Method for preparing phosphatidylserine through continuous enzyme reaction

Abstract

The invention provides a method for preparing phosphatidylserine through continuous enzyme reaction, and belongs to the technical field of biochemical engineering. The method comprises the following steps: dissolving phosphatidylcholine in an organic phase to obtain a phosphatidylcholine solution, and dissolving L-serine in an aqueous solution to obtain an L-serine solution; the method comprises the following steps: mixing a phosphatidylcholine solution and an L-serine solution to obtain an emulsion, feeding the emulsion into a multi-stage continuous stirring tank reactor, and carrying out transesterification under the action of immobilized enzyme to obtain phosphatidylserine. According to the method for preparing phosphatidylserine through the continuous enzyme reaction, steric hindrance of enzyme and a substrate can be reduced, the catalytic efficiency is improved, and the dosage of the enzyme is greatly reduced; high-purity soybean phosphatidylcholine can be catalyzed, and the adverse effect of the high-purity soybean phosphatidylcholine on the reaction is avoided; according to the method, high-efficiency and high-purity continuous production of phosphatidylserine can be realized, the product does not need to be purified additionally, and the reaction time is shortened to be within 6 hours.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a method for preparing phosphatidylserine by continuous enzyme reaction. Background Art

[0002] Phosphatidylserine (PS), also known as serine phospholipids or diacylglycerol phosphoserine (PS), is a ubiquitous phospholipid extracted from the residue left over from soybean oil extraction. It is typically located in the inner layer of cell membranes and is an active substance in these membranes, particularly in brain cells. Its primary function is to improve neuronal function, regulate the conduction of nerve impulses, and enhance memory. Due to its strong lipophilicity, it rapidly crosses the blood-brain barrier and enters the brain, where it soothes vascular smooth muscle cells and increases blood flow to the brain. Conventional PS production utilizes intermittent enzymatic reactions (such as phospholipase D-catalyzed transesterification of phosphatidylcholine with serine). This process is plagued by difficulties in catalyzing the transesterification of high-content soybean phosphatidylcholine, long reaction times, easy inactivation of the enzyme carrier, low reusability, and significant product purity fluctuations. Furthermore, the solvent system is prone to numerous side reactions (such as the formation of phosphatidic acid), requiring multiple purification steps, resulting in low yields, inconsistent yields between batches, high equipment utilization, and high production costs. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for preparing phosphatidylserine by continuous enzymatic reaction, so as to solve the problems of the existing method such as long time consumption, low enzyme catalytic efficiency and low conversion rate.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing phosphatidylserine by continuous enzymatic reaction, comprising the following steps:

[0006] 1) dissolving phosphatidylcholine in an organic phase to obtain a phosphatidylcholine solution, and dissolving L-serine in an aqueous solution to obtain an L-serine solution;

[0007] 2) The phosphatidylcholine solution and the L-serine solution are mixed to obtain an emulsion, and the emulsion is introduced into a multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the action of an immobilized enzyme to obtain phosphatidylserine.

[0008] Preferably, in step 1), the organic phase is ethyl acetate and n-hexane, and the volume ratio of ethyl acetate to n-hexane is 1:2-4.

[0009] Preferably, in step 1) the aqueous solution contains Ca 2 The concentration of + is 10-20 mM, and the pH of the aqueous solution is 3.5-6.5.

[0010] Preferably, in step 1), the volume ratio of the organic phase to the aqueous solution is 1:3-5.

[0011] Preferably, in step 1), the molar ratio of phosphatidylcholine to L-serine is 1:3-5.

[0012] Preferably, the flow rate of the mixed solution in step 2) is 0.5 to 100 mL / min, and the residence time of the mixed solution in the multi-stage continuous stirred tank reactor is 4 to 6 hours.

[0013] Preferably, in step 2), the immobilized enzyme is phospholipase D covalently immobilized on amino-modified porous particles.

[0014] Preferably, in step 2), the mass ratio of the immobilized enzyme to phosphatidylcholine is 1 to 10:1.

[0015] Preferably, the temperature of the transesterification reaction in step 2) is 38-45°C.

