An immobilized lipase and its application

Abstract

The present invention discloses an immobilized lipase and its application. The immobilized lipase disclosed in the present invention is prepared by adding free lipase, an immobilized carrier, a stabilizer and a co-immobilizing agent of biological origin into a buffer solution, stirring under conditions suitable for the immobilized carrier to adsorb the free lipase, and then filtering, washing and drying the obtained solution. The co-immobilizing agent of biological origin used in the present invention has good compatibility with the lipase and is conducive to the dispersion of the lipase in the system, thereby improving the adsorption efficiency of the lipase, and the stabilizer used can ensure the activity of the lipase. The immobilized lipase disclosed in the present invention has a high conversion rate when used in the synthesis of vitamin A palmitate, and has great potential for industrial application.

Description

Technical Field

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

[0002] Lipase exists in various animals, plants and microorganisms in nature, and can catalyze chemical reactions such as hydrolysis, esterification and amidation. At the same time, it also shows efficient catalytic activity in the organic phase, which broadens the application field of the enzyme. After lipase is immobilized, it is conducive to the reuse of the enzyme, thereby reducing the cost of using lipase. Therefore, the immobilization effect of lipase is an important factor affecting the activity and reuse of lipase. The most important factor affecting the immobilization of lipase is that during the immobilization process, the enzyme molecules tend to aggregate and it is difficult to exist in the form of a monolayer, resulting in most of the enzyme molecules not being bound to the carrier during the immobilization process, thereby affecting the immobilization efficiency. Another factor is that the immobilization time is long, during which the structure of the protein molecules is easily destroyed and affects the activity of the enzyme.

[0003] The existing methods add chemical surfactants such as Triton X-100 during the immobilization process to make the enzyme molecules form a monolayer. However, since Triton X-100 is an ether compound, it may have a certain destructive effect on the molecular structure of the protein. At the same time, how to ensure the stability of the enzyme during the immobilization process is currently less studied. Therefore, the development of bio-based surfactants and enzyme structure stabilizers is a necessary condition to ensure the efficient immobilization of lipase. Summary of the invention

[0004] In view of the above problems existing in the prior art, the present invention provides a highly efficient immobilized lipase and characterizes its application in the synthesis of vitamin A palmitate. Specifically, in the preparation method of the immobilized lipase of the present invention, a bio-based co-immobilization agent and a glucosidase substance derived from microalgae are added, which respectively improves the dispersion uniformity of the enzyme in the system and maintains the enzyme structural stability, thereby improving the immobilization efficiency and lipase activity of the lipase. The operation of the present invention is simple, and the immobilized lipase of the present invention is applied to the synthesis reaction of vitamin A palmitate with a high reaction conversion rate.

[0005] To achieve the above objectives, in one aspect, the present invention provides an immobilized lipase, which is obtained by adding free lipase, an immobilization carrier, a stabilizer and a co-immobilization agent of biological origin into a buffer solution, stirring under conditions suitable for the immobilization carrier to adsorb the free lipase, and then filtering, washing and drying the resulting solution.

[0006] In one embodiment of the present invention, in the above-mentioned immobilized lipase, the free lipase is selected from one or more of free lipase from Rhizomucor miehei, free lipase from Humicola Lanuginosa and free lipase from Candida antarctica, preferably free lipase from Rhizomucor miehei. These free lipases can be commercially available or homemade. In the art, the preparation method of these free lipases is well known, and any existing method can be used for preparation, for example, the preparation can be made by the following method: inoculating the bacterial liquid into a culture medium containing basal salts for fermentation, when the dissolved oxygen rises, feeding glycerol, intermittently feeding yeast extract and peptone, controlling the dissolved oxygen and pH, and centrifuging and collecting the supernatant after the fermentation is completed and freeze-dried.

