Method for preparing high-purity phosphatidylserine by batch enzyme catalysis method based on dynamic choline removal

Abstract

The invention provides a method for preparing high-purity phosphatidylserine by a batch enzyme catalysis method based on dynamic choline removal, and belongs to the technical field of enzymatic reaction. According to the method disclosed by the invention, in a reaction system for preparing phosphatidylserine, a byproduct choline is selectively adsorbed by adopting resin. Choline in a reaction system is removed in real time through the integrated adsorption separation module, the enzyme inhibition problem is solved, and meanwhile, the PC conversion rate is larger than or equal to 98% and the PS purity is larger than or equal to 96% in combination with the immobilized enzyme circulation technology; the enzyme can be reused for more than or equal to 20 times (activity retention is more than or equal to 90%); and the choline residual concentration is less than or equal to 0.5 mM (an inhibition threshold value or less).

Description

Technical Field

[0001] The present invention relates to the technical field of enzymatic reactions, and in particular to a method for preparing high-purity phosphatidylserine by a batch enzymatic catalysis method based on the dynamic removal of choline. Background Art

[0002] Phosphatidylserine, also known as complex neuraminic acid (PS), is extracted from the residue left over from soybean oil extraction. It is an active substance in cell membranes, particularly in brain cells. Its primary function is to improve nerve cell function, regulate the conduction of nerve impulses, and enhance memory. Due to its strong lipophilicity, it quickly crosses the blood-brain barrier after absorption and enters the brain, soothing vascular smooth muscle cells and increasing blood flow to the brain.

[0003] In traditional enzyme-catalyzed PS production, the accumulation of the byproduct choline significantly reduces phosphatase D activity through competitive inhibition, leading to a decrease in conversion rate (late-stage conversion rate <80%). In batch processes, choline removal is difficult to achieve in real time, requiring the addition of excess serine (molar ratio >1:6) to drive the reaction equilibrium, significantly increasing costs. Residual choline also accelerates the inactivation of the immobilized enzyme, limiting its reuse (≤10 times). Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of choline inhibition effect and barrier to reuse of immobilized enzyme.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing high-purity phosphatidylserine by a batch enzyme catalysis method based on the dynamic removal of choline. In the reaction system for preparing phosphatidylserine, resin is used to selectively adsorb the byproduct choline.

[0007] Preferably, the reaction system for preparing phosphatidylserine comprises: soybean lecithin, L-serine, magnetically immobilized phosphatase D, an aqueous phase and an organic phase.

[0008] Preferably, the soybean lecithin is a product with a phosphatidylcholine content greater than 90%; the molar ratio of the phosphatidylcholine to L-serine is 1:3-4.

[0009] Preferably, the mass ratio of the soybean lecithin to the magnetically immobilized phosphatase D is 10:1-20, the loading amount of the phosphatase D in the magnetically immobilized phosphatase D is ≥150 mg / g, and the concentration of the phosphatase D is 0.01-5 U / g.

[0010] Preferably, the aqueous phase is an acetate buffer or a citrate buffer at pH 5.8-6.2, containing 0.2-1.5 mM Ca 2 +, the organic phase is ethyl acetate:n-heptane=1:1-3 (v / v).

[0011] Preferably, the resin includes but is not limited to Amberlite IR-120H+, macroporous strong base type I resin or 344; The resin is placed in the reactor outlet circulation pipeline for selectively adsorbing the by-product choline in the reaction solution.

[0012] Preferably, the flow rate of the reaction solution flowing through the resin is 0.8 to 1.6 BV / h.

[0013] Preferably, the present invention provides a method for preparing high-purity phosphatidylserine by a batch enzymatic method based on dynamic removal of choline, comprising the following steps:

[0014] (1) mixing soybean lecithin with the organic phase to obtain a mixed solution 1, and mixing L-serine with the aqueous phase to obtain a mixed solution 2;

[0015] (2) The magnetic immobilized phosphatase D is distributed in the filter disk of the reactor, the resin is placed in the reactor outlet circulation pipeline, the mixed solution 1 and the mixed solution 2 are added, and the reaction is started.

[0016] Preferably, the mass volume ratio of the soybean lecithin to the organic phase is 10g:30-100ml; the mass volume ratio of the L-serine to the aqueous phase is 4.1g:50-150ml; and the loading amount of the resin is 10-100ml / reaction;

[0017] Preferably, the reaction temperature is 42-45° C., and the reaction time is 8-10 h.

