An enzymatic method for the preparation of sodium phosphatidylglycerol

Abstract

The application discloses an enzymatic preparation method of phosphatidylglycerol sodium, which comprises the following steps: A, phosphatidylcholine is subjected to transphosphatidylation with glycerol in a two-phase solvent under the catalysis of immobilized phospholipase D to obtain a crude phosphatidylglycerol reaction solution; B, the crude phosphatidylglycerol reaction solution is separated by using an organic solvent I to obtain separated immobilized phospholipase D and a remaining mixed suspension; C, an organic layer a is separated from the mixed suspension; D, the organic layer a is reacted with an inorganic base to generate a salt to obtain a crude phosphatidylglycerol sodium solution, and after preliminary refining, wet phosphatidylglycerol sodium is obtained; E, the wet phosphatidylglycerol sodium is refined to obtain finished phosphatidylglycerol sodium. Compared with the method reported in the prior art, the method has lower cost, is more efficient and is easy to be industrialized and produced on a large scale.

Description

Technical Field

[0001] This invention relates to the field of phospholipid synthesis technology, specifically to an enzymatic method for preparing sodium phosphatidylglycerol. Background Technology

[0002] Sodium phosphatidylglycerol, such as distearylphosphatidylglycerol sodium (DSPG-Na), is a type of compound used in the synthesis of phospholipids. Compared to natural phospholipids, it has a simpler composition, better thermal stability and antioxidant properties, and is widely used in various emulsions due to its excellent emulsifying properties. Currently, the preparation methods of sodium phosphatidylglycerol are mainly divided into two types: chemical methods and enzymatic methods. In chemical synthesis, the protection and deprotection of functional groups and the selection of phosphorylation reagents result in problems such as long routes, high pollution, and equipment corrosion, and are rarely used in actual industrial production. In enzymatic synthesis, using relatively inexpensive and readily available phosphatidylcholine (PC) as a raw material and phospholipase D (PLD) as a catalyst, it has the characteristics of short routes and high selectivity, and has good prospects for industrialization. In fact, the draft standard for pharmaceutical excipient sodium distearylphosphatidylglycerol also selects the enzymatic method as the source method for pharmaceutical DSPG-Na excipient.

[0003] However, due to its excellent emulsifying properties, sodium phosphatidylglycerol requires an aqueous-organic two-phase system during enzymatic preparation. As the reaction occurs, products are generated and precipitated, emulsification continuously occurs, turning the reaction system into an emulsion, making product separation and immobilized enzyme recovery difficult. To reduce the degree of emulsification and facilitate post-processing, current literature reports generally use large amounts of solvent during the reaction. However, this results in lower reactant concentrations, necessitating a significant increase in glycerol to shift the reaction equilibrium to the right to ensure yield. When preparing phosphatidylglycerol compounds using the methods described in the master's thesis "Development of Phosphatidyl Group Transfer Synthesis of Phosphatidylglycerol by Immobilized Phosphatase D" (Northwest University, 2012) or the journal article "Phosphatidylhydroxyalkanols as Versatile Intermediates in the Synthesis of Headgroup Modified Diacetylenic Phospholipids" (Synthetic Communications, 1992, 22(16): 2293-2304), the amounts of solvent and glycerol used during the reaction are relatively large. In addition, there is the problem of low purity when column chromatography is not used for separation and purification. However, when column chromatography is used for separation and purification, the adsorption effect of the column chromatography packing material leads to low yield, large solvent consumption during column chromatography, and repeated dissolution, heating and concentration operations. These are not conducive to economical, efficient and green large-scale production in industrialization. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide an enzymatic method for the preparation of sodium phosphatidylglycerol suitable for industrial application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] An enzymatic method for preparing sodium phosphatidylglycerol includes the following steps:

[0007] A. Phosphatidylcholine reacts with glycerol in a two-phase solvent via immobilized phospholipase D catalysis to undergo a transphosphatidyl reaction, yielding crude phosphatidylglycerol reaction solution.

[0008] B. Using organic solvent I, the crude phosphatidylglycerol reaction solution is separated to obtain the immobilized phosphatase D and the remaining mixed suspension.

[0009] C. The organic layer a is separated from the mixed suspension;

[0010] D. The organic layer a is added to a sodium-containing inorganic base to undergo a salt-forming reaction, resulting in a crude sodium phosphatidylglycerol solution, which is then preliminarily purified to obtain a wet sodium phosphatidylglycerol product.

[0011] E. The wet sodium phosphatidylglycerol product is purified to obtain the finished sodium phosphatidylglycerol product.

