Synthesis process of 1,3-diacerol and preparation method of lipase used therein

Abstract

The present application relates to a synthesis process of 1,3-diacylglycerol and a method for preparing lipase used therein. The method for preparing lipase comprises the following steps: taking a freeze-dried strain of Antarctic Candida type A and reconstructing it in deionized water to prepare a lipase having a concentration of (0.1-9)×10 9 CFU / ml bacterial liquid; weigh yeast extract, peptone, glucose, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate and dissolve them in deionized water to obtain a culture medium, and then sterilize the culture medium in an autoclave; add the bacterial liquid to the culture medium and culture it in a shaker so that the strain secretes lipase; centrifuge the culture medium in a centrifuge, separate the bacterial liquid and collect the cell precipitate, break the cells, and release the lipase from the cells; use Immobead150 to mix with the lipase solution to obtain an enzyme resin complex, i.e., immobilized enzyme particles. The Antarctic Candida lipase A obtained by this preparation method has a strong specificity for 1,3-diaglycerides and can significantly improve the synthesis efficiency of 1,3-diaglycerides.

Description

Technical Field

[0001] The present application relates to bio-enzyme engineering technology, and specifically to a synthesis process of 1,3-diacerol and a method for preparing lipase used therein. Background Art

[0002] Diacylcerol DAG is a compound generated by the esterification reaction of glycerol and fatty acids. The common isomers are 1,2-diacylcerol and 1,3-diacylcerol. Studies have shown that 1,3-diacylcerol can effectively promote fat metabolism during digestion, help control fat accumulation and protect cardiovascular health. Compared with 1,2-diacylcerol, the absorption pathway of 1,3-diacylcerol outside the small intestine makes it easier to utilize, which reduces the burden on the intestine and therefore has better health benefits. However, in the current process of preparing 1,3-diacylcerol, it is easy to produce more by-products, such as 1,2-diacylcerol and free fatty acids, the yield of 1,3-diacylcerol is low, and the purification process is complicated. Summary of the invention

[0003] In order to overcome the above-mentioned defects, the present application provides a method for preparing lipase. The Antarctic Candida lipase A obtained by the preparation method has strong specificity for 1,3-diacylglycerol and can significantly improve the synthesis efficiency of 1,3-diacylglycerol.

[0004] The technical solution adopted by this application to solve its technical problems is:

[0005] A method for preparing lipase comprises the following steps:

[0006] Activation of strains: freeze-dried strains of Candida antarctica type A were reconstituted in deionized water to a concentration of (0.1-9)×10 9 CFU / ml bacterial solution, and add glucose and mix evenly, and let it stand at 20-35°C for 1-3 hours to promote bacterial activation, wherein the percentage of glucose in the bacterial solution is 0.2-2%;

[0007] Preparation of YPD medium: weigh yeast extract, peptone, glucose, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate, dissolve in deionized water to obtain a medium, adjust the pH value of the medium to 5.5-7.5, and then sterilize the medium in an autoclave, and cool after sterilization to obtain a YPD medium;

[0008] Lipase culture: Add the bacterial solution to the culture medium, and add Tween 80, soybean lecithin and vitamin B to the culture medium. 12 , and cultured in a shaker for 36 to 60 hours to allow the strain to secrete lipase, wherein the culture conditions are: temperature 20 to 40°C, shaker speed 150 to 250 rpm;

[0009] Separation and disruption of enzymes: centrifuge the culture medium to separate the bacterial solution and collect the cell precipitate, use an ultrasonic disruptor to disrupt the cells to release lipase, centrifuge to remove cell debris, and collect the lipase solution;

[0010] Enzyme immobilization: Immobead 150 resin was pretreated with sodium bicarbonate solution, and then the resin was washed several times with phosphate buffer to remove alkaline residues. The lipase solution was then mixed with the resin and reacted for 20 to 30 hours to immobilize the enzyme to obtain an enzyme-resin complex, i.e., immobilized enzyme particles. The immobilized enzyme particles were then soaked in a buffer to improve stability.

[0011] Enzyme protection and freeze-drying treatment: soak the immobilized enzyme particles in a buffer solution containing the first protective agent at a temperature of 2 to 8°C for 8 to 16 hours to allow the protective agent to fully cover the surface of the immobilized enzyme particles. Then, place the immobilized enzyme particles in a drying device for drying. Finally, store the dried immobilized enzyme particles, i.e., Antarctic Candida lipase A, in a moisture-proof package.

[0012] Optionally, in the preparation step of YPD medium, the concentration of each component in the medium is as follows: yeast extract 6-15 g / L, peptone 15-25 g / L, glucose 25-35 g / L, potassium dihydrogen phosphate 0.5-2 g / L, magnesium sulfate heptahydrate 0.1-1 g / L; sterilization conditions: temperature 121±5°C, time 15±5 min; glycerol is added to the sterilized and cooled medium, and the percentage of glycerol in the medium is 0.5-2%.

[0013] Optionally, in the lipase culture step, the volume ratio of the bacterial solution to the culture medium is (0.3-2):19; the bacterial solution is kept at 25-35° C. for 10-15 hours before being added to the culture medium; the percentage of each additive in the culture medium is: Tween 80 0.5-2%, soy lecithin 0.1-0.3% and vitamin B 12 0.01~0.2%, culture conditions: in the first half of the culture, the temperature is 28℃±1℃, and the shaking speed is 180rpm±10rpm; in the second half of the culture, the temperature is 30℃±1℃, and the shaking speed is 220rpm±10rpm.

[0014] Optionally, in the enzyme separation and crushing steps, the centrifugal conditions before crushing are centrifugal force 4000g±500g, temperature 4℃±1℃, time 15min±5min; the crushing conditions are: 2 seconds working, 3 seconds rest, repeated 20 times; before crushing, a second protective agent is added to the culture medium, the second protective agent includes 0.5~1.5% PEG-4000 and 0.2~1% glycerol; the centrifugal conditions after crushing are centrifugal force 10000g±1000g, temperature 4℃±1℃, time 10min±5min; protease inhibitors PMSF, EDTA and sodium azide are added to the lipase solution, the concentration of PMSF is 0.1mM±0.02mM, the EDTA concentration in the lipase solution is 0.05~0.15%, and the sodium azide concentration is 0.005~0.015%, and the lipase solution is stored at 4℃±1℃.

