A composite carrier-immobilized lipase and its preparation method and application

Abstract

The invention discloses a composite carrier-immobilized lipase and a preparation method and application thereof. The preparation method of the composite carrier-immobilized lipase comprises the following steps: S1. reacting a carboxyl-containing polyketone with an acyl chloride reagent to obtain an acyl chloride polyketone, adding triethylamine and 3-thiophenecarboxylic acid to react to obtain a polyketone containing a thiophene substituent; S2. reacting a polyketone containing a thiophene substituent, Fe 3 O 4 The magnetic nanoparticles react with glutaraldehyde solution to obtain a magnetic nanoparticle composite carrier; S3. The magnetic nanoparticle composite carrier, lipase solution and glutaraldehyde solution react to obtain a composite carrier-immobilized lipase; The composite carrier-immobilized lipase prepared by the preparation method of the composite carrier-immobilized lipase of the present invention has high activity and good stability, and when catalyzing the glycerol decomposition reaction to prepare diglyceride, the content of monoglyceride is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biological enzymes, and in particular relates to a composite carrier-immobilized lipase and a preparation method and application thereof. 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 bonding method usually has low lipase catalytic activity, or when used to catalyze the glycerol hydrolysis reaction to prepare diglyceride, a high content of monoglyceride is often obtained. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, a method for preparing a composite carrier-immobilized lipase is provided. The composite carrier-immobilized lipase prepared by the method for preparing a composite carrier-immobilized lipase of the present invention has high activity and good stability, and greatly reduces the content of monoglyceride when catalyzing the glycerol hydrolysis reaction to prepare diglyceride.

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

[0007] S1. reacting a carboxyl-containing polyketone with an acyl chloride reagent to obtain an acyl chloride polyketone, adding triethylamine and 3-thiophenecarboxylic acid to react to obtain a polyketone containing a thiophene substituent;

[0008] S2. The polyketone containing thiophene substituents, Fe3O4 magnetic nanoparticles and glutaraldehyde solution are reacted to obtain a magnetic nanoparticle composite carrier;

[0009] S3. reacting the magnetic nanoparticle composite carrier, lipase solution and glutaraldehyde solution to obtain composite carrier-immobilized lipase.

[0010] In some embodiments of the present invention, in S1, the structure of the carboxyl-containing polyketone is as follows:

[0011]

[0012] Among them, the number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3.

[0013] In some embodiments of the present invention, in S1, the acyl chloride reagent is selected from SOCl2.

[0014] In some embodiments of the present invention, in S1, the mass ratio of the carboxyl-containing polyketone to the acyl chloride reagent is 10-20:1.

[0015] In some embodiments of the present invention, in S1, the mass ratio of the chlorinated polyketone to 3-thiophenecarboxylic acid is 1-5:1.

[0016] In some embodiments of the present invention, in S1, the temperature for reacting the carboxyl-containing polyketone with the acyl chloride reagent is 80-100° C. and the time is 18-24 hours.

[0017] In some embodiments of the present invention, in S1, the mass ratio of triethylamine to 3-thiophenecarboxylic acid is 1:10-20.

[0018] In some embodiments of the present invention, in S1, the temperature of adding triethylamine and 3-thiophenecarboxylic acid to react is 110-130° C. and the time is 12-18 hours.

[0019] In some embodiments of the present invention, in S2, the mass ratio of the polyketone containing thiophene substituents, Fe3O4 magnetic nanoparticles and glutaraldehyde solution is 10-20:3-5:10.

[0020] In some embodiments of the present invention, in S2, the mass concentration of the glutaraldehyde solution is 3-7%.

[0021] In some embodiments of the present invention, in S2, 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 preparation of the Fe3O4 magnetic nanoparticles comprises the following steps:

[0023] 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.

[0024] In some embodiments of the present invention, in S3, the mass ratio of the magnetic nanoparticle composite carrier, the lipase solution and the glutaraldehyde solution is 1:2~5:0.1~0.4.

