Preparation method of Sn-1, 3 specific immobilized lipase

Abstract

The invention discloses a preparation method of Sn-1, 3 specific immobilized lipase, and belongs to the technical field of lipase immobilization. The preparation method specifically comprises the following steps: (1) mixing a water-phase solution and an oil-phase solution, carrying out suspension polymerization reaction, and removing a pore-foaming agent to obtain polymer porous resin; wherein the water-phase solution contains a dispersing agent and salt; the oil phase solution contains a polymerizable monomer, a pore-foaming agent and an initiator; (2) dissolving Sn-1, 3 specific lipase in a buffer solution, centrifuging, and taking supernate to obtain an enzyme solution; and (3) adding polymer porous resin into the enzyme liquid for adsorption, suction filtration, washing and drying. The prepared Sn-1, 3 immobilized lipase is good in specificity and high in enzyme activity, and can be applied to production of a food additive OPO.

Description

Technical Field

[0001] This invention relates to the field of lipase immobilization technology, and more specifically to a method for preparing Sn-1,3-specific immobilized lipase. Background Technology

[0002] 1,3-Dioleoyl-2-palmitoylglycerol (OPO) is a characteristic component of breast milk fat. Breast milk is the ideal food for infants and young children, playing an irreplaceable role in their healthy growth and development. Although breast milk contains only 3.0%–4.5% fat, it provides infants with about 50% of their energy and plays a crucial role in the absorption of various vitamins and minerals, making it very important for infant growth and development. OPO is a novel nutritional enhancer for infant food. When added to infant formula, it can help improve symptoms such as calcium loss and constipation in infants caused by mixed vegetable oils.

[0003] OPO is mostly prepared by lipase-catalyzed transesterification, which rearranges fatty acids on the glycerol molecule. The specific production process includes three main steps: 1) obtaining triglycerides rich in palmitic acid at the Sn-2 position, 2) directional transesterification catalyzed by Sn-1,3 specific lipase, and 3) separation and purification of the product. The most critical step is the Sn-1,3 specific lipase. Immobilizing this key enzyme for continuous and repeated use is an effective way to improve efficiency and reduce costs.

[0004] Therefore, how to immobilize Sn-1,3 specific lipases is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing Sn-1,3 specific immobilized lipase to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing Sn-1,3-specific immobilized lipase specifically includes the following steps:

[0008] (1) Mix the aqueous solution and the oil solution to carry out a suspension polymerization reaction, remove the pore-forming agent, and obtain a polymer porous resin;

[0009] The aqueous solution contains dispersants and salts;

[0010] The oil phase solution contains polymerizable monomers, porogens, and initiators; polymerizable monomers include non-functional monomers and monomers with C4-C6 ... 18 Vinyl functional monomers with side-chain alkyl groups and crosslinking agents;

[0011] (2) Dissolve Sn-1,3 specific lipase in buffer solution, centrifuge, and take the supernatant to obtain enzyme solution;

[0012] (3) Add polymer porous resin to the enzyme solution for adsorption, filter, wash and dry to obtain Sn-1,3 specific immobilized lipase.

[0013] Furthermore, in step (1) above, the dispersant is at least one of natural water-soluble polymer gelatin, synthetic water-soluble polymer gelatin, starch, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, polymethacrylic acid, copolymers of polyacrylic acid and copolymers of acrylates, which can be used alone or in combination, preferably at least one of natural water-soluble polymer gelatin, synthetic water-soluble polymer gelatin and polyvinyl alcohol, more preferably polyvinyl alcohol, and the mass concentration in the aqueous solution is 0.2%-1%.

[0014] The further beneficial effect of the above-mentioned method is that the dispersant selected in this invention can form spherical monomer droplets in the suspension polymerization reaction and carry out the suspension polymerization reaction under the protection of the dispersant.