[0015] Optionally, in the enzyme immobilization step, the Immobead 150 resin is first centrifuged and washed with deionized water, and then pretreated with a 0.05-0.2M sodium bicarbonate solution. The lipase solution and the resin are mixed in a ratio of (0.5-2):10, placed in an environment of 4°C±1°C, the pH is adjusted to 5-7, and the reaction is carried out. During the reaction, a stabilizer and a third protective agent are added to the lipase solution, wherein the stabilizer is a 0.5-2mM CaCl2 solution, and the third protective agent is 0.5-2% PEG-4000 and 0.1-0.5% BSA;

[0016] After the immobilization is completed, the immobilized enzyme particles are immersed in 0.05-0.2M Tris-HCl buffer at 4°C±1°C for 8-16 hours, and then the immobilized enzyme particles are washed by centrifugation with PBS buffer at a centrifugal speed of 2000±500rpm for 5-20 minutes.

[0017] Optionally, in the enzyme protection and freeze-drying treatment steps, the buffer is 0.05-0.2 M Tris-HCl buffer, and the protective agent formula includes: 2-10% glycerol, 10-20% sucrose, 3-8% trehalose and 0.5-5% polyvinyl alcohol;

[0018] The immobilized enzyme particles were dried using a vacuum drying device, and the drying conditions were as follows: the initial temperature was set at 25°C ± 5°C, the vacuum degree was maintained at 40-60 mbar, the drying time was 4-8 hours, and then, while maintaining the vacuum degree, the temperature was slowly raised to 40°C ± 5°C for 4-8 hours, and then further raised to 45°C ± 5°C for 2-5 hours.

[0019] Optionally, an enzyme regeneration step is also included. After each use, the immobilized enzyme particles are washed with 15-25mM phosphate buffer and then immersed in a regeneration solution for 1.5-3h to restore the activity of the enzyme, wherein the regeneration solution includes an aqueous solution of 0.5-2% glycerol, 0.05-0.2% BSA and 3-6mM CaCl2.

[0020] The present application provides a synthesis process of 1,3-diacylglycerol, which uses the Antarctic Candida lipase A prepared by the preparation method of any one of claims 1 to 7 to enzymatically hydrolyze triglycerides to synthesize 1,3-diacylglycerol.

[0021] Optionally, the synthesis process of 1,3-diacerol comprises the following steps:

[0022] First, triglyceride is added to a reaction container, heated to 70-90°C, glycerol is added and stirred evenly, and then Antarctic Candida lipase A is added and mixed, and the reaction is performed for 18-36 hours to obtain a reaction mixture. The reaction mixture is purified by molecular distillation to obtain 1,3-diglyceride, wherein the mass ratio of glycerol to triglyceride is (0.5-2.5):19, the pH of the reaction system is 5.5-7.5, the stirring speed is 150-250rpm, and the amount of Antarctic Candida lipase A added is 1-5% of the total weight of glycerol and triglyceride.

[0023] Optionally, before adding the Candida antarctica lipase A, the reaction vessel is purged with nitrogen for 6 to 12 minutes, and an antioxidant is added to the reaction system during the reaction, wherein the amount of the antioxidant added is 0.005% to 0.02% of the total weight of glycerol and triglycerides.

[0024] The beneficial effects of the present application are as follows: in the present application, the steps of strain selection, enzyme cultivation, enzyme separation, enzyme immobilization and enzyme protection are optimized, and the obtained Antarctic Candida lipase A has a strong specificity for 1,3-diaceryl, which can significantly improve the synthesis efficiency of 1,3-diaceryl, while reducing the generation of by-products such as 1,2-DAG and free fatty acids, ensuring the high yield and high purity of 1,3-diaceryl; at the same time, the obtained Antarctic Candida lipase A can be reused many times through solidification and regeneration treatment, saving resources and reducing costs. The present application synthesizes 1,3-diaceryl through CALA catalysis, which has the advantages of high yield, high purity, low by-product generation, and multiple reuse, and is particularly suitable for the production of food-grade diglycerides. Compared with traditional chemical catalysis methods, the present application not only improves the catalytic efficiency and stability of the enzyme, but also optimizes the production process, reduces energy consumption and the generation of by-products, and has broad industrial application prospects. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0026] A method for preparing lipase comprises the following steps:

[0027] Activation of strains: freeze-dried strains of Candida antarctica type A were reconstituted in deionized water to a concentration of (0.1-9)×10 9 CFU / ml of bacterial solution, and add glucose and mix evenly, and let it stand at 20-35°C for 1-3 hours to promote bacterial activation, wherein the percentage of glucose in the bacterial solution is 0.2-2%; the percentage refers to the mass percentage, the same below, glucose pre-sterilization;

[0028] Preparation of YPD medium: weigh yeast extract, peptone, glucose, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate, dissolve in deionized water to obtain a medium, adjust the pH value of the medium to 5.5-7.5, and then sterilize the medium in an autoclave, and cool after sterilization to obtain a YPD medium;

[0029] Lipase culture: Add the bacterial solution to the culture medium, and add Tween 80, soybean lecithin and vitamin B to the culture medium. 12 , and cultured in a shaker for 36 to 60 hours to allow the strain to secrete lipase, wherein the culture conditions are: temperature 20 to 40°C, shaker speed 150 to 250 rpm;

[0030] Separation and disruption of enzymes: centrifuge the culture medium to separate the bacterial solution and collect the cell precipitate, use an ultrasonic disruptor to disrupt the cells to release lipase, centrifuge to remove cell debris, and collect the lipase solution;

[0031] Enzyme immobilization: Immobead 150 resin was pretreated with sodium bicarbonate solution, and then the resin was washed several times with phosphate buffer to remove alkaline residues. The lipase solution was then mixed with the resin and reacted for 20 to 30 hours to immobilize the enzyme to obtain an enzyme-resin complex, i.e., immobilized enzyme particles. The immobilized enzyme particles were then soaked in a buffer to improve stability.

