Method for producing phosphatidylserine through enzyme catalysis

Abstract

The invention discloses a method for producing phosphatidylserine through enzyme catalysis, and belongs to the technical field of biology, and the method specifically comprises the following steps: in a reaction system of a [BMIM] [BF4] aqueous solution, taking natural phospholipid containing phosphatidylcholine and cephalin as a raw material, adding L-serine, calcium salt and phospholipase D, and carrying out a trans-acylation reaction. By introducing cephalin (4%-10%, preferably 6%-8%) and sphingomyelin (5%-10%) in a specific proportion and combining with a [BMIM] [BF4] ionic liquid system, high efficiency and high purity (the purity gt, 79.6% and the yield gt, 76.4%) of PS production are realized, and the problem that the yield and the purity cannot be considered at the same time in the traditional process is thoroughly solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a method for producing phosphatidylserine by enzyme catalysis. BACKGROUND

[0002] Phosphatidylserine (PS) is a key component of brain nerve cell membranes, has the effects of improving cognitive function and repairing brain injury, and is widely used in infant milk powder, Alzheimer's disease auxiliary treatment drugs and functional foods. At present, the mainstream production in the industry adopts phospholipase D (PLD) to catalyze the transacylation reaction of phosphatidylcholine (PC) and L-serine, and raw materials are easy to obtain and the cost is significantly lower than that of animal brain tissue extraction method.

[0003] In the traditional enzyme catalysis process, there is a significant contradiction between PC conversion rate and PS yield: increasing the PC concentration can increase the PS output, but excessive PC can form micelles to wrap PLD and inhibit enzyme activity; reducing the PC concentration can improve the enzyme efficiency, but it leads to a sharp decrease in the PS yield. At the same time, the residual lysophosphatidylcholine in the reaction system can easily cause side reactions and reduce the purity of the product (literature confirms that when the PC concentration is >60wt%, the PS yield is generally <70%).

[0004] With the increasing requirement of high-end medical food for PS purity to >80%, the existing process cannot meet the requirements of high yield and high purity. Developing a new method to break the antagonistic relationship between PC and enzyme activity has urgent industrial value to meet market demand and reduce production cost. SUMMARY

[0005] The purpose of the present application is to provide a method for producing phosphatidylserine by enzyme catalysis, aiming to improve the content and yield of the product.

[0006] The purpose of the present application can be achieved by the following technical solutions: A method for producing phosphatidylserine by enzyme catalysis, comprising the following steps: In a reaction system of [BMIM][BF4] aqueous solution, natural phospholipids containing phosphatidylcholine and cephalin are used as raw materials, L-serine, calcium salt and phospholipase D are added for transacylation reaction; The mass content of cephalin in the natural phospholipids is 4% to 10%; The natural phospholipids further contain sphingomyelin, and the addition amount of sphingomyelin is 5% to 10% of the mass of the natural phospholipids.

[0007] Further, the mass content of cephalin in the natural phospholipids is 6% to 8%.

[0008] Further, before adding the phospholipase D, lysophosphatidylcholine is added to the system, and the addition amount of lysophosphatidylcholine is 0.1% to 0.3% of the mass of the natural phospholipids.

[0009] Furthermore, in the reaction system: The concentration of the [BMIM][BF4] aqueous solution is 0.8 mol / L; The mass ratio of the natural phospholipids to L-serine is 1:1.2 to 1.5; The amount of calcium salt added is 10% to 20% of the amount of natural phospholipids.

[0010] Furthermore, the temperature of the transacylation reaction is 50–55°C, and the reaction time is 6 hours.

[0011] Furthermore, the amount of phospholipase D added is 40 U / g of natural phospholipids.

[0012] Furthermore, the natural phospholipid is soybean phospholipid; the calcium salt is calcium chloride.

