Immobilized lipase, preparation method and application thereof, and synthesis method of vitamin A palmitate

Abstract

The invention belongs to the technical field of enzyme catalysts, and particularly relates to immobilized lipase, a preparation method and application thereof and a synthesis method of vitamin A palmitate. The immobilized lipase comprises a porous carrier, nano manganous-manganic oxide, nano silicon dioxide and lipase, wherein the nano manganous-manganic oxide and the nano silicon dioxide are loaded on the porous carrier. The method is characterized in that nano manganous-manganic oxide and nano silicon dioxide are added into the immobilized lipase, so that the adsorption effect of the enzyme can be enhanced, the utilization efficiency of the enzyme can be improved, and after the immobilized lipase is subjected to synergistic treatment by adopting the nano manganous-manganic oxide and the nano silicon dioxide, the adsorption stability of the enzyme can be enhanced; therefore, in the reaction of catalytically synthesizing the vitamin A palmitate, more excellent enzyme catalytic activity can be embodied, and the yield of the vitamin A palmitate is obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysts, and in particular relates to an immobilized lipase, a preparation method and application thereof, and a synthesis method of vitamin A palmitate. Background Art

[0002] Lipase is a class of enzymes with multiple catalytic abilities. It can catalyze the hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis of triacylglycerols and other water-insoluble esters. In addition, it also exhibits the activities of some other enzymes, such as phospholipases, lysophospholipases, cholesterol esterases, acyl peptide hydrolases, etc. The different activities of lipases depend on the characteristics of the reaction system. For example, it can promote ester hydrolysis at the oil-water interface, while it can enzymatically synthesize and transesterify in the organic phase. However, the high price of lipase and the problems of recycling and stability limit its industrial application. Immobilizing lipase can solve this problem well. Enzyme immobilization is a technology to improve the stability and reusability of enzymes in the catalytic process. After immobilization, lipase has the advantages of high stability, good continuity, convenient recycling, easy control, repeated use, and reduced cost, which provides technical and economic support for large-scale industrial applications.

[0003] Vitamin A palmitate is a fat-soluble vitamin A derivative that is widely found in nature. Vitamin A palmitate can be converted into an active form of vitamin A in the human body, and plays an important role in maintaining normal vision, promoting growth and development, and enhancing immunity. In addition, vitamin A palmitate also has an antioxidant effect, which helps protect cells from free radical damage. It is an important nutrient that has many benefits for human health.

[0004] At present, the synthesis process of vitamin A palmitate mainly includes chemical method and biological method. For a long time, industrial vitamin A palmitate has been mainly produced by chemical method, using catalysts such as sodium methoxide and Grignard reagent to carry out catalytic reaction under high temperature and high pressure. Not only is the total conversion rate low, but there are also many disadvantages that are difficult to overcome, such as catalyst toxicity, high energy consumption of high temperature and high pressure reaction, many side reactions, dark product color, separation and refining difficulties, etc. At the same time, acid corrodes equipment seriously, which is not conducive to production. With the development of biotechnology, especially the research of enzyme engineering, a new way has been provided for the synthesis of vitamin A palmitate by biocatalysts. Compared with the traditional chemical method, the enzyme synthesis method has the characteristics of mild reaction conditions, strong specificity, few side reactions and high product quality. Immobilized enzyme is a physical or chemical method to combine the enzyme with a water-insoluble macromolecular carrier or embed the enzyme in it, so that the enzyme forms an insoluble gel or semipermeable membrane microcapsule in water to reduce fluidity. Immobilized enzyme is an enzyme application technology developed in the past decade, and has attractive application prospects in industrial production, chemical analysis and medicine. After enzyme immobilization, its stability is generally enhanced, and it is easy to separate from the reaction system, easy to control, and can be used repeatedly. It is convenient to transport and store, and is conducive to automated production. However, existing immobilized lipases generally have the defect that the enzyme immobilization effect is not stable enough, and the enzyme catalytic efficiency is correspondingly reduced, and the scope of use is reduced. This technology still needs further development. Summary of the invention

[0005] The first purpose of the present invention is to overcome the problem of low catalytic efficiency of existing immobilized enzymes and to provide an immobilized lipase with high enzyme catalytic efficiency.

[0006] The second object of the present invention is to provide a method for preparing immobilized lipase.