[0032] Enzyme protection and freeze-drying treatment: soak the immobilized enzyme particles in a buffer solution containing a first protective agent at a temperature of 2 to 8°C for 8 to 16 hours, so that the protective agent fully covers the surface of the immobilized enzyme particles, and then place the immobilized enzyme particles in a drying device for drying, so that the moisture content in the immobilized enzyme particles is less than 3%, and finally store the dried immobilized enzyme particles, i.e., Antarctic Candida lipase A, in a moisture-proof package. In this application, the selection of strains, the cultivation of enzymes, the separation of enzymes, the immobilization of enzymes, and the protection of enzymes are optimized, and the obtained Antarctic Candida lipase A has a strong specificity for 1,3-diacylglycerol, can significantly improve the synthesis efficiency of 1,3-diacylglycerol, and reduce the generation of by-products such as 1,2-DAG and free fatty acids, ensuring the high yield and high purity of 1,3-diacylglycerol; at the same time, the obtained Antarctic Candida lipase A can be reused many times through solidification and regeneration treatment, saving resources and reducing costs.

[0033] In the preparation steps of the YPD medium, the concentrations of the components in the medium are as follows: 6-15 g / L yeast extract, 15-25 g / L peptone, 25-35 g / L glucose, 0.5-2 g / L potassium dihydrogen phosphate, 0.1-1 g / L magnesium sulfate heptahydrate; sterilization conditions: temperature 121±5°C, time 15±5 min; glycerol is added to the sterilized and cooled medium, and the percentage of glycerol in the medium is 0.5-2% to restore the activity of the strain. Glycerol is used as a carbon source auxiliary.

[0034] In the lipase culture step, the volume ratio of bacterial liquid to culture medium is (0.3-2):19; the bacterial liquid is kept at 25-35°C for 10-15 hours before being added to the culture medium; the percentage of each additive in the culture medium is: Tween 80 0.5-2%, soybean lecithin 0.1-0.3% and vitamin B 12 0.01~0.2%, the culture conditions are: in the first half of the culture, the temperature is 28℃±1℃, the shaking speed is 180rpm±10rpm; in the second half of the culture, the temperature is 30℃±1℃, the shaking speed is 220rpm±10rpm. Add Tween 80 In order to promote the secretion of lipase and increase the yield, soybean lecithin is added to assist the secretion of lipase, and vitamin B is added 12 Enhance metabolic activity. During the culture process, the culture temperature is set at about 28°C in the first half to enhance bacterial proliferation, and raised to about 30°C in the second half to optimize the enzyme secretion rate and ensure sufficient oxygen supply. The shaker speed is set at about 180rpm in the first half to maintain sufficient dissolved oxygen; it is increased to 220rpm in the second half to promote uniform distribution of nutrients.

[0035] In the enzyme separation and crushing step, the centrifugal conditions before crushing are centrifugal force 4000g±500g, temperature 4℃±1℃, time 15min±5min, separation of bacterial solution and collection of cell precipitate; crushing conditions are: 2 seconds working, 3 seconds rest, repeated 20 times, release lipase. A cold bath is used to maintain the sample temperature during the process to prevent overheating; before crushing, a second protective agent is added to the culture medium, and the second protective agent includes 0.5-1.5% PEG-4000 and 0.2-1% glycerol; the percentage refers to the mass percentage of PEG-4000 and glycerol in the culture medium, and the protective agent is added to reduce possible enzyme inactivation during the crushing process. The centrifugal conditions after crushing are centrifugal force 10000g±1000g, temperature 4℃±1℃, time 10min±5min; remove cell debris and collect lipase solution. Add protease inhibitors PMSF, EDTA and sodium azide to the lipase solution. The concentration of PMSF is 0.1mM±0.02mM. The lipase solution contains 0.05-0.15% EDTA and 0.005-0.015% sodium azide. Store the lipase solution at 4℃±1℃ to delay enzyme degradation. To prevent the target enzyme from being degraded, add protease inhibitor PMSF, EDTA is used to chelate metal ions to prevent enzyme degradation, and sodium azide is used to inhibit microbial contamination.

[0036] In the enzyme immobilization step, the Immobead 150 resin is first centrifuged and washed with deionized water, and then repeated three times to remove impurities, and then the Immobead 150 resin is pretreated with a 0.05-0.2M sodium bicarbonate solution to improve the binding efficiency of the enzyme and the resin, and the lipase solution and the resin are mixed in a ratio of (0.5-2):10, placed in an environment of 4°C±1°C, and the pH is adjusted to 5-7 for reaction. During the reaction, a stabilizer and a third protective agent are added to the lipase solution, wherein the stabilizer is a 0.5-2mM CaCl2 solution, and the third protective agent is 0.5-2% PEG-4000 and 0.1-0.5% BSA; the amount of polyethylene glycol PEG-4000 and bovine serum albumin BSA refers to the percentage of the lipase solution. The stabilizer is used to enhance the activity and stability of the lipase.

[0037] After the immobilization is completed, the immobilized enzyme particles are immersed in 0.05-0.2M Tris-HCl buffer at 4°C±1°C for 8-16 hours to further improve their structural stability, and then the immobilized enzyme particles are washed by centrifugation with PBS buffer at a centrifugal speed of 2000±500rpm for 5-20 minutes, and washed three times to remove unbound enzyme.

[0038] In the enzyme protection and freeze-drying treatment steps, the buffer is 0.05-0.2M Tris-HCl buffer, and the protective agent formula includes: 2-10% glycerol, 10-20% sucrose, 3-8% trehalose and 0.5-5% polyvinyl alcohol;

[0039] Use vacuum drying equipment to dry the immobilized enzyme particles. The drying conditions are as follows: the initial temperature is set to 25℃±5℃, the vacuum is maintained at 40-60mbar, the drying time is 4-8h, and then the temperature is slowly raised to 40℃±5℃ while maintaining the vacuum for 4-8h, and then further raised to 45℃±5℃ for 2-5h. The temperature should not exceed 50℃ throughout the process to avoid thermal inactivation of the enzyme protein. The moisture content is controlled at 3%, and the particles are finally sealed in moisture-proof packaging for storage. Storage recommendations: Store in a cool, dry environment, avoid exposure to high temperature, humidity or strong light conditions.