[0016] The present invention also provides phosphatidylserine prepared by the method.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The method for preparing phosphatidylserine by continuous enzymatic reaction can reduce steric hindrance between the enzyme and the substrate, improve catalytic efficiency, greatly reduce the amount of enzyme used, and achieve enzyme reuse of ≥20 batches (activity retention >80%); can catalyze high-purity soybean phosphatidylcholine, avoiding the adverse effects of high-purity soybean phosphatidylcholine on the reaction; can achieve efficient (yield >85%) and high-purity (>90%) production of phosphatidylserine without the need for additional product purification, and shortens the reaction time to less than 6 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the apparatus used for preparing phosphatidylserine by continuous enzymatic reaction (1 is an aqueous solution storage tank, 2 is an ethyl acetate and n-hexane mixture storage tank, 3 is a pump, 4 is a pipeline shear, 5 is a multi-stage continuous stirred tank reactor, 6 is an immobilized enzyme, 7 is a separator, 8 is a scraper evaporator, 9 is an ethyl acetate and n-hexane mixture, 10 is a macroporous resin, 11 is phosphatidylserine, 12 is an aqueous solution storage tank, 13 is L-serine and calcium chloride, 14 is calcium chloride, 15 is L-serine, and 16 is phosphatidylcholine). DETAILED DESCRIPTION

[0020] The present invention provides a method for preparing phosphatidylserine by continuous enzymatic reaction, comprising the following steps:

[0021] 1) dissolving phosphatidylcholine in an organic phase to obtain a phosphatidylcholine solution, and dissolving L-serine in an aqueous solution to obtain an L-serine solution;

[0022] 2) The phosphatidylcholine solution and the L-serine solution are mixed to obtain an emulsion, and the emulsion is introduced into a multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the action of an immobilized enzyme to obtain phosphatidylserine.

[0023] In the present invention, phosphatidylcholine is dissolved in an organic phase to obtain a phosphatidylcholine solution, and L-serine is dissolved in an aqueous solution to obtain an L-serine solution. The organic phase is ethyl acetate and n-hexane, and the volume ratio of ethyl acetate to n-hexane is preferably 1:2 to 4, more preferably 1:2.5 to 3.5; the aqueous solution contains Ca 2 The concentration of Ca is preferably 10 to 20 mM, more preferably 12 to 18 mM; 2 + is preferably added in the form of CaCl2; the pH of the aqueous solution is preferably 3.5-6.5, more preferably 4-6; the volume ratio of the organic phase to the aqueous solution is preferably 1:3-5, more preferably 1:3.5-4.5; the molar ratio of the phosphatidylcholine to L-serine is preferably 1:3-5, more preferably 1:3.5-4.5.

[0024] In the present invention, a phosphatidylcholine solution and an L-serine solution are mixed to obtain an emulsion, which is then introduced into a multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the action of an immobilized enzyme to obtain phosphatidylserine. The flow rate of the mixed solution is preferably 0.5 to 100 mL / min, more preferably 1 to 99 mL / min; the residence time of the mixed solution in the multi-stage continuous stirred tank reactor is preferably 4 to 6 hours, more preferably 4.5 to 5.5 hours; the immobilized enzyme is phospholipase D covalently immobilized on amino-modified porous particles, and the preparation method of the immobilized enzyme comprises the following steps: mixing silica, γ-aminopropyltriethoxysilane and phospholipase D, adding water to react, adjusting the pH value, adding ethanol and collecting the precipitate, cross-linking the precipitate with glutaraldehyde, and washing to obtain immobilized phospholipase D (60 U / g to 100 U / g); the reaction temperature is 0°C, and the reaction time is preferably 3 to 5 hours; the silica, γ-aminopropyltriethoxysilane and phospholipase D are mixed, and the reaction mixture is stirred for 3 hours. The mass ratio of the water to the total mass of silica, γ-aminopropyltriethoxysilane and phospholipase D is preferably 5-10:3-5:2-3, more preferably 6-9:4:2.5; the mass ratio of the water to the total mass of silica, γ-aminopropyltriethoxysilane and phospholipase D is preferably 1:2-4, more preferably 1:3; the pH value is preferably 5.0-5.5, more preferably 5.1-5.4; the mass ratio of the ethanol to the total mass of silica, γ-aminopropyltriethoxysilane and phospholipase D is preferably 1:1-10, more preferably 1:2-8; the mass ratio of the precipitate to glutaraldehyde is preferably 1:0.5-5, more preferably 1:0.8-4; the temperature of the cross-linking reaction is preferably -5-10°C, more preferably -3-7°C; the time of the cross-linking reaction is preferably 1-6h, more preferably 2-5h. The method for preparing the immobilized enzyme further includes the following steps: mixing silica gel, γ-aminopropyltriethoxysilane and phospholipase D, adding water for reaction, adjusting the pH value, adding glutaraldehyde for cross-linking reaction, and washing to obtain immobilized phospholipase D (80 U / g to 110 U / g); the mass ratio of the silica gel, γ-aminopropyltriethoxysilane and phospholipase D is preferably 5-10:3 to 5:2-3; the mass ratio of water to the total mass of the silica gel, γ-aminopropyltriethoxysilane and phospholipase D is preferably 1:4-6, more preferably 1:5; the pH value is preferably 5.0-5.5, more preferably 5.1-5.4; the temperature of the cross-linking reaction is preferably 0-10°C, more preferably 1-9°C; and the time of the cross-linking reaction is preferably 2-4 hours, more preferably 3 hours. The mass ratio of the immobilized enzyme to phosphatidylcholine is preferably 1-10:1, more preferably 2-8:1; the temperature of the transesterification reaction is preferably 38-45°C, more preferably 40-43°C.