[0012] The enzymatic preparation method for the above-mentioned sodium phosphatidylglycerol is shown in the flowchart below. Figure 1 As shown, Figure 1 Step (1) indicates that under the catalysis of PLD, phosphatidylcholine reacts with glycerol to undergo a transphosphatidyl reaction to obtain phosphatidylglycerol, and step (2) indicates that phosphatidylglycerol reacts with a sodium-containing inorganic base to form a salt to obtain sodium phosphatidylglycerol.

[0013] In a preferred embodiment of the preparation method of the present invention, in step B, the organic solvent I includes one or more of methanol, ethanol, acetonitrile, and isopropanol; the enzymatic preparation method of sodium phosphatidylglycerol further includes the following steps:

[0014] F. The separated immobilized phospholipase D can be reused after washing with detergent.

[0015] As a preferred embodiment of the preparation method of the present invention, in step A, the two-phase solvent includes an organic phase and an aqueous phase. The operation of the phosphatidyl-transfer reaction includes: adding the organic phase to phosphatidylcholine and stirring until completely dissolved, then adding the glycerol, the aqueous phase, and the immobilized phosphatidyl lipase D, and continuing to stir to react, thereby obtaining the crude phosphatidylglycerol reaction solution; in step B, the operation of separating the immobilized phosphatidyl lipase D includes: adding the organic solvent I to the crude phosphatidylglycerol reaction solution, stirring evenly, and placing it in a separatory device, waiting for the immobilized phosphatidyl lipase D to sink to the bottom, and then releasing the separated immobilized phosphatidyl lipase D from the bottom of the separatory device; in step C, the operation of separating the organic layer a includes: leaving the organic layer a in the separatory device. Organic solvent II is added to the remaining mixed suspension, and the aqueous layer and organic layer are separated by standing to obtain the organic layer a; in step D, the salt formation reaction includes: adding water and sodium-containing inorganic base to the organic layer a and stirring to neutralize it, and obtaining the crude sodium phosphatidylglycerol solution after the reaction is completed; the preliminary purification includes: pouring the crude sodium phosphatidylglycerol solution into a separatory device, standing, separating, releasing the lower organic layer b, and concentrating to obtain the wet sodium phosphatidylglycerol; in step E, the purification includes: heating and dissolving the wet sodium phosphatidylglycerol in a mixed solvent, adding organic solvent III and cooling and stirring to crystallize, filtering to obtain the filter residue after complete crystallization, and vacuum drying the filter residue to obtain the finished sodium phosphatidylglycerol.

[0016] Preferably, in step A, the organic phase includes one or more of ethyl acetate, chloroform, dichloromethane, diethyl ether, and n-hexane; the aqueous phase includes a buffer system and an aqueous solution of a metal chloride, wherein the buffer system includes one or more of a citric acid-sodium citrate buffer system, an acetic acid-sodium acetate buffer system, and a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system, and the metal chloride includes one or more of calcium chloride, sodium chloride, ferric chloride, and ferrous chloride; in step C, the organic solvent II includes one or two of chloroform and dichloromethane; in step D, the sodium-containing inorganic base includes one or more of sodium carbonate, sodium bicarbonate, sodium acetate, sodium citrate, sodium phosphate, sodium methoxide, sodium ethoxide, sodium hydroxide, and sodium tartrate; in step E, the mixed solvent includes a mixture of a halogenated hydrocarbon or alkane and a lower alcohol and water, wherein the halogenated hydrocarbon or alkane includes dichloromethane, chloroform, and C5-C6 hydrocarbons. 10 Alkanes, C5-C 10 One or more of the cycloalkanes, wherein the lower alcohols include one or more of the C1-C4 alcohols, and wherein the organic solvent III includes one or more of the C1-C4 alcohols, acetone, and acetonitrile.

[0017] Preferably, in step A, the mass-to-volume ratio of phosphatidylcholine to the organic phase in the two-phase solvent is 1 g: 1-10 mL, and the volume ratio of the organic phase to the aqueous phase in the two-phase solvent is 0.5-3:1; the molar ratio of phosphatidylcholine to glycerol is 1:10-80; the millimolecular / activity ratio of phosphatidylcholine to immobilized phospholipase D is 1 mmol: 0.2-20 U; and the molar ratio of phosphatidylcholine to metal chloride is 1:0.1-0.5. In step B, the volume-to-mass ratio of the added organic solvent I to the phosphatidylcholine is 2-15 mL: 1 g. In step C, the volume-to-mass ratio of the organic solvent II to the phosphatidylcholine is 1-15 mL: 1 g. In the salt-forming reaction of step D, the molar ratio of phosphatidylcholine to the sodium-containing inorganic base is 1:0.8-2, and the volume-to-mass ratio of water to the phosphatidylcholine is 0.1-4 mL: 1 g.