[0025] In some embodiments of the present invention, in S3, the mass concentration of the lipase solution is 0.5-2 mg / mL.

[0026] In some embodiments of the present invention, in S3, the mass concentration of the glutaraldehyde solution is 3-7%.

[0027] In some embodiments of the present invention, in S3, the reaction temperature is 25-35° C. and the reaction time is 1-2 h.

[0028] Another object of the present invention is to provide a composite carrier-immobilized lipase, including the lipase prepared by the composite carrier-immobilized lipase preparation method.

[0029] Another object of the present invention is to provide a diglyceride for food, which is prepared by catalyzing the glycerol hydrolysis reaction of the lipase immobilized on the composite carrier.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention firstly reacts polyketone containing thiophene substituents with Fe3O4 magnetic nanoparticles to prepare a magnetic nanoparticle composite carrier, and then reacts the magnetic nanoparticle composite carrier with lipase to obtain composite carrier-immobilized lipase; the prepared composite carrier-immobilized lipase has a conjugated structure and hydrogen bonding in its molecular structure, so that the composite carrier-immobilized lipase has high thermal stability and reuse rate, and has high enzyme activity, and when used for catalyzing the glycerol decomposition reaction to prepare diglyceride, the content of monoglyceride is greatly reduced. 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 lipase used in the present invention is all Thermomyces lanuginosus lipase.

[0034] Example 1

[0035] This embodiment provides a method for preparing lipase immobilized on a composite carrier, and the specific preparation steps are as follows:

[0036] 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;

[0037] S2. 10 parts by mass of a carboxyl-containing polyketone was reacted with 1 part by mass of SOCl2 at 80°C for 18h to obtain a polyketone chloride, 0.1 parts by mass of triethylamine and 1 part by mass of 3-thiophenecarboxylic acid were added to 1 part by mass of the polyketone chloride, and the reaction was carried out at 110°C for 12h to obtain a polyketone containing a thiophene substituent;

[0038] The structure of the carboxyl-containing polyketone is shown below:

[0039]

[0040] Among them, the number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3;

[0041] S3. The polyketone containing thiophene substituents 10 parts by mass, Fe3O4 magnetic nanoparticles 3 parts by mass and 10 parts by mass of glutaraldehyde solution at 20 ° C for 10h, the mass concentration of glutaraldehyde solution is 3%, to obtain a magnetic nanoparticle composite carrier;

[0042] S4. 1 part by mass of the magnetic nanoparticle composite carrier, 2 parts by mass of the lipase solution and 0.1 parts by mass of the glutaraldehyde solution were reacted at 25°C for 2h, wherein the mass concentration of the lipase solution was 0.5mg / mL and the mass concentration of the glutaraldehyde solution was 3%, to obtain a composite carrier-immobilized lipase.

[0043] Example 2

[0044] This embodiment provides a method for preparing lipase immobilized on a composite carrier, and the specific preparation steps are as follows:

[0045] 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;

[0046] S2. 20 parts by mass of a carboxyl-containing polyketone was reacted with 1 part by mass of SOCl2 at 100 ° C for 24 hours to obtain a polyketone chloride, 0.25 parts by mass of triethylamine and 1 part by mass of 3-thiophenecarboxylic acid were added to 5 parts by mass of the polyketone chloride, and the reaction was carried out at 130 ° C for 18 hours to obtain a polyketone containing a thiophene substituent;

[0047] The structure of the carboxyl-containing polyketone is shown below:

[0048]

[0049] Among them, the number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3;

[0050] S3. The polyketone containing thiophene substituents 20 parts by mass, Fe3O4 magnetic nanoparticles 5 parts by mass and 10 parts by mass of glutaraldehyde solution at 40 ° C for 5h, the mass concentration of glutaraldehyde solution is 7%, to obtain a magnetic nanoparticle composite carrier;

[0051] S4. 1 part by mass of the magnetic nanoparticle composite carrier, 5 parts by mass of the lipase solution and 0.4 parts by mass of the glutaraldehyde solution were reacted at 35°C for 1 hour, wherein the mass concentration of the lipase solution was 2 mg / mL and the mass concentration of the glutaraldehyde solution was 7%, to obtain a composite carrier-immobilized lipase.