[0015] Furthermore, in step (1) above, the salt is at least one of alkali metal halide (halogen salt), alkali metal sulfate, alkali metal phosphate, alkaline earth metal halide (halogen salt), alkaline earth metal sulfate and alkaline earth metal phosphate, preferably sodium chloride, with a mass concentration of 5%-15% in the aqueous solution.

[0016] The further beneficial effect of the above-mentioned method is that the salt selected in this invention can reduce the solubility of organic matter in water.

[0017] Furthermore, in step (1) above, the non-functional monomer is at least one of styrene, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, acrylonitrile and methacrylonitrile, preferably styrene and methyl methacrylate, and the mass percentage of the polymerizable monomer is 20%-50%.

[0018] The further beneficial effect of the above-mentioned method is that the non-functional monomers selected in this invention have a low price, which can significantly reduce the production cost of polymer porous resins.

[0019] Furthermore, in step (1) above, C4-C 18 The vinyl functional monomer of the side-chain alkyl group is alkyl methacrylate (C4-C5). 18 ) esters and alkyl acrylates (C4-C 18At least one of the following esters, preferably at least one of butyl methacrylate, dodecyl methacrylate and stearic acid methacrylate, in which the mass percentage of the polymerizable monomer is 20%-50%.

[0020] The further beneficial effect of the above-mentioned method is that the C4-C selected by the present invention... 18 Polymer porous resins derived from vinyl functional monomers with side-chain alkyl groups have tunable hydrophobic and hydrophilic regions, and their surface structures are well-matched with Sn-1,3 specific lipases, exhibiting good adsorption and interfacial activation effects.

[0021] Furthermore, in step (1) above, the crosslinking agent is at least one of divinylbenzene, diethylene glycol divinyl ether, butanediol diacrylate, ethylene glycol dimethacrylate, triallyl isocyanurate, di(4-vinylphenyl)methane, 1,2-di(4-vinylphenyl)ethane, 1,3-di(4-vinylphenyl)propane, and 1,4-di(4'-vinylphenoxy)butane, preferably divinylbenzene and ethylene glycol dimethacrylate, and its mass percentage in the polymerizable monomer is 50%-80%.

[0022] The further beneficial effect of the above-mentioned method is that the polymer porous resin prepared by the crosslinking agent selected in this invention has high strength and swelling resistance, which increases the number of cycles of Sn-1,3 specific immobilized lipase.

[0023] Furthermore, in step (1) above, the pore-forming agent is an organic solvent that does not participate in the polymerization reaction and is insoluble or slightly soluble in water, including organic solvents that are poor solvents / swelling agents for the polymer formed. Preferably, it is at least one of toluene, hexane, octane, isooctane, isododecane, methyl ethyl ketone, methyl isobutyl ketone, dichloroethane, dichloropropane, n-butanol and its isomers, n-pentanol and its isomers, n-hexanol and its isomers, and n-octanol and its isomers. More preferably, it is toluene, isobutanol, and methyl isobutyl ketone, and its mass percentage relative to the polymerizable monomer is 100%-250%.

[0024] The further beneficial effect of the above-mentioned method is that the pore structure of the polymer porous resin, including specific surface area, pore volume and average pore size, can be controlled by the type and amount of pore-forming agent. The pore structure and surface properties of the polymer porous resin prepared by the pore-forming agent selected in this invention can be well matched with Sn-1,3 specific lipase.

[0025] Further, in step (1) above, the initiator is at least one of organic peroxide and azo compound, preferably at least one of benzoyl peroxide, dilauryl peroxide, bis(p-chlorobenzoyl) peroxide, dicyclohexyl percarbonate, tert-butyl peroctanoate, tert-butyl peroxy-2-ethylhexanoate, 2,5-bis(2-ethylhexanoylperoxy)-2,5-dimethylhexane, tert-amylperoxy-2-ethylhexane, 2,2'-azobis(isobutyronitrile), and 2,2'-azobis(2-methylisobutyronitrile), more preferably benzoyl peroxide, with a mass percentage of 0.05%-2.5% relative to the polymerizable monomer.