[0025] The present invention also provides phosphatidylserine prepared by the method.

[0026] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1

[0028] Preparation of immobilized enzyme: 500 g of silica, 400 g of γ-aminopropyltriethoxysilane, and 200 g of phospholipase D were added with water in a mass ratio of 1:3 to the total mass of silica, γ-aminopropyltriethoxysilane, and phospholipase D. The pH value was adjusted to 5.2, and the mixture was shaken in a shaker at 0°C for 4 hours. 1500 g of chilled ethanol was then rapidly added dropwise for precipitation. The precipitate was collected and cross-linked with glutaraldehyde in a mass ratio of 1:1 at 5°C for 2 hours. After washing, immobilized phospholipase D (85 U / g) was obtained.

[0029] use Figure 1 The apparatus in the preparation of phosphatidylserine.

[0030] The reaction process is as follows: 14 calcium chloride and 15 L-serine are added to an aqueous solution storage tank 1 to obtain an L-serine aqueous solution, 16 phosphatidylcholine is added to a 2 ethyl acetate and n-hexane mixed solution storage tank to obtain a phosphatidylcholine solution, the L-serine aqueous solution and the phosphatidylcholine solution enter a 4 pipeline shearing through a 3 pump, enter a 5 multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the catalysis of a 6 immobilized enzyme, and are separated by a 7 separator to obtain an aqueous phase and an organic phase, the aqueous phase (water, calcium chloride, L-serine) passes through a 3 pump to reach a 10 macroporous resin adsorption, enters a 12 aqueous solution storage tank while supplementing 13 L-serine and calcium chloride, and then passes through a 3 pump to reach the 1 aqueous solution storage tank to continue the continuous reaction, the organic phase passes through an 8 scraper evaporator to obtain 11 phosphatidylserine and a 9 ethyl acetate and n-hexane mixed solution, the 9 ethyl acetate and n-hexane mixed solution reaches a 2 ethyl acetate and n-hexane mixed solution storage tank while supplementing 16 phosphatidylcholine to continue the continuous reaction.

[0031] The preparation method is as follows:

[0032] 650 g of phosphatidylcholine (92%) was dissolved in an organic phase (the volume ratio of ethyl acetate to n-hexane was 1:3) to obtain a phosphatidylcholine solution. L-serine and calcium chloride were dissolved in an aqueous solution to obtain an L-serine solution. 2The concentration of phosphatidylcholine + was 15 mM, the pH of the aqueous solution was 5, the volume ratio of the organic phase to the aqueous solution was 1:4, and the molar ratio of phosphatidylcholine to L-serine was 1:4. The phosphatidylcholine solution and the L-serine solution were mixed to obtain an emulsion. The emulsion was introduced into a multi-stage continuous stirred tank reactor, where an ester exchange reaction occurred under the action of an immobilized enzyme to produce phosphatidylserine. The flow rate of the mixed solution was 1 mL / min, the residence time of the mixed solution in the multi-stage continuous stirred tank reactor was 5 hours, and the temperature of the ester exchange reaction was 42°C. The phosphatidylserine was spray-dried (inlet air temperature 160°C, outlet air temperature 70°C) to obtain PS powder. Phospholipase D (81 U / g) was immobilized after use.