[0040] The preparation method of lipase also includes an enzyme regeneration step. After each use, the immobilized enzyme particles are washed with 15-25mM phosphate buffer and then soaked in a regeneration solution for 1.5-3 hours to restore the activity of the enzyme. The regeneration solution includes an aqueous solution of 0.5-2% glycerol, 0.05-0.2% BSA and 3-6mM CaCl2. The enzyme used each time is stored in a 50% glycerol and 0.1M Tris-HCl buffer (containing 0.02% NaN3 for preservation) to maintain its stability.

[0041] The enzyme activity verification step is also included. The activity is determined by using the p-NPP substrate method (p-nitrophenyl ester hydrolysis method) to verify the esterase activity of the immobilized lipase to ensure that the enzyme catalytic ability remains efficient. The specific activity of the enzyme (unit: U / mg) is measured before and after lyophilization to evaluate the effect of the lyophilization process on the enzyme activity. The activity loss is within 10%.

[0042] A synthesis process of 1,3-diacerol comprises the following steps:

[0043] First, triglyceride is added to a reaction vessel, heated to 70-90°C, glycerol is added and stirred evenly, glycerol is added gradually and dripped to ensure full mixing and avoid inhibition of enzyme activity by local high concentration of glycerol, then Antarctic Candida lipase A is added and mixed, reacted for 18-36 hours, and a reaction mixture is obtained, and the reaction mixture is purified by molecular distillation to obtain 1,3-diglyceride, wherein the mass ratio of glycerol to triglyceride is (0.5-2.5):19, the pH of the reaction system is 5.5-7.5, the stirring speed is 150-250rpm, and the amount of Antarctic Candida lipase A added is 1-5% of the total weight of glycerol and triglyceride. In this application, Antarctic Candida lipase A (CALA) is used to catalyze the reaction of glycerol and triglyceride to generate 1,3-diglyceride. CALA enzyme has strong 1,3 specificity, can significantly improve the synthesis efficiency of 1,3-diglyceride, and reduce the generation of byproducts such as 1,2-DAG and free fatty acids. By optimizing the reaction conditions, such as temperature, pH value, and the ratio of glycerol to triglycerides, the generation of by-products can be minimized, ensuring high yield and high purity of 1,3-diglycerides. In addition, since the reaction produces almost no free fatty acids, the purification process is simpler, effectively improving production efficiency and economy.

[0044] The present application uses CALA to catalyze the synthesis of 1,3-diacerol, which has the advantages of high yield, high purity, low by-product generation, and multiple reuse, and is particularly suitable for the production of food-grade diglycerol. Compared with traditional chemical catalytic methods, the present application not only improves the catalytic efficiency and stability of the enzyme, but also optimizes the production process, reduces energy consumption and by-product generation, and has broad industrial application prospects.

[0045] This application utilizes the efficient catalytic properties of Antarctic Candida lipase A (CALA) and its optimized culture process. Compared with traditional chemical catalysis, this application significantly reduces the need for high temperature and high pressure conditions by using biological enzymes, thereby reducing equipment costs and energy consumption, and avoiding environmental pollution and subsequent treatment problems caused by strong acid or strong base catalysts. And proposes an optimized culture method for CALA enzyme activity, covering enzyme expression, purification and stability enhancement strategies, so that it can efficiently catalyze the reaction of glycerol and triglycerides under mild conditions (such as 80°C, pH 6.5) to generate 1,3-diaceryl and produce a small amount of by-products, and can be reused many times, greatly saving production costs. This synthesis process has high production efficiency and low by-product generation, and is suitable for many fields such as food, cosmetics and biofuels, providing reliable technical support for the sustainable production of diglycerides.

[0046] Before adding Antarctic Candida lipase A, the reaction vessel is purged with nitrogen for 6 to 12 minutes to discharge oxygen in the reaction vessel and reduce the risk of enzyme activity decreasing due to oxidation. During the reaction, an antioxidant is added to the reaction system in an amount of 0.005% to 0.02% of the total weight of glycerol and triglycerides. The antioxidant is vitamin E. Antioxidants are used to improve the stability of reactants and products. The synthesis process adjusts the reaction conditions, optimizes the enzyme catalytic efficiency, reduces the generation of by-products, and the by-product concentration is less than 10%, the triglyceride conversion rate exceeds 90%, and the monoglyceride and free fatty acid concentrations are less than 6.5%.

[0047] 1. A method for preparing lipase, comprising the following steps:

[0048] Step 1: Activation of strains: Purchase freeze-dried strains of Candida antarctica type A from Sigma-Aldrich Company, take 1 g of freeze-dried strains, reconstitute in 20 mL of sterile deionized water, and prepare a concentration of 1×10 9 CFU / ml of bacterial solution, add 1% glucose (pre-sterilized), ensure that the bacterial solution is evenly mixed, and let it stand at 28°C for 2 hours to promote bacterial activation;

[0049] Step 2: Preparation of YPD medium: The medium components and their contents are: yeast extract 10 g / L, peptone 20 g / L, glucose 30 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, the solvent is water, the pH value is adjusted to 6.5, the medium is sterilized at 121°C for 15 minutes using an autoclave, and after cooling to room temperature, 1% glycerol (carbon source auxiliary) is added to the medium;

[0050] Step 3: Cultivation of lipase: The bacterial solution was cultured at 28°C for 12 hours to restore the activity of the strain, 5 mL of the prepared bacterial solution was added to 95 mL of the culture medium, and cultured in a shaker. In order to promote the secretion of lipase and increase the yield, 1% Tween-80 (purchased from Shanghai McLean Biochemical Technology Co., Ltd.) was added to the culture medium; at the same time, 0.2% soy lecithin (to assist lipase secretion) and 0.05% vitamin B were added. 12 (Enhance metabolic activity);

[0051] The culture conditions are as follows: culture for 48 hours, the culture temperature is set to 28°C in the first 24 hours to enhance bacterial proliferation, and then raised to 30°C to optimize the enzyme secretion rate and ensure sufficient oxygen supply; the shaker speed is set to 180rpm in the first 24 hours to maintain sufficient dissolved oxygen; and increased to 220rpm in the next 24 hours to promote uniform distribution of nutrients.