[0052] Example 3

[0053] This embodiment provides a method for preparing lipase immobilized on a composite carrier, and the specific preparation steps are as follows:

[0054] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 2:1 and the total concentration of 0.5 mol / L were raised to 90°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;

[0055] S2. 15 parts by mass of a carboxyl-containing polyketone was reacted with 1 part by mass of SOCl2 at 90°C for 20 hours to obtain a polyketone chloride, 0.2 parts by mass of triethylamine and 1 part by mass of 3-thiophenecarboxylic acid were added to 3 parts by mass of the polyketone chloride, and the reaction was carried out at 120°C for 15 hours to obtain a polyketone containing a thiophene substituent;

[0056] The structure of the carboxyl-containing polyketone is shown below:

[0057]

[0058] Among them, the number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3;

[0059] S3. The polyketone containing thiophene substituents 15 parts by mass, Fe3O4 magnetic nanoparticles 4 parts by mass and 10 parts by mass of glutaraldehyde solution at 30 ° C for 8h, the mass concentration of glutaraldehyde solution is 5%, to obtain a magnetic nanoparticle composite carrier;

[0060] S4. 1 part by mass of the magnetic nanoparticle composite carrier, 3 parts by mass of the lipase solution and 0.3 parts by mass of the glutaraldehyde solution were reacted at 30°C for 1.5h, wherein the mass concentration of the lipase solution was 1mg / mL and the mass concentration of the glutaraldehyde solution was 5%, to obtain a composite carrier-immobilized lipase.

[0061] Comparative Example 1

[0062] This comparative example provides a method for preparing a composite carrier-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:

[0063] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 2:1 and the total concentration of 0.5 mol / L were raised to 90°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;

[0064] S2. 15 parts by mass of a carboxyl-containing polyketone was reacted with 1 part by mass of SOCl2 at 90°C for 20 hours to obtain a polyketone chloride, 0.2 parts by mass of triethylamine and 1 part by mass of 3-thiophenecarboxylic acid were added to 3 parts by mass of the polyketone chloride, and the reaction was carried out at 120°C for 15 hours to obtain a polyketone containing a thiophene substituent;

[0065] The structure of the carboxyl-containing polyketone is shown below:

[0066]

[0067] Among them, the number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3;

[0068] S3. The polyketone containing thiophene substituents 15 parts by mass, Fe3O4 magnetic nanoparticles 4 parts by mass and 10 parts by mass of glutaraldehyde solution at 30 ° C for 8h, the mass concentration of glutaraldehyde solution is 5%, to obtain a magnetic nanoparticle composite carrier;

[0069] S4. Mix 1 part by mass of the magnetic nanoparticle composite carrier and 3 parts by mass of the lipase solution, wherein the mass concentration of the lipase solution is 1 mg / mL, to obtain composite carrier-immobilized lipase.

[0070] Comparative Example 2

[0071] This comparative example provides a method for preparing a composite carrier-immobilized lipase, which differs from Example 3 only in that 3-thiophenecarboxylic acid is not used to modify the carboxyl-containing polyketone. The specific preparation steps are as follows:

[0072] S1. Under the protection of nitrogen, dissolve ferrous chloride and ferric chloride in water. 2+ and Fe 3+ The molar ratio of 2:1 and the total concentration of 0.5 mol / L were raised to 90°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;

[0073] S2. 15 parts by mass of carboxyl-containing polyketone, 4 parts by mass of Fe3O4 magnetic nanoparticles and 10 parts by mass of glutaraldehyde solution were reacted at 30 ° C for 8h, the mass concentration of glutaraldehyde solution was 5%, to obtain a magnetic nanoparticle composite carrier;

[0074] The structure of the carboxyl-containing polyketone is shown below:

[0075]

[0076] Among them, the number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3;

[0077] S3. 1 part by mass of the magnetic nanoparticle composite carrier, 3 parts by mass of lipase solution and 0.3 parts by mass of glutaraldehyde solution were reacted at 30°C for 1.5h, wherein the mass concentration of the lipase solution was 1mg / mL and the mass concentration of the glutaraldehyde solution was 5%, to obtain a composite carrier-immobilized lipase.