[0033] Example 2

[0034] Preparation of immobilized enzyme: 600 g of silica, 400 g of γ-aminopropyltriethoxysilane, and 200 g of phospholipase D were added with water in a mass ratio of 1:2 to the total mass of silica, γ-aminopropyltriethoxysilane, and phospholipase D. The pH value was adjusted to 5.3, and the mixture was shaken at 0°C for 3 hours. 1500 g of chilled ethanol was then quickly added dropwise for precipitation. The precipitate was collected and cross-linked with glutaraldehyde in a mass ratio of 1:2 at 5°C for 4 hours. After washing, immobilized phospholipase D (90 U / g) was obtained.

[0035] use Figure 1 The apparatus in the preparation of phosphatidylserine.

[0036] The reaction process is as follows: 14 calcium chloride and 15 L-serine are added to an aqueous solution storage tank 1 to obtain an L-serine aqueous solution, 16 phosphatidylcholine is added to a 2 ethyl acetate and n-hexane mixed solution storage tank to obtain a phosphatidylcholine solution, the L-serine aqueous solution and the phosphatidylcholine solution enter a 4 pipeline shearing through a 3 pump, enter a 5 multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the catalysis of a 6 immobilized enzyme, and are separated by a 7 separator to obtain an aqueous phase and an organic phase, the aqueous phase (water, calcium chloride, L-serine) passes through a 3 pump to reach a 10 macroporous resin adsorption, enters a 12 aqueous solution storage tank while supplementing 13 L-serine and calcium chloride, and then passes through a 3 pump to reach the 1 aqueous solution storage tank to continue the continuous reaction, the organic phase passes through an 8 scraper evaporator to obtain 11 phosphatidylserine and a 9 ethyl acetate and n-hexane mixed solution, the 9 ethyl acetate and n-hexane mixed solution reaches a 2 ethyl acetate and n-hexane mixed solution storage tank while supplementing 16 phosphatidylcholine to continue the continuous reaction.

[0037] The preparation method is as follows:

[0038] 750 g of phosphatidylcholine (92%) was dissolved in an organic phase (the volume ratio of ethyl acetate to n-hexane was 1:2) to obtain a phosphatidylcholine solution. L-serine and calcium chloride were dissolved in an aqueous solution to obtain an L-serine solution. 2 The concentration of phosphatidylcholine + was 10 mM, the pH of the aqueous solution was 3.5, the volume ratio of the organic phase to the aqueous solution was 1:3, and the molar ratio of phosphatidylcholine to L-serine was 1:3. The phosphatidylcholine solution and the L-serine solution were mixed to form an emulsion. The emulsion was introduced into a multi-stage continuous stirred tank reactor, where an ester exchange reaction occurred under the action of an immobilized enzyme to produce phosphatidylserine. The flow rate of the mixed solution was 0.5 mL / min, the residence time of the mixed solution in the multi-stage continuous stirred tank reactor was 4 hours, and the temperature of the ester exchange reaction was 38°C. The phosphatidylserine was spray-dried (inlet air temperature 150°C, outlet air temperature 60°C) to obtain PS powder. Phospholipase D (83 U / g) was immobilized after use.

[0039] Example 3

[0040] Preparation of immobilized enzyme: 800 g of silica, 500 g of γ-aminopropyltriethoxysilane, and 200 g of phospholipase D were added with water in a mass ratio of 1:4 to the total mass of silica, γ-aminopropyltriethoxysilane, and phospholipase D. The pH value was adjusted to 5.4, and the mixture was shaken in a shaker at 0° C. for 5 hours. 2000 g of chilled ethanol was then rapidly added dropwise for precipitation. The precipitate was collected and cross-linked with glutaraldehyde in a mass ratio of 1:1.5 at 5° C. for 2 hours. After washing, immobilized phospholipase D (80 U / g) was obtained.

[0041] use Figure 1 The apparatus in the preparation of phosphatidylserine.