[0007] The third object of the present invention is to provide immobilized lipase prepared by the above method.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned immobilized lipase in catalyzing the synthesis of vitamin A palmitate.

[0009] The fifth object of the present invention is to provide a method for synthesizing vitamin A palmitate using the above-mentioned immobilized lipase.

[0010] The immobilized lipase provided by the invention comprises a porous carrier and nano manganese tetraoxide, nano silicon dioxide and lipase loaded on the porous carrier.

[0011] The method for preparing the immobilized lipase provided by the present invention comprises the following steps:

[0012] S1. The porous carrier, nano-manganese tetraoxide and nano-silicon dioxide are mixed in an aqueous ammonia solution and then solid-liquid separation is performed to obtain a solid product and a liquid product;

[0013] S2. The solid product and lipase are mixed evenly in a phosphate buffer solution, and the obtained mixture is subjected to a cross-linking reaction with a cross-linking agent, and then the obtained cross-linking reaction product is filtered, washed with water and dried to obtain an immobilized lipase.

[0014] The method for synthesizing vitamin A palmitate by catalyzing immobilized lipase provided by the present invention comprises subjecting vitamin A acetate and palmitic acid to an enzyme-catalyzed transesterification reaction in the presence of the immobilized lipase to obtain vitamin A palmitate.

[0015] The key of the present invention is to add nano manganese tetraoxide and nano silicon dioxide to the immobilized lipase, thereby enhancing the adsorption effect of the enzyme and improving the utilization efficiency of the enzyme. After the immobilized lipase is synergistically treated with nano manganese tetraoxide and nano silicon dioxide, the adsorption stability of the enzyme can be enhanced, so that it can show better enzyme catalytic activity in the catalytic synthesis of vitamin A palmitate, and the yield of vitamin A palmitate is significantly improved. DETAILED DESCRIPTION

[0016] Any and numerical values ​​disclosed in this article should not be limited to exact values, and should be understood to include values ​​close to these numerical ranges. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed in this article.

[0017] The immobilized lipase provided by the present invention comprises a porous carrier, nano manganese tetraoxide, nano silicon dioxide and lipase loaded on the porous carrier. The mass ratio of the porous carrier, nano manganese tetraoxide and nano silicon dioxide is preferably 1:(0.005-0.01):(0.005-0.01), which is more conducive to improving the adsorption effect and catalytic activity of the lipase, so that the yield of vitamin A palmitate can be further improved. Specifically, based on the content of the porous carrier as 1 part by weight, the content of the nano manganese tetraoxide is preferably 0.005-0.01 part by weight, such as 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 part by weight or any value therebetween; the content of the nano silicon dioxide is preferably 0.005-0.01 part by weight, such as 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 part by weight or any value therebetween. In addition, the mass ratio of the porous carrier to the lipase is preferably 1:(0.01-0.1), such as 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1 parts by weight or any value therebetween.

[0018] The present invention has no particular limitation on the type of porous carrier, and can be any existing material capable of loading lipase formed by closed or through holes, and specific examples thereof include but are not limited to at least one of activated carbon, kaolin, molecular sieve, aluminum oxide, silicon oxide, aluminum silicate, magnesium oxide, porous glass, adsorption resin and ion exchange resin. Among them, the molecular sieve can be listed as at least one of IMF type structure molecular sieve, MFI type structure molecular sieve, Beta type molecular sieve, MOR type structure molecular sieve and FAU type structure molecular sieve, and IMF type structure molecular sieve is particularly preferred. Since the skeleton stability of IMF type structure molecular sieve is higher and the mesoporous structure is richer, it is more conducive to lipase loading as a porous carrier, and the yield of vitamin A palmitate can be further improved. The adsorption resin is preferably a macroporous adsorption resin. The ion exchange resin can be a cation exchange resin and / or an anion exchange resin. The cation exchange resin may be a strong acid cation exchange resin and / or a weak acid cation exchange resin, such as at least one of a strong acid styrene cation exchange resin, a strong acid acrylic cation exchange resin, a weak acid styrene cation exchange resin, and a weak acid acrylic cation exchange resin. The anion exchange resin may be a macroporous anion exchange resin and / or a gel-type anion exchange resin, preferably a macroporous weakly basic styrene anion exchange resin and / or a macroporous weakly basic acrylic anion exchange resin. In addition, the specific surface area of ​​the porous carrier is preferably 10 to 500 m 2 / g, such as 10, 50, 80, 100, 120, 150, 180, 200, 220, 250, 280, 300, 320, 350, 380, 400, 420, 450, 480, 500m 2 / g or any value in between.

