A high-stability immobilized lipase and its catalytic hydrolysis to prepare diglyceride

Abstract

The present invention discloses a high-stability immobilized lipase and a catalytic hydrolysis method thereof for preparing diglyceride. The preparation method of the high-stability immobilized lipase comprises the following steps: S1. reacting a modified acrylate resin represented by formula (I) with Fe 3 O 4 The magnetic nanoparticles react with a glutaraldehyde solution to obtain a magnetic nanoparticle composite carrier; S2. The magnetic nanoparticle composite carrier is activated with an activator for carboxyl groups, and then the lipase solution is reacted with a glutaraldehyde solution to obtain a highly stable immobilized lipase; The present invention subjects the magnetic nanoparticle composite carrier to an activation treatment to obtain an immobilized lipase, and the immobilized lipase has high stability. After being repeatedly used for 20 times, it still retains 91% of the initial enzyme activity, and catalyzes the hydrolysis of triglycerides to obtain a diglyceride containing 35%.

Description

Technical Field

[0001] The invention belongs to the technical field of biological enzymes, and in particular relates to a high-stability immobilized lipase and a catalytic hydrolysis thereof for preparing diglyceride. Background Art

[0002] Diacylglycerol is a functional oil with a similar taste, flavor and energy to ordinary oils, but it does not produce fat accumulation after being ingested in the human body. It has the effect of weight loss and can prevent the occurrence of obesity-related diseases. Therefore, the research and development of diacylglycerol has a good application prospect. The preparation of diacylglycerol mainly includes chemical method and bioenzyme method. The chemical method is carried out under high temperature conditions, with high energy consumption, dark product color, and is not suitable for the production of glycerides rich in polyunsaturated fatty acids. The bioenzyme method has many advantages, such as mild reaction conditions, green process, few by-products, and is suitable for the production of glycerides containing heat-sensitive polyunsaturated fatty acids. Therefore, the bioenzyme method for preparing diacylglycerol has greater prospects.

[0003] Lipase is a kind of biological enzyme. The stability of lipase without carrier immobilization is poor. Therefore, the lipase currently used for preparing diglyceride is often immobilized with a carrier. The main methods for immobilizing lipase include embedding method, adsorption method, cross-linking method, covalent bonding method, etc. Among them, the embedding method and adsorption method are that the enzyme and the carrier are connected through weak interactions. Therefore, although the adsorption method or embedding method can keep the enzyme at a high enzyme activity, 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 method 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 low activity of the immobilized lipase in catalyzing the preparation of diglyceride; in addition, when the immobilized lipase is prepared by covalent binding method, the introduction of the carrier usually results in low catalytic activity of the lipase. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, a method for preparing a highly stable immobilized lipase is provided. The present invention subjects a magnetic nanoparticle composite carrier to an activation treatment to obtain an immobilized lipase. The immobilized lipase has high stability and can retain 91% of the initial enzyme activity after repeated use for 20 times. It catalyzes the hydrolysis of triglycerides to obtain a diglyceride containing 35%.

[0006] The object of the present invention is to provide a method for preparing a highly stable immobilized lipase, comprising the following steps:

[0007] S1. The modified acrylic resin represented by formula (Ⅰ) is reacted with Fe3O4 magnetic nanoparticles and glutaraldehyde solution to obtain a magnetic nanoparticle composite carrier;

[0008] S2. After the carboxyl group of the magnetic nanoparticle composite carrier is activated by an activator, the lipase solution and the glutaraldehyde solution are reacted to obtain a highly stable immobilized lipase;

[0009]

[0010] Formula (I)

[0011] Wherein, x is phenyl, ethyl, vinyl or a cyclic alkyl group having 6 carbon atoms, R1 is H or CH3, and n is 10 to 15.

[0012] In some embodiments of the present invention, the structure of the modified acrylate resin represented by formula (I) is as follows:

[0013]

[0014] Formula (I)

[0015] Wherein, x is phenyl or vinyl, R1 is H or CH3, and n is 10~15.

[0016] In some embodiments of the present invention, in S1, the lipase is selected from at least one of CALB lipase, RML lipase and CALA lipase.

[0017] In some embodiments of the present invention, in S1, the preparation of the Fe3O4 magnetic nanoparticles comprises the following steps:

[0018] 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.

[0019] In some embodiments of the present invention, in S1, the mass ratio of the modified acrylic resin represented by formula (I), Fe3O4 magnetic nanoparticles and glutaraldehyde solution is 5~15:2~6:10.

[0020] In some embodiments of the present invention, in S1, the mass concentration of the glutaraldehyde solution is 3-7%.