[0007] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, the co-immobilizer is derived from a polar glycolipid mixture after purification of microalgae oil, and the preparation method thereof comprises the following steps:

[0008] (1) Adding microalgae biomass to an organic solvent, mixing uniformly at 20 to 50° C. (e.g., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., and 50° C., or a value or range between any two of these values), preferably 35 to 50° C., adjusting the system pressure to 10 to 40 MPa (e.g., 10 MPa, 20 MPa, 30 MPa, and 40 MPa, or a value or range between any two of these values), preferably 30 to 40 MPa, and stirring for 2 to 5 h (e.g., 2 h, 3 h, 4 h, and 5 h). , preferably 3 to 5 hours, so that the microalgae biomass is broken; after the stirring is completed, the supernatant is centrifuged and the centrifugal speed is 5000 to 10000 rpm (for example, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm and 10000 rpm, or a value or range between any two of these values), preferably 8000 to 10000 rpm, to obtain crude algae oil; the supernatant is desolventized under 3 to 10 KPa, preferably 3 to 8 KPa, 45-55 ° C to obtain algae oil solid;

[0009] (2) The algae oil solid is separated by a silica gel column, and the elution solvent is n-heptane:isopropanol = (4-10):1 (v / v) (for example, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 and 10:1, or a value or range between any two of these values), preferably (8-10):1; 1CV (column volume) to 2-3CV (for example, 1 to 2CV, 1 to 2.5CV and 1 to 3CV), preferably 1 to 2.5-3CV (column volume) components are collected; the resulting components are desolvated at 3-10KPa (for example, 3KPa, 4KPa, 5KPa, 6KPa, 7KPa and 8KPa, or a value or range between any two of these values), preferably 3-8KPa, 45-55°C to obtain a co-immobilized agent.

[0010] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, in the method for preparing the co-immobilizer,

[0011] In step (1), the microalgae biomass is microalgae dry algae powder, preferably one or more selected from Spirulina dry algae powder, Chlorella dry algae powder and Phaeodactylum triangularis dry algae powder, more preferably Phaeodactylum triangularis dry algae powder; and / or

[0012] In step (1), the organic solvent is selected from one or more of methanol, chloroform, and n-hexane, preferably methanol; preferably, in step (1), the volume of the organic solvent added per gram of microalgal biomass is 5 to 10 mL (e.g., 5 mL, 6 mL, 7 mL, 8 mL, 9 mL and 10 mL, or a value or range between any two of these values), preferably 8 to 10 mL; and / or

[0013] In step (2), the mass ratio of the algae oil solid to the silica gel is 1:(40-100) (e.g., 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 and 1:100, or a value or range between any two of these values), preferably 1:(40-70);

[0014] The inner diameter of the silica gel column may be, for example, 72 to 100 mm.

[0015] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, the stabilizer is selected from one or more of methyl glucoside, ethyl glucoside and glycerol glucoside, preferably glycerol glucoside.

[0016] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, the immobilization carrier is an adsorption resin, preferably one or more of D3520, Duolite A568 and Amberlite XAD 761 materials, more preferably Duolite A568.

[0017] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, the buffer solution is selected from one or more of Tris-hydrochloric acid buffer and Bis-tris-hydrochloric acid buffer, preferably Tris-hydrochloric acid buffer; the concentration is 0.5-2.0 mol / L (for example, 0.5 mol / L, 1 mol / L, 1.3 mol / L, 1.7 mol / L, 1.9 mol / L and 2.0 mol / L, or a value or range between any two of these values), preferably 0.5-1 mol / L; the pH is 5-8 (for example, 5, 6, 7 and 8), preferably 6-8.

[0018] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, the amount of the buffer solution added is 200-300 mL (e.g., 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL and 300 mL) per gram of free lipase, preferably 250-300 mL; the mass fractions of the co-immobilizer and the stabilizer in the buffer solution are 0.5-3% and 0.1-1%, respectively, preferably 0.5-1.5% and 0.1-0.5%, respectively;

[0019] The mass ratio of the free lipase to the immobilized carrier is: 1:(8-10), preferably 1:(8-9);

[0020] The lipase immobilization conditions are as follows: temperature 20-50°C (for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, or a value or range between any two of these values), preferably 25-35°C; stirring speed 50-100rpm (for example, 50rpm, 80rpm, 100rpm), preferably 50-80rpm; stirring time 12-24h (for example, 12h, 14h, 16h, 18h and 20h), preferably 16-24h.

[0021] In one embodiment of the present invention, in any of the above-mentioned immobilized lipases, filtration and washing are conventional operations, such as filtering with filter paper and washing with the buffer solution, and drying is natural drying at room temperature, such as drying at 20-25°C.

[0022] In another aspect, the present invention provides use of any of the above-mentioned immobilized lipases in catalytic synthesis of vitamin A palmitate.

