Immobilized lipase and application thereof in catalytic preparation of monoglyceride and diglyceride

Abstract

The invention discloses immobilized lipase and application of the immobilized lipase in catalytic preparation of monoglyceride and diglyceride, a preparation method of the immobilized lipase comprises the following steps: S1, reacting a carboxyl-containing high-molecular polymer with Fe3O4 magnetic nanoparticles and a glutaraldehyde solution to obtain a magnetic nanoparticle composite carrier; and S2, activating the magnetic nanoparticle composite carrier with an activator, and reacting with a lipase solution and a glutaraldehyde solution to obtain the immobilized lipase. The method comprises the following steps: immobilizing lipase in a magnetic nanoparticle composite carrier, and activating the magnetic nanoparticle composite carrier to obtain immobilized lipase; the immobilized lipase is used for catalyzing the reaction of glycerol and triglyceride, and the conversion rate of generating monoglyceride and diglyceride through enzymolysis of triglyceride can be greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological enzymes, and in particular relates to an immobilized lipase and an application thereof in catalytic preparation of monoglyceride and diglyceride. Background Art

[0002] Enzymatic transesterification refers to the use of enzyme preparations as catalysts. The enzymatic hydrolysis products of triglycerides mainly include diglycerides (DAG), monoglycerides (MAG), fatty acids (FFA), and glycerol.

[0003] Lipase is a kind of biological enzyme. The stability of lipase without carrier immobilization is poor. Therefore, the lipase currently used to catalyze the enzymatic hydrolysis of triglycerides often needs to be immobilized with a carrier. The main methods for immobilizing lipase include encapsulation, adsorption, cross-linking, covalent bonding, etc. Among them, the encapsulation and adsorption methods are connected between the enzyme and the carrier through weak interactions. Therefore, although the adsorption or encapsulation method can keep the enzyme activity high, the combination of the carrier and the lipase is not strong, and the lipase is easy to fall off the carrier. The covalent bonding method is an effective method for immobilizing lipase. A chemical bond is introduced between the lipase and the carrier, which can stabilize the connection between the lipase and the carrier.

[0004] However, the existing immobilized lipase prepared by covalent binding usually has low reaction activity between the carrier and the lipase, resulting in a small amount of lipase loaded on the carrier, which in turn leads to a low conversion rate of the immobilized lipase in catalyzing the enzymatic hydrolysis of triglycerides to monoglycerides and diglycerides. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, a method for preparing an immobilized lipase is provided. The immobilized lipase prepared by the method for preparing an immobilized lipase of the present invention can be used to catalyze the reaction of glycerol and triglycerides; when catalyzing the enzymatic hydrolysis of triglycerides, the conversion rate of enzymatic hydrolysis of triglycerides to generate monoglycerides and diglycerides can be greatly improved.

[0006] The object of the present invention is to provide a method for preparing immobilized lipase, comprising the following steps: S1. The carboxyl-containing polymer is reacted with Fe3O4 magnetic nanoparticles and glutaraldehyde solution to obtain a magnetic nanoparticle composite carrier; S2. Activating the magnetic nanoparticle composite carrier with an activator, and reacting it with a lipase solution and a glutaraldehyde solution to obtain immobilized lipase.

[0007] In some embodiments of the present invention, the structure of the carboxyl-containing polymer is as follows:

[0008] Wherein, n and m are both positive integers, n:m=1-3:1-5, and the relative molecular weight is 3000-5000.

[0009] In some embodiments of the present invention, the method for preparing the carboxyl-containing polymer comprises the following steps: (1) Mix maleic anhydride and water, add ammonium chloride, heat the reaction, and post-treat to obtain a polysuccinimide intermediate, add a strong alkali aqueous solution to obtain a polysuccinimide intermediate mixed solution; (2) Dissolve 2-aminoethanesulfonic acid and tryptophan in a strong alkaline aqueous solution respectively, add them to the polysuccinimide intermediate mixed solution, heat the reaction, and post-treat to obtain a carboxyl-containing high molecular polymer.

[0010] In some embodiments of the present invention, in (1), the mass ratio of maleic anhydride to ammonium chloride is 1:1.4-2.

[0011] In some embodiments of the present invention, in (1), the temperature of the temperature-raising reaction is 100-200° C., and the time is 0.5-1 hour.