[0018] Preferably, in step A, the phosphatidyl reaction is carried out in a water bath at 25–40°C for 6–10 h, and the metal chloride includes calcium chloride; in step D, the salt formation reaction is carried out at room temperature for 0.5–2 h, and in the preliminary purification, the concentration is carried out until no condensate drips.

[0019] Preferably, in step E, the heating and dissolving are carried out in a water bath at 40-50°C, and the cooling and stirring crystallization are carried out at room temperature with a stirring speed of 20-30 rpm and a crystallization time of 4-5 h; in step E, the vacuum drying is carried out in a vacuum drying oven with a drying temperature of 40-80°C and a drying time of 6-8 h.

[0020] Preferably, in step A, the mass-to-volume ratio of phosphatidylcholine to the organic phase in the two-phase solvent is 1 g: 4-6 mL, and the molar ratio of phosphatidylcholine to glycerol is 1: 30-60.

[0021] As a preferred embodiment of the preparation method of the present invention, the detergent in step F includes one or more of methanol, n-hexane, and purified water.

[0022] Preferably, in step E, the volume ratio of haloalkanes or alkanes to lower alcohols and water in the mixed solvent is (4-16):(4-16):1.

[0023] Preferably, in step E, the organic solvent III includes one or more of C1-C4 alcohols, acetone, and acetonitrile.

[0024] The present invention also provides a method for preparing sodium phosphatidylglycerol by an enzymatic method as described above, wherein the purity of the finished sodium phosphatidylglycerol is greater than 99%.

[0025] The preparation method of this invention reduces the amount of reaction solvent and glycerol used from scratch, improving reaction efficiency and yield. It also provides a solution to the problem of post-reaction product emulsification, and further avoids column purification methods using large amounts of solvent through recrystallization. In addition, this method can reliably achieve the reuse of immobilized PLDs in large-scale production, saving product costs. Compared with methods reported in existing literature, this method is lower in cost, more efficient, and easier to scale up for industrial production.

[0026] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0027] 1. This invention solves the problem of excessive use of reaction solvent and glycerol, which is common in current literature reports. It also solves the problem of difficulty in recovering immobilized enzymes and post-processing purification due to emulsification after the reaction, thus filling a technological gap in the field.

[0028] 2. In the post-processing of the preparation method of the present invention, by continuously adjusting the solvent ratio of the system, immobilized phospholipase D, the reaction aqueous layer, and the saline layer are separated respectively while avoiding re-emulsification. Finally, sodium phosphatidylglycerol with a purity of up to 99% can be obtained through purification.

[0029] 3. The preparation method of the present invention further avoids the column chromatography separation operation that uses a large amount of solvent by recrystallization. By reducing the solvent, the reaction efficiency and purity are improved, and a large amount of waste liquid and waste residue are also avoided, making the entire preparation method greener, more economical and efficient, and more suitable for industrial mass production.

[0030] 4. This invention can also reliably achieve the reuse of immobilized phospholipase D during mass production, thereby saving costs to a great extent. Attached Figure Description

[0031] Figure 1 This is a flowchart illustrating the preparation process of sodium phosphatidylglycerol in this invention.

[0032] Figure 2 The HPLC-ELSD chromatogram results of the sodium phosphatidylglycerol product in Example 1 are shown below.

[0033] Figure 3 The HPLC-ELSD chromatogram results of the sodium phosphatidylglycerol product in Example 2 are shown below.

[0034] Figure 4 The HPLC-ELSD chromatogram of sodium phosphatidylglycerol product in Example 3 is shown. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the reagents and materials used in the following embodiments and comparative examples are all conventional commercially available products.

[0036] The immobilized phospholipase D (PLD) used in the following examples and comparative examples was immobilized using conventional adsorption techniques. PLDs obtained through other conventional immobilization methods are also applicable to this invention.

[0037] Example 1

[0038] Weigh 100g dipalmitoylphosphatidylcholine (DPPC) into a 2L round-bottom flask, add 500ml dichloromethane, and stir until completely dissolved in a 30℃ water bath. Then add 500ml purified water, 600g glycerol, 3.77g anhydrous calcium chloride, 525ul acetic acid, 5.08g sodium acetate, and 90U immobilized phospholipase D (PLD) in sequence. Continue stirring in a 30℃ water bath for 8 hours, then stop the reaction. The reaction system becomes porridge-like.

[0039] Add 1000 ml of methanol to the reaction system, stir well, and place in a separatory funnel. Once the immobilized PLD has settled to the bottom, release the immobilized PLD from the bottom of the funnel. After washing with methanol, n-hexane, and purified water, it can be reused. Add 1000 ml of dichloromethane to the remaining mixed suspension in the funnel, mix well, let stand, and then separate the contents.