[0052] Step 4: Enzyme separation and fragmentation

[0053] 4.1 After 48 hours of culture, the bacterial solution was separated and the cell pellet was collected by centrifugation at 4000g and 4°C for 15 minutes using a Beckman Coulter Allegra X-15R centrifuge. The cells were disrupted using a Qsonica Q700 ultrasonic disruptor, set to 2 seconds on, 3 seconds off, repeated 20 times, to release lipase. A cold bath was used to maintain the sample temperature during the process to prevent overheating. Before ultrasonic disruption, a protective agent (1% PEG-4000 and 0.5% glycerol) was added to the sample to reduce possible enzyme inactivation during the disruption process.

[0054] 4.2 The disrupted solution was centrifuged at 10,000 g, 4°C for 10 minutes to remove cell debris and collect the lipase solution; to prevent the target enzyme from being degraded, the protease inhibitor PMSF (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was added to a final concentration of 0.1 mM. At the same time, 0.1% EDTA (chelating metal ions to prevent enzyme degradation) and 0.01% sodium azide (inhibiting microbial contamination) were added.

[0055] 4.3 Ensure that the lipase solution is always stored at 4°C to delay enzyme degradation.

[0056] Step 5: Enzyme immobilization

[0057] 5.1 Purchase Immobilized beads (Immobead 150) supplied by Sigma-Aldrich, and wash them by centrifugation with deionized water (3000 rpm, 8 minutes), repeating three times to remove impurities.

[0058] 5.2 Use 0.1M Na2HCO3 solution (pH 8.0) to pretreat the resin to improve the binding efficiency of the enzyme to the resin;

[0059] After pretreatment, the cells were washed several times with PBS buffer (pH 7.0) to remove alkaline residues;

[0060] The lipase solution was mixed with the pretreated resin at a ratio of 1:10, placed at 4°C, the pH was adjusted to 6.5, reacted for 24 h, and 1 mM CaCl2 was added to enhance the activity and stability of the lipase, as well as 1% PEG-4000 (polyethylene glycol) and 0.2% BSA (bovine serum albumin) as protective agents;

[0061] After the immobilization, the immobilized enzyme particles were immersed in 0.1 M Tris-HCl buffer at 4°C for 12 hours to further improve their structural stability. The enzyme resin complex was then washed with PBS buffer, centrifuged at 2000 rpm for 10 minutes, and washed three times to remove unbound enzyme.

[0062] Step 6: Enzyme protection and freeze-drying

[0063] 6.1 Soak the immobilized enzyme particles in a buffer containing a protective agent, the protective agent formula includes 5% glycerol, 15% sucrose, 5% trehalose and 2% polyvinyl alcohol (PVA), and mix them thoroughly. The soaking process is left at 4°C for 12 hours to allow the protective agent to fully cover the surface of the enzyme particles, thereby forming a stable protective film to reduce water loss and enzyme activity loss.

[0064] 6.2 Take out the immobilized enzyme particles treated with the protective agent, use a vacuum drying device (Memmert VO49) (or a low-temperature drying device below 50°C) to dry the immobilized enzyme particles, place the immobilized enzyme particles in a vacuum drying oven, set the initial temperature to 25°C, maintain the vacuum degree at 50 mbar, and dry for 6 hours to gradually remove surface moisture.

[0065] While maintaining the vacuum, the temperature was slowly raised to 40°C for 5 hours; then further raised to 45°C and maintained for 3 hours to ensure that the moisture deep in the particles was completely discharged.

[0066] The temperature should not exceed 50℃ during the whole process to avoid thermal inactivation of enzyme protein. The moisture content is controlled at 3%, and the immobilized enzyme particles are sealed in moisture-proof packaging for storage. Storage recommendations: Store in a cool and dry environment, avoid exposure to high temperature, humidity or strong light conditions.

[0067] Step 7: Enzyme activity verification: p-NPP substrate method (p-nitrophenyl ester hydrolysis method) was used for activity determination to verify the esterase activity of the immobilized lipase and ensure that the enzyme catalytic ability remained efficient. The specific activity of the enzyme (unit: U / mg) was measured before and after lyophilization to evaluate the effect of the lyophilization process on the enzyme activity. The activity loss was within 10%.

[0068] 2. Determination of Synthesis Process Parameters of 1,3-Diglycerol

[0069] 1. Determination of glycerol ratio

[0070] The synthesis process of 1,3-diacerol includes the following steps:

[0071] The reaction was carried out using freeze-dried immobilized enzyme particles, namely Antarctic Candida lipase A (CALA), and the amount of enzyme was 2% of the mass of the reaction substrate. In the reaction system, the mass ratio of glycerol to triglyceride is shown in Table 1 below. The reaction was carried out in a solvent-free system, the reaction temperature was 80°C, the pH was 6.5, the reaction time was 24 hours, and the stirring speed was set to 200rpm. The triglyceride was first heated to 80°C to dissolve, and glycerol was gradually added dropwise to ensure sufficient mixing and avoid local high concentrations of glycerol to inhibit enzyme activity. The system was purged with nitrogen for 10 minutes before the reaction to exclude oxygen to reduce the risk of enzyme activity decreasing due to oxidation, and 0.01% antioxidant (vitamin E) was added to the reactants to improve the stability of the substrate and the product.