[0019] In the present invention, the average particle size of the nano manganese tetraoxide is preferably 5nm to 500nm, such as 5nm, 10nm, 20nm, 50nm, 80nm, 100nm, 120nm, 150nm, 180nm, 200nm, 220nm, 250nm, 280nm, 300nm, 320nm, 350nm, 400nm, 450nm, 500nm or any value therebetween. The average particle size of the nano silicon dioxide is preferably 20nm to 200nm, such as 20nm, 50nm, 80nm, 100nm, 120nm, 150nm, 180nm, 200nm or any value therebetween.

[0020] In the present invention, the lipase is preferably selected from at least one fermentation lipase of Candida antarctic, Thermomyces lanuginosus, Rhizomucor miehei, Aspergillus niger, Candida rugosa and Pseudononas sp., and is particularly preferably Candida antarctic lipase B. The inventors of the present invention have found that when the lipase is Candida antarctic lipase B, it can give the immobilized lipase a higher enzyme catalytic efficiency.

[0021] The method for preparing the immobilized lipase provided by the present invention comprises the following steps:

[0022] S1. The porous carrier, nano-manganese tetraoxide and nano-silicon dioxide are mixed in an aqueous ammonia solution and then solid-liquid separation is performed to obtain a solid product and a liquid product;

[0023] S2. The solid product and lipase are mixed evenly in a phosphate buffer solution, and the obtained mixture is subjected to a cross-linking reaction with a cross-linking agent, and then the obtained cross-linking reaction product is filtered, washed with water and dried to obtain an immobilized lipase.

[0024] In the preparation process of the immobilized lipase, in step S1, the mixing method can be to add the porous carrier, nano manganese tetraoxide and nano silicon dioxide into the ammonia solution at the same time and mix them evenly, or to add the porous carrier, nano manganese tetraoxide and nano silicon dioxide into the ammonia solution in any order and mix them evenly, preferably, the porous carrier and nano manganese tetraoxide are stirred and mixed evenly in the ammonia solution, and then the nano silicon dioxide is added for ultrasonic vibration. Wherein, the concentration of the ammonia solution is preferably 20% to 30%, such as 20%, 22%, 24%, 26%, 28%, 30% or any value therebetween. The stirring conditions preferably include a stirring temperature of 30°C to 55°C, such as 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C or any value therebetween; a stirring time of 0.5h to 2h, such as 0.5h, 0.8h, 1h, 1.2h, 1.5h, 1.8h, 2h or any value therebetween. The ultrasonic oscillation conditions preferably include an ultrasonic oscillation temperature of 60°C to 85°C, such as 60°C, 62°C, 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, 82°C, 85°C or any value therebetween; an ultrasonic oscillation time of 3h to 10h, such as 3h, 5h, 8h, 10h or any value therebetween.

[0025] In the preparation process of the immobilized lipase, in step S2, the pH of the phosphate buffer solution is preferably 4 to 7, such as 4, 5, 6, 7 or any value therebetween. The mixing method can be, for example, stirring and mixing. The stirring and mixing temperature is preferably 5°C to 25°C, such as 5°C, 8°C, 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C or any value therebetween; the stirring and mixing time is preferably 1h to 10h, such as 1h, 2h, 4h, 6h, 8h, 10h or any value therebetween.

[0026] In the preparation process of the above-mentioned immobilized lipase, in step S2, the cross-linking agent is preferably glutaraldehyde. The addition amount of the cross-linking agent is preferably 1% to 5% of the mass of the solid product, such as 1%, 2%, 3%, 4%, 5% or any value therebetween. In addition, the conditions of the cross-linking reaction preferably include a temperature of 10°C to 25°C, such as 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C or any value therebetween; and a reaction time of 2h to 10h, such as 2h, 4h, 6h, 8h, 10h or any value therebetween.

[0027] The invention also provides application of the immobilized lipase in catalyzing the synthesis of vitamin A palmitate.

