A method for the enzymatic production of phosphatidylserine

Abstract

The application discloses an enzyme catalysis production method of phosphatidylserine, and belongs to the technical field of biology, and specifically comprises the following steps: in a reaction system of [BMIM][BF4] aqueous solution, taking natural phosphatide containing phosphatidylcholine and cephalin as raw materials, adding L-serine, calcium salt and phospholipase D to carry out a transacylation reaction. By introducing specific proportions of cephalin (4%-10%, preferably 6%-8%) and sphingomyelin (5%-10%), in combination with the [BMIM][BF4] ionic liquid system, the application realizes efficient and high-purity (purity>79.6%, yield>76.4%) production of PS, and completely solves the problem that yield and purity cannot be considered simultaneously in the traditional process.

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 damage, 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 lysophospholipid 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:

[0007] A method for producing phosphatidylserine by enzyme catalysis, comprising the following steps:

[0008] In the 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;

[0009] The mass content of cephalin in the natural phospholipids is 4% to 10%;

[0010] The natural phospholipids further contain sphingomyelin, and the addition amount of sphingomyelin is 5% to 10% of the mass of the natural phospholipids.

[0011] Further, the mass content of cephalin in the natural phospholipids is 6% to 8%.

[0012] Furthermore, before adding phospholipase D, lysophosphatidylcholine is added to the system at an amount of 0.1% to 0.3% of the natural phospholipid content.

[0013] Furthermore, in the reaction system:

[0014] The concentration of the [BMIM][BF4] aqueous solution is 0.8 mol / L;

[0015] The mass ratio of the natural phospholipids to L-serine is 1:1.2 to 1.5;

[0016] The amount of calcium salt added is 10% to 20% of the amount of natural phospholipids.

[0017] Furthermore, the temperature of the transacylation reaction is 50–55°C, and the reaction time is 6 hours.

[0018] Furthermore, the amount of phospholipase D added is 40 U / g of natural phospholipids.

[0019] Furthermore, the natural phospholipid is soybean phospholipid; the calcium salt is calcium chloride.

[0020] Furthermore, after the transacylation reaction is completed, purification treatment is performed:

[0021] 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;

[0022] 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.

[0023] Step S3: The precipitate is dried under reduced pressure at 60-65℃ to obtain the phosphatidylserine product.

[0024] The beneficial effects of this invention are:

[0025] 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:

[0026] (1) Overcoming the antagonistic relationship between PC concentration and enzyme activity to achieve high PS yield:

[0027] 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."

[0028] (2) Significantly improves PS purity to meet the demands of the high-end market:

[0029] The test results of this invention show that the purity of PS is stable at 79.6% to 87.2%. The improvement in purity is due to the addition of sphingomyelin (5% to 10% of the natural phospholipid content) and the purification process, which effectively inhibits the side reactions caused by lysophospholipids.

[0030] (3) Optimizing the ratio of phospholipids is key, directly improving efficiency and product quality:

[0031] In Example 1 of this invention, the effect of phospholipid content (2%–10%) was systematically tested (see Table 1): When phospholipid content was <4% (e.g., 2%), the PS yield was only 67.1%–67.2%, and the purity was 81.4%–81.8%, indicating that low phospholipid content was insufficient to alleviate the PC micelle problem; when the phospholipid content was 4%–10%, the yield and purity increased simultaneously: with 6% phospholipid content, the PS purity was 86.4%–86.5%, and the yield was 83.7%–83.8%; with 8% phospholipid content, the purity was 85.3%–85.5%, and the yield was 86.5%–86.7%; when phospholipid content was >8% (e.g., 10%), the yield and purity decreased slightly (yield 81.2%–81.5%, purity 81.7%–82.0%), but were still better than the traditional process. Data confirms that phospholipids in the range of 4% to 10% (preferably 6% to 8%) can effectively disrupt the PC micelle structure and avoid enzyme inhibition, which is the core improvement of this invention.

[0032] (4) The synergistic effect of sphingomyelin and lysophosphatidylcholine enhances reaction stability:

[0033] This invention, by adding sphingomyelin (5%–10%) and trace amounts of lysophosphatidylcholine (0.1%–0.3%), shows that: sphingomyelin, as an adjuvant, may improve reaction efficiency by stabilizing the PLD conformation or improving substrate dispersibility (e.g., in Example 5, the yield was 87.7% under the condition of 10% sphingomyelin); the addition of lysophosphatidylcholine (0.1%–0.3% in Examples 1–5) may inhibit side reactions.

[0034] (5) The process is highly scalable and suitable for industrial production:

[0035] Examples 2-5 of this invention verified the scale-up effect from small-scale (200 mL) to pilot-scale (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 fixed parameters (e.g., [BMIM][BF4] concentration 0.8 mol / L, PLD addition 40 U / g, temperature 50-55℃, time 6 hours), the method is highly robust, with small fluctuations in yield and purity (yield 76.4%-87.7%, purity 85.1%-87.2%), solving the problem of efficiency decline during scale-up of traditional processes.