[0013] Furthermore, after the transacylation reaction is completed, purification treatment is performed: Step S1: Add an equal volume of 0.8 mol / L [BMIM][BF4] aqueous solution to the crude reaction product, centrifuge and collect the precipitate; Step S2: Add 95% ethanol to the precipitate in a volume equal to 3 times the volume of the [BMIM][BF4] aqueous solution in step S1, stir, and centrifuge to collect the precipitate. Step S3: The precipitate is dried under reduced pressure at 60-65℃ to obtain the phosphatidylserine product.

[0014] The beneficial effects of this invention are: This invention relates to a method for the enzyme-catalyzed production of phosphatidylserine (PS). By optimizing the reaction system and components, it resolves the antagonistic contradiction between phosphatidylcholine (PC) concentration and phospholipase D (PLD) activity in traditional processes. In conventional methods, increasing the PC concentration can increase PS yield, but PC easily forms micelles that encapsulate PLD, inhibiting enzyme activity; conversely, decreasing the PC concentration, while improving enzyme efficiency, leads to a sharp drop in PS yield, and residual lysophospholipids cause side reactions, making it difficult to meet market demands for PS purity >80%. This invention, by introducing a specific ratio of phosphatidylcholine and sphingomyelin into an ionic liquid [BMIM][BF4] aqueous solution system and optimizing reaction parameters, significantly improves PS purity and yield. The specific beneficial effects are analyzed as follows: (1) Overcoming the antagonistic relationship between PC concentration and enzyme activity to achieve high PS yield: Background technology indicates that traditional processes are limited by the conflict between PC and enzyme activity, resulting in PS yields generally below 70%. The method provided by this invention, through the synergistic effect of phospholipids and sphingomyelin, significantly increases the PS yield to 76.4%–87.7% (see Table 1 and Examples 2–5). This effect solves the core problem in traditional processes where "high PC concentration inhibits enzyme activity, and low PC concentration reduces yield."

[0015] (2) Significantly improve the purity of PS to meet the needs of high-end market: The test results of the present application show that the purity of PS is stably at 79.6% to 87.2%, and the improvement of purity is due to the addition of sphingomyelin (5% to 10% of the mass of natural phospholipid) and the purification process, which effectively inhibits the side reaction caused by lysophosphatidylcholine.

[0016] (3) Optimizing the ratio of brain phospholipid is the key to directly improve efficiency and product quality: The system of Example 1 of the present application tests the influence of brain phospholipid content (2% to 10%) (see Table 1): when the brain phospholipid is <4% (such as 2%), the PS yield is only 67.1% to 67.2%, and the purity is 81.4% to 81.8%, indicating that low brain phospholipid is not enough to alleviate the problem of PC micelles; when the brain phospholipid is 4% to 10%, the yield and purity are improved simultaneously: when the brain phospholipid is 6%, the purity of PS is 86.4% to 86.5%, and the yield is 83.7% to 83.8%; when the brain phospholipid is 8%, the purity is 85.3% to 85.5%, and the yield is 86.5% to 86.7%; when the brain phospholipid is >8% (such as 10%), the yield and purity decrease slightly (yield 81.2% to 81.5%, purity 81.7% to 82.0%), but still better than the traditional process. Data confirm that brain phospholipid in the range of 4% to 10% (preferably 6% to 8%) can effectively destroy the structure of PC micelles and avoid enzyme inhibition, which is the core improvement of the present application.

[0017] (4) The synergistic effect of sphingomyelin and lysophosphatidylcholine enhances the stability of the reaction: By adding sphingomyelin (5% to 10%) and trace amounts of lysophosphatidylcholine (0.1% to 0.3%), the test results show that: sphingomyelin as an auxiliary agent, may improve the reaction efficiency by stabilizing the conformation of PLD or improving the dispersion of the substrate (such as in Example 5, under the condition of 10% sphingomyelin, the yield is 87.7%); the addition of lysophosphatidylcholine (0.1% to 0.3% in Examples 1-5) may inhibit the side reaction.

