizer.

[0007] Enzymes, as biocatalysts, possess significant advantages such as high efficiency, specificity, and mild reaction conditions, making the enzymatic synthesis of isosorbide esters increasingly attractive. However, existing enzymatic synthesis techniques for isosorbide esters still have some problems, such as poor enzyme stability, easy inactivation during the reaction leading to decreased catalytic efficiency; unsatisfactory enzyme-substrate compatibility affecting the reaction; and difficulties in enzyme recovery and reuse after the reaction, increasing production costs. Therefore, it is necessary to further improve and optimize the enzymatic synthesis methods for isosorbide esters. Summary of the Invention

[0008] To address the above problems, this invention provides a method for the enzymatic synthesis of isosorbide esters, the operation steps of which are as follows, in parts by mass:

[0009] Construction of S1 reaction system: In the reaction vessel, add 8-12 parts isosorbide, 18-30 parts fatty acid, 1-3 parts immobilized lipase in sequence, and then add 80-120 parts solvent. Stir with a paddle stirrer at a speed of 200-300r / min for 15-30 minutes to make the reaction system uniformly mixed.

[0010] S2 reaction conditions control: The reaction system is placed in a constant temperature shaking water bath and reacted for 12-36 hours at 40-60℃ and shaking speed of 150-250r / min.

[0011] S3 Product Separation and Enzyme Recovery: After the reaction, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed 3-5 times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate at a volume ratio of (5-10):1) as the eluent at a flow rate of 1-5 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0012] The immobilized lipase was prepared by reacting an epoxy-based carrier, a cross-linking agent, a buffer solution, and the lipase, followed by vacuum filtration and freeze-drying.

[0013] The crosslinking agent is composed of 4-epoxypropoxycarbazole and triethylamine.

[0014] As a preferred embodiment of the present invention, the fatty acid is one of oleic acid, stearic acid, and lauric acid.

[0015] As a preferred embodiment of the present invention, the solvent is n-hexane or tert-butanol.

[0016] As a preferred embodiment of the present invention, the pressure of the S3 vacuum distillation is 10-20 mmHg and the temperature is 40-50℃.

[0017] As a preferred embodiment of the present invention, the method for preparing the immobilized lipase is as follows, according to parts by mass:

[0018] Enzyme covalent immobilization and primary network construction: 80-100 parts of epoxy-based support were placed in a reactor equipped with a stirrer and a temperature control system. 15-25 parts of crosslinking agent and 1000-1200 parts of buffer solution with a pH of 7.8-8.2 were added. The mixture was stirred at a stirring rate of 300-400 r / min and reacted at 55-70℃ for 3-6 hours. After the reaction was completed, the mixture was cooled to room temperature, and 8-12 parts of lipase were added. The mixture was reacted at 25-30℃ for 8-14 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at a temperature of -50~-40℃ and a pressure of 10-20 Pa for 12-24 hours to obtain immobilized lipase.

[0019] As a preferred embodiment of the present invention, the crosslinking agent is composed of 12-20 parts of 4-epoxypropoxycarbazole and 0.5-1.5 parts of triethylamine.

[0020] As a preferred embodiment of the present invention, the epoxy-based support is selected from one of ES-101, ES-102, and ES-103.

[0021] As a preferred embodiment of the present invention, the buffer solution is one of phosphate buffer, tris-HCl, and glycine buffer.

[0022] As a preferred embodiment of the present invention, the lipase is one of the lipases derived from Candida albicans, Aspergillus oryzae, and Aspergillus niger.

[0023] Reaction Mechanism: This invention uses an epoxy-based carrier as the carrier and specific epoxy-based carriers, cross-linking agents, and reaction conditions to firmly fix lipase on the carrier, forming a stable structure. This effectively prevents lipase loss during the reaction process and improves the stability and lifespan of lipase.