[0018] The present invention has the following beneficial effects:

[0019] (1) The present invention utilizes a resin (preferably a cation exchange resin) to selectively adsorb choline (Kd = 120 mL / g), dynamically removing choline and maintaining the choline concentration in the reaction solution ≤ 0.3 mM to eliminate the inhibitory effect of choline on the enzymatic reaction. In a comparative experiment without choline removal, the conversion rate dropped to 75% in the late reaction phase, while the present invention achieved a conversion rate of >98% throughout the entire reaction.

[0020] (2) The present invention combines magnetic immobilized enzyme microspheres with adsorption columns to achieve dual circulation control of enzyme and choline. After the enzyme is reused 20 times, the activity is retained at 92%.

[0021] (3) The real-time removal of choline shifts the reaction equilibrium to the right, and the amount of serine is reduced to a molar ratio of 1:3, while the traditional process requires 1:6. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] 1. Raw materials and reaction system

[0025] Raw materials: high-purity soybean (PC≥90%), L-serine.

[0026] Solvent system: aqueous phase (pH 5.8-6.2 acetic acid-sodium acetate buffer (0.2M), containing 0.8mM Ca 2 +), organic phase (ethyl acetate:n-heptane=1:2 v / v).

[0027] Preparation of immobilized phospholipase D: Silica, magnetite powder, γ-aminopropyltriethoxysilane, and phospholipase D solution (mass ratio of 5:3:5:2) were shaken at 0°C for 4 hours. Ethanol chilled at -10°C was then rapidly added dropwise to precipitate the solution. The precipitate was collected and cross-linked with glutaraldehyde at a mass ratio of 2.5:1. After washing, immobilized phospholipase D was obtained. Phospholipase D was immobilized on magnetic mesoporous silica microspheres (particle size 50-100 μm, pore size 10 nm, surface modified with carboxyl groups) with an enzyme loading of ≥150 mg / g.

[0028] Strong acid cation exchange resin: Amberlite IR-120H+ type, the resin can be regenerated by acid and alkali elution and reused ≥50 times.

[0029] 2. Operation process

[0030] Take 100 g of soybean lecithin with a soybean phosphatidylcholine content of 92%, add 300 ml of organic phase (ethyl acetate:n-heptane=1:2 v / v), stir and dissolve, and set aside.

[0031] Add 41g of L-serine and 10g of calcium chloride to 500ml of 0.2M sodium acetate-acetate buffer, stir and dissolve to obtain an L-serine solution for later use. The molar ratio of soybean phosphatidylcholine to L-serine is 1:3.2.

[0032] Take 150g of immobilized enzyme microspheres (0.2u / g), evenly distribute them on the filter disk, cover the filter disk and fix it. Set aside.

[0033] Take the activated macroporous resin, wet pack it into a column with a volume of 500 ml, drain the liquid, and place it in the circulation pipeline at the outlet of the reactor for standby use.

[0034] Connect the reaction apparatus to the circulation apparatus, add L-serine solution, place the immobilized enzyme microspheres at the interface of the serine aqueous solution, start stirring (300 rpm), raise the temperature to 45°C, add the organic phase containing soybean lecithin, and react. The reaction solution is passed through the adsorption column at a rate of 0.8-1.6 BV / h. The reaction is carried out for 9 hours (the choline concentration is monitored every 30 minutes). After the reaction is completed, the immobilized enzyme microspheres are recovered by magnetic separation (recovery rate >99%), and the organic phase is separated by centrifugation (6000 rpm, 10 minutes). The organic phase is concentrated by vacuum evaporation (50°C, -0.08 MPa), and the organic solvent is recovered until the residue becomes viscous. 300 ml of water is added and stirred, and after filtering, 200 ml of anhydrous ethanol is added to the filter residue, stirred, and filtered. The filter residue is dried under reduced pressure at 60°C for 4 hours to obtain the finished PS product.

[0035] After measurement: PC conversion rate: 98.6%;

[0036] PS purity: 91% (HPLC);

[0037] Choline residue: 0.28 mM (ion chromatography);

[0038] The enzyme activity retention after 20 reuses was 91.3%.

[0039] Example 2 (pilot scale-up)

[0040] Equipment: 200 L reactor, adsorption column (20 L volume) connected in series, other conditions were scaled up according to Example 1;

[0041] Production capacity: The single batch output of PS is about 185kg, with an average of 2 batches per day, and the regeneration cycle of the choline adsorption column is 10 batches.

[0042] Compared with the traditional process cost: serine consumption is reduced by 40%, and the enzyme cost ratio is reduced from 35% to 12%.