[0036] (6) The purification process is simplified and efficient:

[0037] Compared to traditional solvent extraction methods, this invention uses ionic liquids to improve product separation efficiency through selective precipitation.

[0038] In summary, this invention achieves high efficiency and high purity (purity >79.6%, yield >76.4%) in PS production by introducing specific proportions of phospholipids (4%–10%, preferably 6%–8%) and sphingomyelin (5%–10%), combined with the [BMIM][BF4] ionic liquid system, thus completely solving the problem of yield and purity being mutually exclusive in traditional processes. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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. Furthermore, unless otherwise specified, the raw materials, reagents, or devices used in the following embodiments can be obtained from conventional commercial channels or by existing known methods.

[0040] Example 1

[0041] A method for enzyme-catalyzed production of phosphatidylserine includes the following steps:

[0042] Add 50g of soybean lecithin (75% phosphatidylcholine content and 2%, 4%, 6%, 8%, and 10% phospholipid content), 5g of sphingomyelin, 75g of L-serine, and 10g of calcium chloride to 200mL of 0.8mol / L [BMIM][BF4] aqueous solution. Stir at 500rpm until homogeneous and heat to 50-55℃. Then add 0.1g of lysophosphatidylcholine and stir at 50℃ for 10min (500rpm). Add 4mL of phospholipase D (40U / g) and react for 6h. After the reaction is complete, centrifuge to collect the crude product. Add 200mL of 0.8mol / L [BMIM][BF4] aqueous solution to the crude product and stir. Centrifuge again to collect the precipitate. Add 600mL of 95% ethanol to the precipitate and stir for 15min. Centrifuge again to collect the precipitate. Dry the precipitate under reduced pressure at 65℃ to obtain the product. Detect the product content and calculate the yield. The results are shown in Table 1 below.

[0043] Table 1

[0044]

[0045] Example 2

[0046] Add 30g of soybean lecithin (75.8% phosphatidylcholine and 8.43% cephalin), 3g of sphingomyelin, and 45g of [BMIM][BF4] to 120mL of 0.8mol / L aqueous solution. L-serine and 6g of calcium chloride were stirred at 500 rpm until homogeneous and heated to 52℃. Then, 0.06g of lysophosphatidylcholine was added and stirred at 50℃ for 10 min (500 rpm). 2.4mL of phospholipase D (40U / g) was added and the reaction was allowed to proceed for 6 h. After the reaction was complete, the crude product was collected by centrifugation. 120mL of 0.8mol / L [BMIM][BF4] aqueous solution was added to the crude product and stirred. The precipitate was collected by centrifugation. 360mL of 95% ethanol was added to the precipitate and stirred for 15 min. The precipitate was collected by centrifugation again and dried under reduced pressure at 65℃ to obtain the product. The product content was determined and the yield was calculated. The phosphatidylserine content in the product was 87.2%, and the yield was 84.3%.

[0047] Example 3

[0048] Add 250g of soybean lecithin (70.5% phosphatidylcholine and 8.89% cephalin), 25g of sphingomyelin, and 375g of [BMIM][BF4] to 1L of 0.8mol / L [BMIM][BF4] aqueous solution. L-serine and 50g calcium chloride were stirred at 500rpm until homogeneous and heated to 52℃. Then, 0.5g lysophosphatidylcholine was added and stirred at 50℃ for 10min (500rpm). 16mL of phospholipase D (40U / g) was added and the reaction was allowed to proceed for 6h. After the reaction was complete, the crude product was collected by centrifugation. 1L of 0.8mol / L [BMIM][BF4] aqueous solution was added to the crude product and stirred. The precipitate was collected by centrifugation. 3L of 95% ethanol was added to the precipitate and stirred for 15min. The precipitate was collected by centrifugation again and dried under reduced pressure at 65℃ to obtain the product. The product content was detected and the yield was calculated. The phosphatidylserine content in the product was 85.7%, and the yield was 76.4%.

[0049] Example 4

[0050] Add 375g of soybean lecithin (73.2% phosphatidylcholine and 7.14% cephalin), 37.5g of sphingomyelin, 552.5g of L-serine, and 75g of calcium chloride to 1.5L of 0.8mol / L [BMIM][BF4] aqueous solution. Stir at 500rpm until homogeneous and heat to 52℃. Then add 0.75g of lysophosphatidylcholine and stir at 50℃ for 10min (500rpm). Add 24mL of phospholipase D (40U / g) and react for 6h. After reaction, centrifuge to collect the crude reaction product. Add 1.5L of [BMIM][BF4] aqueous solution to the crude reaction product. The precipitate was collected by stirring a 0.8 mol / L [BMIM][BF4] aqueous solution, centrifuging, adding 4.5 L of 95% ethanol, stirring for 15 min, centrifuging again, and drying the precipitate under reduced pressure at 65 °C to obtain the product. The product content was detected and the yield was calculated. The phosphatidylserine content in the product was 85.1%, and the yield was 77.5%.