[0024] The enzymatic synthesis method for isosorbide esters of the present invention has the following significant advantages compared with the prior art:

[0025] 1. The carbazole group enhances the binding strength between the carrier and the enzyme. Its active epoxy groups can covalently react with the amino and thiol groups of lipase in an alkaline buffer environment, forming stable chemical bonds, preventing enzyme detachment during the reaction and improving reusability. Simultaneously, as part of the cross-linking agent, it constructs a primary network structure with the epoxy-based carrier. The rigid carbazole ring increases steric hindrance in the network, forming a dense three-dimensional structure that protects the enzyme's active site from external interference.

[0026] 2. The carbazole group helps improve the stability of immobilized enzymes. Its hydrophobic structure can interact with the hydrophobic regions of lipases to form a local hydrophobic microenvironment, enhance the rigidity of the enzyme protein, improve its tolerance to factors such as temperature and pH, reduce thermal denaturation, and extend its service life. Furthermore, the network of carbazole groups on the surface of the immobilized enzyme can hinder the contact between the protease and the enzyme molecule, reduce the risk of enzyme degradation, and enable the immobilized enzyme to maintain high activity in continuous reactions.

[0027] 3. Facilitates enzyme recovery and reuse: After the reaction is complete, the immobilized lipase can be separated from the reaction product by simple filtration and can be directly reused after washing.

[0028] 4. Green and environmentally friendly: The entire synthesis process operates under mild reaction conditions, avoiding harsh conditions such as high temperature and high pressure, thus reducing energy consumption. Furthermore, the organic solvent can be recovered and recycled through vacuum distillation, and no large amounts of waste liquid are generated, meeting the requirements of green chemistry development. Detailed Implementation

[0029] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments, is provided below.

[0030] Example 1: A method for the enzymatic synthesis of isosorbide esters, the operation steps of which are as follows:

[0031] Construction of S1 reaction system: In the reaction vessel, add 8g isosorbide, 18g fatty acid, 1g immobilized lipase in sequence, then add 80g solvent, and stir with a paddle stirrer at a speed of 200r / min for 15 minutes to make the reaction system uniformly mixed.

[0032] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 12 hours at 40℃ and shaking speed of 150r / min.

[0033] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed three times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the eluent at a flow rate of 1 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0034] The fatty acid mentioned is oleic acid.

[0035] The solvent is n-hexane.

[0036] The pressure of the S3 vacuum distillation is 10 mmHg and the temperature is 40℃.

[0037] The method for preparing the immobilized lipase is as follows:

[0038] Enzyme covalent immobilization and primary network construction: 80g of epoxy-based support was placed in a reactor equipped with a stirrer and a temperature control system. 15g of cross-linking agent and 1000g of pH 7.8 buffer solution were added. The mixture was stirred at 300r / min and reacted at 55℃ for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, and 8g of lipase was added. The reaction was continued at 25℃ for 8 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at -50℃ and 10Pa for 12 hours to obtain immobilized lipase.

[0039] The crosslinking agent consists of 12g of 4-epoxypropoxycarbazole and 0.5g of triethylamine.

[0040] The epoxy-based support is selected from ES-101.

[0041] The buffer solution is a phosphate buffer.

[0042] The lipase mentioned is a lipase derived from Candida albicans.

[0043] Example 2: A method for the enzymatic synthesis of isosorbide esters, the operation steps of which are as follows:

[0044] Construction of S1 reaction system: In the reaction vessel, 9g isosorbide, 22g fatty acid, 2g immobilized lipase were added in sequence, followed by 90g solvent. The mixture was stirred at 250r / min for 20 minutes using a paddle stirrer to ensure that the reaction system was mixed evenly.

[0045] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 20 h at 45℃ and shaking speed of 200 r / min;

[0046] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed four times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 6:1 as the eluent at a flow rate of 2 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0047] The fatty acid mentioned is stearic acid.

[0048] The solvent is n-hexane.

[0049] The pressure of the S3 vacuum distillation is 15 mmHg, and the temperature is 45℃.

