Method for preparing phosphatidyl ethanolamine synthetic phospholipids under catalysis of phospholipase D

Abstract

The invention relates to the field of synthetic phospholipids, and discloses a method for preparing phosphatidyl ethanolamine synthetic phospholipids under catalysis of phospholipase D. The method comprises the following steps: A, completely dissolving PC in an organic solvent to obtain a PC solution; b, adding ethanolamine into the buffer solution, and adjusting the pH value in an ice bath; c, mixing a metal salt solution with phospholipase D to obtain a fermentation enzyme solution, pouring a buffer solution containing ethanolamine, and uniformly mixing to obtain a water phase; d, mixing the PC solution with a water phase to uniformly disperse PC, and carrying out hydrolysis reaction to obtain a PE crude product reaction solution; e, standing for layering to obtain a water layer, extracting the water layer, and concentrating the obtained organic layer to obtain a PE crude product concentrate; f, refining to obtain a PE crude product I; g, performing column chromatography separation and vacuum concentration to obtain a PE crude product II with the purity not lower than 98%; f, drying to obtain a PE finished product. The method is simple in process, good in enzyme specificity, mild in reaction condition, environment-friendly, high in yield and beneficial to large-scale production of enterprises.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthetic phospholipids, in particular to a method for preparing synthetic phosphatidylethanolamine using phospholipase D catalysis. BACKGROUND

[0002] Synthetic phosphatidylethanolamine is mainly used in liposome injection of doxorubicin hydrochloride and irinotecan hydrochloride, which can make the cationic liposome membrane better fusion, high stability, small cytotoxicity, and its application in drug carriers such as long-circulating liposomes, micelles, and long-circulating nanoparticles has developed rapidly in recent years. The existing chemical synthesis method for preparing synthetic phosphatidylethanolamine has the problems of high difficulty, long process, catalyst residue, poor stability, and high cost. Compared with chemical synthesis, enzyme catalysis is easier to implement, and the by-products are less than chemical synthesis. Moreover, due to the selectivity of enzymes, the reaction time is shorter and the efficiency is higher. Therefore, using enzymes for catalytic conversion will be an ideal way to obtain high-quality synthetic phospholipids.

[0003] Phospholipase D (PLD) is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester bonds and base exchange reactions. As a product of catalytic hydrolysis activity, PLD contains a hydroxyl binding acceptor that reacts with phosphatidyl to form an intermediate product that can catalyze the exchange of the polar end group of phosphatidylcholine (PC) to synthesize other rare phospholipids. Deng Yangmin et al. (Chemical Industry Progress, 2017, 36(07): 2601-2606) determined the optimal conditions for phospholipase D catalyzing the synthesis of phosphatidylethanolamine from phosphatidylcholine and ethanolamine in an organic solvent-water two-phase system, which were reaction temperature 28℃, pH 5.5, and substrate molar ratio (ethanolamine / phosphatidylcholine) 20:1. Under these conditions, the yield of phosphatidylethanolamine was 87.2%, but this method required a large amount of flammable organic solvent diethyl ether to dissolve PC, which was costly and dangerous. Chen Kequan et al. (CN106479993A, 2017-03-08) invented a patent method for phospholipase D catalyzing the synthesis of phosphatidylethanolamine from phosphatidylcholine, which included the preparation of phospholipase D and the optimization of enzyme production conditions, and the establishment of a catalytic system, which could achieve efficient synthesis of phosphatidylethanolamine. However, the yield of phosphatidylethanolamine was less than 70%. Liu Yihan et al. (CN114891765B, 2023-12-19) used genetic engineering technology to invent a high-activity phospholipase D, and effectively prepared phosphatidylethanolamine using the enzyme. The yield of phosphatidylethanolamine in this patent was low, and the production cost of high-activity phospholipase D was high, which was not conducive to large-scale production of phosphatidylethanolamine.

[0004] Currently, there are still few reports on the preparation of synthetic phosphatidylethanolamine from PC-based synthetic phospholipids with a single structure and composition through enzyme catalysis. SUMMARY

[0005] The present application aims at solving the problems of large amount of organic solvent, low yield and high cost in the reaction of catalytic synthesis of structural phosphatidylethanolamine by phospholipase D.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] A method for preparing phosphatidylethanolamine synthetic phospholipid by using phospholipase D catalysis, characterized in that it comprises the following steps:

[0008] A. Dissolving phosphatidylcholine synthetic phospholipid completely in an organic solvent to obtain a phosphatidylcholine synthetic phospholipid solution;

[0009] B. Adding ethanolamine into a buffer solution and mixing uniformly, and adjusting pH value in an ice bath to obtain a buffer solution containing ethanolamine;

[0010] C. Mixing a metal salt solution and phospholipase D uniformly to obtain a fermentation enzyme solution, and mixing the fermentation enzyme solution and the buffer solution containing ethanolamine uniformly to obtain an aqueous phase;

[0011] D. Mixing the phosphatidylcholine synthetic phospholipid solution and the aqueous phase to uniformly disperse the phosphatidylcholine synthetic phospholipid, and performing a hydrolysis reaction to obtain a phosphatidylethanolamine synthetic phospholipid crude product reaction liquid;