[0023] In another aspect, the present invention provides an enzymatic synthesis method of vitamin A palmitate, comprising the step of using any of the above-mentioned immobilized lipases as a catalyst to catalyze the reaction of raw materials vitamin A acetate and palmitic acid to generate vitamin A palmitate.

[0024] In one embodiment of the present invention, in the enzymatic synthesis method of vitamin A palmitate, the molar ratio of vitamin A acetate to palmitic acid is 1:(1-1.2) (e.g., 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.16, 1:1.17, 1:1.18, 1:1.19, 1:1.2, or a value or range between any two of these values), preferably 1:(1.1-1.15); the mass ratio of the immobilized lipase to the vitamin A acetate is 1:(40-100) (for example, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 and 1:100, or a value or range between any two of these values), preferably 1:(40-60); the reaction solvent is one or more of n-hexane, n-heptane and n-octane, preferably n-octane; the reaction temperature is 30-60°C (for example, 35°C, 40°C, 45°C, 50°C, 55°C and 60°C), preferably 30-45°C, and the reaction time is 6-10h (for example, 6h, 7h, 8h, 9h and 10h), preferably 8-10h.

[0025] The beneficial effects of the present invention are:

[0026] The invention provides a high-activity lipase immobilization method. The microalgae oil separation extract is mainly composed of polar glycolipids, and has hydrophilic and hydrophobic amphiphilic properties in its chemical structure. The microalgae oil separation extract can be used as a bio-based co-immobilizer. On the one hand, the microalgae oil separation extract has good biocompatibility with enzyme molecules, and on the other hand, the enzyme molecules can be guaranteed to be in a monolayer form during the immobilization process, thereby improving the enzyme immobilization efficiency; reducing the occurrence of enzyme desorption during use, and increasing the number of enzyme reuses; adding glucosides during the immobilization process can further maintain the activity of the lipase, making it difficult to be inactivated during the immobilization process, and better ensuring its catalytic effect.

[0027] The method of the present invention is simple to operate, and has high protein adsorption rate and lipase activity, which are respectively up to 99.2% and 11038U / g. It is applied to the synthesis reaction of vitamin A palmitate and has a high conversion rate, which can be as high as 99.6%. The enzyme also maintains good stability during the application process, and has great potential for industrial application. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0029] Spirulina dry algae powder, Chlorella dry algae powder and Phaeochromis triangularis dry algae powder are products of Shandong Boran Spirulina Biological Co., Ltd., Jinan Shenghe Chemical Co., Ltd. and Shanghai Guangyu Biotechnology Co., Ltd. respectively.

[0030] The free lipase from Rhizomucor miehei, the free lipase from Humicola Lanuginosa and the free lipase from Candida antarctica are produced by fermentation using Rhizomucor miehei, Humicola Lanuginosa and Candida antarctica respectively by the following method:

[0031] The bacterial liquid (OD600 = 20-40) was inoculated into a 100L fermenter containing a basal salt medium (1g / L calcium sulfate, 18g / L potassium sulfate, 15g / L magnesium sulfate, 4.2g / L potassium hydroxide, 2.7% (v / v) phosphoric acid, 4% (v / v) glycerol) at a 10% (v / v) inoculation rate, at a temperature of 30°C, a rotation speed of 800rpm, and a ventilation volume of 8L / min; when the dissolved oxygen rose to 35%, 60% (v / v) glycerol was added at a flow rate of about 2mL / (min·L), the time is 6 to 8 hours; yeast extract and peptone are added every 12 hours, the amount of yeast extract added is 1% (w / v, g / 100ml), and the amount of peptone added is 1.5% (w / v, g / 100ml); the dissolved oxygen is controlled within the range of 20% to 30% by adjusting the ventilation volume, and the pH is controlled at about 5.5 by adding ammonia water; after culturing for 140 hours, the fermentation liquid supernatant is collected by centrifugation at 4°C and 12000rpm for 5 minutes, and freeze-dried for later use.

[0032] Tween 80, Triton X-100, methyl glucoside, ethyl glucoside and glyceryl glucoside are all products of Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0033] The analytical standards SQDG (thioisosorhamnosyl diacylglyceride), MGDG (monogalactosyl diacylglyceride) and DGDG (digalactosyl diacylglyceride) are all products of Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0034] Silica gel, D3520 (ion exchange macroporous adsorption resin), Duolite A568 (weakly alkaline phenolic anion exchange resin) and Amberlite XAD 761 (ion exchange macroporous adsorption resin) are all products of Xi'an Lanxiao Technology New Materials Co., Ltd.