[0026] Furthermore, in step (1) above, the temperature of the suspension polymerization reaction depends on the decomposition temperature of the initiator and the boiling point of the porogen or the azeotropic point of the porogen with water, preferably 60-100℃, more preferably 80℃, and the time is 8h. The stirring speed in the suspension polymerization reaction has a significant impact on the particle size. In principle, smaller particles are obtained at higher stirring speeds. By adjusting the stirring speed, those skilled in the art can control the particle size of the beaded polymer within the desired range, with the bead diameter preferably 100-1000μm, more preferably 150-710μm. After the suspension polymerization reaction is completed, the porogen can be removed from the polymer spheres by distillation or washing, so that the content of residual monomer or porogen meets the requirements of GB / T24395.

[0027] Furthermore, in step (2) above, the buffer solution is a glycine-sodium hydroxide buffer solution; the concentration of the enzyme solution is 10-80 mg / mL, and the pH value is 9-10.

[0028] Furthermore, in step (3) above, the adsorption temperature is 30°C and the time is 1-4 hours, preferably 2 hours; the drying method is freeze drying.

[0029] Furthermore, in step (3) above, the immobilization of Sn-1,3 specific lipase adopts a simple adsorption method: Sn-1,3 specific lipase powder is weighed and dissolved in glycine-sodium hydroxide buffer solution, impurities are removed by centrifugation, the supernatant (i.e., the enzyme solution) is taken, added to a polymer porous resin, and shaken in a 30°C water bath for 2 hours for adsorption. The enzyme solution is removed by filtration, and the mixture is washed several times with buffer solution and freeze-dried to obtain Sn-1,3 specific immobilized lipase. Those skilled in the art can adjust the enzyme solution concentration based on the target adsorption amount. The filtrate can be recycled. If the loading of Sn-1,3 specific lipase is not as expected, the immobilization operation can be repeated multiple times.

[0030] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The preparation method of this invention includes the preparation of a polymer porous resin (lipase immobilization carrier) and the immobilization of Sn-1,3 specific lipase. First, a polymer porous resin containing long alkyl side chains is prepared by suspension polymerization. The resulting carrier resin has a suitable specific surface area and pore size, a low surface activation energy, and a high adsorption capacity for Sn-1,3 specific lipase, and can be used directly without any pretreatment. Then, Sn-1,3 specific lipase is dissolved in a buffer solution, and the above-mentioned carrier resin is placed in the enzyme solution for adsorption. After adsorption, the resin is washed and dried to obtain Sn-1,3 specific immobilized lipase. The obtained immobilized lipase has higher enzyme activity and Sn-1,3 specificity than free lipase, and also exhibits better operational stability.

[0032] 2. The Sn-1,3 immobilized lipase prepared by this invention has good specificity and high enzyme activity, and can be used in the production of food additive OPO. Attached Figure Description

[0033] Figure 1 The image shows a cross-sectional SEM image of the polymer porous resin prepared in Example 1.

[0034] Figure 2 An image of the polymer porous resin prepared in Example 2;

[0035] Figure 3 The results of mercury porosimetry tests on the polymer porous resins prepared in Examples 1-2 are shown.

[0036] Figure 4 This is a comparison of the enzyme activities in Examples 1-2 and Comparative Examples 1-3;

[0037] Figure 5 This is a comparison of the highest conversion rates of each enzyme in Examples 1-2 and Comparative Examples 1-3. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] The preparation method of Sn-1,3 specific immobilized lipase includes the following steps:

[0041] (1) Add 600mL of pure water and 4g of polyvinyl alcohol to a 2L flask equipped with a condenser, stirrer and thermometer, heat to 80℃, and after the polyvinyl alcohol in the flask dissolves, add 30g of sodium chloride. Once completely dissolved, it becomes an aqueous solution.

[0042] (2) Weigh 90.0g of styrene, 60.0g of dodecyl methacrylate, 50.0g of divinylbenzene with a purity of 80%, 200g of isobutanol and 1.8g of benzoyl peroxide with a purity of 75%, mix them evenly to obtain the oil phase solution.