[0040] The lower layer of mixed organic suspension was released and placed in a beaker. 100 ml of water and 14.43 g of sodium carbonate were added, and the mixture was stirred at room temperature for 1 hour to neutralize. During the stirring process, the lower organic layer gradually became clear. After the reaction was completed, the neutralized solution was poured into a separatory funnel, allowed to stand, and then separated.

[0041] The lower organic layer was released and concentrated until no condensation dripped. 500 ml of a mixed solvent (chloroform:methanol:water = 60:25:4) was added in a 50°C water bath and stirred until dissolved. After complete dissolution, 400 ml of methanol was added, and the mixture was cooled to room temperature and stirred at 20 rpm for 4 hours to crystallize. After complete crystallization, the residue was filtered and dried in a vacuum drying oven at 60°C for 6 hours to obtain 90.20 g of a white solid, which was the finished product, sodium dipalmitoylphosphatidylglycerol (DPPG-Na), with a yield of 90.2% and a purity of 100.0%. HPLC-ELSD results are as follows. Figure 2 As shown.

[0042] Example 2

[0043] Weigh 100g of dishuranophosphatidylcholine (DEPC) into a 2L round-bottom flask, add 400ml of ethyl acetate, and stir to dissolve in a 40℃ water bath. Then add 500ml of purified water, 400g of glycerol, 5.6g of ferric chloride, 500ul of acetic acid, 5.0g of sodium acetate, and 60U of immobilized PLD in sequence. Continue stirring in a 40℃ water bath for 10 hours, then stop the reaction. The reaction system becomes porridge-like.

[0044] Add 500 ml of acetonitrile to the reaction system, stir well, and place in a separatory funnel. Once the immobilized PLD has settled to the bottom, release the immobilized PLD from the bottom of the funnel. After washing with methanol, n-hexane, and purified water, it can be reused. Add 1000 ml of chloroform to the remaining mixed suspension in the funnel, mix well, let stand, and then separate the contents.

[0045] The lower layer of mixed organic suspension was released and placed in a beaker. 100 ml of water and 11.50 g of anhydrous sodium citrate were added, and the mixture was stirred at room temperature for 2 hours to neutralize. During the stirring process, the lower organic layer gradually became clear. After the reaction was completed, the neutralized solution was poured into a separatory funnel, allowed to stand, and then separated.

[0046] The lower organic layer was released and concentrated until no condensation dripped. 500 ml of a mixed solvent (chloroform:methanol:water = 60:60:6) was added in a 40°C water bath and stirred until dissolved. After complete dissolution, 400 ml of methanol was added, and the mixture was cooled to room temperature and stirred at 20 rpm for 4 hours to crystallize. After complete crystallization, the residue was filtered and dried in a vacuum drying oven at 40°C for 7 hours to obtain 93.11 g of a white solid, which was the finished product, sodium diergoside phosphatidylglycerol (DEPG-Na), with a yield of 93.1% and a purity of 99.65%. HPLC-ELSD results are as follows. Figure 3 As shown.

[0047] Example 3

[0048] Weigh 100g of distearate phosphatidylcholine (DSPC) into a 2L round-bottom flask, add 400ml of chloroform, and stir to dissolve in a 40℃ water bath. Then add 500ml of purified water, 400g of glycerol, 5.6g of ferric chloride, 500ul of acetic acid, 5.0g of sodium acetate, and 60U of immobilized PLD in sequence. Continue stirring and reacting in a 40℃ water bath for 10 hours, then stop the reaction. The reaction system becomes porridge-like.

[0049] Add 500 ml of methanol to the reaction system, stir well, and place in a separatory funnel. Once the immobilized PLD has settled to the bottom, release the immobilized PLD from the bottom of the funnel. After washing with methanol, n-hexane, and purified water, it can be reused. Add 1000 ml of chloroform to the remaining mixed suspension in the funnel, mix well, let stand, and then separate the contents.

[0050] The lower layer of mixed organic suspension was released and placed in a beaker. 100 ml of water and 11.55 g of sodium bicarbonate were added, and the mixture was stirred at room temperature for 2 hours to neutralize. During the stirring process, the lower organic layer gradually became clear. After the reaction was completed, the neutralized solution was poured into a separatory funnel, allowed to stand, and then separated.