[0042] The reaction process is as follows: 14 calcium chloride and 15 L-serine are added to an aqueous solution storage tank 1 to obtain an L-serine aqueous solution, 16 phosphatidylcholine is added to a 2 ethyl acetate and n-hexane mixed solution storage tank to obtain a phosphatidylcholine solution, the L-serine aqueous solution and the phosphatidylcholine solution enter a 4 pipeline shearing through a 3 pump, enter a 5 multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the catalysis of a 6 immobilized enzyme, and are separated by a 7 separator to obtain an aqueous phase and an organic phase, the aqueous phase (water, calcium chloride, L-serine) passes through a 3 pump to reach a 10 macroporous resin adsorption, enters a 12 aqueous solution storage tank while supplementing 13 L-serine and calcium chloride, and then passes through a 3 pump to reach the 1 aqueous solution storage tank to continue the continuous reaction, the organic phase passes through an 8 scraper evaporator to obtain 11 phosphatidylserine and a 9 ethyl acetate and n-hexane mixed solution, the 9 ethyl acetate and n-hexane mixed solution reaches a 2 ethyl acetate and n-hexane mixed solution storage tank while supplementing 16 phosphatidylcholine to continue the continuous reaction.

[0043] The preparation method is as follows:

[0044] 800 g of phosphatidylcholine (92%) was dissolved in an organic phase (the volume ratio of ethyl acetate to n-hexane was 1:4) to obtain a phosphatidylcholine solution. L-serine and calcium chloride were dissolved in an aqueous solution to obtain an L-serine solution. 2 The concentration of phosphatidylcholine + was 20 mM, the pH of the aqueous solution was 6.5, the volume ratio of the organic phase to the aqueous solution was 1:5, and the molar ratio of phosphatidylcholine to L-serine was 1:5. The phosphatidylcholine solution and the L-serine solution were mixed to obtain an emulsion. The emulsion was introduced into a multi-stage continuous stirred tank reactor, where an ester exchange reaction occurred under the action of an immobilized enzyme to produce phosphatidylserine. The flow rate of the mixed solution was 100 mL / min, the residence time of the mixed solution in the multi-stage continuous stirred tank reactor was 6 hours, and the temperature of the ester exchange reaction was 45°C. The phosphatidylserine was spray-dried (inlet air temperature 170°C, outlet air temperature 80°C) to obtain PS powder. Phospholipase D (72 U / g) was immobilized after use.

[0045] Example 4

[0046] Preparation of immobilized enzyme: 600 g of silica gel, 400 g of γ-aminopropyltriethoxysilane, and 250 g of phospholipase D were mixed, water was added, and the mass ratio of water to the total mass of silica gel, γ-aminopropyltriethoxysilane, and phospholipase D was 1:5. The pH value was adjusted to 5.2, and the mixture was shaken in a shaker at 0°C for 2 hours. 3000 g of chilled ethanol was then quickly added dropwise for precipitation. The precipitate was collected and cross-linked with glutaraldehyde at a mass ratio of 1:2 at 2°C for 3 hours. After washing, immobilized phospholipase D (98 U / g) was obtained.

[0047] use Figure 1 The apparatus in the preparation of phosphatidylserine.

[0048] The reaction process is as follows: 14 calcium chloride and 15 L-serine are added to an aqueous solution storage tank 1 to obtain an L-serine aqueous solution, 16 phosphatidylcholine is added to a 2 ethyl acetate and n-hexane mixed solution storage tank to obtain a phosphatidylcholine solution, the L-serine aqueous solution and the phosphatidylcholine solution enter a 4 pipeline shearing through a 3 pump, enter a 5 multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the catalysis of a 6 immobilized enzyme, and are separated by a 7 separator to obtain an aqueous phase and an organic phase, the aqueous phase (water, calcium chloride, L-serine) passes through a 3 pump to reach a 10 macroporous resin adsorption, enters a 12 aqueous solution storage tank while supplementing 13 L-serine and calcium chloride, and then passes through a 3 pump to reach the 1 aqueous solution storage tank to continue the continuous reaction, the organic phase passes through an 8 scraper evaporator to obtain 11 phosphatidylserine and a 9 ethyl acetate and n-hexane mixed solution, the 9 ethyl acetate and n-hexane mixed solution reaches a 2 ethyl acetate and n-hexane mixed solution storage tank while supplementing 16 phosphatidylcholine to continue the continuous reaction.