[0028] The present invention also provides a method for synthesizing vitamin A palmitate by catalysis of immobilized lipase, which comprises subjecting vitamin A acetate and palmitic acid to an enzyme-catalyzed ester exchange reaction in the presence of the immobilized lipase to obtain vitamin A palmitate.

[0029] In the above-mentioned process of immobilized lipase-catalyzed synthesis of vitamin A palmitate, the molar ratio of vitamin A acetate to palmitic acid is preferably 1:(1.5-5), such as 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5 or any value therebetween.

[0030] In the process of synthesizing vitamin A palmitate catalyzed by the immobilized lipase, the amount of the immobilized lipase is preferably 1 to 3 times the mass of vitamin A acetate, such as 1, 1.5, 2, 2.5, 3 times or any value therebetween.

[0031] In the above-mentioned process of immobilized lipase-catalyzed synthesis of vitamin A palmitate, the conditions for the enzyme-catalyzed ester exchange reaction preferably include a temperature of 15°C to 55°C, such as 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C or any value therebetween; and a time of 3h to 12h, such as 3h, 4h, 6h, 8h, 10h, 12h or any value therebetween.

[0032] In the process of synthesizing vitamin A palmitate catalyzed by immobilized lipase, the enzyme-catalyzed transesterification reaction usually needs to be carried out in the presence of an organic solvent. Specific examples of the organic solvent include, but are not limited to, at least one of n-hexane, cyclohexane, petroleum ether, n-heptane, n-octane, n-nonane, and n-decane.

[0033] The method for synthesizing vitamin A palmitate catalyzed by immobilized lipase generally further comprises separating the solid and liquid of the obtained reaction product after the enzyme-catalyzed transesterification reaction is completed, and then concentrating the obtained filtrate to obtain vitamin A palmitate.

[0034] The present invention will be described in detail below through examples.

[0035] In the following examples and comparative examples, 3A molecular sieve was purchased from Changge Gerun Purification Materials Co., Ltd.; D101 macroporous adsorption resin was purchased from Langfang Miaoyang Chemical Co., Ltd., with a specific surface area of ​​≥550m 2 / g; Alumina was purchased from Guangzhou Changyu Chemical Co., Ltd., with a specific surface area of ​​76m 2 / g; Nano manganese tetraoxide was purchased from Zhejiang Zhitai Nano Micro New Materials Co., Ltd. with the item number ZT and a particle size of 40nm-50nm; Nano silicon dioxide was purchased from Shanghai Naio Nano Technology Co., Ltd. with the item number NO-O-001-1 and an average particle size of 20nm; Aspergillus niger lipase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd. with the item number FDG-2208 and an enzyme activity of 100,000 U / g; Antarctic Candida lipase B was Lipozyme CALBL from Novozymes; Thermomyces lanuginosus lipase was purchased from Wuxi Weilan Biotechnology Co., Ltd. with the product model lipase FM-10 and an enzyme activity of 100,000 U / g.

[0036] Preparation Example 1 Immobilized lipase and its preparation method

[0037] S1. 10 g of 3A molecular sieve and 0.1 g of nano manganese tetraoxide were added to 100 mL of 25% ammonia solution, stirred at 55 ° C for 0.5 h, and then 0.1 g of nano silicon dioxide was added and ultrasonically oscillated at 20 KHz and 65 ° C for 5 h, and the solid was filtered;

[0038] S2. Add the solid obtained in the above step and 1 g of Aspergillus niger lipase to 100 mL of phosphate buffer with a pH of 7, stir at 10°C for 5 h, then add 2% glutaraldehyde and stir at 15°C for 10 h, filter, wash with water 3 times and then dry to obtain immobilized lipase K1.

[0039] Preparation Example 2 Immobilized lipase and its preparation method

[0040] S1. 10 g of D101 macroporous adsorption resin and 0.05 g of nano manganese tetraoxide were added to 100 mL of 28% ammonia solution, stirred at 30 ° C for 2 h, and then 0.05 g of nano silicon dioxide was added and ultrasonically oscillated at 20 KHz and 85 ° C for 3 h, and the solid was filtered;

[0041] S2. Add the solid obtained in the above step and 0.1 g of Candida antarctica lipase B to 100 mL of pH 5 phosphate buffer, stir at 25°C for 1 h, then add 1% glutaraldehyde and stir at 25°C for 5 h, filter, wash with water 3 times and dry to obtain immobilized lipase K2.