[0012] E. Letting the phosphatidylethanolamine synthetic phospholipid crude product reaction liquid stand to separate into a water layer, taking the water layer to perform extraction, and taking the obtained organic layer to perform concentration to obtain a phosphatidylethanolamine synthetic phospholipid crude product concentrate;

[0013] F. Performing refinement on the phosphatidylethanolamine synthetic phospholipid crude product concentrate to obtain a phosphatidylethanolamine synthetic phospholipid crude product I;

[0014] G. Performing column chromatography separation on the phosphatidylethanolamine synthetic phospholipid crude product I, and performing concentration under reduced pressure to obtain a phosphatidylethanolamine synthetic phospholipid crude product II;

[0015] H. Drying the phosphatidylethanolamine synthetic phospholipid crude product II to obtain a phosphatidylethanolamine synthetic phospholipid finished product.

[0016] Preferably, in step B, the pH value is adjusted by using concentrated hydrochloric acid, the pH value is adjusted to 5-6, and the ice bath temperature comprises 0-5℃.

[0017] Preferably, in step A, the mass-volume ratio of the phosphatidylcholine synthetic phospholipid to the organic solvent is 1g:40-50mL; in step B, the mass-volume ratio of the phosphatidylcholine synthetic phospholipid to the ethanolamine is 1g:12-15mL, and the mass-volume ratio of the phosphatidylcholine synthetic phospholipid to the buffer is 1g:40-50mL; in step B, the pH value is adjusted with concentrated hydrochloric acid, and the process of adjusting with concentrated hydrochloric acid includes slowly adding concentrated hydrochloric acid while stirring, and the volume ratio of the concentrated hydrochloric acid to the ethanolamine is 1:1.25-1.30; in step C, the mass-volume ratio of the phospholipase D to the metal salt solution is 1g:10-20mL, and the mass ratio of the phospholipase D to the phosphatidylcholine synthetic phospholipid is 1:1-2; in step D, the hydrolysis reaction time is 12-18 hours, and the temperature is 25-45℃.

[0018] Preferably, in step A, the organic solvent includes one or more of diethyl ether, dichloromethane, and trichloromethane, and the buffer includes one or more of acetate buffer, citrate buffer, and phosphate buffer; in step C, the metal salt solution is an aqueous solution of a metal salt, and the metal salt includes one or more of calcium chloride, sodium chloride, potassium chloride, iron chloride, and zinc chloride; and the phospholipase D is prepared by fermentation of Streptomyces.

[0019] Preferably, in step A, the concentration of the buffer is 0.2mol / L, and the pH value is 5.6; in step C, the specific activity of the phospholipase D is 8-20U / mg, and the concentration of the metal salt solution is 0.2mol / L.

[0020] Preferably, in step E, the extraction operation includes: standing the crude phosphatidylethanolamine synthetic phospholipid reaction solution to separate layers, separating the water layer, and extracting with an extractant to obtain an organic layer, repeating the operation 3 times, and combining the organic layers to obtain a crude phosphatidylethanolamine synthetic phospholipid extraction solution; the concentration operation includes: drying the crude phosphatidylethanolamine synthetic phospholipid extraction solution with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a crude phosphatidylethanolamine synthetic phospholipid concentrate; in step F, the refining operation includes: stirring and beating the crude phosphatidylethanolamine synthetic phospholipid concentrate with a refining solvent, filtering under reduced pressure, repeating the beating 2 times, further concentrating and drying the filter residue to obtain a crude phosphatidylethanolamine synthetic phospholipid I; in step G, the column chromatography separation operation includes: subjecting the crude phosphatidylethanolamine synthetic phospholipid I to silica gel column chromatography separation with a complex eluent; and in step H, the drying method includes vacuum drying.

[0021] Preferably, in step E, the extractant includes one or more of diethyl ether, dichloromethane, trichloromethane; the total amount of the extractant is in a volume-mass ratio of 60 mL:1 g with respect to the phosphatidylcholine synthetic phospholipid; in step F, the refining solvent includes one or more of ethyl acetate, acetone; the mass-volume ratio of the phosphatidylethanolamine synthetic phospholipid crude concentrate to the refining solvent is 1 g:5-7 mL; the beating time is 0.5-1 h; in step G, the complex eluent is a mixture of a halogenated alkane and a short-chain alcohol, the volume ratio of the halogenated alkane to the short-chain alcohol is 7:1-7:3; the halogenated alkane includes one or more of trichloromethane, dichloromethane; the short-chain alcohol includes one or more of methanol, ethanol, isopropyl alcohol; in step H, the vacuum drying operation includes: placing the phosphatidylethanolamine synthetic phospholipid crude product II in a vertical vacuum vibration dryer, and vacuum drying at a temperature of 50-70°C, a vacuum degree of 0.08-0.1 MPa for 4-8 hours.

[0022] Preferably, in step C, before pouring the fermentation enzyme solution into the buffer containing ethanolamine, the ice bath in step B is removed.