[0050] The method for preparing the immobilized lipase is as follows:

[0051] Enzyme covalent immobilization and primary network construction: 85g of epoxy-based support was placed in a reactor equipped with a stirrer and a temperature control system, and 18g of cross-linking agent and 1050g of pH 8 buffer solution were added. The mixture was stirred at 350r / min and reacted at 60℃ for 4 hours. After the reaction was completed, the mixture was cooled to room temperature, and 9g of lipase was added. The reaction was continued at 25℃ for 10 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at -45℃ and 15Pa for 16 hours to obtain immobilized lipase.

[0052] The crosslinking agent consists of 14g of 4-epoxypropoxycarbazole and 0.8g of triethylamine.

[0053] The epoxy-based support is selected from ES-102.

[0054] The buffer solution is tris-HCl.

[0055] The lipase mentioned is a lipase derived from Aspergillus oryzae.

[0056] Example 3: A method for the enzymatic synthesis of isosorbide esters, the operation steps of which are as follows:

[0057] Construction of S1 reaction system: In the reaction vessel, 11g isosorbide, 27g fatty acid, 2g immobilized lipase were added in sequence, followed by 110g solvent. The mixture was stirred at 250r / min for 25 minutes using a paddle stirrer to ensure that the reaction system was mixed evenly.

[0058] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 30 h at 55℃ and shaking speed of 200 r / min;

[0059] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered using a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed four times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 8:1 as the eluent at a flow rate of 4 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0060] The fatty acid mentioned is stearic acid.

[0061] The solvent is tert-butanol.

[0062] The pressure of the S3 vacuum distillation is 15 mmHg, and the temperature is 45℃.

[0063] The method for preparing the immobilized lipase is as follows:

[0064] Enzyme covalent immobilization and primary network construction: 95g of epoxy-based support was placed in a reactor equipped with a stirrer and a temperature control system, and 23g of cross-linking agent and 1150g of pH 8 buffer solution were added. The mixture was stirred at 350r / min and reacted at 65℃ for 5 hours. After the reaction was completed, the mixture was cooled to room temperature, and 11g of lipase was added. The reaction was continued at 30℃ for 12 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at -45℃ and 15Pa for 20 hours to obtain immobilized lipase.

[0065] The crosslinking agent consists of 18g of 4-epoxypropoxycarbazole and 1.3g of triethylamine.

[0066] The epoxy-based support is selected from ES-102.

[0067] The buffer solution is tris-HCl.

[0068] The lipase mentioned is a lipase derived from Aspergillus oryzae.

[0069] Example 4: A method for the enzymatic synthesis of isosorbide esters, the operation steps of which are as follows:

[0070] Construction of S1 reaction system: In the reaction vessel, 12g isosorbide, 30g fatty acid, 3g immobilized lipase were added in sequence, followed by 120g solvent. The mixture was stirred at 300r / min for 30 minutes using a paddle stirrer to ensure that the reaction system was mixed evenly.

[0071] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 36 hours at 60℃ and shaking speed of 250r / min.

[0072] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed five times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:1 as the eluent at a flow rate of 5 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0073] The fatty acid mentioned is lauric acid.

[0074] The solvent is tert-butanol.

[0075] The pressure of the S3 vacuum distillation is 20 mmHg, and the temperature is 50°C.

[0076] The method for preparing the immobilized lipase is as follows:

[0077] Enzyme covalent immobilization and primary network construction: 100g of epoxy-based support was placed in a reactor equipped with a stirrer and a temperature control system. 25g of cross-linking agent and 1200g of pH 8.2 buffer solution were added. The mixture was stirred at 400r / min and reacted at 70℃ for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, and 12g of lipase was added. The reaction was continued at 30℃ for 14 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at -40℃ and 20Pa for 24 hours to obtain immobilized lipase.

[0078] The crosslinking agent is composed of 20g of 4-epoxypropoxycarbazole and 1.5g of triethylamine.