[0042] Preparation Example 3 Immobilized lipase and its preparation method

[0043] S1. 10 g of aluminum oxide and 0.08 g of nano manganese tetraoxide were added to 100 mL of 30% ammonia solution, stirred at 40 ° C for 1 h, and then 0.08 g of nano silicon dioxide was added and ultrasonically oscillated at 20 KHz and 70 ° C for 6 h, and the solid was filtered;

[0044] S2. Add the solid obtained in the above step and 0.5 g of Thermomyces lanuginosus lipase to 100 mL of pH 4 phosphate buffer, stir at 25°C for 1 h, then add 5% glutaraldehyde and stir at 20°C for 8 h, filter, wash with water 3 times and dry to obtain immobilized lipase K3.

[0045] Preparation Example 4 Immobilized lipase and preparation method thereof

[0046] Immobilized lipase was prepared according to the method of Preparation Example 1, except that the amount of nano manganese tetraoxide was adjusted to 0.01 g. The other conditions were the same as those of Preparation Example 1, and immobilized lipase K4 was obtained.

[0047] Preparation Example 5 Immobilized lipase and its preparation method

[0048] Immobilized lipase was prepared according to the method of Preparation Example 1, except that the amount of nano-silica was adjusted to 0.01 g. The other conditions were the same as those of Preparation Example 1, to obtain immobilized lipase K5.

[0049] Preparation Example 6 Immobilized lipase and its preparation method

[0050] Immobilized lipase was prepared according to the method of Preparation Example 2, except that the amount of Candida antarctica lipase B was replaced by the same weight portion of Thermomyces lanuginosus lipase, and the other conditions were the same as those of Preparation Example 2 to obtain immobilized lipase K6.

[0051] Comparative Preparation Example 1 Immobilized lipase and its preparation method

[0052] Immobilized lipase was prepared according to the method of Preparation Example 2, except that nano-silicon dioxide was not added. The remaining steps were the same as those of Preparation Example 2. The specific process is as follows:

[0053] S1. Add 10 g of D101 macroporous adsorption resin and 0.05 g of nano manganese tetraoxide to 100 mL of 28% ammonia solution, stir at 30 ° C for 2 h, and then ultrasonically oscillate at 20 KHz and 85 ° C for 3 h, and filter to obtain a solid;

[0054] S2. Add the solid obtained in the above step and 0.1 g of Candida antarctica lipase B to 100 mL of pH 5 phosphate buffer, stir at 25°C for 1 h, then add 1% glutaraldehyde and stir at 25°C for 5 h, filter, wash with water 3 times and dry to obtain immobilized lipase K2-1.

[0055] Comparative Preparation Example 2 Immobilized lipase and its preparation method

[0056] Immobilized lipase was prepared according to the method of Preparation Example 2, except that nano manganese tetraoxide was not added. The remaining steps were the same as those of Preparation Example 2. The specific process is as follows:

[0057] S1. Add 10 g of D101 macroporous adsorption resin to 100 mL of 28% ammonia solution, stir at 30 ° C for 2 h, then add 0.05 g of nano-silica and ultrasonically oscillate at 20 KHz and 85 ° C for 3 h, and filter to obtain a solid;

[0058] S2. Add the solid obtained in the above step and 0.1 g of Candida antarctica lipase B to 100 mL of pH 5 phosphate buffer, stir at 25°C for 1 hour, then add 1% glutaraldehyde and stir at 25°C for 5 hours, filter, wash with water 3 times and dry to obtain immobilized lipase K2-2.

[0059] Comparative Preparation Example 3 Immobilized lipase and its preparation method

[0060] Immobilized lipase was prepared according to the method of Preparation Example 2, except that nano manganese tetraoxide and nano silicon dioxide were not added. The remaining steps were the same as those of Preparation Example 2. The specific process is as follows:

[0061] S1. Add 10 g of D101 macroporous adsorption resin to 100 mL of 28% ammonia solution, stir at 30 ° C for 2 h, then ultrasonically oscillate at 20 KHz and 85 ° C for 3 h, and filter to obtain a solid;

[0062] S2. Add the solid obtained in the above step and 0.1 g of Candida antarctica lipase B to 100 mL of pH 5 phosphate buffer, stir at 25°C for 1 h, then add 1% glutaraldehyde and stir at 25°C for 5 h, filter, wash with water 3 times and dry to obtain immobilized lipase K2-3.