[0023] Preferably, the complete dissolution in step A, the uniform mixing in step B, and the uniform dispersion in step D are all by stirring.

[0024] Preferably, the phosphatidylcholine includes one or more of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), diarachidoylphosphatidylcholine (DEPC).

[0025] The application also provides a phosphatidylethanolamine synthetic phospholipid product obtained by any of the above methods for preparing phosphatidylethanolamine synthetic phospholipids catalyzed by phospholipase D, characterized in that the purity of the phosphatidylethanolamine synthetic phospholipid product is not less than 98%.

[0026] The application can obtain high-purity and high-yield phosphatidylethanolamine, which is closely related to the feeding sequence. The feeding sequence must be to first prepare a buffer containing ethanolamine, then mix the fermentation enzyme solution with the buffer containing ethanolamine uniformly to form an aqueous phase, and finally pour the aqueous phase into the prepared PC synthetic phospholipid solution to start the reaction timing.

[0027] The effect of feeding sequence on phospholipase D-catalyzed reactions can be analyzed from several aspects. First, the correct feeding sequence helps to maintain the appropriate concentrations of substrates and products in the reaction system, which is crucial for avoiding substrate inhibition and product inhibition. If ethanolamine is added too early, it may compete with phosphatidylcholine for the active site of phospholipase D, leading to the formation of dead-end complexes and inhibiting the generation of the desired product PE. Second, the appropriate feeding sequence is beneficial for controlling the pH and ionic strength of the reaction, which is critical for maintaining the optimal activity state of phospholipase D. As disclosed research shows, phospholipase D exhibits the highest catalytic activity at pH 5.5. Therefore, the pH of the reaction system should be adjusted first, and then the substrates should be added gradually to ensure the stability of the reaction environment. Finally, the appropriate feeding sequence can also reduce the occurrence of side reactions and improve the purity of the product. For example, adding precise amounts of substrates gradually can reduce non-specific reactions caused by excess substrates, thereby improving the purity of the target product PE.

[0028] The effect of ice bath on phospholipase D-catalyzed reactions cannot be ignored. First, ice bath can lower the temperature of the reaction system, helping to control the reaction rate. Since the reaction catalyzed by phospholipase D is usually exothermic, too low a temperature may inhibit enzyme activity, while moderate low temperature can slow down the reaction, making the reaction easier to control, thereby improving the yield and purity of the product. Second, ice bath helps to reduce enzyme inactivation caused by temperature. In actual operation, preheating or precooling the reaction system to the appropriate temperature through ice bath can avoid the loss of enzyme activity caused by too high or too low temperature. In addition, ice bath also helps to reduce the formation of bubbles during the reaction, which is very important for maintaining the uniformity and stability of the reaction system.

[0029] When preparing the above-mentioned buffer containing ethanolamine, the amount of concentrated hydrochloric acid should be controlled to maintain the pH of the buffer system at 5-6. If too much hydrochloric acid is added, on the one hand, it will reduce the catalytic activity of phospholipase D, and on the other hand, it will lead to the generation of degradation products lysophosphatidylethanolamine (LPE) in the subsequent phosphatidylethanolamine synthetic phospholipid ester bond enzymolysis. Therefore, too strong acidity is not conducive to the preparation of phosphatidylethanolamine synthetic phospholipid, and concentrated hydrochloric acid should be added slowly while stirring, because the dropwise addition process will release a lot of heat, and too fast dropwise addition will cause the solution to boil.

[0030] During the purification of phosphatidylethanolamine, refining and column chromatography can remove most of the phospholipase D and pigments, playing a role in purification, ensuring that the purity of phosphatidylethanolamine synthetic phospholipid is not less than 98%. Column chromatography should not use ternary eluent containing water, on the one hand, it is easy to cause the water content of the product to exceed the standard, on the other hand, silica gel is easy to absorb water, leading to poor column chromatography effect. The final vacuum drying can ensure that the solvent residue and microbial test results meet the quality standard requirements.

[0031] In the preparation of fermentation enzyme solutions, metal cations of metal salts are essential for phospholipase D, playing a role in activating phospholipase D. However, excessive metal cations will inhibit the activity of phospholipase D.

[0032] The phosphatidylethanolamine includes one or more of distearyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), and disqualoyl phosphatidylethanolamine (DEPE).

[0033] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0034] 1. Compared with chemical synthesis, the enzymatic synthesis of the present invention has obvious advantages: (1) it is environmentally friendly, (2) the reaction conditions are mild, (3) the enzyme has good specificity, (4) the process steps are short, and (5) the yield is high. Therefore, the present invention is easy to realize large-scale production.

[0035] 2. The process of this invention prepares phosphatidylethanolamine from phosphatidylcholine to synthesize phospholipids. Compared with the existing enzymatic synthesis technology, it has a high yield and can obtain high-purity (not less than 98%) phosphatidylethanolamine, which significantly improves product quality and stability. At the same time, it helps to improve the quality of formulations and has obvious advantages in production efficiency.