[0043] (3) Add the oil phase solution to the flask, start stirring, heat to 80℃ for suspension polymerization reaction for 8 hours, switch to distillation mode and heat to 90℃, and gradually heat to 96℃ to remove isobutanol by distillation, remove polyvinyl alcohol by hot water washing, remove residual isobutanol by ethanol washing, sieve and collect polymer spheres with a particle size of 150-710μm, which is the polymer porous resin.

[0044] (4) Weigh 3.2g of Sn-1,3 specific lipase powder and dissolve it in 300mL of glycine-sodium hydroxide buffer solution. Centrifuge to remove impurities and take the supernatant to obtain the enzyme solution.

[0045] (5) Add polymer porous resin to the enzyme solution, shake in a 30°C water bath shaker for 2 hours for adsorption, filter to remove the enzyme solution, wash several times with buffer solution, freeze dry to obtain Sn-1,3 specific immobilized lipase.

[0046] The cross-sectional SEM image of the polymer porous resin prepared in Example 1 is shown below. Figure 1 As shown. By Figure 1 It can be seen that the pore size of the polymer porous resin (microspheres) is... BET testing showed its specific surface area to be 40m². 2 / g.

[0047] Example 2

[0048] The preparation method of Sn-1,3 specific immobilized lipase includes the following steps:

[0049] (1) Add 600mL of pure water and 6g of hydroxyethyl cellulose to a 2L flask equipped with a condenser, stirrer and thermometer, heat to 80℃, and after the hydroxyethyl cellulose in the flask dissolves, add 30g of sodium chloride. Once completely dissolved, it becomes an aqueous solution.

[0050] (2) Weigh 90.0g of methyl methacrylate, 63.0g of stearate methacrylate, 57.0g of ethylene glycol dimethacrylate (80% purity), 400g of toluene, and 1.8g of benzoyl peroxide (75% purity), mix them evenly to form the oil phase solution.

[0051] (3) Add the oil phase solution to the flask, start stirring, heat to 80℃ for suspension polymerization reaction for 8 hours, switch to distillation mode and heat to 88℃, and gradually heat to 96℃ to remove toluene by distillation, wash with hot water to remove hydroxyethyl cellulose, wash with ethanol to remove residual toluene, sieve and collect polymer balls with a particle size of 150-710μm, which is the polymer porous resin.

[0052] (4) Weigh 3.2 mg of Sn-1,3 specific lipase powder and dissolve it in 300 mL of glycine-sodium hydroxide buffer solution. Centrifuge to remove impurities and take the supernatant to obtain the enzyme solution.

[0053] (5) Add polymer porous resin to the enzyme solution, shake in a 30°C water bath shaker for 2 hours for adsorption, filter to remove the enzyme solution, wash several times with buffer solution, freeze dry to obtain Sn-1,3 specific immobilized lipase.

[0054] The image of the polymer porous resin prepared in Example 2 is shown below. Figure 2 As shown. By Figure 2 It can be seen that the pore size of the polymer porous resin (microspheres) is... BET testing showed its specific surface area to be 60m². 2 / g.

[0055] The mercury porosimetry results of the polymer porous resins prepared in Examples 1-2 are as follows: Figure 3 As shown. By Figure 3 It can be seen that the pore size of the obtained resin is mainly distributed in the macropore region (>50nm).

[0056] Example 3

[0057] The performance testing method for the Sn-1,3 specific immobilized lipase (immobilized enzyme A) prepared in Example 1 specifically includes the following steps:

[0058] (1) Weigh 7.5g of stearyl palmitate and 17.5g of oleic acid, dissolve them at 60℃, and then weigh 1.5g of the Sn-1,3 specific immobilized lipase prepared in Example 1 and add it to the substrate. React for 8h.