[0072] Table 1: Experimental data under different glycerol ratios (temperature 80°C, reaction time 24 hours)

[0073] Glycerol concentration (%) 1,3-Diglyceride (%) 1,2-Diglyceride (%) Monoglyceride (%) Triglyceride (%) Free fatty acids (%) 3 55 15 15 14 1 4 58 17 12 12.2 0.8 5 66 17 10 6.5 0.5 6 62 18 12 7.3 0.7 7 54 16 15 14.2 0.8

[0074] According to Table 1, when the glycerol concentration is 5%, that is, the mass ratio of glycerol to triglyceride is 1:19, the content of 1,3-diaceryl is the highest, and the content of monoglyceride and free fatty acid reaches the lowest when the glycerol concentration is 5%, reflecting a higher reaction efficiency.

[0075] 2. Determination of reaction temperature

[0076] The synthesis process of 1,3-diacerol includes the following steps:

[0077] The immobilized enzyme particles after freeze-drying, namely Antarctic Candida lipase A (CALA), were used for the reaction, and the amount of enzyme was 2% of the mass of the reaction substrate. In the reaction system, the mass ratio of glycerol to triglyceride was 1:19, and the reaction was carried out in a solvent-free system. The reaction temperature was shown in Table 2, the pH was 6.5, the reaction time was 24 hours, and the stirring speed was set to 200rpm. The triglyceride was first heated and dissolved, and glycerol was gradually added dropwise to ensure sufficient mixing and avoid the inhibition of enzyme activity by local high concentrations of glycerol. The system was purged with nitrogen for 10 minutes before the reaction to exclude oxygen to reduce the risk of enzyme activity decreasing due to oxidation, and 0.01% antioxidant (vitamin E) was added to the reactants to improve the stability of the substrate and the product.

[0078] Table 2: Experimental data at different reaction temperatures (glycerol ratio is 5%, reaction time is 24 hours)

[0079] Temperature(℃) 1,3-Diglyceride (%) 1,2-Diglyceride (%) Monoglyceride (%) Triglyceride (%) Free fatty acids (%) 40 42 18 20 18.8 1.2 60 54 16 18 11 1.0 80 66 17 10 6.5 0.5 100 61 19 12 7.2 0.8 120 59 16 15 9.2 0.8

[0080] 3. Determination of reaction time

[0081] The synthesis process of 1,3-diacerol includes the following steps:

[0082] The reaction was carried out using freeze-dried immobilized enzyme particles, namely Antarctic Candida lipase A (CALA), and the amount of enzyme was 2% of the mass of the reaction substrate. In the reaction system, the mass ratio of glycerol to triglyceride was 1:19, the reaction was carried out in a solvent-free system, the reaction temperature was 80°C, the pH was 6.5, the reaction time was shown in Table 3, and the stirring speed was set to 200rpm. The triglyceride was first heated and dissolved, and glycerol was gradually added dropwise to ensure sufficient mixing and avoid the inhibition of enzyme activity by local high concentrations of glycerol. The system was purged with nitrogen for 10 minutes before the reaction to exclude oxygen to reduce the risk of enzyme activity decreasing due to oxidation, and 0.01% antioxidant (vitamin E) was added to the reactants to improve the stability of the substrate and the product.

[0083] Table 3: Experimental data at different reaction times (temperature 80°C, glycerol ratio 5%)

[0084] Time (h) 1,3-Diglyceride (%) 1,2-Diglyceride (%) Monoglyceride (%) Triglyceride (%) Free fatty acids (%) 2 40 10 25 23.2 1.8 18 63 17 12 7.1 0.9 24 67 16 10 6.5 0.5 30 65 17 10 7.2 0.8 36 65 17 10 6.9 1.1

[0085] As shown in Table 3, when the reaction time of 1,3-diacerol is 24 hours, the concentration of converted diglyceride is the highest and the concentration of free fatty acids is the lowest.

[0086] Within 24 hours, about 90% of triglycerides are converted into diglycerides, the residual amount of triglycerides is only 10%, and the concentration of by-products (free fatty acids) is 0.3mmol / L, accounting for less than 3% of the total reactants. Compared with the traditional catalytic method, the latter has a triglyceride conversion rate of 60% to 75%, and more by-products (including monoglycerides and free fatty acids) are generated, which may be as high as 15% to 20%. Under the conditions of 80°C and pH 6.5, the present application achieved a conversion rate of 90%, the proportion of monoglycerides generated was low, and the by-products were reduced, indicating that lipase has higher reaction efficiency and selectivity. The advantage of this process is that the reaction conditions are mild, the enzyme catalysis method can reduce energy consumption and equipment costs, and has high industrialization potential.

[0087] 3. Comparison before and after molecular distillation purification

[0088] The synthesis process of 1,3-diacerol includes the following steps:

[0089] The immobilized enzyme particles after freeze-drying, namely Antarctic Candida lipase A (CALA), were used for the reaction, and the amount of enzyme was 2% of the mass of the reaction substrate. In the reaction system, the mass ratio of glycerol to triglyceride was 1:19, the reaction was carried out in a solvent-free system, the reaction temperature was 80°C, the pH was 6.5, the reaction time was 24h, and the stirring speed was set to 200rpm. The triglyceride was first heated and dissolved, and glycerol was gradually added dropwise to ensure full mixing and avoid the inhibition of enzyme activity by local high concentrations of glycerol. The system was purged with nitrogen for 10 minutes before the reaction to exclude oxygen to reduce the risk of enzyme activity decreasing due to oxidation, and 0.01% antioxidant (vitamin E) was added to the reactant to improve the stability of the substrate and the product. The products before and after purification were compared, as shown in Table 4.

[0090] Table 4: Component content before molecular distillation purification (unit: %)

[0091] Element Concentration before purification, % Concentration after purification, % 1,2-Diglyceride 17 16 1,3-Diglyceride 63 74 Monoglyceride 10 6 Free fatty acids 2 0.05 Triglycerides 5 3.95

[0092] Through the purification step of molecular distillation, the concentration of diglyceride increased from 8.2mmol / L to 9.2mmol / L, and the purity increased by 12.2%. The concentration of monoglyceride decreased from 1.0mmol / L to 0.1mmol / L, and the free fatty acid decreased from 0.3mmol / L to 0.05mmol / L, which was almost completely removed, proving the high efficiency of molecular distillation in removing impurities. The triglyceride was reduced to 0.2, ensuring the high purity of the final product.