[0049] The preparation method is as follows:

[0050] 800 g of phosphatidylcholine (92%) was dissolved in an organic phase (the volume ratio of ethyl acetate to n-hexane was 1:3) to obtain a phosphatidylcholine solution. L-serine and calcium chloride were dissolved in an aqueous solution to obtain an L-serine solution. 2 The concentration of phosphatidylcholine + was 15 mM, the pH of the aqueous solution was 5, the volume ratio of the organic phase to the aqueous solution was 1:4, and the molar ratio of phosphatidylcholine to L-serine was 1:4. The phosphatidylcholine solution and the L-serine solution were mixed to obtain an emulsion. The emulsion was introduced into a multi-stage continuous stirred tank reactor, where an ester exchange reaction occurred under the action of an immobilized enzyme to produce phosphatidylserine. The flow rate of the mixed solution was 1 mL / min, the residence time of the mixed solution in the multi-stage continuous stirred tank reactor was 5 hours, and the temperature of the ester exchange reaction was 42°C. The phosphatidylserine was spray-dried (inlet air temperature 160°C, outlet air temperature 70°C) to obtain PS powder. Phospholipase D (92 U / g) was immobilized after use.

[0051] Example 5

[0052] Preparation of immobilized enzyme: 500 g of silica gel, 500 g of γ-aminopropyltriethoxysilane, and 300 g of phospholipase D were mixed, water was added, and the mass ratio of water to the total mass of silica gel, γ-aminopropyltriethoxysilane, and phospholipase D was 1:4. The pH value was adjusted to 5.2, and the mixture was shaken in a shaker at 0°C for 3 hours. 4000 g of chilled ethanol was then rapidly added dropwise for precipitation. The precipitate was collected and cross-linked with glutaraldehyde at a mass ratio of 1:2 at 3°C for 2 hours. After washing, immobilized phospholipase D (110 U / g) was obtained.

[0053] use Figure 1The apparatus in the preparation of phosphatidylserine.

[0054] The reaction process is as follows: 14 calcium chloride and 15 L-serine are added to an aqueous solution storage tank 1 to obtain an L-serine aqueous solution, 16 phosphatidylcholine is added to a 2 ethyl acetate and n-hexane mixed solution storage tank to obtain a phosphatidylcholine solution, the L-serine aqueous solution and the phosphatidylcholine solution enter a 4 pipeline shearing through a 3 pump, enter a 5 multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the catalysis of a 6 immobilized enzyme, and are separated by a 7 separator to obtain an aqueous phase and an organic phase, the aqueous phase (water, calcium chloride, L-serine) passes through a 3 pump to reach a 10 macroporous resin adsorption, enters a 12 aqueous solution storage tank while supplementing 13 L-serine and calcium chloride, and then passes through a 3 pump to reach the 1 aqueous solution storage tank to continue the continuous reaction, the organic phase passes through an 8 scraper evaporator to obtain 11 phosphatidylserine and a 9 ethyl acetate and n-hexane mixed solution, the 9 ethyl acetate and n-hexane mixed solution reaches a 2 ethyl acetate and n-hexane mixed solution storage tank while supplementing 16 phosphatidylcholine to continue the continuous reaction.

[0055] The preparation method is as follows:

[0056] 950 g of phosphatidylcholine (92%) was dissolved in an organic phase (the volume ratio of ethyl acetate to n-hexane was 1:2) to obtain a phosphatidylcholine solution. L-serine and calcium chloride were dissolved in an aqueous solution to obtain an L-serine solution. 2 The concentration of phosphatidylcholine + was 10 mM, the pH of the aqueous solution was 3.5, the volume ratio of the organic phase to the aqueous solution was 1:3, and the molar ratio of phosphatidylcholine to L-serine was 1:3. The phosphatidylcholine solution and the L-serine solution were mixed to form an emulsion. The emulsion was introduced into a multi-stage continuous stirred tank reactor, where an ester exchange reaction occurred under the action of an immobilized enzyme to produce phosphatidylserine. The flow rate of the mixed solution was 0.5 mL / min, the residence time of the mixed solution in the multi-stage continuous stirred tank reactor was 4 hours, and the temperature of the ester exchange reaction was 38°C. The phosphatidylserine was spray-dried (inlet air temperature 150°C, outlet air temperature 60°C) to obtain PS powder. Phospholipase D (99 U / g) was immobilized after use.