[0018] (5) The process has strong scalability and is suitable for industrial production: The Examples 2-5 in the present application verify the scale-up effect from small test (200 mL) to pilot test (10 L): Example 2 (120 mL system): PS purity 87.2%, yield 84.3%; Example 5 (10 L system): PS purity 85.9%, yield 87.7%. The results show that, under the fixed parameters (such as [BMIM][BF4] concentration 0.8 mol / L, PLD addition amount 40 U / g, temperature 50-55°C, time 6 hours), the method has high robustness, small fluctuations in yield and purity (yield 76.4%-87.7%, purity 85.1%-87.2%), and solves the problem of efficiency decline in traditional process scale-up.

[0019] (6) Simplified and efficient purification process: Compared with the traditional solvent extraction method, the present application uses ionic liquid to improve the separation efficiency of the product by selective precipitation.

[0020] Summary: The present application realizes efficient and high-purity PS production (purity > 79.6%, yield > 76.4%) by introducing specific proportions of cephalin (4%-10%, preferably 6%-8%) and sphingomyelin (5%-10%) in combination with [BMIM][BF4] ionic liquid system, and completely solves the problem of incompatible yield-purity in traditional process. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. Meanwhile, the raw materials, reagents or devices used in the following embodiments, if not specifically stated, can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0022] Example 1 A method for enzymatic production of phosphatidylserine, comprising the following steps: To 200 mL of 0.8 mol / L aqueous [BMIM][BF4] solution, 50 g of soybean phospholipid with 75% phosphatidylcholine content and 2%, 4%, 6%, 8%, and 10% content of cephalin, respectively, 5 g of sphingomyelin, 75 g of L-serine, and 10 g of calcium chloride were added, and stirred at 500 rpm and heated to 50-55°C. Then, 0.1 g of lysophosphatidylcholine was added, and dissolved by stirring at 50°C (500 rpm, 10 min). Then, 4 mL of phospholipase D (40 U / g) was added, and reacted for 6 h. After completion of the reaction, the reaction crude product was collected by centrifugation. To the reaction crude product, 200 mL of 0.8 mol / L aqueous [BMIM][BF4] solution was added and stirred, and then the precipitate was collected by centrifugation. To the precipitate, 600 mL of 95% ethanol was added, stirred for 15 min, and then the precipitate was collected by centrifugation. The precipitate was dried at 65°C under reduced pressure to obtain the product. The content of the product was measured and the yield was calculated. The results are shown in Table 1 below.

[0023] Table 1

[0024] Example 2 To 120 mL of 0.8 mol / L aqueous [BMIM][BF4] solution, 30 g of soybean phospholipid with 75.8% phosphatidylcholine content and 8.43% content of cephalin, 3 g of sphingomyelin, 45 g of L-serine, and 6 g of calcium chloride were added, and stirred at 500 rpm and heated to 52°C. Then, 0.06 g of lysophosphatidylcholine was added, and dissolved by stirring at 50°C (500 rpm, 10 min). Then, 2.4 mL of phospholipase D (40 U / g) was added, and reacted for 6 h. After completion of the reaction, the reaction crude product was collected by centrifugation. To the reaction crude product, 120 mL of 0.8 mol / L aqueous [BMIM][BF4] solution was added and stirred, and then the precipitate was collected by centrifugation. To the precipitate, 360 mL of 95% ethanol was added, stirred for 15 min, and then the precipitate was collected by centrifugation. The precipitate was dried at 65°C under reduced pressure to obtain the product. The content of the product was measured and the yield was calculated. The content of phosphatidylserine in the product was 87.2%, and the yield was 84.3%.