[0093] 4. Example of Synthesis Process of 1,3-Diglyceride

[0094] 1. Embodiment 1

[0095] Preparation method: Add 190g of triglyceride (pressed rapeseed oil), 2g of Antarctic Candida lipase A (CALA), and 10g of food-grade glycerol to a 1L three-necked flask equipped with a nitrogen addition tube, a thermometer, and a vacuum pump, heat to 80°C, start stirring, and react for 24 hours at a stirring speed of 200r / min, with the pH maintained at 6.5. After the reaction, filter out Antarctic Candida lipase A (CALA) to obtain a reaction mixture, and the parameters obtained by Waters' high performance liquid chromatograph (HPLC-RID) are shown in Table 5:

[0096] Table 5

[0097]

[0098] 2. Embodiment 2:

[0099] Preparation method: 198 g of triglyceride (pressed rapeseed oil), 2 g of Antarctic Candida lipase A (CALA), and 2 g of food-grade glycerol were added to a 1L three-necked flask equipped with a nitrogen addition tube, a thermometer, and a vacuum pump, and the temperature was raised to 40°C and stirred. The stirring speed was 200 r / min, and the reaction was carried out for 6 hours, and the pH was maintained at 6.5. After the reaction, the Antarctic Candida lipase A (CALA) was filtered out to obtain a reaction mixture. The parameters obtained by the high performance liquid chromatograph (HPLC-RID) of Waters Company are shown in Table 6:

[0100] Table 6

[0101]

[0102] 3. Embodiment 3:

[0103] Preparation method: 180 g of triglyceride (pressed rapeseed oil), 2 g of Antarctic Candida lipase A (CALA), and 20 g of food-grade glycerol were added to a 1L three-necked flask equipped with a nitrogen addition tube, a thermometer, and a vacuum pump, and the temperature was raised to 120°C and stirred. The stirring speed was 200 r / min, and the reaction was carried out for 36 hours, and the pH was maintained at 6.5. After the reaction, the Antarctic Candida lipase A (CALA) was filtered out to obtain a reaction mixture. The parameters obtained by the high performance liquid chromatograph (HPLC-RID) of Waters Company are shown in Table 7:

[0104] Table 7

[0105]

[0106] 4. Embodiment 4:

[0107] Preparation method: Add 192g of triglyceride (pressed rapeseed oil), 2g of Antarctic Candida A lipase (CALB), and 8g of food-grade glycerol to a 1L three-necked flask equipped with a nitrogen addition tube, a thermometer, and a vacuum pump, heat to 70 degrees, start stirring, and react for 20 hours at a stirring speed of 200r / min, with the pH maintained at 6.5. After the reaction, filter out the Antarctic Candida A lipase (CALB) to obtain a reaction mixture. The parameters obtained by Waters' high performance liquid chromatograph (HPLC-RID) are shown in Table 8:

[0108] Table 8

[0109]

[0110] 5. Embodiment 5:

[0111] Preparation method: Add 200g of triglyceride (pressed rapeseed oil), 2g of Antarctic Candida A-type lipase (CALB), and 12g of food-grade glycerol to a 1L three-necked flask equipped with a nitrogen addition tube, a thermometer, and a vacuum pump, raise the temperature to 90 degrees, start stirring, and react for 28 hours at a stirring speed of 200r / min, with the pH maintained at 6.5. After the reaction, filter out the Antarctic Candida A-type lipase (CALB) to obtain a reaction mixture, and the parameters obtained by the high performance liquid chromatograph (HPLC-RID) of Waters Company are shown in Table 9:

[0112] Table 9

[0113]

[0114] 5. Reusability of Candida antarctica lipase A (CALA)

[0115] The enzyme catalytic effects under different reuse times are shown in Table 10

[0116] Table 10

[0117] Reuse times Diacylceride (%) Monoglyceride (%) Free fatty acids (%) Triglyceride (%) 1 83 10 0.5 6.5 50 82.5 10.2 0.5 6.8 100 82 10.3 0.5 7.2 150 81.8 10.4 0.5 7.3 200 78 11 0.6 10.4 250 72 12 0.6 15.4

[0118] As shown in Table 10, Antarctic Candida lipase A (CALA) has extremely high reusability under the conditions of 80°C, 24 hours, and 5% glycerol. Even after the 150th reuse, the enzyme activity remains at a high level. The enzyme activity began to decline significantly after 200 reuses, the diglyceride rate decreased by 3.8%, and the free fatty acid increased by 0.5%. After 250 times, the catalytic effect was greatly reduced, the diglyceride ratio decreased by 6%, and the triglyceride ratio increased by 4.5%. It is recommended to replace the new enzyme when the reuse reaches about 200 times to ensure production efficiency.

[0119] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these modifications and improvements are all within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for preparing lipase, characterized in that: The steps include: Activation of strains: freeze-dried strains of Candida antarctica type A were reconstituted in deionized water to a concentration of (0.1-9)×10 9 CFU / ml bacterial solution, and add glucose and mix evenly, and let it stand at 20-35°C for 1-3 hours to promote bacterial activation, wherein the percentage of glucose in the bacterial solution is 0.2-2%; Preparation of YPD medium: weigh yeast extract, peptone, glucose, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate, dissolve in deionized water to obtain a medium, adjust the pH value of the medium to 5.5-7.5, and then sterilize the medium in an autoclave, and cool after sterilization to obtain a YPD medium; Lipase culture: Add the bacterial solution to the culture medium, and add Tween 80, soybean lecithin and vitamin B to the culture medium. 12 , and cultured in a shaker for 36 to 60 hours to allow the strain to secrete lipase, wherein the culture conditions are: temperature 20 to 40°C, shaker speed 150 to 250 rpm; Separation and disruption of enzymes: centrifuge the culture medium to separate the bacterial solution and collect the cell precipitate, use an ultrasonic disruptor to disrupt the cells to release lipase, centrifuge to remove cell debris, and collect the lipase solution; Enzyme immobilization: Immobead 150 resin was pretreated with sodium bicarbonate solution, and then the resin was washed several times with phosphate buffer to remove alkaline residues. The lipase solution was then mixed with the resin and reacted for 20 to 30 hours to immobilize the enzyme to obtain an enzyme-resin complex, i.e., immobilized enzyme particles. The immobilized enzyme particles were then soaked in a buffer to improve stability. Enzyme protection and freeze-drying treatment: soak the immobilized enzyme particles in a buffer solution containing the first protective agent at a temperature of 2 to 8°C for 8 to 16 hours to allow the protective agent to fully cover the surface of the immobilized enzyme particles. Then, place the immobilized enzyme particles in a drying device for drying. Finally, store the dried immobilized enzyme particles, i.e., Antarctic Candida lipase A, in a moisture-proof package.