[0021] In some embodiments of the present invention, in S1, the reaction temperature is 20-40° C. and the reaction time is 5-10 h.

[0022] 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, N-hydroxysuccinimide, and dicyclohexylcarbodiimide.

[0023] In some embodiments of the present invention, in S2, the mass ratio of the magnetic nanoparticle composite carrier to the activator is 1:1-5.

[0024] 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 1:3~6:0.2~0.5.

[0025] In some embodiments of the present invention, in S2, the mass concentration of the lipase solution is 0.5-2 mg / mL.

[0026] In some embodiments of the present invention, in S2, the mass concentration of the glutaraldehyde solution is 3-7%.

[0027] In some embodiments of the present invention, in S2, the activation temperature is 30-45° C. and the activation time is 12-18 h.

[0028] In some embodiments of the present invention, in S2, the reaction temperature is 25-35° C. and the reaction time is 1-2 h.

[0029] Another object of the present invention is to provide a high-stability immobilized lipase, including one prepared by the preparation method of the high-stability immobilized lipase.

[0030] Another object of the present invention is to provide a diglyceride for food, which is prepared by catalyzing the glycerol hydrolysis reaction of the high-stability immobilized lipase.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention first reacts a modified acrylic resin with Fe3O4 magnetic nanoparticles and glutaraldehyde to prepare a magnetic nanoparticle composite carrier, activates the magnetic nanoparticle composite carrier, and then reacts with lipase to obtain a highly stable immobilized lipase; the present invention activates the magnetic nanoparticle composite carrier so that a larger amount of lipase is loaded on the carrier, thereby improving the catalytic activity of the immobilized lipase in preparing diglyceride, catalyzing the hydrolysis of triglyceride, and obtaining a diglyceride containing 35%.

[0033] (2) The molecular structure of the high-stability immobilized lipase prepared by the present invention contains a conjugated structure and a hydrogen bond, which makes the high-stability immobilized lipase have high thermal stability and reuse rate, and has high enzyme activity. After being reused 20 times, the immobilized lipase still maintains 91% of the initial enzyme activity. DETAILED DESCRIPTION

[0034] 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.

[0035] In the following examples, CALB lipase, RML lipase or CALA lipase are all commercially available, with an enzyme activity of 6000 U / g. The enzyme activity is determined according to the national standard GB / T23535-2009, which is defined as the amount of lipase required to catalyze the hydrolysis of fat to produce 1 μmol of titratable fatty acids per minute at 40°C and pH 7.5. The modified acrylate resins shown in formula (I) used in the following examples and comparative examples are all from Guangdong Boxing New Materials Technology Co., Ltd., and relevant records are available in Chinese patent CN118290718A.

[0036] Example 1

[0037] This embodiment provides a method for preparing a highly stable immobilized lipase, and the specific preparation steps are as follows:

[0038] 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;

[0039] S2. 5 parts by mass of a modified acrylic resin represented by formula (Ⅰ) and 6 parts by mass of Fe3O4 magnetic nanoparticles and 10 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 20 ° C for 10 h to obtain a magnetic nanoparticle composite carrier;

[0040] S3. 1 part by mass of the magnetic nanoparticle composite carrier and 1 part by mass of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed, activated at 30°C for 18 hours, 3 parts by mass of CALB lipase solution and 0.5 parts by mass of glutaraldehyde solution were added, the mass concentration of the CALB lipase solution was 0.5 mg / mL, and the mass concentration of the glutaraldehyde solution was 7%, reacted at 25°C for 2 hours, magnetically separated the immobilized lipase, washed with water and dried to obtain a highly stable immobilized lipase;

[0041]

[0042] Formula (I)

[0043] Among them, n is 10~15.

[0044] The enzyme activity of the high-stability immobilized lipase was determined to be 5700 U / g, and the enzyme activity recovery rate was about 95%. After the high-stability immobilized lipase was reused 20 times, its enzyme activity was determined to be reduced to 5460 U / g, and the enzyme activity recovery rate was about 91%.