[0035] Vitamin A acetate crystals are products of Zhejiang Medicine Co., Ltd.

[0036] Palmitic acid is a product of Aladdin.

[0037] Immobilized lipase Novozym 435 is a product of Novozymes.

[0038] The analytical test methods used in the following examples are as follows:

[0039] 1) Detection and quantification of polar glycolipid mixtures:

[0040] a. Prepare standard solutions of SQDG (sulphoisosorhamnose diacylglyceride, 0-200 ppm), MGDG (monogalactose diacylglyceride, 0-200 ppm) and DGDG (digalactose diacylglyceride, 0-200 ppm) with methanol respectively.

[0041] b. Take about 1 mg of the co-immobilizer to prepare a polar glycolipid mixture sample (mass is m), add an appropriate amount of methanol to re-dissolve and perform quantitative analysis and detection.

[0042] c. High performance liquid chromatograph, Agilent LC-1200, chromatographic column: HILIC column (4.6mm×150mm, 3.5μm, XBridge, Waters, USA), column temperature: 30℃, detector: ELSD. Mobile phase A is acetonitrile: water (95:5v / v, 2.5mM ammonium formate, 0.02% formic acid); mobile phase B is acetonitrile: water (50:50v / v, 2.5mM ammonium formate, 0.02% formic acid). The initial mobile phase is 100% A, then linearly changes to 50% A within 20min, then returns to the initial mobile phase within 1min and maintains for 6min, flow rate: 1ml / min, sample volume: 10μL. The retention times of SQDG of MGDG, DGDG are 6.4min, 11.8min and 16.4min, respectively.

[0043] d. Calculate the concentration of the corresponding substances according to the standard curve and the peak area of ​​SQDG, MGDG and DGDG in the sample and calculate the proportion of each substance in the polar glycolipid mixture (c 折算 *v / m*100%, where c 折算are the concentrations of SQDG, MGDG and DGDG respectively converted according to the standard curve, in mg / L; v is the total volume of the sample after dilution, in L; m is the total mass of the polar glycolipid mixture, in mg). The total mass percentage is the sum of the three ratios.

[0044] 2) Protein content and protein adsorption rate detection

[0045] The protein content was detected using the Bio-Tech BCA protein concentration assay kit.

[0046] The protein adsorption rate was calculated as follows: (X1-X2) / X1*100%, wherein X1 was the protein content in the enzyme solution before adsorption (i.e., after adding the buffer solution and before adding the resin, glucoside and co-immobilizer), expressed in mg / mL; X2 was the protein content in the enzyme solution after adsorption (i.e., after adding the resin, glucoside and co-immobilizer and stirring the reaction), expressed in mg / mL.

[0047] 3) Enzyme activity detection

[0048] a. Take 320 μL of water, 8 g of lauric acid and 2.4 g of n-propanol in a 50 mL conical flask, preheat it in a constant temperature shaker (60°C, 200 rpm) for 10 min, and then add about 30 mg of immobilized lipase (mass is m).

[0049] b. Continue the reaction at 60°C and 200 rpm for 20 min, take a sample and dilute it appropriately with n-heptane for enzyme activity detection.

[0050] c. Prepare standard solutions of lauric acid (0-30mM), n-propanol (0-30mM) and propyl laurate (0-7.5mM) with n-heptane respectively; the samples were detected by gas chromatograph: Agilent GC7890B, capillary column: Restek Stabilwax DA column (15m×0.25mm, 0.25μm), and FID detector. The injection volume was 1μL, the carrier gas was nitrogen, and the flow rate was 2mL / min. The injector temperature was 280℃, the detector temperature was 280℃, and the heating program was:

[0051] 0~2min:60℃

[0052] 2~6min: 60-260℃, heating 50℃ / min

[0053] 6~8min:250℃

[0054] Gas chromatography will separate n-propanol, propyl laurate and lauric acid with corresponding retention times of 1.2 min, 5.1 min and 6.8 min, respectively.

[0055] d. Calculate the concentration of the corresponding substances and the lauric acid conversion rate (C = (A1-A2) / A1*100%, wherein A1 is the concentration of lauric acid before the reaction (i.e., after preheating and before adding the immobilized lipase), in mM; A2 is the concentration of lauric acid after the reaction, in mM) according to the standard curve and the peak areas of n-propanol, propyl laurate and lauric acid in the sample.