[0059] (2) Samples were taken at 2h, 4h, 6h and 8h respectively, with 60μL of sample taken each time. 5mL of n-hexane was added to the sample and mixed well. 2mL of the mixed sample was then put into a sample bottle for GC detection.

[0060] (3) The conversion rate was tested according to the method of national standard GB30604-2015. The final results of 1,3-dioleoyl-2-palmitoylglycerol triglyceride (OPO) conversion rate and Sn-2 content were as follows: 2h, OPO conversion rate 57.3%, Sn-2 content 67%; 4h, OPO conversion rate 62%, Sn-2 content 70.9%; 6h, OPO conversion rate 61.1%, Sn-2 content 69.5%; 8h, OPO conversion rate 60.3%, Sn-2 content 68.2%.

[0061] (4) Enzyme activity was detected according to the method of national standard GB / T 23535-2009, and the enzyme activity result was 850u / g.

[0062] Example 4

[0063] Example 2 describes a performance testing method for the Sn-1,3-specific immobilized lipase (immobilized enzyme B) prepared, which specifically includes the following steps:

[0064] (1) Weigh 7.5g of stearyl palmitate and 17.5g of oleic acid, dissolve them at 60°C, and then weigh 1.5g of the Sn-1,3 specific immobilized lipase prepared in Example 2 and add it to the substrate. React for 8 hours.

[0065] (2) Samples were taken at 2h, 4h, 6h and 8h respectively, with 60μL of sample taken each time. 5mL of n-hexane was added to the sample and mixed well. 2mL of the mixed sample was then put into a sample bottle for GC detection.

[0066] (3) The detection was carried out in accordance with the method of national standard GB30604-2015. The final results of the conversion rate of 1,3-dioleoyl-2-palmitoylglycerol triglyceride (OPO) and the Sn-2 content were as follows: 2h, OPO conversion rate 57.7% and Sn-2 content 68%; 4h, OPO conversion rate 62.6% and Sn-2 content 71.9%; 6h, OPO conversion rate 62% and Sn-2 content 70.6%; 8h, OPO conversion rate 60.9% and Sn-2 content 69%.

[0067] (4) Enzyme activity was detected according to the method of national standard GB / T 23535-2009, and the enzyme activity result was 900u / g.

[0068] Comparative Example 1

[0069] The performance testing method for commercially available immobilized enzyme Lipase DF "Amano" IM specifically includes the following steps:

[0070] (1) Weigh 7.5g of stearyl palmitate and 17.5g of oleic acid, dissolve them at 60℃, then weigh 1.5g of commercially available immobilized enzyme LipaseDF "Amano" IM and add it to the substrate. React for 8h.

[0071] (2) Samples were taken at 2h, 4h, 6h and 8h respectively, with 60μL of sample taken each time. 5mL of n-hexane was added to the sample and mixed well. 2mL of the mixed sample was then put into a sample bottle for GC detection.

[0072] (3) The detection was carried out in accordance with the method of national standard GB30604-2015. The final results of the conversion rate of 1,3-dioleoyl-2-palmitoylglycerol triglyceride (OPO) and the Sn-2 content were as follows: 2h, OPO conversion rate 56.5% and Sn-2 content 65.8%; 4h, OPO conversion rate 60.5% and Sn-2 content 68.2%; 6h, OPO conversion rate 59.3% and Sn-2 content 66.9%; 8h, OPO conversion rate 58.4% and Sn-2 content 65.8%.

[0073] (4) Enzyme activity was detected according to the method of national standard GB / T 23535-2009, and the enzyme activity result was 750u / g.

[0074] Comparative Example 2

[0075] Commercially available immobilized enzymes The performance testing method for TL IM specifically includes the following steps:

[0076] (1) Weigh 7.5g of stearyl palmitate and 17.5g of oleic acid, dissolve them at 60℃, and then weigh 1.5g of commercially available immobilized enzyme. TL IM was added to the substrate and the reaction was allowed to proceed for 8 hours.