[0045] Example 2

[0046] This embodiment provides a method for preparing a highly stable immobilized lipase, and the specific preparation steps are as follows:

[0047] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:2 and the total concentration of 1 mol / L were raised to 100°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;

[0048] S2. 15 parts by mass of a modified acrylic resin represented by formula (Ⅰ) was mixed with 2 parts by mass of Fe3O4 magnetic nanoparticles and 10 parts by mass of a glutaraldehyde solution, the mass concentration of the glutaraldehyde solution was 7%, and the reaction was carried out at 40 ° C for 5h to obtain a magnetic nanoparticle composite carrier;

[0049] S3. 1 part by mass of the magnetic nanoparticle composite carrier and 5 parts by mass of N-hydroxysuccinimide were mixed, activated at 45°C for 12h, 6 parts by mass of RML lipase solution and 0.2 parts by mass of glutaraldehyde solution were added, the mass concentration of the RML lipase solution was 2mg / mL, the mass concentration of the glutaraldehyde solution was 3%, the reaction was carried out at 35°C for 1h, the immobilized lipase was magnetically separated, washed with water and dried to obtain a highly stable immobilized lipase;

[0050]

[0051] Formula (I)

[0052] Among them, n is 10~15.

[0053] The enzyme activity of the high-stability immobilized lipase was determined to be 5750 U / g, and the enzyme activity recovery rate was about 96%. After the high-stability immobilized lipase was reused 20 times, its enzyme activity was determined to be reduced to 5520 U / g, and the enzyme activity recovery rate was about 92%.

[0054] Example 3

[0055] This embodiment provides a method for preparing a highly stable immobilized lipase, and the specific preparation steps are as follows:

[0056] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:2 and the total concentration of 1 mol / L were raised to 100°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;

[0057] S2. 15 parts by mass of a modified acrylic resin represented by formula (Ⅰ) was mixed with 2 parts by mass of Fe3O4 magnetic nanoparticles and 10 parts by mass of a glutaraldehyde solution, the mass concentration of the glutaraldehyde solution was 7%, and the reaction was carried out at 40 ° C for 5h to obtain a magnetic nanoparticle composite carrier;

[0058] S3. 1 part by mass of the magnetic nanoparticle composite carrier and 5 parts by mass of N-hydroxysuccinimide were mixed, activated at 45°C for 12h, 6 parts by mass of RML lipase solution and 0.2 parts by mass of glutaraldehyde solution were added, the mass concentration of the RML lipase solution was 2mg / mL, the mass concentration of the glutaraldehyde solution was 3%, the reaction was carried out at 35°C for 1h, the immobilized lipase was magnetically separated, washed with water and dried to obtain a highly stable immobilized lipase;

[0059]

[0060] Formula (I)

[0061] Among them, n is 10~15.

[0062] The enzyme activity of the high-stability immobilized lipase was measured to be 5820 U / g, and the enzyme activity recovery rate was about 97%; after the high-stability immobilized lipase was reused 20 times, its enzyme activity was measured to be reduced to 5640 U / g, and the enzyme activity recovery rate was about 94%.

[0063] Example 4

[0064] This embodiment provides a method for preparing a highly stable immobilized lipase, and the specific preparation steps are as follows:

[0065] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:2 and the total concentration of 1 mol / L were raised to 100°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;

[0066] S2. 15 parts by mass of a modified acrylic resin represented by formula (Ⅰ) was mixed with 2 parts by mass of Fe3O4 magnetic nanoparticles and 10 parts by mass of a glutaraldehyde solution, the mass concentration of the glutaraldehyde solution was 7%, and the reaction was carried out at 40 ° C for 5h to obtain a magnetic nanoparticle composite carrier;

[0067] S3. 1 part by mass of the magnetic nanoparticle composite carrier and 5 parts by mass of N-hydroxysuccinimide were mixed, activated at 45°C for 12h, 6 parts by mass of RML lipase solution and 0.2 parts by mass of glutaraldehyde solution were added, the mass concentration of the RML lipase solution was 2mg / mL, the mass concentration of the glutaraldehyde solution was 3%, the reaction was carried out at 35°C for 1h, the immobilized lipase was magnetically separated, washed with water and dried to obtain a highly stable immobilized lipase;

[0068]

[0069] Formula (I)

[0070] Among them, n is 10~15.

[0071] The enzyme activity of the high-stability immobilized lipase was measured to be 5880 U / g, and the enzyme activity recovery rate was about 98%. After the high-stability immobilized lipase was reused 20 times, its enzyme activity was measured to be reduced to 5700 U / g, and the enzyme activity recovery rate was about 95%.

