A method for recovering L-serine from a phosphatidylserine enzyme synthesis reaction solution
By using porous media for capillary phase separation during the enzymatic preparation of phosphatidylserine, the problems of high L-serine loss and high energy consumption during recovery were solved, achieving low-cost and high-efficiency L-serine recovery.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHAANXI ANSAIFEITE TECH CO LTD
- Filing Date
- 2023-12-22
- Publication Date
- 2026-06-19
AI Technical Summary
In the existing enzymatic preparation of phosphatidylserine, the loss of L-serine is too large, and the recovery process is energy-intensive and costly, making it difficult to effectively reduce the loss.
A porous medium was added to an aqueous solution of L-serine before crystallization. The supersaturation concentration of L-serine was reduced by utilizing capillary phase separation, thereby reducing ethanol consumption. L-serine was then recovered through solid-liquid separation.
It reduced the energy consumption required for crystallization, decreased ethanol consumption, improved the recovery rate of L-serine, and reduced production costs.
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Figure CN117776948B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of L-serine recovery, and in particular to a method for recovering L-serine based on a phosphatidylserine enzymatic synthesis reaction solution. Background Technology
[0002] Phosphatidylserine (PS), a rare natural phospholipid, is one of the three essential phospholipids for the human body and a second-generation brain-boosting product after soy lecithin. PS is widely found in both plants and animals and plays a vital physiological role. In the human body, PS is abundant in the brain, accounting for 10-20% of the total phospholipids. It is the most abundant acidic phospholipid in the cell membranes of nerve fibers in the brain, and can be rapidly absorbed by the body. It crosses the blood-brain barrier to regulate the functional state of key cell membrane proteins, thus improving brain function, repairing brain damage, enhancing immunity, and improving memory.
[0003] Phosphatidylserine can be prepared by physical extraction, chemical synthesis, and enzyme-catalyzed synthesis. Enzyme catalysis has the advantages of mild reaction conditions (usually room temperature and pressure), good reaction selectivity, and high conversion rate, and is currently the mainstream process for the industrial preparation of phosphatidylserine.
[0004] The enzymatic synthesis of PS uses phosphatidylcholine (PC) and L-serine as substrates, catalyzed by phospholipase D. Theoretically, PC and L-serine react in a 1:1 molar ratio, but in practice, the actual amount of L-serine used can be up to 100 times the theoretical value (molar ratio). This is because: firstly, besides the main PC enzymatic reaction to PS, there is a side reaction of PC hydrolysis to phosphatidic acid (PA), which reduces the yield of phosphatidylserine; secondly, the product choline exhibits feedback inhibition, both preventing the reaction equilibrium from shifting to the right and reducing the conversion rate of phosphatidylserine, as well as the rate of the enzymatic reaction. Therefore, production practice mainly overcomes these drawbacks by increasing the amount of the relatively inexpensive second substrate, L-serine, as a substrate. This results in the actual amount of L-serine used far exceeding the theoretical value, leading to excessive L-serine loss. For example, Chinese Patent 200810017261.9 and Jiang Bo et al. (Purification and Catalytic Preparation of Phosphatidylserine by Phospholipase D, China Oils and Fats 2020, 45: 97-101). Therefore, in the production process of PS, the recovery and reuse of L-serine is very important for reducing costs and increasing efficiency.
[0005] For the enzymatic preparation of phosphatidylserine, the recovery and reuse of L-serine refers to both recovering L-serine from the reaction solution and removing the byproduct choline. Crystallization of L-serine can achieve this. The L-serine-containing solution is first decolorized with activated carbon, the decolorized solution is concentrated until crystals precipitate, cooled, and then crystallized with ethanol and filtered to obtain the L-serine product. Chinese patent CN 201310508490.1 mentions concentrating the L-serine eluent to 30%, adding an equal volume of 80% ethanol, stirring evenly, slowly cooling to 5°C, and allowing it to stand for at least 6 hours to allow L-serine to fully crystallize. Filtering yields the L-serine product, with an L-serine yield of 79.6%.
[0006] Feng Juanjuan (Research on Phosphatidylserine Catalytic Synthesis Process Based on Effective Utilization of L-Serine, 2016, Northwest University) used ethanol recrystallization to recover excess L-serine in the PLD-catalyzed soybean lecithin to PS synthesis reaction system. This method requires anhydrous ethanol and a relatively low temperature. The recovery rate of L-serine can reach about 90%, and the amount of choline impurities in the recovered L-serine is 0.56%. Although the recovery and reuse of L-serine can be achieved, the cost is still relatively high.