2. The method for preparing lipase according to claim 1, characterized in that: In the preparation steps of YPD culture medium, the concentrations of the components in the culture medium are as follows: yeast extract 6-15 g / L, peptone 15-25 g / L, glucose 25-35 g / L, potassium dihydrogen phosphate 0.5-2 g / L, magnesium sulfate heptahydrate 0.1-1 g / L; sterilization conditions: temperature 121±5°C, time 15±5 min; glycerol is added to the sterilized and cooled culture medium, and the percentage of glycerol in the culture medium is 0.5-2%.

3. The method for preparing lipase according to claim 1, characterized in that: In the lipase culture step, the volume ratio of bacterial liquid to culture medium is (0.3-2):19; the bacterial liquid is kept at 25-35°C for 10-15 hours before being added to the culture medium; the percentage of each additive in the culture medium is: Tween 80 0.5-2%, soybean lecithin 0.1-0.3% and vitamin B 12 0.01~0.2%, culture conditions: in the first half of the culture, the temperature is 28℃±1℃, and the shaking speed is 180rpm±10rpm; in the second half of the culture, the temperature is 30℃±1℃, and the shaking speed is 220rpm±10rpm.

4. The method for preparing lipase according to claim 1, characterized in that: In the enzyme separation and crushing step, the centrifugal conditions before crushing are centrifugal force 4000g±500g, temperature 4℃±1℃, time 15min±5min; the crushing conditions are: 2 seconds working, 3 seconds rest, repeated 20 times; before crushing, a second protective agent is added to the culture medium, the second protective agent includes 0.5-1.5% PEG-4000 and 0.2-1% glycerol; the centrifugal conditions after crushing are centrifugal force 10000g±1000g, temperature 4℃±1℃, time 10min±5min; protease inhibitors PMSF, EDTA and sodium azide are added to the lipase solution, the concentration of PMSF is 0.1mM±0.02mM, the EDTA in the lipase solution is 0.05-0.15%, and the sodium azide is 0.005-0.015%, and the lipase solution is stored in an environment of 4℃±1℃.

5. The method for preparing lipase according to claim 1, characterized in that: In the enzyme immobilization step, the Immobead 150 resin is first centrifuged and washed with deionized water, and then pretreated with a 0.05-0.2M sodium bicarbonate solution. The lipase solution and the resin are mixed in a ratio of (0.5-2):10, placed in an environment of 4°C±1°C, the pH is adjusted to 5-7, and a reaction is carried out. During the reaction, a stabilizer and a third protective agent are added to the lipase solution, wherein the stabilizer is a 0.5-2mM CaCl2 solution, and the third protective agent is 0.5-2% PEG-4000 and 0.1-0.5% BSA; After the immobilization is completed, the immobilized enzyme particles are immersed in 0.05-0.2M Tris-HCl buffer at 4°C±1°C for 8-16 hours, and then the immobilized enzyme particles are washed by centrifugation with PBS buffer at a centrifugal speed of 2000±500rpm for 5-20 minutes.

6. The method for preparing lipase according to claim 1, characterized in that: In the enzyme protection and freeze-drying treatment steps, the buffer is 0.05-0.2M Tris-HCl buffer, and the protective agent formula includes: 2-10% glycerol, 10-20% sucrose, 3-8% trehalose and 0.5-5% polyvinyl alcohol; The immobilized enzyme particles were dried using a vacuum drying device, and the drying conditions were as follows: the initial temperature was set at 25°C ± 5°C, the vacuum degree was maintained at 40-60 mbar, the drying time was 4-8 hours, and then, while maintaining the vacuum degree, the temperature was slowly raised to 40°C ± 5°C for 4-8 hours, and then further raised to 45°C ± 5°C for 2-5 hours.

7. The method for preparing lipase according to claim 1, characterized in that: The method also includes an enzyme regeneration step. After each use, the immobilized enzyme particles are washed with 15-25mM phosphate buffer and then immersed in a regeneration solution for 1.5-3 hours to restore the activity of the enzyme. The regeneration solution includes an aqueous solution of 0.5-2% glycerol, 0.05-0.2% BSA and 3-6mM CaCl2.

8. A process for synthesizing 1,3-diacerol, characterized in that: The Antarctic Candida lipase A prepared by the preparation method of any one of claims 1 to 7 is used to enzymatically hydrolyze triglycerides to synthesize 1,3-diglyceride.

9. The synthesis process of 1,3-diacerol according to claim 8, characterized in that: The steps include: First, triglyceride is added to a reaction container, heated to 70-90°C, glycerol is added and stirred evenly, and then Antarctic Candida lipase A is added and mixed, and the reaction is performed for 18-36 hours to obtain a reaction mixture. The reaction mixture is purified by molecular distillation to obtain 1,3-diglyceride, wherein the mass ratio of glycerol to triglyceride is (0.5-2.5):19, the pH of the reaction system is 5.5-7.5, the stirring speed is 150-250rpm, and the amount of Antarctic Candida lipase A added is 1-5% of the total weight of glycerol and triglyceride.

10. The synthesis process of 1,3-diacerol according to claim 9, characterized in that: Before adding the Antarctic Candida lipase A, the reaction container is purged with nitrogen for 6 to 12 minutes. During the reaction, an antioxidant is added to the reaction system in an amount of 0.005% to 0.02% of the total weight of glycerol and triglycerides.

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