[0063] Example 1 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0064] The substrate vitamin A acetate 1g (3mmol), 2.34g (9mmol) palmitic acid, 3g immobilized lipase K1 and 100mL n-hexane were mixed, stirred and reacted at a temperature of 35°C for 8h, the immobilized lipase was separated by filtration, and the filtrate was evaporated under reduced pressure to recover n-hexane to obtain a light yellow oil. The light yellow oil was analyzed according to the method of USP28 of the United States Pharmacopoeia, and the results showed that the content of vitamin A palmitate was 96.3%, and the pure yield was 98.5%.

[0065] Example 2 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0066] The substrate vitamin A acetate 1g (3mmol), 1.15g (4.5mmol) palmitic acid, 1g immobilized lipase K2 and 100mL petroleum ether were mixed, reacted at a temperature of 50°C for 3h, the immobilized lipase was separated by filtration, and the filtrate was evaporated under reduced pressure to recover the petroleum ether to obtain a light yellow oil. The light yellow oil was analyzed according to the method of USP28 of the United States Pharmacopoeia, and the results showed that the content of vitamin A palmitate was 95.9%, and the pure yield was 97.8%.

[0067] Example 3 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0068] The substrate vitamin A acetate 1g (3mmol), 3.85g (15mmol) palmitic acid, 2g immobilized lipase K3 and 100mL petroleum ether were mixed, stirred and reacted at a temperature of 15°C for 12h, the immobilized lipase was separated by filtration, and the filtrate was evaporated under reduced pressure to recover the petroleum ether to obtain a light yellow oil. The light yellow oil was analyzed according to the method of USP28 of the United States Pharmacopoeia, and the results showed that the content of vitamin A palmitate was 96.1%, and the pure yield was 98.2%.

[0069] Example 4 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0070] Vitamin A palmitate was synthesized by using immobilized lipase according to the method of Example 1, except that immobilized lipase K1 was replaced by immobilized lipase K4 of the same weight, and the other conditions were the same as those of Example 1, to obtain a light yellow oil. The light yellow oil was analyzed according to the method of United States Pharmacopoeia USP28, and the results showed that the content of vitamin A palmitate was 91.8%, and the pure yield was 96.4%.

[0071] Example 5 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0072] Vitamin A palmitate was synthesized by using immobilized lipase according to the method of Example 1, except that immobilized lipase K1 was replaced by immobilized lipase K5 of the same weight, and the other conditions were the same as those of Example 1, to obtain a light yellow oil. The light yellow oil was analyzed according to the method of United States Pharmacopoeia USP28, and the results showed that the content of vitamin A palmitate was 90.9%, and the pure yield was 95.7%.

[0073] Example 6 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0074] Vitamin A palmitate was synthesized by using immobilized lipase according to the method of Example 2, except that immobilized lipase K1 was replaced by immobilized lipase K6 of the same weight, and the other conditions were the same as those of Example 2, to obtain a light yellow oil. The light yellow oil was analyzed according to the method of United States Pharmacopoeia USP28, and the results showed that the content of vitamin A palmitate was 91.2%, and the pure yield was 96.1%.

[0075] Comparative Example 1 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0076] Vitamin A palmitate was synthesized by using immobilized lipase according to the method of Example 2, except that immobilized lipase K2 was replaced by immobilized lipase K2-1 of the same weight, and the other conditions were the same as those of Example 1, and finally a light yellow oil was obtained. The light yellow oil was analyzed according to the method of United States Pharmacopoeia USP28, and the results showed that the content of vitamin A palmitate was 86.7%, and the pure yield was 86.4%.

[0077] Comparative Example 2 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0078] Vitamin A palmitate was synthesized by using immobilized lipase according to the method of Example 2, except that immobilized lipase K2 was replaced by immobilized lipase K2-2 of the same weight, and the other conditions were the same as those of Example 1, and finally a light yellow oil was obtained. The light yellow oil was analyzed according to the method of United States Pharmacopoeia USP28, and the results showed that the content of vitamin A palmitate was 79.9%, and the pure yield was 82.7%.