[0079] The epoxy-based support is selected from ES-103.

[0080] The buffer solution is a glycine buffer solution.

[0081] The lipase mentioned is a lipase derived from Aspergillus niger.

[0082] Comparative Example 1: The enzymatic synthesis method for isosorbide esters, the operation steps of which are as follows:

[0083] Construction of S1 reaction system: In the reaction vessel, add 8g isosorbide, 18g fatty acid, 1g lipase, and then add 80g solvent. Stir with a paddle stirrer at 200r / min for 15 minutes to make the reaction system uniformly mixed.

[0084] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 12 hours at 40℃ and shaking speed of 150r / min.

[0085] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered through a Buchner funnel to separate the lipase. The lipase was washed three times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the eluent at a flow rate of 1 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0086] The fatty acid mentioned is oleic acid.

[0087] The solvent is n-hexane.

[0088] The pressure of the S3 vacuum distillation is 10 mmHg and the temperature is 40℃.

[0089] The lipase mentioned is a lipase derived from Candida albicans.

[0090] Comparative Example 2: The enzymatic synthesis method for isosorbide esters, the operation steps of which are as follows:

[0091] Construction of S1 reaction system: In the reaction vessel, add 8g isosorbide, 18g fatty acid, 1g immobilized lipase in sequence, then add 80g solvent, and stir with a paddle stirrer at a speed of 200r / min for 15 minutes to make the reaction system uniformly mixed.

[0092] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 12 hours at 40℃ and shaking speed of 150r / min.

[0093] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed three times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the eluent at a flow rate of 1 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0094] The fatty acid mentioned is oleic acid.

[0095] The solvent is n-hexane.

[0096] The pressure of the S3 vacuum distillation is 10 mmHg and the temperature is 40℃.

[0097] The method for preparing the immobilized lipase is as follows:

[0098] Enzyme covalent immobilization and primary network construction: 15g of crosslinking agent was placed in a reactor equipped with a stirrer and a temperature control system, and 1000g of buffer solution with a pH of 7.8 was added. The mixture was stirred at a stirring rate of 300r / min and reacted at 55℃ for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, and 8g of lipase was added. The mixture was then reacted at 25℃ for another 8 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at -50℃ and 10Pa for 12 hours to obtain immobilized lipase.

[0099] The crosslinking agent consists of 12g of 4-epoxypropoxycarbazole and 0.5g of triethylamine.

[0100] The buffer solution is a phosphate buffer.

[0101] The lipase mentioned is a lipase derived from Candida albicans.

[0102] Comparative Example 3: The enzymatic synthesis method for isosorbide esters, the operation steps of which are as follows:

[0103] Construction of S1 reaction system: In the reaction vessel, add 8g isosorbide, 18g fatty acid, 1g immobilized lipase in sequence, then add 80g solvent, and stir with a paddle stirrer at a speed of 200r / min for 15 minutes to make the reaction system uniformly mixed.

[0104] S2 reaction conditions control: The reaction system was placed in a constant temperature shaking water bath and reacted for 12 hours at 40℃ and shaking speed of 150r / min.

[0105] S3 Product Separation and Enzyme Recovery: After the reaction was completed, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed three times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1 as the eluent at a flow rate of 1 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester.

[0106] The fatty acid mentioned is oleic acid.

[0107] The solvent is n-hexane.

[0108] The pressure of the S3 vacuum distillation is 10 mmHg and the temperature is 40℃.

[0109] The method for preparing the immobilized lipase is as follows:

[0110] Enzyme covalent immobilization and primary network construction: 80g of epoxy-based support was placed in a reaction vessel equipped with a stirrer and a temperature control system, and 1000g of pH 7.8 buffer solution was added. The mixture was stirred at 300r / min and reacted at 55℃ for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, and 8g of lipase was added. The reaction was continued at 25℃ for 8 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at -50℃ and 10Pa for 12 hours to obtain immobilized lipase.