[0056] e. The enzyme activity calculation formula is: enzyme activity (U / g) = N*C / (20*m); where N is the number of moles of lauric acid initially added, in μmol; C is the conversion rate of lauric acid; 20 is the reaction time, in min; m is the mass of immobilized enzyme, in g. The amount of enzyme required to generate 1 μmol of propyl laurate per minute under standard reaction conditions is defined as 1U.

[0057] 4) Vitamin A palmitate reaction test

[0058] a. Prepare standard solutions of vitamin A acetate (0-1000 ppm) and vitamin A palmitate (0-1000 ppm) with methanol respectively.

[0059] b. Take the sample after the synthesis reaction of vitamin A palmitate and dilute it with methanol appropriately for later use.

[0060] c. Detect the sample and the standard solution sample in a high performance liquid chromatograph. The instrument model is Agilent LC-1200, the chromatographic column is Agilent SB-AQ (4.6×250mm, 5μm), the detection wavelength is 326nm; the detection temperature is 40℃; the mobile phase is isopropanol: water = 95:1 (v / v); the flow rate is 1mL / min; the sample volume is 2μL. The retention times of vitamin A acetate and vitamin A palmitate are 12.6min and 26.4min respectively.

[0061] d. Calculate the concentration of the corresponding substances and the conversion rate of vitamin A acetate according to the standard curve and the peak areas of vitamin A acetate and vitamin A palmitate in the sample (C = (A1-A2) / A1*100%, wherein A1 is the concentration of vitamin A acetate before the reaction (i.e., after adding reaction solvents such as n-octane, n-hexane or n-heptane and palmitic acid to vitamin A acetate, and before adding immobilized lipase), in ppm; A2 is the concentration of vitamin A acetate after the reaction, in ppm).

[0062] Example 1: Preparation of co-immobilization agent

[0063] Spirulina-derived co-immobilizer-co-immobilizer A:

[0064] (1) Preparation of Spirulina algae oil solid: Weigh 300g of Spirulina dry algae powder, add it to a 5L stainless steel pressure reactor, add 2.4L of n-hexane, slowly heat to 35°C, adjust the system pressure to 10MPa, and stir for 3h. After stirring, centrifuge the material at 8000rpm to obtain the supernatant, i.e., crude algae oil. Remove the solvent from the obtained supernatant at 4KPa and 50°C. After the solvent is completely removed, obtain algae oil solid, weighing about 32.4g.

[0065] (2) Collection of target eluted components: Weigh about 800g of silica gel, mix and stir with 3L of n-heptane: isopropanol = 4:1 (v / v) mixed solution until there are no bubbles, and fill the separation column (the inner diameter of the glass chromatography column is 72mm, and the effective column volume is 3.3L), with a column volume of about 2.4L. Take 20g of algae oil solid in step (1), re-dissolve it with 20mL of n-heptane: isopropanol = 4:1 (v / v) mixed solution, and load it into the filling separation column. And elute with n-heptane: isopropanol = 4:1 (v / v) mixed solution, record the volume of the collected components, combine the components corresponding to the elution volume of 2.4 to 4.8L, and remove the solvent under 4KPa and 50°C. After the solvent is completely removed, the co-immobilization agent A is obtained, weighing about 7.8g.

[0066] After quantitative analysis by high performance liquid chromatography, the total mass percentage of polar glycolipids in the co-immobilizer A was 99.3%, of which MGDG was 52.2%, DGDG was 24.0%, and SQDG was 23.1%.

[0067] Chlorella-derived co-immobilizer-co-immobilizer B:

[0068] (1) Preparation of Chlorella algae oil solid: Weigh 300g of Chlorella dry algae powder, add it to a 3L stainless steel pressure reactor, add 1.5L chloroform, maintain the temperature at 20°C, adjust the system pressure to 30MPa, and stir for 5h. After stirring, centrifuge the material at 10000rpm to obtain the supernatant, i.e., crude algae oil. Remove the solvent from the obtained supernatant at 8KPa and 50°C. After the solvent is completely removed, obtain algae oil solid, weighing about 63.5g.