[0078] Example 4

[0079] This embodiment provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0080] 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier 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, and the content of monoglyceride was 4.14%, and the content of diglyceride was 46.7%.

[0081] Example 5

[0082] This embodiment provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0083] 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier 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, and the content of monoglyceride was 4.32%, and the content of diglyceride was 48.3%.

[0084] Example 6

[0085] This embodiment provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0086] 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier 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, and the content of monoglyceride was 4.41%, and the content of diglyceride was 47.1%.

[0087] Comparative Example 3

[0088] This comparative example provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0089] 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier 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, and the content of monoglyceride was 8.13%, and the content of diglyceride was 35.9%.

[0090] Comparative Example 4

[0091] This comparative example provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0092] 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were evenly mixed, 0.5 parts by mass of the composite carrier 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, and the content of monoglyceride was 7.97%, and the content of diglyceride was 37.3%.

[0093] Comparative Example 5

[0094] This comparative example provides a method for preparing diglyceride catalyzed by lipase, and the specific preparation steps are as follows:

[0095] 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of lipase were added, the mixture was reacted at 200 rpm and 50°C for 12 hours, and the mixture was filtered to obtain a product. The content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD, and the content of monoglyceride was 10.48%, and the content of diglyceride was 33.5%.

[0096] Example 7

[0097] This embodiment provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

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

[0099] S2: 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of composite carrier immobilized lipase after high-temperature heat treatment was added, the mixture was reacted at 200 rpm and 50°C for 12 hours, and the product was filtered to obtain the product; the content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD, and the content of monoglyceride was 3.98%, and the content of diglyceride was 43.6%.

[0100] Example 8

[0101] This embodiment provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

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

[0103] S2: 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier immobilized lipase after high-temperature heat treatment was added, the mixture was reacted at 200 rpm and 50°C for 12 hours, and the product was filtered to obtain the product; the content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD, and the content of monoglyceride was 4.13%, and the content of diglyceride was 45.0%.

[0104] Example 9

[0105] This embodiment provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

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

[0107] S2: 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier immobilized lipase after high-temperature heat treatment was added, the mixture was reacted at 200 rpm and 50°C for 12 hours, and the product was filtered to obtain the product; the content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD, and the content of monoglyceride was 4.24%, and the content of diglyceride was 44.4%.

[0108] Comparative Example 6

[0109] This comparative example provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0110] S1. The composite carrier-immobilized lipase prepared in Comparative Example 1 was placed in a water bath at 60°C for 0.5h to obtain a composite carrier-immobilized lipase after high temperature heat treatment;

[0111] S2: 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of the composite carrier immobilized lipase after high-temperature heat treatment was added, the mixture was reacted at 200 rpm and 50°C for 12 hours, and the product was filtered to obtain the product; the content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD, and the content of monoglyceride was 6.24%, and the content of diglyceride was 24.6%.

[0112] Comparative Example 7

[0113] This comparative example provides a composite carrier-immobilized lipase-catalyzed preparation of diglyceride, and the specific preparation steps are as follows:

[0114] S1. The composite carrier-immobilized lipase prepared in Comparative Example 2 was placed in a water bath at 60°C for 0.5h to obtain a composite carrier-immobilized lipase after high temperature heat treatment;

[0115] S2: 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol were uniformly mixed, 0.5 parts by mass of composite carrier immobilized lipase after high-temperature heat treatment was added, the mixture was reacted at 200 rpm and 50°C for 12 hours, and the product was filtered to obtain the product; the content of monoglyceride and diglyceride in the product was analyzed by HPLC-ELSD, and the content of monoglyceride was 6.04%, and the content of diglyceride was 27.8%.