[0051] The lower organic layer was released and concentrated until no condensation dripped. 500 ml of a mixed solvent (chloroform:methanol:water = 60:60:6) was added in a 40°C water bath and stirred until dissolved. After complete dissolution, 400 ml of methanol was added, and the mixture was cooled to room temperature and stirred at 20 rpm for 4 hours to crystallize. After complete crystallization, the residue was filtered and dried in a vacuum drying oven at 40°C for 8 hours to obtain 90.15 g of a white solid, which was the finished product, sodium distearate phosphatidylglycerol (DSPG-Na), with a yield of 90.2% and a purity of 99.72%. HPLC-ELSD results are as follows. Figure 4 As shown.

[0052] Example 4

[0053] Weigh 100g of dilauroyl phosphatidylcholine (DLPC) into a 2L round-bottom flask, add 400ml of dichloromethane and 200ml of chloroform, and stir in a 35℃ water bath until completely dissolved. Then add 500ml of purified water, 350g of glycerol, 2.0g of sodium chloride, 530ul of acetic acid, 5.10g of sodium acetate, and 30U of immobilized PLD in sequence. Continue stirring in a 35℃ water bath for 6 hours, then stop the reaction. The reaction system becomes porridge-like.

[0054] Add 1200 ml of ethanol to the reaction system, stir well, and place in a separatory funnel. After the immobilized PLD settles to the bottom, release the immobilized PLD from the bottom of the funnel. After washing with methanol, n-hexane, and purified water, it can be reused. Add 1000 ml of chloroform to the remaining mixed suspension in the funnel, mix well, let stand, and then separate the contents.

[0055] The lower layer of mixed organic suspension was released and placed in a beaker. 200 ml of water and 5.5 g of sodium hydroxide were added, and the mixture was stirred at room temperature for 0.5 h to neutralize. During the stirring process, the lower organic layer gradually became clear. After the reaction was completed, the neutralized solution was poured into a separatory funnel, allowed to stand, and then separated.

[0056] The lower organic layer was released and concentrated until no condensate dripped. 500 ml of a mixed solvent (chloroform:methanol:water = 45:25:3) was added in a 40°C water bath and stirred to dissolve. After complete dissolution, 400 ml of methanol was added and the mixture was cooled to room temperature and stirred at 30 rpm for 5 hours to crystallize. After complete crystallization, the residue was filtered and dried in a vacuum drying oven at 60°C for 6 hours to obtain 88.10 g of a white solid, which was the finished product, sodium dilauroyl phosphatidylglycerol (DLPG-Na), with a yield of 88.1% and a purity of 99.83%.

[0057] Example 5

[0058] Weigh 100g dipalmitoylphosphatidylcholine (DPPC) into a 2L round-bottom flask, add 500ml dichloromethane, and stir until completely dissolved in a 30℃ water bath. Then add 500ml purified water, 600g glycerol, 3.77g anhydrous calcium chloride, 525ul acetic acid, 5.08g sodium acetate, and 90U immobilized PLD in sequence. Continue stirring in a 30℃ water bath for 8 hours, then stop the reaction. The reaction system becomes porridge-like.

[0059] Add 1500 ml of methanol to the reaction system, stir well, and place in a separatory funnel. Once the immobilized PLD has settled to the bottom, release the immobilized PLD from the bottom of the funnel. After washing with methanol, n-hexane, and purified water, it can be reused. Add 1000 ml of dichloromethane to the remaining mixed suspension in the funnel, mix well, let stand, and then separate the contents.

[0060] The lower layer of mixed organic suspension was released and placed in a beaker. 100 ml of water and 14.43 g of sodium carbonate were added, and the mixture was stirred at room temperature for 1 hour to neutralize. During the stirring process, the lower organic layer gradually became clear. After the reaction was completed, the neutralized solution was poured into a separatory funnel, allowed to stand, and then separated.

[0061] The lower organic layer was released and concentrated until no condensate dripped. 500 ml of a mixed solvent (chloroform:methanol:water = 60:25:4) was added in a 50°C water bath and stirred to dissolve. After complete dissolution, 200 ml of methanol was added and the mixture was cooled to room temperature and stirred at 20 rpm for 4 hours to crystallize. After complete crystallization, the residue was filtered and dried in a vacuum drying oven at 60°C for 6 hours to obtain 86.23 g of a white solid, which was the finished product, sodium dipalmitoylphosphatidylglycerol (DPPG-Na), with a yield of 86.2% and a purity of 100.0%.

[0062] Comparative Example 1

[0063] Weigh 10g dipalmitoylphosphatidylcholine (DPPC) into a 1L round-bottom flask, add 330ml of diethyl ether, and stir until completely dissolved in a 30℃ water bath. Then add 500ml purified water, 120g glycerol, 3.77g anhydrous calcium chloride, 525ul acetic acid, 5.08g sodium acetate, and 9U immobilized phospholipase D (PLD) in sequence. Continue stirring in a 30℃ water bath for 8 hours, then stop the reaction. The reaction system separates into two layers, and the reaction product is partially emulsified.