[0012] In some embodiments of the present invention, in (1), the strong base is selected from at least one of sodium hydroxide and potassium hydroxide.

[0013] In some embodiments of the present invention, in (1), the mass concentration of the strong alkali aqueous solution is 8-12%.

[0014] In some embodiments of the present invention, in (2), the mass ratio of 2-aminoethanesulfonic acid, tryptophan and polysuccinimide intermediate is 1.3~1.7:1:3~5.

[0015] In some embodiments of the present invention, in (2), the temperature of the temperature-raising reaction is 25-35° C., and the time is 18-26 hours.

[0016] In some embodiments of the present invention, in (2), the mass concentration of the strong alkali aqueous solution is 8-12%.

[0017] In some embodiments of the present invention, in S1, the mass ratio of the carboxyl-containing high molecular polymer, Fe3O4 magnetic nanoparticles and glutaraldehyde solution is 4-9:3-7:5.

[0018] In some embodiments of the present invention, in S1, the mass concentration of the glutaraldehyde solution is 3-7%.

[0019] In some embodiments of the present invention, in S1, the reaction temperature is 25-40° C. and the reaction time is 6-12 h.

[0020] In some embodiments of the present invention, in S1, the lipase is selected from at least one of CALB lipase, TLL lipase and CALA lipase.

[0021] In some embodiments of the present invention, in S1, the preparation of the Fe3O4 magnetic nanoparticles comprises the following steps: Under the protection of nitrogen, ferrous chloride and ferric chloride are dissolved in water. 2+ and Fe 3+ The molar ratio is 1~2:1~2, the total concentration is 0.1~1 mol / L, the temperature is raised to 80~100℃, stirred and ammonia water is added dropwise to a pH of 9~11, the solid-liquid separation is performed, the mixture is washed with water and dried to obtain Fe3O4 magnetic nanoparticles.

[0022] In some embodiments of the present invention, in S2, the mass ratio of the magnetic nanoparticle composite carrier, the lipase solution and the glutaraldehyde solution is 1:4~8:0.3~0.7.

[0023] In some embodiments of the present invention, in S2, the mass concentration of the lipase solution is 0.5-2 mg / mL.

[0024] In some embodiments of the present invention, in S2, the mass concentration of the glutaraldehyde solution is 3-7%.

[0025] In some embodiments of the present invention, in S2, the activator is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dicyclohexylcarbodiimide.

[0026] In some embodiments of the present invention, in S2, the mass ratio of the magnetic nanoparticle composite carrier to the activator is 1:3-6.

[0027] In some embodiments of the present invention, in S2, the activation temperature is 35-50° C. and the activation time is 10-20 h.

[0028] In some embodiments of the present invention, in S2, the reaction temperature is 25-35° C. and the reaction time is 2-3 h.

[0029] Another object of the present invention is to provide an immobilized lipase, including one obtained by the preparation method of the immobilized lipase.

[0030] Another object of the present invention is to provide the use of the immobilized lipase in the preparation of monoglycerides and diglycerides for food.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The present invention activates the magnetic nanoparticle composite carrier so that the amount of lipase loaded on the carrier is large, and the activity of the immobilized lipase in catalyzing the enzymatic hydrolysis of triglycerides is improved. The immobilized lipase is used to catalyze the reaction of glycerol and triglycerides; when catalyzing the enzymatic hydrolysis of triglycerides, the conversion rate of the enzymatic hydrolysis of triglycerides to generate monoglycerides and diglycerides is greatly improved; the molecular structure of the prepared immobilized lipase contains a conjugated structure and a hydrogen bond effect, so that the immobilized lipase has a higher thermal stability. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] The CALB lipase, TLL lipase and CALA lipase used in the present invention are all commercially available.

[0034] The carboxyl-containing polymers used in the following examples and comparative examples are all from Shandong Chambroad Holding Group Co., Ltd. The specific structure of the carboxyl-containing polymer is the modified polyaspartic acid in Example 2 of Chinese patent CN118756144B.