[0116] Comparative Example 8

[0117] This comparative example provides a method for preparing diglyceride catalyzed by lipase, and the specific preparation steps are as follows:

[0118] S1. The lipase is placed in a water bath at 60°C for 0.5h to obtain a high temperature heat treated lipase;

[0119] S2: 70.4 parts by mass of rapeseed oil and 1.84 parts by mass of glycerol are uniformly mixed, 0.5 parts by mass of lipase after high-temperature heat treatment is added, the mixture is reacted at 200 rpm and 50°C for 12 hours, and the product is filtered to obtain the product; the content of monoglyceride and diglyceride in the product is analyzed by HPLC-ELSD, and the content of monoglyceride is 7.99%, and the content of diglyceride is 16.7%.

[0120] By comparing Examples 4 to 6 and Comparative Examples 3 to 5, it can be seen that the content of diglycerol prepared by catalysis of the lipases in Examples 4 to 6 of the present invention can reach more than 46.7%, while the content of diglycerol prepared by catalysis of the lipases in Comparative Examples 3 to 5 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 catalytic preparation of diglycerol.

[0121] By comparing Examples 8 to 10 and Comparative Examples 6 to 8, 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 content of diglycerol prepared by the lipases of Examples 8 to 10 remains substantially unchanged, while the thermal stability of the lipases of Comparative Examples 1 to 2 or without carrier solidification is low, and the content of diglycerol prepared by the lipases of Comparative Examples 6 to 8 is much lower than that of Examples 8 to 10. It can be seen that the lipases of Examples 1 to 3 of the present invention have high thermal stability.

[0122] 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 lipase immobilized on a composite carrier, characterized in that: The steps include: S1. reacting a carboxyl-containing polyketone with SOCl2 in a mass ratio of 10 to 20:1 to obtain an acyl chloride polyketone, adding triethylamine and 3-thiophenecarboxylic acid to react to obtain a polyketone containing a thiophene substituent; S2. reacting a polyketone containing a thiophene substituent and Fe3O4 magnetic nanoparticles with a glutaraldehyde solution in a mass ratio of 10-20:3-5:10 at a temperature of 25-35°C for 1-2 hours to obtain a magnetic nanoparticle composite carrier; S3. reacting a magnetic nanoparticle composite carrier, a lipase solution and a glutaraldehyde solution in a mass ratio of 1:2~5:0.1~0.4 at a temperature of 25~35°C for 1~2h to obtain a composite carrier-immobilized lipase; In S1, the structure of the carboxyl-containing polyketone is as follows: The number average molecular weight of the carboxyl-containing polyketone is 50,000 to 100,000 g / mol, and the PDI is less than 1.3; the mass ratio of the chlorinated polyketone to 3-thiophenecarboxylic acid is 1 to 5:1; the temperature for the reaction of the carboxyl-containing polyketone with the chlorination reagent is 80 to 100°C, and the time is 18 to 24 hours; the temperature for the reaction of adding triethylamine and 3-thiophenecarboxylic acid is 110 to 130°C, and the time is 12 to 18 hours.

2. The method for preparing the composite carrier-immobilized lipase according to claim 1, characterized in that: S2, the mass concentration of the glutaraldehyde solution is 3-7%.

3. The method for preparing the composite carrier-immobilized lipase according to claim 1, characterized in that: In S3, the mass concentration of the lipase solution is 0.5-2 mg / mL; The mass concentration of the glutaraldehyde solution is 3-7%.

4. A composite carrier-immobilized lipase, characterized in that: The lipase immobilized on the composite carrier is prepared by the preparation method of any one of claims 1 to 3.

5. Use of the composite carrier-immobilized lipase according to claim 4 in the preparation of diglyceride for food.

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