[0025] Example 3 Into 1 L of 0.8 mol / L aqueous [BMIM][BF4] solution, 250 g of soybean phospholipid with phosphatidylcholine content of 70.5% and plasmalogen content of 8.89%, 25 g of sphingomyelin, 375 g of L-serine and 50 g of calcium chloride were added, stirred uniformly at 500 rpm and heated to 52°C, then 0.5 g of lysophosphatidylcholine was added, dissolved by stirring at 50°C (500 rpm, 10 min), then 16 mL of phospholipase D (40 U / g) was added, reacted for 6 h, after completion, the reaction crude product was collected by centrifugation, 1 L of 0.8 mol / L aqueous [BMIM][BF4] solution was added to the reaction crude product and stirred, then the precipitate was collected by centrifugation, 3 L of 95% ethanol was added to the precipitate, stirred for 15 min, then the precipitate was collected by centrifugation, and the precipitate was dried at 65°C under reduced pressure to obtain the product, the content of the product was detected and the yield was calculated, and the detection showed that the content of phosphatidylserine in the product was 85.7% and the yield was 76.4%.

[0026] Example 4 Into 1.5 L of 0.8 mol / L aqueous [BMIM][BF4] solution, 375 g of soybean phospholipid with phosphatidylcholine content of 73.2% and plasmalogen content of 7.14%, 37.5 g of sphingomyelin, 552.5 g of L-serine and 75 g of calcium chloride were added, stirred uniformly at 500 rpm and heated to 52°C, then 0.75 g of lysophosphatidylcholine was added, dissolved by stirring at 50°C (500 rpm, 10 min), then 24 mL of phospholipase D (40 U / g) was added, reacted for 6 h, after completion, the reaction crude product was collected by centrifugation, 1.5 L of 0.8 mol / L aqueous [BMIM][BF4] solution was added to the reaction crude product and stirred, then the precipitate was collected by centrifugation, 4.5 L of 95% ethanol was added to the precipitate, stirred for 15 min, then the precipitate was collected by centrifugation, and the precipitate was dried at 65°C under reduced pressure to obtain the product, the content of the product was detected and the yield was calculated, and the detection showed that the content of phosphatidylserine in the product was 85.1% and the yield was 77.5%.

[0027] Example 5 To 10L 0.8mol / L of [BMIM][BF4] aqueous solution, 2.5kg of soybean phospholipid with phosphatidylcholine content of 76.3% and plasmalogen content of 6.02%, 250g of sphingomyelin, 3.75kg of L-serine and 500g of calcium chloride were added, and stirred at 500rpm and heated to 52℃, then 5g of lysophosphatidylcholine was added, and dissolved at 50℃ (500rpm, 10min), then 160mL of phospholipase D (40U / g) was added, and reacted for 6h, after completion, the reaction crude product was collected by centrifugation, 10L of 0.8mol / L [BMIM][BF4] aqueous solution was added to the reaction crude product and stirred, then the precipitate was collected by centrifugation, 30L of 95% ethanol was added to the precipitate, stirred for 15min, then the precipitate was collected by centrifugation, and the precipitate was dried at 65℃ under reduced pressure to obtain the product, the product was detected and the yield was calculated, and the detection showed that the phosphatidylserine content in the product was 85.9%, and the yield was 87.7%.

[0028] Principle analysis of the application: The core principle of the application is to utilize the remolding reaction microenvironment of the cerbroside-sphingomyelin-ionic liquid ternary synergistic system to break through the inhibition of PC micelles on PLD. The specific mechanism is inferred according to the data of the examples: (1) The role of cerbroside: Example 1 shows that the reaction is optimized when the cerbroside is 4%-10%. The cerbroside may be embedded in the PC micelles (the background technology indicates that PC is easy to form micelles), destroy the dense structure, and reduce the PLD wrapping (for example, when the cerbroside is 8%, the yield is 86.7%); when it is too low (2%), the micelles inhibit obviously (the yield is 67.2%), and when it is too high (10%), a competitive substrate may be introduced, resulting in a slight decrease in efficiency.

[0029] (2) The auxiliary function of sphingomyelin: Sphingomyelin (5%-10%) may enhance the thermal stability of PLD (reaction temperature 50-55℃), and the specific reference is Example 5 (yield 87.7%) and Example 3 (still maintains a yield of 76.4% in large scale). As an amphoteric molecule, sphingomyelin may promote the contact of the substrate (PC and L-serine) with the enzyme by adjusting the interfacial tension.