[0035] Example 1 This embodiment provides a method for preparing immobilized lipase, and the specific preparation steps are as follows: S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:1 and the total concentration of 0.1 mol / L were raised to 80°C, stirred and ammonia water was added dropwise until the pH value was 9-11, solid-liquid separation was performed, washed with water and dried to obtain Fe3O4 magnetic nanoparticles; S2. 4 parts by mass of a carboxyl-containing polymer were mixed with 7 parts by mass of Fe3O4 magnetic nanoparticles and 5 parts by mass of a glutaraldehyde solution, the mass concentration of the glutaraldehyde solution was 7%, and the reaction was carried out at 25 ° C for 12h to obtain a magnetic nanoparticle composite carrier; S3. Mix 10 parts by mass of the magnetic nanoparticle composite carrier and 30 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, activate at 35°C for 20 hours, add 40 parts by mass of CALB lipase solution and 7 parts by mass of glutaraldehyde solution, the mass concentration of CALB lipase solution is 0.5 mg / mL, and the mass concentration of glutaraldehyde solution is 7%, react at 25°C for 3 hours, magnetically separate the immobilized lipase, wash with water and dry to obtain the immobilized lipase.

[0036] Example 2 This embodiment provides a method for preparing immobilized lipase, and the specific preparation steps are as follows: S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:1 and the total concentration of 0.1 mol / L were raised to 80°C, stirred and ammonia water was added dropwise until the pH value was 9-11, solid-liquid separation was performed, washed with water and dried to obtain Fe3O4 magnetic nanoparticles; S2. 9 parts by mass of a carboxyl-containing polymer and 3 parts by mass of Fe3O4 magnetic nanoparticles and 5 parts by mass of a glutaraldehyde solution were mixed, the mass concentration of the glutaraldehyde solution was 3%, and the reaction was carried out at 40 ° C for 6h to obtain a magnetic nanoparticle composite carrier; S3. Mix 10 parts by mass of the magnetic nanoparticle composite carrier and 60 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, activate at 50°C for 10 hours, add 80 parts by mass of CALB lipase solution and 3 parts by mass of glutaraldehyde solution, the mass concentration of CALB lipase solution is 2 mg / mL, and the mass concentration of glutaraldehyde solution is 3%, react at 35°C for 2 hours, magnetically separate the immobilized lipase, wash with water and dry to obtain the immobilized lipase.

[0037] Example 3 This embodiment provides a method for preparing immobilized lipase, and the specific preparation steps are as follows: S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:1 and the total concentration of 0.1 mol / L were raised to 80°C, stirred and ammonia water was added dropwise until the pH value was 9-11, solid-liquid separation was performed, washed with water and dried to obtain Fe3O4 magnetic nanoparticles; S2. 7 parts by mass of a carboxyl-containing polymer and 5 parts by mass of Fe3O4 magnetic nanoparticles and 5 parts by mass of a glutaraldehyde solution were mixed, the mass concentration of the glutaraldehyde solution was 5%, and the reaction was carried out at 35 ° C for 8h to obtain a magnetic nanoparticle composite carrier; S3. Mix 10 parts by mass of the magnetic nanoparticle composite carrier and 40 parts by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, activate at 40°C for 15 hours, add 60 parts by mass of CALB lipase solution and 5 parts by mass of glutaraldehyde solution, the mass concentration of CALB lipase solution is 1.5 mg / mL, and the mass concentration of glutaraldehyde solution is 5%, react at 30°C for 2.5 hours, magnetically separate the immobilized lipase, wash with water and dry to obtain the immobilized lipase.

[0038] Comparative Example 1 This comparative example provides a method for preparing immobilized lipase, which is different from Example 3 only in that the magnetic nanoparticle composite carrier is directly mixed with the lipase without being covalently linked. The specific preparation steps are as follows: S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:1 and the total concentration of 0.1 mol / L were raised to 80°C, stirred and ammonia water was added dropwise until the pH value was 9-11, solid-liquid separation was performed, washed with water and dried to obtain Fe3O4 magnetic nanoparticles; S2. 7 parts by mass of a carboxyl-containing polymer and 5 parts by mass of Fe3O4 magnetic nanoparticles and 5 parts by mass of a glutaraldehyde solution were mixed, the mass concentration of the glutaraldehyde solution was 5%, and the reaction was carried out at 35 ° C for 8h to obtain a magnetic nanoparticle composite carrier; S3. Mix 10 parts by mass of the magnetic nanoparticle composite carrier and 60 parts by mass of CALB lipase solution, wherein the mass concentration of the CALB lipase solution is 1.5 mg / mL, and dry to obtain immobilized lipase.