[0111] The epoxy-based support is selected from ES-101.

[0112] The buffer solution is a phosphate buffer.

[0113] The lipase mentioned is a lipase derived from Candida albicans.

[0114] Purity test of isosorbide esters: determined by gas chromatography-mass spectrometry.

[0115] Table 1: Test results of each embodiment and comparative example

[0116]

[0117] Based on the data analysis of the above examples and comparative examples, the present invention can efficiently and environmentally prepare high-purity isosorbide esters, and has good application prospects.

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for the enzymatic synthesis of isosorbide esters, characterized in that: The operating steps are as follows, according to the mass fraction: Construction of S1 reaction system: In the reaction vessel, add 8-12 parts isosorbide, 18-30 parts fatty acid, 1-3 parts immobilized lipase in sequence, and then add 80-120 parts solvent. Stir with a paddle stirrer at a speed of 200-300r / min for 15-30 minutes to make the reaction system uniformly mixed. S2 reaction conditions control: The reaction system is placed in a constant temperature shaking water bath and reacted for 12-36 hours at 40-60℃ and shaking speed of 150-250r / min. S3 Product Separation and Enzyme Recovery: After the reaction, the reaction mixture was filtered through a Buchner funnel to separate the immobilized lipase. The immobilized lipase was washed 3-5 times with solvent and used in the next batch of reaction. The filtrate was distilled under reduced pressure to remove the solvent and obtain the crude product. The crude product was purified by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate at a volume ratio of (5-10):1) as the eluent at a flow rate of 1-5 mL / min. The eluent containing the target product was collected and concentrated under reduced pressure to obtain high-purity isosorbide ester. The immobilized lipase was prepared by reacting an epoxy-based carrier, a cross-linking agent, a buffer solution, and the lipase, followed by vacuum filtration and freeze-drying. The crosslinking agent is composed of 4-epoxypropoxycarbazole and triethylamine; The method for preparing the immobilized lipase is as follows, according to parts by mass: Enzyme covalent immobilization and primary network construction: 80-100 parts of epoxy-based support were placed in a reactor equipped with a stirrer and a temperature control system. 15-25 parts of crosslinking agent and 1000-1200 parts of buffer solution with a pH of 7.8-8.2 were added. The mixture was stirred at a stirring rate of 300-400 r / min and reacted at 55-70℃ for 3-6 hours. After the reaction was completed, the mixture was cooled to room temperature, and 8-12 parts of lipase were added. The reaction was continued at 25-30℃ for 8-14 hours. The mixture was then vacuum filtered, and the filtered solid was freeze-dried at a temperature of -50~-40℃ and a pressure of 10-20 Pa for 12-24 hours to obtain immobilized lipase. The crosslinking agent consists of 12-20 parts of 4-epoxypropoxycarbazole and 0.5-1.5 parts of triethylamine.

2. The method for enzymatic synthesis of isosorbide esters according to claim 1, characterized in that: The fatty acid mentioned is one of oleic acid, stearic acid, and lauric acid.

3. The method for enzymatic synthesis of isosorbide esters according to claim 1, characterized in that: The solvent is n-hexane or tert-butanol.

4. The method for enzymatic synthesis of isosorbide ester according to claim 1, characterized in that: The pressure of the S3 vacuum distillation is 10-20 mmHg, and the temperature is 40-50℃.

5. The method for enzymatic synthesis of isosorbide esters according to claim 1, characterized in that: The epoxy-based support is selected from one of ES-101, ES-102, and ES-103.

6. The method for enzymatic synthesis of isosorbide esters according to claim 1, characterized in that: The buffer solution is one of phosphate buffer, tris-HCl, or glycine buffer.

7. The method for enzymatic synthesis of isosorbide esters according to claim 1, characterized in that: The lipase mentioned is derived from one of the lipases of Candida albicans, Aspergillus oryzae, and Aspergillus niger.

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