[0072] Comparative Example 1

[0073] This comparative example provides a method for preparing a highly stable immobilized lipase, which is different from Example 2 only in that the magnetic nanoparticle composite carrier is not activated. The specific preparation steps are as follows:

[0074] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 1:2 and the total concentration of 1 mol / L were raised to 100°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;

[0075] S2. 15 parts by mass of a modified acrylic resin represented by formula (Ⅰ) was mixed with 2 parts by mass of Fe3O4 magnetic nanoparticles and 10 parts by mass of a glutaraldehyde solution, the mass concentration of the glutaraldehyde solution was 7%, and the reaction was carried out at 40 ° C for 5h to obtain a magnetic nanoparticle composite carrier;

[0076] S3. 6 parts by mass of RML lipase solution and 0.2 parts by mass of glutaraldehyde solution were added to 1 part by mass of the magnetic nanoparticle composite carrier, the mass concentration of the RML lipase solution was 2 mg / mL, the mass concentration of the glutaraldehyde solution was 3%, and the reaction was carried out at 35°C for 1h, and the immobilized lipase was magnetically separated, washed with water and dried to obtain a highly stable immobilized lipase;

[0077]

[0078] Formula (I)

[0079] Among them, n is 10~15.

[0080] The enzyme activity of the high-stability immobilized lipase was determined to be 5100 U / g, and the enzyme activity recovery rate was about 85%. After the high-stability immobilized lipase was reused 20 times, its enzyme activity was determined to be reduced to 4800 U / g, and the enzyme activity recovery rate was about 80%.

[0081] Example 5

[0082] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0083] 30 parts by mass of rapeseed oil and 15 parts by mass of water were uniformly mixed, 0.9 parts by mass of the high-stability immobilized lipase prepared in Example 1 was added, the mixture was reacted at 200 rpm and 45° C. for 12 h, and the product was filtered to obtain the product; the diglyceride content of the product was analyzed by HPLC-ELSD, and the diglyceride content was 35%.

[0084] Example 6

[0085] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0086] 30 parts by mass of rapeseed oil and 15 parts by mass of water were evenly mixed, 1.8 parts by mass of the high-stability immobilized lipase prepared in Example 2 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and the product was filtered to obtain the product; the diglyceride content of the product was analyzed by HPLC-ELSD, and the diglyceride content was 39%.

[0087] Example 7

[0088] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0089] 30 parts by mass of rapeseed oil and 15 parts by mass of water were evenly mixed, 1.8 parts by mass of the high-stability immobilized lipase prepared in Example 3 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and the product was filtered to obtain the product; the diglyceride content of the product was analyzed by HPLC-ELSD, and the diglyceride content was 43%.

[0090] Example 8

[0091] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0092] 30 parts by mass of rapeseed oil and 15 parts by mass of water were evenly mixed, 1.8 parts by mass of the high-stability immobilized lipase prepared in Example 4 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and the product was filtered to obtain the product; the diglyceride content of the product was analyzed by HPLC-ELSD, and the diglyceride content was 45%.

[0093] Comparative Example 2

[0094] This comparative example provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0095] 30 parts by mass of rapeseed oil and 15 parts by mass of water were evenly mixed, 1.8 parts by mass of the high-stability immobilized lipase prepared in Comparative Example 1 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and the product was filtered to obtain the product; the diglyceride content of the product was analyzed by HPLC-ELSD, and the diglyceride content was 26%.

[0096] Example 9

[0097] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0098] S1. The high stability immobilized lipase prepared in Example 1 was placed in a water bath at 60° C. for 0.5 h to obtain a high stability immobilized lipase after high temperature heat treatment;

[0099] S2. 30 parts by mass of rapeseed oil and 15 parts by mass of water were uniformly mixed, 0.9 parts by mass of the high-stability immobilized lipase obtained after high-temperature heat treatment in step S1 was added, the mixture was reacted at 200 rpm and 45° C. for 12 h, and filtered to obtain a product; the product was analyzed by HPLC-ELSD to determine the diglyceride content, and the diglyceride content was 31%.

[0100] Example 10

[0101] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0102] S1. The high stability immobilized lipase prepared in Example 2 was placed in a water bath at 60° C. for 0.5 h to obtain a high stability immobilized lipase after high temperature heat treatment;

[0103] S2. 30 parts by mass of rapeseed oil and 15 parts by mass of water were uniformly mixed, 1.8 parts by mass of the high-stability immobilized lipase obtained after high-temperature heat treatment in step S1 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and filtered to obtain a product; the product was analyzed by HPLC-ELSD to determine the diglyceride content, and the diglyceride content was 35%.

[0104] Embodiment 11

[0105] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0106] S1. The high stability immobilized lipase prepared in Example 3 was placed in a water bath at 60° C. for 0.5 h to obtain a high stability immobilized lipase after high temperature heat treatment;

[0107] S2. 30 parts by mass of rapeseed oil and 15 parts by mass of water were uniformly mixed, 1.8 parts by mass of the high-stability immobilized lipase obtained after high-temperature heat treatment in step S1 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and filtered to obtain a product; the product was analyzed by HPLC-ELSD to determine the diglyceride content, and the diglyceride content was 41%.