[0036] 3. The process of this invention for preparing phosphatidylethanolamine phospholipids from phosphatidylcholine achieves similar or even higher yields compared to existing enzymatic synthesis techniques, which have lower enzyme costs and fewer process steps. The yields of various phosphatidylethanolamine phospholipids are not less than 80% and even close to 90%, ensuring product quality while reducing production costs. Therefore, this invention has high economic value.

[0037] 4. Currently, there are few reports on the preparation of phosphatidylethanolamine synthetic phospholipids by enzyme catalysis, starting from PC-type synthetic phospholipids with a defined structure and simple composition. This invention fills this technological gap in the field. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the reaction process in the preparation of DSPE in Example 1;

[0039] Figure 2 This is a schematic diagram of the reaction process in the DPPE preparation process of Example 2;

[0040] Figure 3 This is a schematic diagram of the reaction process in the preparation of DOPE in Example 3;

[0041] Figure 4 This is a schematic diagram of the reaction process in the DEPE preparation process of Example 4;

[0042] Figure 5 The liquid chromatogram of the finished DSPE in Example 1;

[0043] Figure 6 The liquid chromatogram of the finished DPPE in Example 2;

[0044] Figure 7 The liquid chromatogram of the finished DOPE in Example 3;

[0045] Figure 8 The image shows the liquid chromatogram of the finished DEPE product from Example 4.

[0046] Figure 9 The liquid chromatogram of the finished DSPE in Comparative Example 3;

[0047] Figure 10 The image shows the liquid chromatogram of the finished DSPE product from Comparative Example 4. Detailed Implementation

[0048] 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 and accompanying drawings. Unless otherwise specified, the reagents and materials used in the following examples and comparative examples are commercially available products.

[0049] Example 1

[0050] The reaction route is as follows Figure 1 As shown. 10 g (12.66 mmol) of synthesized phospholipid DSPC was weighed into a 2 L round-bottom flask, and 400 mL of chloroform was added. Stirring was started until the DSPC was completely dissolved to obtain a DSPC solution. In a 1 L beaker, 400 mL of acetate-sodium acetate buffer (0.2 mol / L, pH = 5.6) and 120 mL of ethanolamine were added. The beaker was placed in a water bath at 0–5 °C, and stirring was started. Concentrated hydrochloric acid was slowly added dropwise to the buffer until the pH reached 5–6, consuming a total of 150 mL of concentrated hydrochloric acid. In a 250 mL beaker, 100 mL of CaCl2 solution (0.2 mol / L) and 5 g of phospholipase D (20 U / mg) were added. The mixture was stirred thoroughly to form a fermentation enzyme solution. The water bath was removed, and the fermentation enzyme solution was mixed thoroughly with the buffer containing ethanolamine to form an aqueous phase. Finally, the aqueous phase was slowly poured into the DSPC solution. The reaction temperature was 30 °C, and the reaction was stirred for 12 h.

[0051] After the reaction was completed, the reaction solution was allowed to stand and separate into layers. The aqueous layer was extracted three times with chloroform (200 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 12.2 g of crude DSPE concentrate. The concentrate was stirred and slurried with 60 mL of ethyl acetate for 1 h, filtered under reduced pressure, and the slurry was repeated twice. The filter residue was further concentrated and dried to obtain crude DSPE I. Crude DSPE I was subjected to silica gel column chromatography, eluted with chloroform-methanol (7:1 to 7:3), and concentrated under reduced pressure to obtain 9.1 g of crude DSPE II. Crude DSPE II was vacuum dried in a vertical vacuum vibrating dryer at a temperature of 50–70 °C and a vacuum degree of 0.08–0.1 MPa for 5 h to obtain 8.6 g of finished DSPE product (purity 99.80%, yield 90.7%). Figure 5 As shown, the purity of DSPE in the finished product was determined by HPLC, and the purity reached 99.80% (external standard method). Yield (mass%) = Change in DSPC / Initial amount of DSPC × 100%.

[0052] Example 2

[0053] The reaction route is as follows Figure 2 As shown. 10 g (13.62 mmol) of synthesized phospholipid DPPC was weighed into a 2 L round-bottom flask, and 400 mL of chloroform was added. Stirring was started to completely dissolve the DPPC, obtaining a DPPC solution. In a 1 L beaker, 400 mL of acetate-sodium acetate buffer (0.2 mol / L, pH = 5.6) and 120 mL of ethanolamine were added. The beaker was placed in a water bath at 0–5 °C, and stirring was started. Concentrated hydrochloric acid was slowly added dropwise to the buffer until the pH reached 5–6, consuming a total of 151 mL of concentrated hydrochloric acid. In a 250 mL beaker, 100 mL of CaCl2 solution (0.2 mol / L) and 5 g of phospholipase D (20 U / mg) were added, and the mixture was stirred thoroughly to form a fermentation enzyme solution. The water bath was removed, and the fermentation enzyme solution was mixed thoroughly with the buffer containing ethanolamine to form an aqueous phase. Finally, the aqueous phase was slowly poured into the DPPC solution. The reaction temperature was 30 °C, and the reaction was stirred for 12 h.

