阅读说明：本技术 一种高纯度甘油磷酸二酯的制备方法 (Preparation method of high-purity diglyceride phosphate ) 是由 王方华 王永华 闵雪珂 杨博 王卫飞 于 2021-01-15 设计创作，主要内容包括：本发明属于酶的基因工程技术领域,公开了一种高纯度甘油磷酸二酯的制备方法,以溶血磷脂为原料,以金属离子螯合剂处理的pfGDPD为催化剂,在水相体系中水解溶血磷脂,得到甘油磷酸二酯产物；所述pfGDPD的氨基酸序列为SEQID No:1。本发明从溶血磷脂出发,通过单一酶法水解一步可制备获得高纯度的甘油磷酸二酯(如GPC,GPE,GPI等),转化率接近100％。本发明为甘油磷酸二酯的生产提供了一种新的思路和方法。(The invention belongs to the technical field of enzyme genetic engineering, and discloses a preparation method of high-purity diglyceride phosphate, which takes lysophospholipid as a raw material and pfGDPD treated by a metal ion chelating agent as a catalyst to hydrolyze the lysophospholipid in a water phase system to obtain a diglyceride phosphate product; the amino acid sequence of the pfGDPD is SEQID No: 1. The invention starts from lysophospholipid, can prepare high-purity diglyceride phosphate (such as GPC, GPE, GPI and the like) by one-step single enzymatic hydrolysis, and has the conversion rate close to 100 percent. The invention provides a new idea and method for the production of diglyceride phosphate.)

1. A preparation method of high-purity diglyceride phosphate is characterized in that lysophospholipid is used as a raw material, pfGDPD treated by a metal ion chelating agent is used as a catalyst, and the lysophospholipid is hydrolyzed in an aqueous phase system to obtain a diglyceride phosphate product; the amino acid sequence of the pfGDPD is SEQ ID No. 1.

2. The process according to claim 1, wherein the high-purity diglyceride of glycerophosphate is prepared by dissolving lysophospholipid in a buffer solution having a pH of 8.0, and adding pfGDPD treated with a chelating agent to the reaction system at a temperature of 30 to 90 ℃ to conduct an enzymatic hydrolysis reaction.

3. The method according to claim 2, wherein the buffer solution is a boric acid buffer.

4. The method according to claim 1, wherein the lysophospholipid is one or more of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidylglycerol.

5. The method according to claim 2, wherein the lysophospholipid is one or more of lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidylglycerol.

6. The method according to claim 1 or 2 or 3 or 4 or 5, wherein the treatment temperature of the metal ion chelating agent is 4-90 ℃, the treatment time is 1-12h, and the concentration of the chelating agent is 10-50 mM.

7. The process according to claim 6, wherein the chelating agent is one or more selected from the group consisting of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid and salts thereof, citric acid and salts thereof, tartaric acid and salts thereof, and gluconic acid and salts thereof.

8. The preparation method of claim 6, wherein the conditions of the enzymolysis reaction are 30-50 ℃, 4-48 h, and 300 +/-200 r/min of stirring.

9. The preparation method of claim 7, wherein the conditions of the enzymolysis reaction are 30-50 ℃, 4-48 h, and 300 +/-200 r/min of stirring.

Technical Field

The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a method for obtaining a high-purity catalytic product of diglyceride phosphate by using an enzyme method.

Background

Diglyceride phosphate is a water-soluble small molecular substance normally existing in human body. Glycerol Phosphatidylcholine (GPC), Glycerol Phosphatidylethanolamine (GPE), Glycerol Phosphatidylinositol (GPI), and the like are common. Diacylglycerol phosphate is a product of phospholipid metabolism and plays an important physiological role in the human body. In the case of GPC, the most important physiological function of GPC in vivo is to cross the blood brain barrier, providing the necessary source of choline for the synthesis of acetylcholine and Phospholipids (PC). Clinical research shows that GPC has obvious curative effect on cerebral ischemic stroke, Alzheimer's disease and multiple cerebral infarction type dementia. The traditional Chinese medicine composition is clinically used for improving the liver protection effect of toxic liver injury and the like; meanwhile, GPC also has various obvious effects of preventing aging, reducing blood fat, strengthening brain and the like, and is increasingly emphasized as research and application of health products and pharmaceutical preparations, and the development of a preparation method of high-purity diglyceride phosphate has important value.