[0039] Comparative Example 2 This comparative example provides a method for preparing immobilized lipase, which is different from Example 3 only in that the magnetic nanoparticle composite carrier is not activated. The specific preparation steps are as follows: S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:1 and the total concentration of 0.1 mol / L were raised to 80°C, stirred and ammonia water was added dropwise until the pH value was 9-11, solid-liquid separation was performed, washed with water and dried to obtain Fe3O4 magnetic nanoparticles; S2. 7 parts by mass of a carboxyl-containing polymer and 5 parts by mass of Fe3O4 magnetic nanoparticles and 5 parts by mass of a glutaraldehyde solution were mixed, the mass concentration of the glutaraldehyde solution was 5%, and the reaction was carried out at 35 ° C for 8h to obtain a magnetic nanoparticle composite carrier; S3. Add 60 parts by mass of CALB lipase solution and 5 parts by mass of glutaraldehyde solution to 10 parts by mass of the magnetic nanoparticle composite carrier, the mass concentration of CALB lipase solution is 1.5 mg / mL, the mass concentration of glutaraldehyde solution is 5%, react at 30°C for 2.5h, magnetically separate the immobilized lipase, wash with water and dry to obtain immobilized lipase.

[0040] Example 4 This embodiment provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Example 1 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 61%, and the content of diglyceride was 30.5%, that is, the conversion rate of triglyceride was 91.5%.

[0041] Example 5 This embodiment provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Example 2 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 62%, and the content of diglyceride was 31.2%, that is, the conversion rate of triglyceride was 93.2%.

[0042] Example 6 This embodiment provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Example 3 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 61.6%, and the content of diglyceride was 30.9%, that is, the conversion rate of triglyceride was 92.5%.

[0043] Comparative Example 3 This comparative example provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: 10 mmol of triglyceride and 4 mmol of glycerol were evenly mixed, 0.5 mmol of immobilized lipase prepared in Comparative Example 1 was added, the mixture was reacted at 200 rpm and 50° C. for 12 h, and the product was filtered. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 43.7%, and the content of diglyceride was 18.9%, that is, the conversion rate of triglyceride was 62.6%.

[0044] Comparative Example 4 This comparative example provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: 10 mmol of triglyceride and 4 mmol of glycerol were evenly mixed, 0.5 mmol of immobilized lipase prepared in Comparative Example 2 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 50.8%, and the content of diglyceride was 20.3%, that is, the conversion rate of triglyceride was 71.1%.

[0045] Example 7 This embodiment provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: S1. The immobilized lipase prepared in Example 1 was placed in a water bath at 60° C. for 0.5 h to obtain an immobilized lipase after high temperature heat treatment; S2. 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Example 1 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered to obtain the product. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 56.4%, and the content of diglyceride was 28.6%, that is, the conversion rate of triglyceride was 85%.

[0046] Example 8 This embodiment provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: S1. The immobilized lipase prepared in Example 2 was placed in a water bath at 60° C. for 0.5 h to obtain an immobilized lipase after high temperature heat treatment; S2. 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Example 2 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered to obtain the product. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 57.5%, and the content of diglyceride was 28.9%, that is, the conversion rate of triglyceride was 86.4%.

[0047] Example 9 This embodiment provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: S1. The immobilized lipase prepared in Example 3 was placed in a water bath at 60° C. for 0.5 h to obtain an immobilized lipase after high temperature heat treatment; S2. 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Example 3 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered to obtain the product. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 57.2%, and the content of diglyceride was 28.1%, that is, the conversion rate of triglyceride was 85.3%.

[0048] Comparative Example 5 This comparative example provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: S1. The immobilized lipase prepared in Comparative Example 1 was placed in a water bath at 60° C. for 0.5 h to obtain an immobilized lipase after high temperature heat treatment; S2. 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Comparative Example 1 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered to obtain the product. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 31.3%, and the content of diglyceride was 10.4%, that is, the conversion rate of triglyceride was 41.7%.

[0049] Comparative Example 6 This comparative example provides a method for preparing monoglyceride and diglyceride by catalysis of immobilized lipase, and the specific preparation steps are as follows: S1. The immobilized lipase prepared in Comparative Example 2 was placed in a water bath at 60° C. for 0.5 h to obtain an immobilized lipase after high temperature heat treatment; S2. 10 mmol of triglyceride and 4 mmol of glycerol were mixed evenly, 0.5 mmol of immobilized lipase prepared in Comparative Example 2 was added, the mixture was reacted at 200 rpm and 50°C for 12 h, and the product was filtered to obtain the product. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD. The content of monoglyceride was 39.7%, and the content of diglyceride was 13.6%, that is, the conversion rate of triglyceride was 53.3%.