[0043] Comparative Example 1

[0044] To a 100mL beaker, add 50mL of pH 5 acetic acid-sodium acetate buffer, 10g of phosphatidylcholine (PC60) and L-serine at a molar ratio of 1:10, and 1g of calcium chloride. The mixture was heated to 45°C. After the substrates were completely dissolved, 500U of phospholipase D enzyme solution was added. The reaction was allowed to proceed for 5 hours at 250rpm. After completion of the reaction, the PS yield and content were measured. The PS yield was 65%, and the PS content was 65%.

[0045] Comparative Example 2

[0046] To a 100mL beaker, add 50mL of pH 5 acetic acid-sodium acetate buffer, 10g of phosphatidylcholine (PC90) and L-serine in a molar ratio of 1:10, and 1g of calcium chloride. The mixture is heated to 45°C. After the substrates are completely dissolved, 900U of phospholipase D enzyme solution is added. The reaction is allowed to proceed for 8 hours at 250rpm. However, little reaction occurs, and the product cannot be isolated. This is because the higher the purity of the phosphatidylcholine in the soybean lecithin, the greater the steric hindrance. This results in the inability of phospholipase D to catalyze the hydrolysis of the ester bond between phosphate and choline in the phospholipid molecule (releasing choline) and base exchange.

[0047] As can be seen from the above examples, the present invention solves the problem of enzyme inhibition by removing choline from the reaction system in real time through an integrated adsorption separation module. At the same time, combined with immobilized enzyme recycling technology, it achieves a PC conversion rate of ≥98% and a PS purity of ≥96%; the enzyme is reused ≥20 times (activity retention ≥90%); and the residual choline concentration is ≤0.5 mM (below the inhibition threshold).

[0048] In summary, the present invention has the following advantages:

[0049] 1. High efficiency conversion: after the choline inhibition is released, the reaction conversion rate increases by more than 20%;

[0050] 2. Low cost: Reduce the amount of serine and the frequency of enzyme supplementation, and reduce the overall cost by 30%;

[0051] 3. Green production: Choline adsorption resin can be recycled and the solvent recovery rate is >95%, which meets environmental protection requirements.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing high-purity phosphatidylserine by a batch enzymatic method based on dynamic removal of choline, characterized in that: In the reaction system for preparing phosphatidylserine, resin is used to selectively adsorb the by-product choline.

2. The method according to claim 1, wherein The reaction system for preparing phosphatidylserine comprises: soybean lecithin, L-serine, magnetically immobilized phosphatase D, an aqueous phase and an organic phase.

3. The method according to claim 2, wherein The soybean lecithin is a product with a phosphatidylcholine content greater than 90%; the molar ratio of the phosphatidylcholine to L-serine is 1:3-4.

4. The method according to claim 3, wherein The mass ratio of the soybean lecithin to the magnetically immobilized phosphatase D is 10:1-20, the loading amount of the phosphatase D in the magnetically immobilized phosphatase D is ≥150 mg / g, and the concentration of the phosphatase D is 0.01-5 U / g.

5. The method according to claim 4, wherein The aqueous phase is a pH 5.8-6.2 acetate buffer or citrate buffer containing 0.2-1.5 mM Ca 2 +, the organic phase is ethyl acetate:n-heptane=1:1-3 (v / v).

6. The method according to claim 5, wherein The resin includes but is not limited to Amberlite IR-120H+, macroporous strong base type I resin or 344; The resin is placed in the reactor outlet circulation pipeline for selectively adsorbing the by-product choline in the reaction solution.

7. The method according to claim 6, wherein The flow rate of the reaction solution flowing through the cation exchange resin is 0.8 to 1.6 BV / h.

8. The method according to any one of claims 1 to 7, wherein: The steps include: (1) mixing soybean lecithin with the organic phase to obtain a mixed solution 1, and mixing L-serine with the aqueous phase to obtain a mixed solution 2; (2) The magnetic immobilized phosphatase D is distributed in the filter disk of the reactor, the resin is placed in the reactor outlet circulation pipeline, the mixed solution 1 and the mixed solution 2 are added, and the reaction is started.

9. The method according to claim 8, wherein The mass volume ratio of the soybean lecithin to the organic phase is 10g:30-100ml; the mass volume ratio of the L-serine to the aqueous phase is 4.1g:50-150ml; and the loading amount of the resin is 10-100ml / reaction.

10. The method according to claim 9, wherein The reaction temperature is 42-45° C., and the reaction time is 8-10 h.