[0064] The reaction solution was allowed to stand and separated, retaining the upper organic layer. 70 ml of methanol was added to the organic layer, followed by the dissolution of 1.4 g of sodium carbonate in 10 ml of water, which was then added to the organic layer. The mixture was then stirred at room temperature for 1 hour to neutralize. After neutralization, the mixture was allowed to stand again and separated, retaining the upper organic layer.

[0065] The organic layer was concentrated at 45°C and repeatedly evaporated to dryness with ethanol (otherwise it would affect the column chromatography separation effect). It was then completely dissolved in 50 ml of mixed solvent (chloroform:methanol:water = 65:25:4). The column was packed with 300 g silica gel by dry packing and the sample was loaded to start column chromatography. First, it was eluted with a chloroform:methanol mixed solvent of 60:30, and then with a chloroform:methanol mixed solvent of 30:30. The fraction was monitored by TLC thin layer chromatography. The fraction of pure DPPG target compound was collected and concentrated in a round bottom flask. In the end, a total of 3300 ml of chloroform and 2600 ml of methanol were used.

[0066] The pure DPPG sample in the round-bottom flask was transferred to a petri dish and dried in a vacuum drying oven at 45°C to obtain 6.23 g.

[0067] Table 1 shows a comparison of solvent usage and effects between the preparation routes of Comparative Example 1 and Example 1.

[0068] Table 1 Comparison of the effects of different preparation routes

[0069]

[0070] As shown in Table 1, even with a higher phosphatidylcholine feed amount in Example 1 compared to Comparative Example 1, the solvent usage in Example 1 (3.4 L of organic solvent for 100 g PC) was significantly less than that in Comparative Example 1 (equivalent to 63 L of organic solvent for 100 g PC). However, the yield of Example 1 (90.2%) was much higher than that of Comparative Example 1 (62.3%), and the purity of the product in Example 1 was closer to 100%. Therefore, compared to Comparative Example 1, the preparation route and method of Example 1 avoided the problem of using a large amount of solvent in column chromatography and solved the emulsification problem, making the reaction more efficient.

[0071] Comparative Example 2

[0072] Weigh 100g of distearate phosphatidylcholine (DSPC) into a 2L round-bottom flask, add 400ml of chloroform, and stir to dissolve in a 40℃ water bath. Then add 500ml of purified water, 400g of glycerol, 5.6g of ferric chloride, 500ul of acetic acid, 5.0g of sodium acetate, and 60U of immobilized PLD in sequence. Continue stirring and reacting in a 40℃ water bath for 10 hours, then stop the reaction. The reaction system becomes porridge-like.

[0073] Add 500 ml of methanol to the reaction system, stir well, and place in a separatory funnel. Once the immobilized PLD has settled to the bottom, release the immobilized PLD from the bottom of the funnel. After washing with methanol, n-hexane, and purified water, it can be reused. Add 1000 ml of chloroform to the remaining mixed suspension in the funnel, mix well, let stand, and then separate the contents.

[0074] The lower layer of mixed organic suspension was released and placed in a beaker. 500 ml of water and 11.55 g of sodium bicarbonate were added, and the mixture was stirred and neutralized at room temperature for 2 hours. After the reaction was completed, the neutralized solution was poured into a separatory funnel and observed after standing. It was found that a small part of the bottom organic layer could be clarified, but most of the emulsion appeared in the middle and a small amount of water layer appeared on the top, which was not conducive to the post-processing of the product.

[0075] Comparative Example 3

[0076] Weigh 100g of distearate phosphatidylcholine (DSPC) into a 2L round-bottom flask, add 400ml of chloroform, and stir to dissolve in a 40℃ water bath. Then add 500ml of purified water, 400g of glycerol, 5.6g of ferric chloride, 500ul of acetic acid, 5.0g of sodium acetate, and 60U of immobilized PLD in sequence. Continue stirring and reacting in a 40℃ water bath for 10 hours, then stop the reaction. The reaction system becomes porridge-like.

[0077] Adding 500 ml of methanol and 1000 ml of chloroform to the reaction system at once, stirring thoroughly, and then allowing it to stand in a separatory funnel for observation revealed that a small portion of the lower organic layer was clear, but the middle layer consisted mostly of emulsion containing a large amount of immobilized enzyme, with a small amount of water layer at the top. The product state did not allow for separation of the product and immobilized enzyme, nor for post-processing.

[0078] Table 2 shows the differences in preparation steps and effects between Comparative Examples 2 and 3 and Example 1.