At present, the preparation method of the diglyceride of glycerophosphate mainly comprises 3 methods, namely a biological tissue direct extraction method, and the method has the advantages of complex process, low yield and high cost; secondly, the lecithin is prepared by hydrolysis, the method has simple process and low cost, but the post-treatment is quite complicated, the product purity is low, and particularly the problem of environmental protection cannot be solved. And thirdly, chemical synthesis, namely, preparation from basic chemical raw materials, but the steps are various, the process is complex, and the yield is low. The production of glycerophosphorylcholine from D-mannitol by a series of reactions was only 38.6% (based on D-mannitol) as an example. Therefore, it is urgently needed to develop a new preparation method, improve the product purity, reduce the production cost and simplify the production procedure, thereby promoting the wide application of the diglyceride phosphate.

Glycerol phosphodiesterase (GDPD; EC 3.1.4.46) is a class of enzymes that catalyzes the hydrolysis of the 3 '-5' phosphodiester bond of glycerol phosphodiesters. The inventors succeeded in constructing recombinant strains of Escherichia coli having a diacylglycerol Phosphodiesterase (pfGDPD) derived from Pyrococcus furiosus (Pyrococcus furiosus) and a method for producing an enzyme protein, which is capable of hydrolyzing a lysophospholipid substrate in addition to a diacylglycerol phosphate substrate, in previous studies (Wang FH, Lai LH, Liu YH, Yang B, Wang YH. Expression and Characterization of the characteristics of a Novel Glycerophosphodiester phosphor from Pyrococcus furiosus DSM 3638 at potas lysophosphoipase D activity.int.J.mol.Sci.2016,17(6), 831.). However, in the case of an experiment for hydrolyzing a lysophospholipid substrate, two products, i.e., a diglyceride phosphate (e.g., GPC, GPE, GPI, etc.) and a lysophosphatidic acid (LPA), are produced, and the use thereof for the preparation of a high-purity diglyceride phosphate is still not ideal.

Disclosure of Invention

Based on the fact that pfGDPD found by experiments has the characteristic of ester bond hydrolysis, the invention provides a brand-new preparation method of the diglyceride phosphate, starting from lysophospholipid, the high-purity diglyceride phosphate (such as GPC, GPE, GPI and the like) can be prepared by one-step single enzymatic hydrolysis, and the conversion rate is close to 100%.

The technical scheme of the invention is as follows:

a method for preparing high-purity diglyceride phosphate comprises hydrolyzing lysophospholipid in water phase system with lysophospholipid as raw material and metal ion chelating agent treated phosphodiesterase (pfGDPD) from Pyrococcus furiosus as enzyme catalyst to obtain diglyceride phosphate product; wherein the amino acid sequence of the pfGDPD is SEQID No. 1, and the nucleic acid sequence thereof is SEQID No. 2. Dissolving lysophospholipid substrate in buffer solution with pH8.0, adding pfGDPD treated by metal ion chelating agent into reaction system at 30-90 deg.C, and performing enzymolysis reaction to obtain high-purity diglyceride.

Preferably, the buffer solution is a boric acid buffer.

Preferably, the lysophospholipid is any one or more than two of Lysophosphatidylcholine (LPC), Lysophosphatidylethanolamine (LPE), Lysophosphatidylserine (LPS), Lysophosphatidylinositol (LPI), and Lysophosphatidylglycerol (LPG).

Preferably, the temperature of the treatment of the metal ion chelating agent is 4-90 ℃, the treatment time is 1-12h, and the concentration of the chelating agent is 10-50 mM.

Preferably, the chelating agent is one or more of ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetate (EDTA-2Na), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DPTA) and salts thereof, Citric Acid (CA) and salts thereof, Tartaric Acid (TA) and salts thereof, and Gluconic Acid (GA) and salts thereof.

Preferably, the conditions of the enzymolysis reaction are that the temperature is 30-50 ℃, the time is 4-48 h, and the stirring is carried out at 300 +/-200 r/min.

Compared with the prior art, the invention has the following beneficial effects:

the invention uses the diglyceride phosphodiesterase (pfGDPD) from Pyrococcus furiosus treated by a metal ion chelating agent as an enzyme catalyst, and can prepare high-purity diglyceride phosphate (such as GPC, GPE, GPI and the like) by single enzymatic hydrolysis from lysophospholipid, does not contain impurity LPA, and has the conversion rate close to 100%.