[0007] These processes all require concentrating the L-serine solution to a certain degree of supersaturation before adding high-concentration ethanol for crystallization. The concentration process is energy-intensive and can easily lead to the decomposition and deterioration of L-serine. Therefore, it is necessary to develop a recovery method that can reduce energy consumption or ethanol consumption and avoid the deterioration of L-serine.
[0008] To address this problem, the present invention proposes a method for recovering L-serine from a phosphatidylserine enzymatic synthesis reaction solution. Summary of the Invention
[0009] The purpose of this invention is to provide a method for recovering L-serine based on a phosphatidylserine enzymatic synthesis reaction solution, so as to solve the problems mentioned in the background art.
[0010] To achieve the above objectives, the present invention provides the following technical solution:
[0011] A method for recovering L-serine based on a phosphatidylserine enzymatic synthesis reaction solution includes the following steps:
[0012] S1. Phosphatidylcholine and L-serine are subjected to an enzymatic reaction catalyzed by phospholipase to obtain a mixed aqueous solution containing L-serine and choline.
[0013] S2. Add porous media and alcohol to the aqueous solution of L-serine and choline obtained in S1 to precipitate crystals and obtain a solid mixture of L-serine and porous media.
[0014] S3. The solid mixture of S2 is separated into L-serine and a porous medium solid mixture by solid-liquid separation;
[0015] S4. Dissolve the L-serine and porous medium solid mixture obtained in S3 in water, and then separate the solid and liquid to obtain an L-serine solution.
[0016] In a further embodiment, the content of phosphatidylcholine in S1 is 25-60%, and the organic solvent used is ethyl acetate, chloroform, or diethyl ether.
[0017] In a further embodiment, the enzymatic reaction in S1 includes dissolving phosphatidylcholine in an organic solvent to obtain a first mixture, dissolving serine in a buffer solution to obtain a second mixture, and finally mixing the first mixture and the second mixture together before adding phospholipase and reacting.
[0018] In a further embodiment, the phospholipase is phospholipase D.
[0019] In a further embodiment, the porous medium in S2 may be macroporous silica gel, resin, zeolite, or molecular sieve.
[0020] In a further embodiment, the alcohol in S2 may be ethanol or isopropanol.
[0021] In a further embodiment, the solid mixture in S4 is washed with deionized water to dissolve the L-serine and remove the insoluble porous media.
[0022] Compared with the prior art, the beneficial effects of the present invention are:
[0023] The crystallization of L-serine typically involves concentrating an aqueous solution of L-serine to a supersaturated state, followed by gradual crystallization by lowering the temperature. This invention introduces a porous medium before crystallization, causing capillary phase separation (CPS) within the medium. CPS reduces the supersaturated L-serine concentration required for crystallization, or, at the same supersaturated L-serine concentration, requires less ethanol. Therefore, this process reduces the energy consumption for concentration required for crystallization, lowers ethanol consumption, achieves a higher recovery rate, and the porous medium added throughout the process can be recycled, reducing the production cost of phosphatidylserine. Attached Figure Description
[0024] Figure 1 Comparison of thin-layer chromatography (TLC) detection of PS synthesized from new L-serine and recovered L-serine via enzymatic reaction. Detailed Implementation
[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0026] Example 1
[0027] In this embodiment of the invention, a method for recovering L-serine based on a phosphatidylserine enzymatic synthesis reaction solution includes the following steps:
[0028] 10g of phosphatidylcholine (25% concentration) was dissolved in 50mL of ethyl acetate. 84g of L-serine was then dissolved in 240mL of acetate buffer (pH 5.6). The two solutions were mixed thoroughly, and then 5mL of phospholipase D (120U / mL activity) was added. The mixture was reacted at 40℃ for 6 hours. After the reaction, the aqueous phase was separated using a separatory funnel. The residual organic solvent in the aqueous phase was removed by centrifugation to obtain an aqueous solution containing L-serine and choline. The L-serine concentration in the aqueous solution was adjusted to 30%.
[0029] Take 200ml of the aqueous phase and divide it into two equal portions;
[0030] Traditional crystallization method: One sample was prepared using conventional crystallization methods. 100 mL of 80% ethanol (v / v) was added and stirred until homogeneous. The mixture was then cooled to 10°C and allowed to stand for 6 hours to crystallize naturally. The L-serine crystals were obtained by filtration and dissolved in 120 mL of deionized water to obtain an L-serine aqueous solution. The contents of L-serine and choline were determined. The recovered L-serine content was 24.6 g, and the choline content was 36.8 mg.