[0054] After the reaction was completed, the reaction solution was allowed to stand and separate into layers. The aqueous layer was extracted three times with chloroform (200 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 12.5 g of crude DPPE concentrate. The crude DPPE concentrate was stirred and slurried with 60 mL of ethyl acetate for 1 h, filtered under reduced pressure, and the slurry was repeated twice. The filter residue was further concentrated and dried to obtain crude DPPE I. Crude DPPE I was subjected to silica gel column chromatography, eluted with chloroform-methanol (7:1 to 7:3), and concentrated under reduced pressure to obtain 8.8 g of crude DPPE II. Crude DPPE II was vacuum dried in a vertical vacuum vibrating dryer at a temperature of 50–70 °C and a vacuum degree of 0.08–0.1 MPa for 6 hours to obtain 8.2 g of finished DPPE (purity 99.61%, yield 86.7%). The purity of DPPE in the finished product was determined according to the method in Example 1. The liquid chromatogram of the finished product is shown below. Figure 6 As shown. Yield (mass%) = Change in DPPC / Initial DPPC × 100%.

[0055] Example 3

[0056] The reaction route is as follows Figure 3 As shown. 10 g (12.72 mmol) of synthesized phospholipid DOPC was weighed into a 2 L round-bottom flask, and 500 mL of chloroform was added. Stirring was started to completely dissolve the DOPC, obtaining a DOPC solution. In a 1 L beaker, 400 mL of citrate-sodium citrate buffer (0.2 mol / L, pH = 5.6) and 150 mL of ethanolamine were added. The beaker was placed in a water bath at 0–5 °C, and stirring was started. Concentrated hydrochloric acid was slowly added dropwise to the buffer until the pH reached 5–6, consuming a total of 187 mL of concentrated hydrochloric acid. In a 250 mL beaker, 100 mL of CaCl2 solution (0.2 mol / L) and 10 g of phospholipase D (20 U / mg) were added, and the mixture was stirred thoroughly to form a fermentation enzyme solution. The water bath was removed, and the fermentation enzyme solution was mixed thoroughly with the buffer containing ethanolamine to form an aqueous phase. Finally, the aqueous phase was slowly poured into the DOPC solution. The reaction temperature was 30 °C, and the reaction was stirred for 16 h.

[0057] After the reaction was completed, the reaction solution was allowed to stand and separate into layers. The aqueous layer was extracted three times with chloroform (200 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.3 g of crude DOPE concentrate. The crude DOPE concentrate was stirred and slurried with 50 mL of ethyl acetate for 1 h, filtered under reduced pressure, and the slurry was repeated twice. The filter residue was further concentrated and dried to obtain crude DOPE I. Crude DOPE I was subjected to silica gel column chromatography, eluted with chloroform-methanol (7:1 to 7:3), and concentrated under reduced pressure to obtain 8.3 g of crude DOPE II. Crude DOPE II was vacuum dried in a vertical vacuum vibrating dryer at a temperature of 50–70 °C and a vacuum degree of 0.08–0.1 MPa for 7 hours to obtain 7.9 g of finished DOPE (purity 99.32%, yield 83.8%). The purity of DOPE in the finished product was determined according to the method in Example 1. The liquid chromatogram of the finished product is shown below. Figure 7 As shown. Yield (mass%) = Change in DOPC / Initial DOPC × 100%.

[0058] Example 4

[0059] The reaction route is as follows Figure 4 As shown. 10 g (11.13 mmol) of synthesized phospholipid DEPC was weighed into a 2 L round-bottom flask, and 500 mL of chloroform was added. Stirring was started to completely dissolve the DEPC, obtaining a DEPC solution. In a 1 L beaker, 400 mL of citrate-sodium citrate buffer (0.2 mol / L, pH = 5.6) and 150 mL of ethanolamine were added. The beaker was placed in a water bath at 0–5 °C, and stirring was started. Concentrated hydrochloric acid was slowly added dropwise to the buffer until the pH reached 5–6, consuming a total of 186 mL of concentrated hydrochloric acid. In a 250 mL beaker, 100 mL of CaCl2 solution (0.2 mol / L) and 10 g of phospholipase D (20 U / mg) were added, and the mixture was stirred thoroughly to form a fermentation enzyme solution. The water bath was removed, and the fermentation enzyme solution was mixed thoroughly with the buffer containing ethanolamine to form an aqueous phase. Finally, the aqueous phase was slowly poured into the DEPC solution. The reaction temperature was 30 °C, and the reaction was stirred for 16 h.