[0069] (2) Collection of target eluted components: Weigh about 1400g of silica gel, mix and stir with 5L of n-heptane:isopropanol = 8:1 (v / v) mixed solution until there are no bubbles, and fill the separation column (the inner diameter of the glass chromatography column is 92mm, and the effective column volume is 6.6L), with a column volume of about 4.2L. Take 20g of algae oil solid in step (1), re-dissolve it with 20mL of n-heptane:isopropanol = 8:1 (v / v) mixed solution, and load it into the filling separation column. Elute with n-heptane:isopropanol = 8:1 (v / v) mixed solution, record the volume of the collected components, combine the components corresponding to the elution volume of 4.2 to 10.5L, and remove the solvent under 8KPa and 50°C. After the solvent is completely removed, the co-immobilization agent B is obtained, weighing about 9.7g.

[0070] After quantitative analysis by high performance liquid chromatography, the total mass percentage of polar glycolipids in the co-immobilizer B was 99.4%, of which MGDG was 54.7%, DGDG was 22.6%, and SQDG was 22.1%.

[0071] Co-immobilizer from Phaeodactylum triangularis - Co-immobilizer C:

[0072] (1) Preparation of solid algae oil from P. triangularis: Weigh 300 g of dry algae powder from P. triangularis, add it to a 5 L stainless steel pressure reactor, add 3 L of methanol, slowly heat to 50 ° C, adjust the system pressure to 40 MPa, and stir for 2 h. After stirring, centrifuge the material at 5000 rpm to obtain the supernatant, i.e., crude algae oil. Remove the solvent from the obtained supernatant at 3 KPa and 50 ° C. After the solvent is completely removed, obtain algae oil solid, weighing about 79.8 g.

[0073] (2) Collection of target eluted components: Weigh about 2000g of silica gel, and mix and stir with 7L of n-heptane: isopropanol = 10:1 (v / v) mixed solution until there are no bubbles, and fill the separation column (the inner diameter of the glass chromatography column is 100mm, and the effective column volume is 9.4L), and the column volume is about 6L. Take 20g of algae oil solid in step (1), re-dissolve it with 20mL of n-heptane: isopropanol = 10:1 (v / v) mixed solution, and load it into the filling separation column. And elute with n-heptane: isopropanol = 10:1 (v / v) mixed solution, record the volume of the collected components, combine the components corresponding to the elution volume of 6 to 18L, and remove the solvent under 3KPa and 50°C. After the solvent is completely removed, the co-immobilization agent C is obtained, weighing about 11.6g.

[0074] After quantitative analysis by high performance liquid chromatography, the total mass percentage of polar glycolipids in the co-immobilizer C was 99.5%, of which MGDG was 56.1%, DGDG was 23.1%, and SQDG was 20.3%.

[0075] Example 2: Lipase immobilization

[0076] Lipase Immobilization-Enzyme A

[0077] Weigh 1g of free lipase from Candida antarctica, add 200mL of 2mol / L, pH5.0 Bis-tris-HCl buffer, then add 10g of D3520 resin, 2g of methyl glucoside and 6g of co-immobilizer A, respectively, and stir at 50°C and 100rpm for 12h. After stirring, filter the solution, wash the resin once with 2mol / L, pH5.0 Bistris-HCl buffer, and dry the resin naturally to obtain immobilized lipase A.

[0078] The results showed that the protein adsorption rate was 98.5% and the activity of immobilized lipase A was 10653 U / g.

[0079] Lipase Immobilization-Enzyme B

[0080] 1 g of free lipase from Humicola Lanuginosa was weighed, 300 mL of 1 mol / L, pH 8.0 Tris-HCl buffer was added, and then 9 g of Amberlite XAD 761 resin, 0.3 g of ethyl glucoside and 1.5 g of co-immobilizer B were added respectively, and stirred at 20°C and 80 rpm for 16 h. After stirring, the solution was filtered, and the resin was washed once with 1 mol / L, pH 8.0 Tris-HCl buffer, and the resin was dried naturally to obtain immobilized lipase B.

[0081] The results showed that the protein adsorption rate was 98.8% and the activity of immobilized lipase B was 10846 U / g.

[0082] Lipase Immobilization-Enzyme C

[0083] Weigh 1g of free lipase from Rhizomucor miehei, add 250mL of 0.5mol / L, pH 6.0 Tris-HCl buffer, then add 8g of Duolite A568 resin, 1.25g of glycerol glucoside and 3.75g of co-immobilizer C, respectively, and stir at 35°C and 50rpm for 24h. After stirring, filter the solution, wash the resin once with 0.5mol / L, pH 6.0 Tris-HCl buffer, and dry the resin naturally to obtain immobilized lipase C.