[0050] By comparing Examples 4 to 6 and Comparative Examples 3 to 4, it can be seen that the conversion rate of the lipases in Examples 4 to 6 of the present invention to generate monoglycerides and diglycerides by catalyzing the enzymatic hydrolysis of triglycerides can reach more than 91.5%, while the conversion rate of the lipases in Comparative Examples 3 to 4 to generate monoglycerides and diglycerides by catalyzing the enzymatic hydrolysis of triglycerides is much lower than that in Examples 4 to 6. It can be seen that the lipases in Examples 1 to 3 of the present invention have higher enzyme activities for catalyzing the enzymatic hydrolysis of triglycerides to prepare monoglycerides and diglycerides.

[0051] By comparing Examples 7 to 9 and Comparative Examples 5 to 6, it can be seen that the lipases of Examples 1 to 3 of the present invention have high thermal stability. After being treated at 60° C. for 0.5 h, the conversion rate of preparing monoglyceride and diglyceride by enzymatic hydrolysis of triglyceride catalyzed by the lipases of Examples 7 to 9 is basically unchanged, while the thermal stability of the lipases of Comparative Examples 1 to 2 is relatively low, and the conversion rate of preparing monoglyceride and diglyceride by enzymatic hydrolysis of triglyceride catalyzed by the lipases of Comparative Examples 5 to 6 is much lower than that of Examples 7 to 9. It can be seen that the lipases of Examples 1 to 3 of the present invention have high thermal stability.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that after reading the specification of this application, the technicians can still modify or replace the specific implementation mode of the present invention with equivalents, but these modifications or changes do not deviate from the scope of protection of the pending claims of the present application.

Claims

1. A method for preparing immobilized lipase, characterized in that: The steps include: S1. The carboxyl-containing polymer is reacted with Fe3O4 magnetic nanoparticles and glutaraldehyde solution to obtain a magnetic nanoparticle composite carrier; S2. The magnetic nanoparticle composite carrier is activated by an activator, and then reacted with a lipase solution and a glutaraldehyde solution to obtain an immobilized lipase; In S1, the structure of the carboxyl-containing polymer is as follows: Wherein, n and m are both positive integers, n:m=1-3:1-5, and the relative molecular weight is 3000-5000; In S2, the lipase is selected from at least one of CALB lipase, TLL lipase and CALA lipase; In S2, the activation temperature is 35-50°C and the activation time is 10-20h; In S2, the reaction temperature is 25-35°C and the reaction time is 2-3h.

2. The method for preparing immobilized lipase according to claim 1, characterized in that: In S1, the mass ratio of the carboxyl-containing high molecular polymer, Fe3O4 magnetic nanoparticles and glutaraldehyde solution is 4-9:3-7:

5.

3. The method for preparing immobilized lipase according to claim 1, characterized in that: In S1, the mass concentration of the glutaraldehyde solution is 3-7%.

4. The method for preparing immobilized lipase according to claim 1, characterized in that: In S1, the reaction temperature is 25-40°C and the reaction time is 6-12 hours.

5. The method for preparing immobilized lipase according to claim 1, characterized in that: In S2, the mass ratio of the magnetic nanoparticle composite carrier, the lipase solution and the glutaraldehyde solution is 1:4-8:0.3-0.

7.

6. The method for preparing immobilized lipase according to claim 1, characterized in that: In S2, the mass concentration of the lipase solution is 0.5-2 mg / mL; The mass concentration of the glutaraldehyde solution is 3-7%.

7. The method for preparing immobilized lipase according to claim 1, characterized in that: In S2, the activator is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dicyclohexylcarbodiimide.

8. The method for preparing immobilized lipase according to claim 1, characterized in that: In S2, the mass ratio of the magnetic nanoparticle composite carrier to the activator is 1:3-6.

9. An immobilized lipase, characterized in that: The immobilized lipase is prepared by the preparation method of any one of claims 1 to 8.

10. Use of the immobilized lipase according to claim 9 in the preparation of monoglycerides and diglycerides for food.

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