[0060] After the reaction, the reaction solution was allowed to stand and separate into layers. The aqueous layer was extracted three times with chloroform (200 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.4 g of crude DEPE concentrate. The crude DEPE concentrate was stirred and slurried with 50 mL of ethyl acetate for 1 hour, filtered under reduced pressure, and the slurry was repeated twice. The residue was further concentrated and dried to obtain crude DEPE I. Crude DEPE I was subjected to silica gel column chromatography, eluted with chloroform-methanol (7:1 to 7:3), and concentrated under reduced pressure to obtain 8.0 g of crude DEPE II. Crude DEPE II was vacuum dried in a vertical vacuum vibrating dryer at a temperature of 50–70 °C and a vacuum degree of 0.08–0.1 MPa for 8 hours to obtain 7.5 g of DEPE product (purity 99.14%, yield 78.8%). The purity of DEPE in the product was determined according to the method in Example 1. The liquid chromatogram of the product is shown below. Figure 8 As shown. Yield (mass%) = Change in DEPC / Initial DEPC × 100%.

[0061] Comparative Example 1

[0062] The chemical synthesis of DSPE was carried out using a conventional synthetic method: At 15°C, a 150 mL solution of PC13 (31.1 g, 0.25 mol) in dichloromethane was added to a 1000 mL solution of 61.0 g imidazole (0.90 mol), followed by a 100 mL solution of triethylamine in dichloromethane (100 mL triethylamine, 250 mL dichloromethane). The reaction was allowed to proceed for 15 minutes. Over 1 hour, 44 g of a self-made glyceryl distearate phosphorous acid solution was slowly added dropwise. The reaction was monitored by TLC until completion. The reaction was quenched with water / pyridine, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and the mother liquor was evaporated to dryness. 47 g of a yellow solid was obtained and directly added to the next step. The product from the previous step was dissolved in 500 mL of LMF / CH2Cl2 (1:1 v / v), and 30 g of pentanoyl chloride (0.21 mol) was slowly added dropwise at 0°C. The reaction was allowed to proceed for 30 minutes, followed by 4 hours at room temperature. Then, 13 g of ethanolamine (0.21 mol) was added, and the reaction was carried out for 2 hours. The reaction was confirmed by TLC, and the product was extracted with dichloromethane to obtain 45.2 g of crude product. The above reaction product was added to a mixed solution of 200 mL of 5% hydrogen peroxide, 15 mL of acetic acid, and 100 mL of dichloromethane, and stirred at room temperature for 40 minutes. The mixture was then extracted with dichloromethane, eluted by silica gel column chromatography with chloroform-methanol (10:1–3:1), yielding 41.0 g of DSPE, with a yield of 77%. Yield (mol%) = (amount of DSPE / amount of glyceryl distearate phosphorous acid) × 100%.

[0063] A comparison of Example 1 and Comparative Example 1 shows that Comparative Example 1 requires the self-made distearate glyceryl phosphorous acid and the use of flammable and explosive hydrogen peroxide, which is lengthy and complex with poor atom economy. In contrast, the production process of the present invention is simple, the reaction conditions are mild, it is environmentally friendly, and the yield is high. Synthetic phospholipids DSPE with a purity of not less than 98% can be obtained in a single pot, which is beneficial for large-scale production by enterprises.

[0064] Comparative Example 2

[0065] The catalytic synthesis of phosphatidylethanolamine is a two-way reaction system. 0.05 g of ethanolamine was added to 1 mL of acetate buffer (0.2 M, pH 5.5) containing 0.05 mg / mL phospholipase D. The mixture was stirred in an ice bath and blotted for 60 min to obtain a solution containing the enzyme-substrate complex (blotase). Then, 12 g / L phosphatidylcholine was added to the above solution containing the enzyme-substrate complex (blotase), and the mixture was placed at 30 °C with shaking at 200 rpm. The high-performance liquid chromatography (HPLC) method for the determination of phosphatidylcholine and phosphatidylethanolamine was performed using a Waters 2535 HPLC system. The chromatographic column was a Waters Xbridge Hilic (4.6 mm × 250 mm, 5 μm). Mobile phase A was methanol-water-glacial acetic acid-triethylamine (85:15:0.45:0.05), and mobile phase B was n-hexane-isopropanol-mobile phase A (20:48:32). Gradient elution was used, the column temperature was 30 °C, and ELSD detection was performed. The final yield of phosphatidylethanolamine was 66.5% after 10 h of reaction.

[0066] Comparative Example 3

[0067] The reaction route is as follows Figure 1 As shown. 10 g (12.66 mmol) of synthesized phospholipid DSPC was weighed into a 2 L round-bottom flask, and 400 mL of chloroform was added. Stirring was started to completely dissolve the DSPC, obtaining a DSPC solution. 120 mL of ethanolamine was added to 3636 mL of acetate buffer (0.2 M, pH 5.5) containing phospholipase D at a concentration of 0.05 mg / mL. The mixture was incubated on ice with gentle stirring for 60 min to obtain a solution containing the enzyme-substrate complex (blotase). Finally, the solution containing the enzyme-substrate complex (blotase) was slowly poured into the DSPC solution. The reaction was carried out at 30 °C with stirring for 12 h.