[0079] Comparative Example 3 Synthesis of Vitamin A Palmitate Catalyzed by Immobilized Lipase

[0080] Vitamin A palmitate was synthesized by catalysis of immobilized lipase according to the method of Example 2, except that immobilized lipase K2 was replaced by immobilized lipase K2-3 of the same weight, and the other conditions were the same as those of Example 1, and finally a light yellow oil was obtained. The light yellow oil was analyzed according to the method of United States Pharmacopoeia USP28, and the results showed that the content of vitamin A palmitate was 75.0%, and the pure yield was 78.6%.

[0081] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. An immobilized lipase, characterized in that: The immobilized lipase comprises a porous carrier, nano manganese tetraoxide, nano silicon dioxide and lipase loaded on the porous carrier.

2. The immobilized lipase according to claim 1, characterized in that The mass ratio of the porous carrier, nano manganese tetraoxide and nano silicon dioxide is 1:(0.005-0.01):(0.005-0.01); the mass ratio of the porous carrier and lipase is 1:(0.01-0.1).

3. The immobilized lipase according to claim 1 or 2, characterized in that: The porous carrier is selected from at least one of activated carbon, kaolin, molecular sieve, alumina, silicon oxide, aluminum silicate, magnesium oxide, porous glass, adsorption resin and ion exchange resin; Preferably, the average particle size of the nano manganese tetraoxide is 5nm to 500nm; Preferably, the average particle size of the nano-silicon dioxide is 20nm to 200nm; Preferably, the lipase is selected from at least one lipase obtained by fermentation of Candida antarctic, Thermomyces lanuginosus, Rhizomucor miehei, Aspergillus niger, Candida rugosa and Pseudononas sp.

4. A method for preparing immobilized lipase, characterized in that: The method comprises the following steps: S1. The porous carrier, nano-manganese tetraoxide and nano-silicon dioxide are mixed in an aqueous ammonia solution and then solid-liquid separation is performed to obtain a solid product and a liquid product; S2. The solid product and lipase are mixed evenly in a phosphate buffer solution, and the obtained mixture is subjected to a cross-linking reaction with a cross-linking agent, and then the obtained cross-linking reaction product is filtered, washed with water and dried to obtain an immobilized lipase.

5. The method for preparing immobilized lipase according to claim 4, characterized in that: In step S1, the mixing method includes stirring and mixing the porous carrier and nano manganese tetraoxide in an ammonia solution, and then adding nano silicon dioxide for ultrasonic vibration; Preferably, the concentration of the aqueous ammonia solution is 20% to 30%; Preferably, the stirring conditions include a stirring temperature of 30°C to 55°C and a stirring time of 0.5h to 2h; Preferably, the ultrasonic oscillation conditions include an ultrasonic oscillation temperature of 60° C. to 85° C. and an ultrasonic oscillation time of 3 h to 10 h.

6. The method for preparing immobilized lipase according to claim 4, characterized in that: In step S2, the pH value of the phosphate buffer solution is 4 to 7; Preferably, the mixing method is stirring and mixing, and the stirring and mixing temperature is 5°C to 25°C and the time is 1h to 10h; Preferably, the cross-linking agent is glutaraldehyde; Preferably, the amount of the cross-linking agent added is 1% to 5% of the mass of the solid product; Preferably, the conditions of the cross-linking reaction include a temperature of 10° C. to 25° C. and a reaction time of 2 h to 10 h.

7. Immobilized lipase prepared by the method according to any one of claims 4 to 6.

8. Use of the immobilized lipase according to any one of claims 1 to 3 and 7 in catalytic synthesis of vitamin A palmitate.

9. A method for synthesizing vitamin A palmitate catalyzed by immobilized lipase, characterized in that: The method comprises subjecting vitamin A acetate and palmitic acid to an enzyme-catalyzed transesterification reaction in the presence of the immobilized lipase described in any one of claims 1 to 3 and 7 to obtain vitamin A palmitate.

10. The method for synthesizing vitamin A palmitate catalyzed by immobilized lipase according to claim 9, characterized in that: The molar ratio of vitamin A acetate to palmitic acid is 1:(1.5-5); Preferably, the amount of the immobilized lipase is 1 to 3 times the mass of vitamin A acetate; Preferably, the conditions for the enzyme-catalyzed transesterification reaction include a temperature of 15° C. to 55° C. and a time of 3 h to 12 h.