[0077] (2) Samples were taken at 2h, 4h, 6h and 8h respectively, with 60μL of sample taken each time. 5mL of n-hexane was added to the sample and mixed well. 2mL of the mixed sample was then put into a sample bottle for GC detection.

[0078] (3) The detection was carried out in accordance with the method of national standard GB30604-2015. The final results of the conversion rate of 1,3-dioleoyl-2-palmitoylglycerol triglyceride (OPO) and the Sn-2 content were as follows: 2h, OPO conversion rate 55.1% and Sn-2 content 65.3%; 4h, OPO conversion rate 58.5% and Sn-2 content 66.6%; 6h, OPO conversion rate 57.9% and Sn-2 content 65.5%; 8h, OPO conversion rate 55.4% and Sn-2 content 63.1%.

[0079] (4) Enzyme activity was detected according to the method of national standard GB / T 23535-2009, and the enzyme activity result was 650u / g.

[0080] Comparative Example 3

[0081] Commercially available immobilized enzymes The performance testing method for RM IM specifically includes the following steps:

[0082] (1) Weigh 7.5g of stearyl palmitate and 17.5g of oleic acid, dissolve them at 60℃, and then weigh 1.5g of commercially available immobilized enzyme. RM IM was added to the substrate and reacted for 8 hours.

[0083] (2) Samples were taken at 2h, 4h, 6h and 8h respectively, with 60μL of sample taken each time. 5mL of n-hexane was added to the sample and mixed well. 2mL of the mixed sample was then put into a sample bottle for GC detection.

[0084] (3) The detection was carried out in accordance with the method of national standard GB30604-2015. The final results of the conversion rate of 1,3-dioleoyl-2-palmitoylglycerol triglyceride (OPO) and the Sn-2 content were as follows: 2h, OPO conversion rate 54.5% and Sn-2 content 64.3%; 4h, OPO conversion rate 56.9% and Sn-2 content 65.3%; 6h, OPO conversion rate 57.5% and Sn-2 content 66.5%; 8h, OPO conversion rate 56.4% and Sn-2 content 65.1%.

[0085] (4) Enzyme activity was detected according to the method of national standard GB / T 23535-2009, and the enzyme activity result was 700u / g.

[0086] Performance testing

[0087] 1. Enzyme activity test

[0088] Enzyme activity tests were performed on the Sn-1,3 immobilized lipases prepared in Examples 1-2 and other commercially available immobilized lipases in Comparative Examples 1-3, and the enzyme activity was compared. The specific procedures are as follows:

[0089] (1) The enzyme activity of the above immobilized lipase was tested according to the national standard GB / T 23535-2009.

[0090] (2) Substrate preparation:

[0091] Weigh 40g of polyvinyl alcohol (degree of polymerization 1750±50) (accurate to 0.1g), add 800mL of water, heat in boiling water, stir until completely dissolved, cool and bring the volume to 1000mL. Filter with clean double-layered gauze and keep the filtrate for later use.

[0092] Take 150 mL of the above filtrate, add 50 mL of olive oil, and process with a high-speed homogenizer for 6 min (processed twice, 5 min apart, 3 min each time) to obtain a milky white PVA emulsion. This solution should be prepared fresh before use.

[0093] Phosphate buffer solution (pH 7.5): Weigh out 1.96 g of potassium dihydrogen phosphate and 39.62 g of disodium hydrogen phosphate dodecahydrate, dissolve them in water, and bring the volume to 500 mL. If necessary, adjust the pH of the solution to 7.5 ± 0.05.

[0094] Prepare a 0.05 mol / L sodium hydroxide standard solution. When using it, dilute it accurately by 10 times.

[0095] Prepare 10 g / L of phenolphthalein indicator.

[0096] (3) Analysis steps

[0097] Weigh 2g of enzyme sample. Take two 100mL Erlenmeyer flasks and add 4mL of substrate solution and 5mL of phosphate buffer to each flask (blank A) and sample flask (sample B). Add 15mL of 95% ethanol to flask A and preheat in a water bath at 40℃±0.2℃ for 5min. Then add 1g of immobilized enzyme to each flask (A) and B, mix immediately, and start timing. After reacting for 15min, immediately add 15mL of 95% ethanol to flask B to stop the reaction and remove the flask.