[0068] After the reaction was completed, the reaction solution was allowed to stand and separate into layers. The aqueous layer was extracted three times with chloroform (200 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.5 g of crude DSPE concentrate. The crude DSPE concentrate was stirred and slurried with 60 mL of ethyl acetate for 1 h, filtered under reduced pressure, and the slurry was repeated twice. The filter residue was further concentrated and dried to obtain crude DSPE I. Crude DSPE I was subjected to silica gel column chromatography, eluted with chloroform-methanol (7:1 to 7:3), and concentrated under reduced pressure to obtain 7.7 g of crude DSPE II. Crude DSPE II was vacuum dried in a vertical vacuum vibrating dryer at a temperature of 50–70 °C and a vacuum degree of 0.08–0.1 MPa for 5 h to obtain 7.2 g of finished DSPE product (purity 99.35%, yield 76.0%). The liquid chromatogram of the finished product is shown below. Figure 9 As shown.

[0069] A comparison of Example 1 with Comparative Examples 2 and 3 shows that: Comparative Example 2 also uses the phospholipase D method to synthesize phosphatidylethanolamine, but the PE yield obtained by this method is no more than 70%; the only difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses an imprinting enzyme process, but the PE yield obtained by this method is only 76%; Example 1 uses a process that comprehensively improves the catalytic activity of phospholipase D, which can obtain synthetic phospholipid DSPE with a purity of not less than 98% in a yield of about 90%, which is beneficial for large-scale production by enterprises.

[0070] Comparative Example 4

[0071] The reaction route is as follows Figure 1 As shown. 10 g (12.66 mmol) of synthesized phospholipid DSPC was weighed into a 2 L round-bottom flask, and 400 mL of chloroform was added. Stirring was started until the DSPC was completely dissolved to obtain a DSPC solution. In a 1 L beaker, 400 mL of acetate-sodium acetate buffer (0.2 mol / L, pH = 5.6) and 120 mL of ethanolamine were added. The beaker was placed in a water bath at 0–5 °C, and stirring was started. Concentrated hydrochloric acid was slowly added dropwise to the buffer until the pH reached 5–6, consuming a total of 150 mL of concentrated hydrochloric acid. In a 250 mL beaker, 100 mL of CaCl2 solution (0.2 mol / L) and 5 g of phospholipase D (20 U / mg) were added. The mixture was stirred thoroughly to form a fermentation enzyme solution. The water bath was removed, and the fermentation enzyme solution was mixed thoroughly with the buffer containing ethanolamine to form an aqueous phase. Finally, the aqueous phase was slowly poured into the DSPC solution. The reaction temperature was 30 °C, and the reaction was stirred for 12 h.

[0072] After the reaction was completed, the reaction solution was allowed to stand and separate into layers. The aqueous layer was separated and extracted three times with chloroform (200 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 12.2 g of crude DSPE. The crude DSPE was then vacuum dried in a vertical vacuum vibrating dryer at a temperature of 50–70 °C and a vacuum degree of 0.08–0.1 MPa for 5 hours to obtain 5.5 g of finished DSPE (purity 63.33%, yield 58.7%). The purity of DSPE in the finished product was determined according to the method in Example 1. The liquid chromatogram of the finished product is shown below. Figure 10 As shown. Yield (mass%) = Change in DSPC / Initial DSPC × 100%.

[0073] The product data comparison between Example 1 and Comparative Example 4 is shown in Table 1:

[0074] Table 1

[0075]

[0076] As can be seen from Table 1, one of the advantages of the preparation method of the present invention is that the purification and column chromatography process can remove most of the impurities, thereby achieving purification and obtaining phosphatidylethanolamine synthetic phospholipids with a purity of not less than 98%. Example 1 shows a significant improvement in the purity of the finished product compared to Comparative Example 4.

[0077] 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, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for producing synthetic phospholipids using phospholipase D catalysis, characterized by, The method comprises the following steps: A. completely dissolving phosphatidylcholine synthetic phospholipids in an organic solvent to obtain a phosphatidylcholine synthetic phospholipid solution; B. adding ethanolamine into a buffer and mixing uniformly, adjusting the pH value in an ice bath to obtain a buffer containing ethanolamine; C. mixing a metal salt solution and phospholipase D uniformly to obtain a fermentation enzyme solution, mixing the fermentation enzyme solution and the buffer containing ethanolamine uniformly to obtain an aqueous phase; D. mixing the phosphatidylcholine synthetic phospholipid solution and the aqueous phase to uniformly disperse the phosphatidylcholine synthetic phospholipids, and performing a hydrolysis reaction to obtain a crude product of phosphatidylethanolamine synthetic phospholipids; E. standing and separating the crude product of phosphatidylethanolamine synthetic phospholipids to obtain an aqueous layer, extracting the aqueous layer, and concentrating the obtained organic layer to obtain a crude product concentrate of phosphatidylethanolamine synthetic phospholipids; F. refining the crude product concentrate of phosphatidylethanolamine synthetic phospholipids to obtain a crude product I of phosphatidylethanolamine synthetic phospholipids; G. performing column chromatography separation on the crude product I of phosphatidylethanolamine synthetic phospholipids, and performing vacuum concentration to obtain a crude product II of phosphatidylethanolamine synthetic phospholipids; H. drying the crude product II of phosphatidylethanolamine synthetic phospholipids to obtain a finished product of phosphatidylethanolamine synthetic phospholipids.