[0051] Example 5

[0052] Add 2.5 kg of soybean lecithin (76.3% phosphatidylcholine and 6.02% cephalin), 250 g of sphingomyelin, and 3.75 kg of [BMIM][BF4] to 10 L of 0.8 mol / L [BMIM][BF4] aqueous solution. L-serine and 500g calcium chloride were stirred at 500rpm until homogeneous and heated to 52℃. Then, 5g of lysophosphatidylcholine was added and stirred at 50℃ for 10min (500rpm). 160mL of phospholipase D (40U / g) was added and the reaction was allowed to proceed for 6h. After the reaction was completed, the crude product was collected by centrifugation. 10L of 0.8mol / L [BMIM][BF4] aqueous solution was added to the crude product and stirred. The precipitate was collected by centrifugation. 30L of 95% ethanol was added to the precipitate and stirred for 15min. The precipitate was collected by centrifugation again and dried under reduced pressure at 65℃ to obtain the product. The product content was detected and the yield was calculated. The phosphatidylserine content in the product was 85.9%, and the yield was 87.7%.

[0053] Principle analysis of the invention:

[0054] The core principle of this invention lies in utilizing a ternary synergistic system of phospholipids-sphingomyelin-ionic liquid to reshape the reaction microenvironment, thereby overcoming the inhibition of PLD by PC micelles. The specific mechanism is inferred from the data in the embodiments:

[0055] (1) Effect of phosphatidylcholine: Example 1 shows that the reaction is optimized when phosphatidylcholine is present at 4%–10%. Phosphatidylcholine may embed into PC micelles (background art indicates that PC is prone to forming micelles), disrupting its dense structure and reducing PLD encapsulation (e.g., yield of 86.7% when phosphatidylcholine is present at 8%); micelle inhibition is significant when the concentration is too low (2%) (yield of 67.2%), and competition from substrates may be introduced when the concentration is too high (10%), resulting in a slight decrease in efficiency.

[0056] (2) Auxiliary function of sphingomyelin: Sphingomyelin (5%–10%) may enhance the thermal stability of PLD (reaction temperature 50–55°C), as specifically seen in Example 5 (yield 87.7%) and Example 3 (yield maintained at 76.4% even on a large scale). As an amphoteric molecule, sphingomyelin may promote the contact between substrates (PC and L-serine) and enzymes by regulating interfacial tension.

[0057] (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. This concentration was used in Examples 1-5, and the purity of PS was >79.6% in all cases, while the enzyme was easily inactivated in the traditional aqueous system. Ionic liquid is also beneficial for selectively precipitating impurities during the purification stage (e.g., the purity of PS after purification in Example 4 was 85.1%).

[0058] (4) Regulation of trace amounts of lysophosphatidylcholine: Adding 0.1% to 0.3% may neutralize the side effects of residual lysophosphatidylcholine.

[0059] (5) Optimization of calcium salt and temperature parameters: Calcium salt (10% to 20%) is used as a cofactor to stabilize enzyme activity; temperature of 50 to 55℃ (used uniformly in the examples) balances reaction rate and enzyme stability, and avoids high temperature inactivation.

[0060] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the enzyme-catalyzed production of phosphatidylserine, characterized in that, Includes the following steps: In the reaction system of [BMIM][BF4] aqueous solution, soybean lecithin containing phosphatidylcholine and cephalin was used as raw material, and L-serine, calcium salt and phospholipase D were added to carry out the transacylation reaction. Before adding phospholipase D, add lysophosphatidylcholine to the system at an amount of 0.1% to 0.3% of the soybean phospholipid content. The soybean phospholipids contain 4% to 10% by mass; The raw materials also include sphingomyelin, and the amount of sphingomyelin added is 5% to 10% of the soybean phospholipid content.

2. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, The soybean phospholipids contain 6% to 8% cephalin by mass.

3. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, In the reaction system: The concentration of the [BMIM][BF4] aqueous solution is 0.8 mol / L; The mass ratio of soybean phospholipids to L-serine is 1:1.2 to 1.5; The amount of calcium salt added is 10% to 20% of the soybean phospholipid content.

4. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, The transacylation reaction was carried out at a temperature of 50–55°C for 6 hours.

5. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, The amount of phospholipase D added is 40 U / g soybean phospholipid.

6. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, The calcium salt is calcium chloride.

7. The method for enzyme-catalyzed production of phosphatidylserine according to claim 1, characterized in that, After the transacylation reaction is completed, purification 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.

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