[0098] Add two drops of phenolphthalein indicator to both the blank and sample solutions, and titrate with sodium hydroxide standard solution until a faint red color remains for 30 seconds, which is the titration endpoint. Record the volume of sodium hydroxide standard solution consumed.

[0099] (4) Enzyme activity is calculated using the following formula:

[0100]

[0101] In the formula:

[0102] X1—Enzyme activity of the sample, u / g;

[0103] V1—The volume of sodium hydroxide standard solution consumed during sample titration, in milliliters;

[0104] V2—The volume of sodium hydroxide standard solution consumed during blank titration, in milliliters;

[0105] C—Concentration of sodium hydroxide standard solution, in moles per liter;

[0106] 1.00 mL of 50-0.05 mol / L sodium hydroxide solution is equivalent to 50 μmol of fatty acid;

[0107] n1—the dilution factor of the sample;

[0108] 0.05 — Conversion factor for sodium hydroxide standard solution concentration;

[0109] —Reaction time 15 min, calculated as 1 min.

[0110] The results are expressed as integers.

[0111] (5) The immobilized enzyme samples were tested according to the above experimental steps, and the results are as follows: Figure 4 As shown. By Figure 4 It can be seen that immobilized enzymes A and B have significantly improved enzyme activity compared to the three commercially available immobilized enzymes.

[0112] 2. Specificity test

[0113] Specificity tests were performed on the Sn-1,3 immobilized lipases prepared in Examples 1-2 and other commercially available immobilized lipases in Comparative Examples 1-3. The specific procedures are as follows:

[0114] (1) Testing was conducted according to the method of national standard GB30604-2015.

[0115] Reagents and materials: n-hexane (chromatographic grade), heptadecanoglycerate standard (purity 99%), 1,3-dioleoyl-2-palmitoylglycerate standard (purity 99%), palmitate triglyceride standard (purity 99%), nitrogen (purity 99.999%).

[0116] Instruments and equipment: Gas chromatograph equipped with FID flame ionization detector.

[0117] Chromatographic conditions:

[0118] ① Chromatographic column: 100% dimethyl polysiloxane high-temperature resistant chromatographic column (column length 15m, inner diameter 0.25mm, film thickness 0.1um) or a chromatographic column with equivalent performance.

[0119] ②Inlet temperature 320℃, detector temperature 370℃.

[0120] ③Programmed temperature rise: 300℃, hold for 3 min, then rise to 380℃ at a rate of 20℃ / min and hold for 10 min.

[0121] ④ Use nitrogen as the carrier gas in constant pressure mode. Injection volume: 1 μL; resolution should be greater than 1.5.

[0122] Analysis steps:

[0123] Preparation of standard solutions

[0124] ① Heptadecanoglycerate standard solution (2 mg / mL): Weigh 100 mg of heptadecanoglycerate standard into a 50 mL volumetric flask, add an appropriate amount of n-hexane to dissolve, and dilute to the mark, then shake well.

[0125] ② 1,3-Dioleoyl-2-palmitoylglycerol standard solution (1 mg / mL): Weigh 100 mg of 1,3-dioleoyl-2-palmitoylglycerol standard into 100 mL of volumetric precipitate, add an appropriate amount of n-hexane to dissolve, and dilute to the mark, then shake well.

[0126] ③ Palmitate triglyceride standard solution (1 mg / mL): Weigh 100 mg of palmitate triglyceride standard into 100 mL of volumetric precipitate, add an appropriate amount of n-hexane to dissolve, dilute to the mark, and shake well.

[0127] ④ Mixed standard solution (0.2 mg / mL): Weigh 1.0 mL of heptadecanoglycerate standard solution, 2.0 mL of 1,3-dioleoyl-2-palmitoylglycerate standard solution and 2.0 mL of palmitoglycerate standard solution into a 10 mL volumetric flask, add an appropriate amount of n-hexane to dilute to the mark, and shake well.