[0057] Example 6

[0058] Preparation of immobilized enzyme: 1000 g of silica gel, 300 g of γ-aminopropyltriethoxysilane, and 200 g of phospholipase D were mixed, water was added, and the mass ratio of the water to the total mass of the silica gel, γ-aminopropyltriethoxysilane, and phospholipase D was 1:6. The pH value was adjusted to 5.4, and the mixture was shaken in a -5°C shaker for 2 hours. 2000 g of chilled ethanol was then rapidly added dropwise for precipitation. The precipitate was collected and cross-linked with glutaraldehyde in a mass ratio of 1:4 at 2°C for 4 hours. After washing, immobilized phospholipase D (93 U / g) was obtained.

[0059] use Figure 1 The apparatus in the preparation of phosphatidylserine.

[0060] The reaction process is as follows: 14 calcium chloride and 15 L-serine are added to an aqueous solution storage tank 1 to obtain an L-serine aqueous solution, 16 phosphatidylcholine is added to a 2 ethyl acetate and n-hexane mixed solution storage tank to obtain a phosphatidylcholine solution, the L-serine aqueous solution and the phosphatidylcholine solution enter a 4 pipeline shearing through a 3 pump, enter a 5 multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the catalysis of a 6 immobilized enzyme, and are separated by a 7 separator to obtain an aqueous phase and an organic phase, the aqueous phase (water, calcium chloride, L-serine) passes through a 3 pump to reach a 10 macroporous resin adsorption, enters a 12 aqueous solution storage tank while supplementing 13 L-serine and calcium chloride, and then passes through a 3 pump to reach the 1 aqueous solution storage tank to continue the continuous reaction, the organic phase passes through an 8 scraper evaporator to obtain 11 phosphatidylserine and a 9 ethyl acetate and n-hexane mixed solution, the 9 ethyl acetate and n-hexane mixed solution reaches a 2 ethyl acetate and n-hexane mixed solution storage tank while supplementing 16 phosphatidylcholine to continue the continuous reaction.

[0061] The preparation method is as follows:

[0062] 950 g of phosphatidylcholine (92%) was dissolved in an organic phase (the volume ratio of ethyl acetate to n-hexane was 1:4) to obtain a phosphatidylcholine solution. L-serine and calcium chloride were dissolved in an aqueous solution to obtain an L-serine solution. 2 The concentration of phosphatidylcholine + was 20 mM, the pH of the aqueous solution was 6.5, the volume ratio of the organic phase to the aqueous solution was 1:5, and the molar ratio of phosphatidylcholine to L-serine was 1:5. The phosphatidylcholine solution and the L-serine solution were mixed to obtain an emulsion. The emulsion was introduced into a multi-stage continuous stirred tank reactor, where an ester exchange reaction occurred under the action of an immobilized enzyme to produce phosphatidylserine. The flow rate of the mixed solution was 100 mL / min, the residence time of the mixed solution in the multi-stage continuous stirred tank reactor was 6 hours, and the temperature of the ester exchange reaction was 45°C. The phosphatidylserine was spray-dried (inlet air temperature 170°C, outlet air temperature 80°C) to obtain PS powder. Phospholipase D (84 U / g) was immobilized after use.

[0063] Comparative Example 1

[0064] To the reactor, 400 mL of acetic acid-sodium acetate buffer with a pH of 5 was added, followed by 126 g of phosphatidylcholine (PC purity 60%) and 105 g of L-serine (the molar ratio of which was selected to be 1:100), 10 g of calcium chloride, and 1600 ml of an organic phase (ethyl acetate and n-hexane in a volume ratio of 1:3). The temperature was raised to 45° C. After the substrate was completely dissolved, 30 g (6300 U) of phospholipase D solution was added. The reaction was continued for 5 hours at a speed of 250 rpm. After the reaction was completed, the conversion rate and purity of PS were detected.

[0065] Comparative Example 2

[0066] To the reactor, 400 mL of acetic acid-sodium acetate buffer with a pH of 5 was added, followed by 85 g of phosphatidylcholine (PC purity 90%) and 105 g of L-serine (molar ratio of 1:100), 10 g of calcium chloride, and 1600 ml of an organic phase (ethyl acetate and n-hexane volume ratio of 1:3). The temperature was raised to 45° C. After the substrate was completely dissolved, 60 g (12600 U) of phospholipase D solution was added. The reaction was continued for 8 hours at a speed of 250 rpm. After the reaction was completed, the conversion rate and purity of PS were detected.

[0067] Experimental Example 1

[0068] The conversion rate and purity of PS in Example 1 and Comparative Examples 1 and 2 were determined. Conversion rate = weight of phosphatidylserine / weight of phosphatidylcholine. Purity was determined by HPLC gradient elution followed by evaporative light flash detection.