[0084] The protein adsorption rate was found to be 99.2% and the activity of immobilized lipase C was 11038 U / g.

[0085] Example 3: Synthesis of Vitamin A Palmitate and Lipase Recycling

[0086] The air in the three-necked flask was replaced with nitrogen, 10 g of vitamin A acetate was accurately weighed and placed in a 250 mL three-necked flask, 100 mL of n-octane, 8.59 g of palmitic acid, and 0.25 g of immobilized lipase A were added, and the mixture was stirred and reacted for 10 hours at 35°C. After the reaction was completed, the conversion rate of vitamin A acetate in the reaction was detected to be 99.6%.

[0087] After the reaction, the reaction system was filtered to obtain immobilized lipase A, which was added to the reaction system according to the ratio of the above reactants. The recovered immobilized lipase A was added to the reaction system again. The same conditions were repeated 20 times. The reaction conversion rate (i.e., the conversion rate of vitamin A acetate) is shown in Table 1.

[0088] Table 1 Reaction effect of immobilized lipase A after 20 applications

[0089]

[0090] As shown in Table 1, the immobilized lipase A still maintained a high catalytic activity after 20 applications, indicating that the lipase activity was stable.

[0091] Example 4: Synthesis of Vitamin A Palmitate

[0092] The air in the three-necked flask was replaced with nitrogen, 10 g of vitamin A acetate was accurately weighed and placed in a 250 mL three-necked flask, 100 mL of n-hexane, 8.98 g of palmitic acid, and 0.17 g of immobilized lipase B were added, and the mixture was stirred at 45°C for 8 hours. After the reaction was completed, the conversion rate of vitamin A acetate in the reaction was detected to be 99.5%.

[0093] Example 5: Synthesis of Vitamin A Palmitate

[0094] The air in the three-necked flask was replaced with nitrogen, 10 g of vitamin A acetate was accurately weighed and placed in a 250 mL three-necked flask, 100 mL of n-heptane, 9.37 g of palmitic acid, and 0.1 g of immobilized lipase C were added, and the mixture was stirred at 60° C. for 6 hours. After the reaction was completed, the conversion rate of vitamin A acetate in the reaction was detected to be 99.4%.

[0095] Comparative Example 1

[0096] Immobilized lipase D was prepared by referring to the method of "Immobilized lipase - enzyme C" in Example 2, except that no co-immobilizer C was added, and other operations were the same. The protein adsorption rate was 82.1% and the enzyme activity was 7964.8 U / g.

[0097] Vitamin A palmitate was prepared by referring to the method of Example 5, except that the immobilized lipase C was replaced by an equal amount of immobilized lipase D, and the other operations were the same. After the reaction was completed, the conversion rate of vitamin A acetate was 78.5%.

[0098] Comparative Example 2

[0099] Immobilized lipase E was prepared by referring to the method of "Immobilized lipase - enzyme C" in Example 2, except that glycerol glucoside was not added, and other operations were the same. After testing, the protein adsorption rate was 97.8% and the enzyme activity was 9893.8 U / g.

[0100] Vitamin A palmitate was prepared by referring to the method of Example 5, except that the immobilized lipase C was replaced by an equal amount of immobilized lipase E, and the other operations were the same. After the reaction was completed, the conversion rate of vitamin A acetate was detected to be 95.2%. The lipase application experiment was conducted, and it was found that the conversion rate of vitamin A acetate dropped to 68.9% after the fifth application.

[0101] Comparative Example 3

[0102] Immobilized lipase F was prepared by referring to the method of "Immobilized Lipase - Enzyme C" in Example 2, except that the co-immobilizer C and glycerol glucoside were not added, and the other operations were the same. After testing, the protein adsorption rate was 81.2% and the enzyme activity was 7058.2 U / g.

[0103] Vitamin A palmitate was prepared by referring to the method of Example 5, except that the immobilized lipase C was replaced by an equal amount of immobilized lipase F, and the other operations were the same. After the reaction was completed, the conversion rate of vitamin A acetate was 70.6%.