[0128] Sample solution preparation: Weigh 20 mg of the sample and place it in a 25 mL volumetric flask. Add a small amount of n-hexane to dissolve and shake well. Dilute to the mark with n-hexane and shake well.

[0129] Determination: Under specified chromatographic conditions, inject 1 μL each of the standard solution and the sample solution into the chromatograph, and record the peak areas of the components in the standard solution and the sample solution. Calculate the results based on the peak area ratio. Figure 5 As shown. By Figure 5 It can be seen that immobilized enzyme B has the highest conversion rate, followed by immobilized enzyme A, which shows a significant improvement in specificity compared to the three commercial immobilized enzymes.

[0130] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing Sn-1,3-specific immobilized lipase, characterized in that, Specifically, the following steps are included: (1) Mix the aqueous solution and the oil solution to carry out a suspension polymerization reaction, remove the pore-forming agent, and obtain a polymer porous resin; The aqueous solution contains a dispersant and a salt; The oil phase solution contains polymerizable monomers, pore-forming agents, and initiators; the polymerizable monomers include non-functional monomers with C4-C64 content. 18 Vinyl functional monomers with side-chain alkyl groups and crosslinking agents; (2) Dissolve Sn-1,3 specific lipase in buffer solution, centrifuge, and take the supernatant to obtain enzyme solution; (3) Add polymer porous resin to the enzyme solution for adsorption, filter, wash and dry to obtain the Sn-1,3 specific immobilized lipase.

2. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the dispersant is at least one of natural water-soluble polymer gelatin, synthetic water-soluble polymer gelatin, starch, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, polymethacrylic acid, copolymers of polyacrylic acid and copolymers of acrylates, and has a mass concentration of 0.2%-1% in the aqueous solution.

3. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the salt is at least one of alkali metal halide, alkali metal sulfate, alkali metal phosphate, alkaline earth metal halide, alkaline earth metal sulfate and alkaline earth metal phosphate, and has a mass concentration of 5%-15% in the aqueous solution.

4. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the non-functional monomer is at least one of styrene, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, acrylonitrile and methacrylonitrile, and its mass percentage in the polymerizable monomer is 20%-50%.

5. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the one with C4-C 18 The vinyl functional monomer of the side-chain alkyl group is alkyl methacrylate (C4-C5). 18 ) esters and alkyl acrylates (C4-C 18 At least one of the esters, comprising 20%-50% by mass of the polymerizable monomer.

6. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the crosslinking agent is at least one of divinylbenzene, diethylene glycol divinyl ether, butanediol diacrylate, ethylene glycol dimethacrylate, triallyl isocyanurate, di(4-vinylphenyl)methane, 1,2-di(4-vinylphenyl)ethane, 1,3-di(4-vinylphenyl)propane, and 1,4-di(4'-vinylphenoxy)butane, and its mass percentage in the polymerizable monomer is 50%-80%.

7. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the porogen is at least one of toluene, hexane, octane, isooctane, isododecane, methyl ethyl ketone, methyl isobutyl ketone, dichloroethane, dichloropropane, n-butanol and its isomers, n-pentanol and its isomers, n-hexanol and its isomers, and n-octanol and its isomers, and the mass percentage relative to the polymerizable monomer is 100%-250%.

8. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (1), the initiator is at least one of organic peroxide and azo compound, and its mass percentage relative to the polymerizable monomer is 0.05%-2.5%.

9. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (2), the buffer solution is a glycine-sodium hydroxide buffer solution; the concentration of the enzyme solution is 10-80 mg / mL, and the pH value is 9-10.

10. The method for preparing a Sn-1,3-specific immobilized lipase according to claim 1, characterized in that, In step (3), the adsorption temperature is 30°C and the time is 1-4 hours; the drying method is freeze drying.

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