[0069] Experimental results: as shown in Table 1.

[0070] Table 1 Yield, content and purity of PS with different treatments

[0071] PS conversion rate % PS purity % Example 1 95.3 92 Comparative Example 1 76 72 Comparative Example 2 30 84

[0072] As can be seen from Table 1, the PS conversion rate and purity of Example 1 are much higher than those of Comparative Examples 1 and 2. Comparative Examples 1 and 2 cannot achieve continuous reaction using conventional methods, and the corresponding PS conversion rate and purity are greatly reduced. The PS conversion rate of Comparative Example 2 is lower than that of Comparative Example 1. The reason is that the higher the PC purity, the more difficult it is to catalyze the conversion of phosphatidylcholine into phosphatidylserine, and the enzyme cleaves the phosphodiester bond to produce choline. The higher the PC content, the higher the free choline concentration and the greater the steric hindrance.

[0073] Experimental Example 2

[0074] Comparison of reaction time and enzyme usage times of the batch process of Example 1 and Comparative Example 2

[0075] Experimental results: as shown in Table 2.

[0076] Table 2 Comparison results of Example 1 and Comparative Example 2

[0077] Reaction time Number of times the enzyme is used Example 1 5h Multiple uses of immobilized enzymes Intermittent process of comparative example 2 8h Phospholipase D is added once for use

[0078] As shown in Table 2, the preparation method of the present invention reduces reaction time by over 30% compared to a batch process. In a batch process, the enzyme is added all at once and distributed throughout the aqueous phase. In contrast, the preparation method of Example 1 of the present invention positions the immobilized enzyme at the reaction interface of a multi-stage continuous stirred tank reactor for multiple recycling. This increases enzyme concentration compared to a batch process, and immobilization facilitates enzyme separation and reuse, significantly reducing enzyme cost and reaction time.

[0079] From the above examples, comparative examples and experimental examples, it can be seen that the method for preparing phosphatidylserine by continuous enzymatic reaction of the present invention can reduce the steric hindrance between the enzyme and the substrate, improve the catalytic efficiency, and greatly reduce the amount of enzyme used; it can catalyze high-purity soybean phosphatidylcholine and avoid the adverse effects of high-purity soybean phosphatidylcholine on the reaction; it can achieve efficient and high-purity continuous production of phosphatidylserine, and the reaction time is shortened to less than 6 hours.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing phosphatidylserine by continuous enzymatic reaction, characterized in that: The following steps are involved: 1) dissolving phosphatidylcholine in an organic phase to obtain a phosphatidylcholine solution, and dissolving L-serine in an aqueous solution to obtain an L-serine solution; 2) The phosphatidylcholine solution and the L-serine solution are mixed to obtain an emulsion, and the emulsion is introduced into a multi-stage continuous stirred tank reactor to undergo an ester exchange reaction under the action of an immobilized enzyme to obtain phosphatidylserine.

2. The method according to claim 1, characterized in that In step 1), the organic phase is ethyl acetate and n-hexane, and the volume ratio of ethyl acetate to n-hexane is 1:2-4.

3. The method according to claim 1, characterized in that Step 1) Ca in the aqueous solution 2 The concentration of + is 10-20 mM, and the pH of the aqueous solution is 3.5-6.

5.

4. The method according to claim 1, wherein In step 1), the volume ratio of the organic phase to the aqueous solution is 1:3-5.

5. The method according to claim 1, characterized in that In step 1), the molar ratio of phosphatidylcholine to L-serine is 1:3-5.

6. The method according to claim 1, characterized in that Step 2) The flow rate of the mixed solution is 0.5 to 100 mL / min, and the residence time of the mixed solution in the multi-stage continuous stirred tank reactor is 4 to 6 hours.

7. The method according to claim 1, characterized in that Step 2) The immobilized enzyme is phospholipase D covalently immobilized on amino-modified porous particles.

8. The method according to claim 1 or 7, characterized in that In step 2), the mass ratio of the immobilized enzyme to phosphatidylcholine is 1 to 10:

1.

9. The method according to claim 1, characterized in that The temperature of the transesterification reaction in step 2) is 38-45°C.

10. Phosphatidylserine prepared by the method according to any one of claims 1 to 9.

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