[0108] Example 12

[0109] This embodiment provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability. The specific preparation steps are as follows:

[0110] S1. The high stability immobilized lipase prepared in Example 4 was placed in a water bath at 60° C. for 0.5 h to obtain a high stability immobilized lipase after high temperature heat treatment;

[0111] S2. 30 parts by mass of rapeseed oil and 15 parts by mass of water were uniformly mixed, 1.8 parts by mass of the high-stability immobilized lipase obtained after high-temperature heat treatment in step S1 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and filtered to obtain a product; the product was analyzed by HPLC-ELSD to determine the diglyceride content, and the diglyceride content was 43%.

[0112] Comparative Example 3

[0113] This comparative example provides a method for preparing diglyceride by catalysis of immobilized lipase with high stability, and the specific preparation steps are as follows:

[0114] S1. The high stability immobilized lipase prepared in Comparative Example 1 was placed in a water bath at 60° C. for 0.5 h to obtain a high stability immobilized lipase after high temperature heat treatment;

[0115] S2. 30 parts by mass of rapeseed oil and 15 parts by mass of water were uniformly mixed, 1.8 parts by mass of the high-stability immobilized lipase obtained after high-temperature heat treatment in step S1 was added, the mixture was reacted at 200 rpm and 45° C. for 24 h, and filtered to obtain a product; the product was analyzed by HPLC-ELSD to determine the diglyceride content, and the diglyceride content was 18%.

[0116] By comparing Examples 5 to 8 and Comparative Example 2, it can be seen that the content of diglyceride prepared by catalysis of the lipases in Examples 5 to 8 of the present invention can reach more than 35%, while the content of diglyceride prepared by catalysis of the lipase in Comparative Example 2 is much lower than that in Examples 5 to 8. It can be seen that the lipases in Examples 1 to 4 of the present invention have higher enzyme activities for catalytic preparation of diglyceride.

[0117] By comparing Examples 9 to 12 and Comparative Example 3, it can be seen that the immobilized lipases of Examples 1 to 4 of the present invention have high thermal stability. After being treated at 60° C. for 0.5 h, the content of diglycerol prepared by the immobilized lipases of Examples 9 to 12 remains substantially unchanged, while the immobilized lipase of Comparative Example 1 whose carrier is not activated has low thermal stability, and the content of diglycerol prepared by the catalysis of Comparative Example 3 is much lower than that of Examples 9 to 12. It can be seen that the lipases of Examples 1 to 4 of the present invention have high thermal stability.

[0118] 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. reacting the modified acrylic resin represented by formula (I) with Fe3O4 magnetic nanoparticles and glutaraldehyde solution, wherein the mass concentration of the glutaraldehyde solution is 3 to 7%, the reaction temperature is 20 to 40°C, and the reaction time is 5 to 10 hours to obtain a magnetic nanoparticle composite carrier; S2. activating the carboxyl group of the magnetic nanoparticle composite carrier with an activator, the activation temperature is 30-45°C, the time is 12-18h, reacting with a lipase solution and a glutaraldehyde solution, the mass concentration of the lipase solution is 0.5-2mg / mL, the mass concentration of the glutaraldehyde solution is 3-7%, the reaction temperature is 25-35°C, the time is 1-2h, and immobilized lipase is obtained; Formula (I) Wherein, x is phenyl, ethyl, vinyl or a cyclic alkyl group having 6 carbon atoms, R1 is H or CH3, and n is 10 to 15; In S1, the mass ratio of the modified acrylic resin represented by formula (I), Fe3O4 magnetic nanoparticles and glutaraldehyde solution is 5-15:2-6:10; In S2, the lipase is selected from at least one of CALB lipase, RML lipase and CALA lipase; In S2, the activator is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; In S2, the mass ratio of the magnetic nanoparticle composite carrier to the activator is 1:1-5; In S2, the mass ratio of the magnetic nanoparticle composite carrier, the lipase solution and the glutaraldehyde solution is 1:3-6:0.2-0.

5.

2. The method for preparing immobilized lipase according to claim 1, characterized in that: The structure of the modified acrylate resin represented by formula (I) is as follows: Formula (I) Wherein, x is phenyl or vinyl, R1 is H or CH3, and n is 10~15.

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

4. Use of the immobilized lipase as claimed in claim 3 in the preparation of diglycerides for food.

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