[0104] Comparative Example 4

[0105] Immobilized lipase G was prepared by referring to the method of "Immobilization of lipase - enzyme C" in Example 2, except that the co-immobilizer C was replaced with an equal mass of Triton X-100, and the other operations were the same. After testing, the protein adsorption rate was 86.3% and the enzyme activity was 8961.4 U / g.

[0106] Vitamin A palmitate was prepared by referring to the method of Example 5, except that the immobilized lipase C was replaced by an equal amount of immobilized lipase G, and the other operations were the same. After the reaction was completed, the conversion rate of vitamin A acetate was 88.2%.

[0107] Comparative Example 5

[0108] Immobilized lipase H was prepared by referring to the method of "Immobilization of lipase - enzyme C" in Example 2, except that the co-immobilizer C was replaced with an equal mass of Tween 80, and the other operations were the same. After testing, the protein adsorption rate was 87.2% and the enzyme activity was 9061.3 U / g.

[0109] Vitamin A palmitate was prepared by referring to the method of Example 5, except that the immobilized lipase C was replaced by an equal amount of immobilized lipase H, and the other operations were the same. After the reaction was completed, the conversion rate of vitamin A acetate was 89.7%.

[0110] Comparative Example 6

[0111] Vitamin A palmitate was prepared by referring to the method of Example 3, except that the immobilized lipase A was replaced with an equal mass of commercial lipase Novozym 435, and the other operations were the same. After the reaction was completed, the conversion rate of vitamin A acetate was detected to be 98.4%. The lipase application experiment was conducted, and it was found that the conversion rate of vitamin A acetate dropped to 86.9% after the fifth application.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An immobilized lipase, which is prepared by adding free lipase, an immobilization carrier, a stabilizer and a co-immobilization agent of biological origin into a buffer solution, stirring under conditions suitable for the immobilization carrier to adsorb the free lipase, and then filtering, washing and drying the resulting solution; The stabilizer is selected from one or more of methyl glucoside, ethyl glucoside and glycerol glucoside; The co-immobilizing agent is derived from a polar glycolipid mixture after purification of microalgae oil, and the preparation method thereof comprises the following steps: (1) adding an organic solvent to microalgae biomass, stirring at 20 to 50° C. and a system pressure of 10 to 40 MPa to break the microalgae biomass; centrifuging after stirring to obtain a supernatant, i.e., crude algae oil; and removing the solvent from the supernatant to obtain algae oil solid; (2) The algae oil solid is separated by a silica gel column, and the elution solvent is n-heptane:isopropanol = (4-10): 1 (v / v); 1CV to 2-3CV (column volume) components are collected; and the obtained components are freed from the solvent to obtain the co-immobilization agent.

2. The immobilized lipase according to claim 1, characterized in that: The free lipase is selected from one or more of the free lipase derived from Rhizomucor miehei, the free lipase derived from Humicola Lanuginosa and the free lipase derived from Candida antarctica.

3. The immobilized lipase according to claim 1, characterized in that: In the preparation method of the co-immobilizing agent, In step (1), the microalgae biomass is dry microalgae powder; and / or In step (1), the organic solvent is selected from one or more of methanol, chloroform and n-hexane; and / or In step (2), the mass ratio of the algae oil solid to the silica gel in the silica gel column is 1:(40-100).

4. The immobilized lipase according to claim 1, characterized in that: The immobilization carrier is an adsorption resin.

5. The immobilized lipase according to claim 1, characterized in that: The buffer solution is selected from one or more of Tris-hydrochloric acid buffer and Bis-tris-hydrochloric acid buffer.

6. The immobilized lipase according to any one of claims 1 to 5, characterized in that: The amount of the buffer solution is 200-300 mL per gram of free lipase; and / or The mass fractions of the co-immobilizer and the stabilizer in the buffer solution are 0.5-3% and 0.1-1%, respectively; and / or The mass ratio of the free lipase to the immobilized carrier is 1:(8-10); and / or The conditions suitable for the immobilized carrier to adsorb the free lipase are: temperature of 20-50° C.; stirring speed of 50-100 rpm; and stirring time of 12-24 h.

7. Use of the immobilized lipase according to any one of claims 1 to 6 in catalytic synthesis of vitamin A palmitate.

8. An enzymatic synthesis method of vitamin A palmitate, comprising the step of using the immobilized lipase according to any one of claims 1 to 6 as a catalyst to catalyze the reaction of raw materials vitamin A acetate and palmitic acid to produce vitamin A palmitate.

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