[0030] (3) The key role of [BMIM][BF4] ionic liquid: As a reaction medium, its aqueous solution (0.8 mol / L) may maintain the conformational activity of PLD. The concentration is used uniformly in Examples 1-5, and the purity of PS is all >79.6%, while the enzyme is easy to be inactivated in the traditional aqueous phase system. The ionic liquid is also beneficial to the selective precipitation of impurities in the purification stage (for example, the purity of PS after purification in Example 4 is 85.1%).

[0031] (4) The regulation of trace lysophosphatidylcholine: The addition amount of 0.1%-0.3% may neutralize the side reaction of residual lysophosphatidylcholine.

[0032] (5) Optimization of calcium salt and temperature parameters: calcium salt (10%~20%) as cofactor to stabilize enzyme activity; temperature 50~55℃ (uniformly used in examples) to balance reaction rate and enzyme stability, avoiding inactivation at high temperature.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; for example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It must be noted that as used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Terms such as "comprise", "comprising", "comprises", "include", "including", "includes", "contain", "containing", "contains", "characterized by" and the like are to be understood as open-ended terms (i.e., conforming to the meaning

[0034] While the embodiments of the application have been shown and described herein, it is to be understood that the application is not limited to these embodiments. Rather, many modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the application as defined by the appended claims and their equivalents.

Claims

1. A method for the enzymatic production of phosphatidylserine, characterized in that, The method comprises the following steps: In a reaction system of [BMIM][BF4] aqueous solution, taking natural phospholipid containing phosphatidylcholine and cephalin as raw material, adding L-serine, calcium salt and phospholipase D to carry out transacylation reaction; The mass content of cephalin in the natural phospholipid is 4%-10%; The natural phospholipid further contains sphingomyelin, and the addition amount of sphingomyelin is 5%-10% of the mass of the natural phospholipid.

2. The method of claim 1, wherein the enzyme catalyzes the production of phosphatidylserine, and wherein the enzyme is a phosphatidylserine synthase. The mass content of cephalin in the natural phospholipid is 6%-8%.

3. The method of claim 1, wherein the enzyme catalyzing the production of phosphatidylserine is a phosphatidylserine synthase. Before adding phospholipase D, add lysophosphatidylcholine to the system, and the addition amount of lysophosphatidylcholine is 0.1%-0.3% of the mass of the natural phospholipid.

4. The method of claim 1, wherein the enzyme catalyzing the production of phosphatidylserine is a phosphatidylserine synthase. In the reaction system: The concentration of the [BMIM][BF4] aqueous solution is 0.8 mol / L; The mass ratio of the natural phospholipid to L-serine is 1:1.2-1.5; The addition amount of the calcium salt is 10%-20% of the mass of the natural phospholipid.

5. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, The temperature of the transacylation reaction is 50-55 DEG C, and the reaction time is 6 hours.

6. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, The addition amount of the phospholipase D is 40 U / g of the natural phospholipid.

7. The method of claim 1, wherein the enzyme is a phosphatidylserine synthase. The natural phospholipid is soybean phospholipid, and the calcium salt is calcium chloride.

8. The method of claim 1, wherein the enzyme catalyzing the production of phosphatidylserine is a phosphatidylserine synthase. After the transacylation reaction is completed, purification treatment is carried out: Step S1, add 0.8 mol / L [BMIM][BF4] aqueous solution with the same volume as the reaction system to the reaction crude product, and centrifuge to obtain the precipitate; Step S2, add 95% ethanol with a volume of 3 times the volume of the [BMIM][BF4] aqueous solution in step S1 to the precipitate, stir and then centrifuge to obtain the precipitate; Step S3, dry the precipitate at 60-65 DEG C under reduced pressure to obtain phosphatidylserine product.

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