[0079] Table 2 Comparison of preparation steps

[0080]

[0081]

[0082] As shown in Table 2, in Comparative Example 3, after the conversion to phosphatidyl, the product was directly extracted without first separating the immobilized PLD. The lower layer contained only a small amount of organic layer and the top layer contained only a small amount of water layer, while the middle emulsion contained a large amount of PLD. Therefore, it was impossible to separate the product from the immobilized enzyme and to perform post-processing of the product. In Example 3, the product prepared by the method of the present invention can be separated from the immobilized PLD after the conversion to phosphatidyl, which facilitates post-processing. Therefore, the yield and purity of the final product obtained are high.

[0083] As shown in Table 2, excessive water usage in the salt formation reaction will cause re-emulsification: In Comparative Example 2, the volume-to-mass ratio of water to phosphatidylcholine in the salt formation reaction was 5 mL: 1 g. After salt formation, there was only a small amount of organic layer at the bottom and only a small amount of water layer at the top, with emulsions appearing in the middle, which is not conducive to post-processing of the product; In Example 3, when the volume-to-mass ratio of water to phosphatidylcholine in the salt formation reaction was within the range of 0.1 to 4 mL: 1 g, the separation effect after salt formation was good.

[0084] Therefore, it is shown that the preparation method of the present invention solves the problem of difficulty in recovering immobilized enzymes and post-processing purification of products due to emulsification or re-emulsification after the transphosphatidyl reaction, and can also realize the reuse of immobilized phosphatase D.

[0085] Experiment 1: Solvent Screening Experiment

[0086] The phosphatidyl-converting reaction was carried out according to the feeding ratio of Example 3. After 10 hours of reaction, the reaction system was in the form of porridge. The porridge-like reaction system was divided into 10 equal portions (each portion was obtained by reacting 10g of distearylphosphatidylcholine), each portion being 150ml. The effects of adding different reagents on the separating enzyme and subsequent treatment were investigated, and the results are shown in Table 3 below.

[0087] Table 3. Analysis of Major Solvents

[0088]

[0089]

[0090] As shown in Table 3, common transacylation reactions reported in the literature usually result in the enzyme being located between two phases, requiring multiple treatments for separation and washing. This invention, by studying specific types and proportions of solvents, solves the reaction emulsification problem while allowing the enzyme to precipitate from the reaction mixture to the bottom without affecting subsequent processing, making it suitable for industrial production.

[0091] Example 1: Experiment on the reuse of recycled enzymes

[0092] 100g of dipalmitoylphosphatidylcholine was weighed into a 2L round-bottom flask, 500ml of dichloromethane was added, and the mixture was stirred and dissolved completely in a 30℃ water bath. Then, 500ml of purified water, 600g of glycerol, 3.77g of anhydrous calcium chloride, 525ul of acetic acid, 5.08g of sodium acetate, and the immobilized PLD recovered in Example 1 were added in sequence. The reaction was continued to be stirred in a 30℃ water bath for 8 hours. Thin-layer chromatography was performed on the sample, and the reaction results were consistent with those in Example 1. The results showed that the recovered PLD enzyme was basically the same as that used for the first time.

[0093] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An enzymatic method for preparing sodium phosphatidylglycerol, characterized in that, Includes the following steps: A. Phosphatidylcholine reacts with glycerol in a two-phase solvent via immobilized phospholipase D catalysis to undergo a transphosphatidyl reaction, yielding crude phosphatidylglycerol reaction solution. B. Using organic solvent I, the crude phosphatidylglycerol reaction solution is separated to obtain the immobilized phosphatase D and the remaining mixed suspension. C. The organic layer a is separated from the mixed suspension; D. The organic layer a is added to a sodium-containing inorganic base to undergo a salt-forming reaction, resulting in a crude sodium phosphatidylglycerol solution, which is then preliminarily purified to obtain a wet sodium phosphatidylglycerol product. E. The wet sodium phosphatidylglycerol product is purified to obtain the finished sodium phosphatidylglycerol product.

2. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 1, characterized in that, In step B, the organic solvent I includes one or more of methanol, ethanol, acetonitrile, and isopropanol; the enzymatic preparation method of sodium phosphatidylglycerol further includes the following steps: F. The separated immobilized phospholipase D can be reused after washing with detergent.

3. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 1, characterized in that, In step A, the two-phase solvent includes an organic phase and an aqueous phase. The operation of the phosphatidyl-transfer reaction includes: adding the organic phase to phosphatidylcholine and stirring until completely dissolved, then adding the glycerol, the aqueous phase, and the immobilized phosphatidyllipase D, and continuing to stir to react, thereby obtaining the crude phosphatidylglycerol reaction solution; In step B, the operation of separating the immobilized phosphatidyllipase D includes: adding the organic solvent I to the crude phosphatidylglycerol reaction solution, stirring evenly, and placing it in a separatory device, waiting for the immobilized phosphatidyllipase D to sink to the bottom, and then releasing the separated immobilized phosphatidyllipase D from the bottom of the separatory device; In step C, the operation of separating the organic layer a includes: the remaining mixed suspension in the separatory device In step D, organic solvent II is added, and the mixture is allowed to stand to separate the aqueous and organic layers, resulting in organic layer a. The salt-forming reaction in step D includes adding water and a sodium-containing inorganic base to organic layer a, followed by stirring and neutralization. After the reaction is complete, a crude sodium phosphatidylglycerol solution is obtained. The preliminary purification process includes pouring the crude sodium phosphatidylglycerol solution into a separatory apparatus, allowing it to stand, separating the layers, releasing the lower organic layer b, and concentrating to obtain a wet sodium phosphatidylglycerol product. In step E, the purification process includes heating and dissolving the wet sodium phosphatidylglycerol product in a mixed solvent. After complete dissolution, organic solvent III is added, and the mixture is cooled and stirred to crystallize. After complete crystallization, the residue is filtered, and the residue is vacuum-dried to obtain the finished sodium phosphatidylglycerol product.

4. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 3, characterized in that, In step A, the organic phase includes one or more of ethyl acetate, chloroform, dichloromethane, diethyl ether, and n-hexane; the aqueous phase includes a buffer system and an aqueous solution of a metal chloride, wherein the buffer system includes one or more of a citrate-sodium citrate buffer system, an acetate-sodium acetate buffer system, and a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system, and the metal chloride includes one or more of calcium chloride, sodium chloride, ferric chloride, and ferrous chloride; in step C, the organic solvent II includes one or two of chloroform and dichloromethane; in step D, the sodium-containing inorganic base includes one or more of sodium carbonate, sodium bicarbonate, sodium acetate, sodium citrate, sodium phosphate, sodium methoxide, sodium ethoxide, sodium hydroxide, and sodium tartrate; in step E, the mixed solvent includes a mixture of a halogenated hydrocarbon or alkane and a lower alcohol and water, wherein the halogenated hydrocarbon or alkane includes dichloromethane, chloroform, and C5-C6 hydrocarbons. 10 Alkanes, C5-C 10 The lower alcohols include one or more of the cycloalkanes, and the organic solvent III includes one or more of the C1-C4 alcohols, acetone, and acetonitrile.

5. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 3, characterized in that, In step A, the mass-to-volume ratio of phosphatidylcholine to the organic phase in the two-phase solvent is 1 g: 1–10 mL, and the volume ratio of the organic phase to the aqueous phase in the two-phase solvent is 0.5–3:1; the molar ratio of phosphatidylcholine to glycerol is 1:10–80; the millimolecular / activity ratio of phosphatidylcholine to immobilized phospholipase D is 1 mmol: 0.2–20 U; the molar ratio of phosphatidylcholine to metal chloride is 1:0.1–0.5; in step B, the volume-to-mass ratio of the added organic solvent I to the phosphatidylcholine is 2–15 mL: 1 g; in step C, the volume-to-mass ratio of the organic solvent II to the phosphatidylcholine is 1–15 mL: 1 g; in the salt-forming reaction of step D, the molar ratio of phosphatidylcholine to the sodium-containing inorganic base is 1:0.8–2, and the volume-to-mass ratio of water to the phosphatidylcholine is 0.1–4 mL: 1 g.

6. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 3, characterized in that, In step A, the phosphatidyl reaction is carried out in a water bath at 25–40°C for 6–10 h, and the metal chloride includes calcium chloride; in step D, the salt formation reaction is carried out at room temperature for 0.5–2 h.

7. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 3, characterized in that, In step E, the heating and dissolving are carried out in a water bath at 40-50°C, and the cooling and stirring crystallization are carried out at room temperature with a stirring speed of 20-30 rpm and a crystallization time of 4-5 h. In step E, the vacuum drying is carried out in a vacuum drying oven at a drying temperature of 40-80°C and a drying time of 6-8 h.

8. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 5, characterized in that, In step A, the mass-to-volume ratio of phosphatidylcholine to the organic phase in the two-phase solvent is 1 g: 4–6. m L The molar ratio of phosphatidylcholine to glycerol is 1:30 to 60.

9. The enzymatic preparation method of sodium phosphatidylglycerol according to claim 2, characterized in that, The detergent mentioned in step F includes one or more of methanol, n-hexane, and purified water.

10. A sodium phosphatidylglycerol prepared by an enzymatic method according to any one of claims 1 to 9.

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