[0031] The recovery process of this invention is as follows: First, 10g of Amberlite FPC11 (DOW Chemical) cationic dry resin is added to another sample, followed by 100mL of 80% ethanol (v / v). After stirring evenly, the mixture is cooled to 10℃ and allowed to stand for 6 hours to crystallize naturally. The mixture is then filtered to obtain a mixture of L-serine crystals and resin. The solid mixture is washed with 120mL of deionized water, and the water-insoluble resin is removed by filtration to obtain an L-serine aqueous solution. The contents of L-serine and choline are then measured. The recovered L-serine content is 38.8g, and the choline content is 40.5mg. The results of L-serine recovery using the conventional crystallization method and the method of this invention are shown in the table below:
[0032]
[0033] Example 2
[0034] 10g of 60% phosphatidylcholine was dissolved in 50mL of ethyl acetate, and 240mL of acetate buffer (pH 5.6) was added. After stirring until homogeneous, 1.6g of CaCl2 and 84g of L-serine were added, and the mixture was stirred until homogeneous. Then, 5mL of phospholipase D (enzyme activity 120U / mL) was added. The reaction was maintained at 40℃ for 20h. After the reaction was completed, the aqueous phase containing L-serine and choline was obtained by centrifugation.
[0035] After adding 30g of macroporous silica gel and 200mL of 80% ethanol (v / v) to the aqueous phase and stirring until homogeneous, the mixture was allowed to stand at 10℃ for 6 hours to crystallize naturally. The mixture was then filtered to obtain a mixture of L-serine crystals and silica gel. The solid mixture of L-serine and silica gel was washed with 200mL of deionized water, and the water-insoluble silica gel was removed by filtration to obtain an aqueous solution of L-serine. The content of L-serine was determined by the ninhydrin method, and the recovered L-serine content was 78g, while the choline content was 72.8mg.
[0036] Example 3
[0037] The effects of the enzymatic reaction of recovered L-serine to prepare phosphatidylserine are as follows:
[0038] Weigh out two 10g portions of phosphatidylcholine (60% purity) and dissolve them separately in 50mL of ethyl acetate. One portion used fresh L-serine, and the other used recycled L-serine. Other reaction procedures were the same as in Example 1. The reaction was then investigated. After the reaction, organic phase samples were analyzed by TLC using chloroform:methanol:ammonia:water = 10:4:0.5:0.4 (V / V) as the developing solvent and phosphomolybdic acid as the colorimetric reagent. The synthesis of phosphatidylserine using fresh L-serine and recycled L-serine was compared. Figure 1 As shown, there is no significant difference in reaction between the new L-serine and the recovered L-serine, therefore the recovered L-serine can be reused.
[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.
[0040] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. A method for recovering L-serine from a phosphatidylserine enzymatic synthesis reaction solution, characterized in that: Includes the following steps: S1. Phosphatidylcholine and L-serine are subjected to an enzymatic reaction catalyzed by phospholipase to obtain a mixed aqueous solution containing L-serine and choline. S2. Add porous media and alcohol to the mixed aqueous solution of L-serine and choline obtained in S1 to precipitate crystals and obtain a solid mixture of L-serine and porous media, wherein the porous media is cationic dry resin or macroporous silica gel. S3. The solid mixture of S2 is separated into L-serine and a porous medium solid mixture by solid-liquid separation; S4. Dissolve the L-serine and porous medium solid mixture obtained in S3 in water, and then separate the solid and liquid to obtain an L-serine solution.
2. The method for recovering L-serine from an enzymatic synthesis reaction solution based on phosphatidylserine according to claim 1, characterized in that: The content of phosphatidylcholine in S1 is 25-60%.
3. The method for recovering L-serine based on the phosphatidylserine enzymatic synthesis reaction solution according to claim 1, characterized in that: The enzymatic reaction in S1 involves dissolving phosphatidylcholine in an organic solvent to obtain a first mixture, then dissolving serine in a buffer solution to obtain a second mixture, and finally mixing the first and second mixtures together, adding phospholipase, and reacting.
4. The method for recovering L-serine based on the phosphatidylserine enzymatic synthesis reaction solution according to claim 1, characterized in that: The phospholipase is phospholipase D.
5. The method for recovering L-serine based on the phosphatidylserine enzymatic synthesis reaction solution according to claim 1, characterized in that: The alcohols used in S2 are ethanol and isopropanol.
6. The method for recovering L-serine based on the phosphatidylserine enzymatic synthesis reaction solution according to claim 1, characterized in that: The solid mixture in S4 is washed with deionized water to dissolve L-serine and remove insoluble porous media.