2. The method for producing synthetic phospholipids using phospholipase D according to claim 1, wherein In step B, the pH value is adjusted by using concentrated hydrochloric acid, the pH value is adjusted to 5-6, and the ice bath temperature comprises 0-5℃.

3. The method for producing synthetic phospholipids using phospholipase D according to claim 1, wherein In step A, the mass-volume ratio of the phosphatidylcholine synthetic phospholipids to the organic solvent comprises 1g:40-50mL; in step B, the mass-volume ratio of the phosphatidylcholine synthetic phospholipids to the ethanolamine comprises 1g:12-15mL, and the mass-volume ratio of the phosphatidylcholine synthetic phospholipids to the buffer comprises 1g:40-50mL; in step B, the pH value is adjusted by using concentrated hydrochloric acid, and the process of adjusting by using concentrated hydrochloric acid comprises slowly adding concentrated hydrochloric acid while stirring, the volume ratio of the concentrated hydrochloric acid to the ethanolamine comprises 1:1.25-1.30; in step C, the mass-volume ratio of the phospholipase D to the metal salt solution comprises 1g:10-20mL, and the mass ratio of the phospholipase D to the phosphatidylcholine synthetic phospholipids comprises 1:1-2; in step D, the hydrolysis reaction time comprises 12-18 hours, and the temperature comprises 25-45℃.

4. The method for producing synthetic phospholipids using phospholipase D according to claim 1, wherein In step A, the organic solvent comprises one or more of diethyl ether, dichloromethane, and trichloromethane, and the buffer comprises one or more of acetate buffer, citrate buffer, and phosphate buffer; in step C, the metal salt solution is an aqueous solution of a metal salt, the metal salt comprises one or more of calcium chloride, sodium chloride, potassium chloride, iron chloride, and zinc chloride, and the phospholipase D is prepared by fermentation of streptomyces.

5. The method for producing synthetic phospholipids by using phospholipase D according to claim 1, wherein In step A, the concentration of the buffer is 0.2mol / L, and the pH value is 5.6; in step C, the specific activity of the phospholipase D comprises 8-20U / mg, and the concentration of the metal salt solution comprises 0.2mol / L.

6. The method for producing synthetic phospholipids by using phospholipase D according to claim 1, wherein, In step E, the extracting operation includes: allowing the crude synthetic phospholipid of the phosphatidylethanolamine to stand and separate into layers, separating the water layer and extracting with an extractant to obtain an organic layer, repeating the operation for 3 times, and combining the organic layers to obtain the crude synthetic phospholipid of the phosphatidylethanolamine extract; the concentrating operation includes: drying the crude synthetic phospholipid of the phosphatidylethanolamine extract with anhydrous sodium sulfate and concentrating under reduced pressure to obtain the crude synthetic phospholipid of the phosphatidylethanolamine concentrate; in step F, the refining operation includes: stirring and beating the crude synthetic phospholipid of the phosphatidylethanolamine concentrate with a refining solvent, filtering under reduced pressure, repeating the beating for 2 times, and further concentrating and drying the filter residue to obtain the crude synthetic phospholipid of the phosphatidylethanolamine I; in step G, the column chromatography separation operation includes: subjecting the crude synthetic phospholipid of the phosphatidylethanolamine I to silica gel column chromatography separation with a composite eluent; and in step H, the drying method includes vacuum drying.

7. The method for producing synthetic phospholipids by catalysis of phospholipase D according to claim 6, wherein, In step E, the extractant includes one or more of diethyl ether, dichloromethane and trichloromethane; the total amount of the extractant is 60 mL per 1 g of the volume / mass ratio of the synthetic phospholipid of the phosphatidylcholine; in step F, the refining solvent includes one or more of ethyl acetate and acetone; the mass / volume ratio of the crude synthetic phospholipid of the phosphatidylethanolamine concentrate to the refining solvent is 1 g: 5-7 mL, and the beating time is 0.5-1 h; in step G, the composite eluent is a mixture of a halogenated alkane and a short-chain alcohol, and the volume ratio of the halogenated alkane to the short-chain alcohol is 7:1-7:3; the halogenated alkane includes one or more of trichloromethane and dichloromethane, and the short-chain alcohol includes one or more of methanol, ethanol and isopropyl alcohol; and in step H, the vacuum drying time is 4-8 hours.

8. The method for producing synthetic phospholipids by catalysis of phospholipase D according to claim 1, wherein, In step C, the ice bath in step B is removed before the fermentation enzyme solution is poured into the buffer containing ethanolamine. ​ 9. The method for producing synthetic phospholipids by catalysis of phospholipase D according to claim 1, wherein, The phosphatidylcholine includes one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine and diarachidonoylphosphatidylcholine. ​ 10. A synthetic phosphatidylethanolamine product obtained by the method of any one of claims 1 to 9 using phospholipase D catalysis to produce synthetic phosphatidylethanolamine products, characterized by, The purity of the finished synthetic phospholipid of the phosphatidylethanolamine is not less than 98%.

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