Drawings

FIG. 1 is a NMR spectrum of the untreated wild-type pfGDPD hydrolyzed lysophospholipid of example 1.

FIG. 2 is a NMR spectrum of hydrolyzed lysophospholipids after treatment with the wild-type pfGDPD metal ion chelator of example 2.

FIG. 3 is a mass spectrum of hydrolyzed lysophospholipid after treatment with the wild-type pfGDPD metal ion chelator of example 2.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1: treated purified pfGDPD hydrolyzed lysophospholipids

(1) Metal ion chelator treatment of pfGDPD: EDTA-2Na was added to the purified pfGDPD at a final concentration of 20mM, and the mixture was allowed to stand at 4 ℃ for 2 hours.

(2) pfGDPD after EDTA-2Na treatment hydrolyzed lysophospholipids: lysophosphatidylcholine LPC (16:0) was dissolved in borate buffer (pH 8.0), and pfGDPD treated in step (1) was added and reacted at 30 ℃ for 4 hours at 300r/min in a constant temperature shaker.

The reaction product was subjected to NMR examination, and the results showed that (FIG. 2) wild-type pfGDPD treated with a metal ion chelating agent had a significantly enhanced ability to catalyze hydrolysis of lysophospholipid substrate, and that the reaction system contained only GPC, and no formation of LPA (16:0) or residue of LPC (16:0) was detected, and the purity of the formed diglyceride phosphate (GPC) was high.

Mass spectrometry of the reaction samples of this example showed (FIG. 3) that only GPC was detected, confirming 100% conversion to diglycerol phosphate (GPC) during the reaction.

Example 2

Determination of the formation of GPC by hydrolysis of lysophospholipids after treatment of pfGDPD with different Metal ion chelators: disodium ethylenediaminetetraacetate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid and its sodium salt, citric acid and its sodium salt, tartaric acid and its sodium salt, gluconic acid and its sodium salt, each of which was added to the purified pfGDPD at a final concentration of 20mM, were allowed to stand at 4 ℃ for 2 hours, and then the thus-treated pfGDPD was taken out to hydrolyze lysophospholipids, respectively, and the reaction system and reaction conditions were the same as those in example 1. And (3) performing nuclear magnetic resonance detection on the reacted product, wherein results show that the treatment effects of different metal ion chelating agents are not obviously different, and the conversion rate of diglyceride phosphate (GPC) generated in the reaction process can reach 100%.

Comparative example: untreated purified pfGDPD hydrolyzed lysophospholipids

The raw material is lysophosphatidylcholine (16:0LPC)

Lysophosphatidylcholine LPC (16:0) was dissolved in borate buffer (pH 8.0), and purified pfGDPD was added and reacted at 30 ℃ for 4 hours at 300r/min in a constant temperature shaker. The reaction product was taken and subjected to NMR examination, and the results showed that (FIG. 1) untreated wild-type pfGDPD had poor ability to catalyze hydrolysis of lysophospholipid substrate, and that the reaction system contained not only GPC but also LPA and unreacted substrate LPC (16:0), and that the conversion rate to diglyceride phosphate GPC was within 5%.

Sequence listing

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Lys Asp Arg Asp Ala Ala Lys Glu Val Ala Arg Ile Val Ser Glu Asn

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atggtaggta acccaatgtg ggagcgtgat aagatcatcg tcctgggtca tcgtggttac 60

atggcaaagt acccggagaa cagcctgctg tctatccgta aagctattga agcaggtgca 120

gatggtgttg aaatcgatgt atggctgtct aaagacaaca aagtgatcct gatgcacgac 180

gaaaccatcg accgtacctc taacctgaaa ggccgtcaga aagaaatgac gctggaggaa 240

ctgaagaaag cgaacatcgg tatgggcgaa cgtatcccga ctctggaaga agtattcgaa 300

atcctgccga aagacgctct gctgaacatc gaaatcaaag accgtgacgc tgctaaagaa 360

gtcgcccgta ttgtctctga gaacaacccg gaacgtgtta tgatctcttc ctttgacatc 420

gaagcgctgc gtgaataccg caaatacgac gacactacta tcatgggtct gctggtagat 480

aaagaagaaa ccgtgccgct gatccctaaa ctgaaagaga aactgaacct gtggagcgtt 540

aacgtgccga tggaagcgat tccgattatt ggcttcgaga agacctacca agcgatcaaa 600

tgggtgcgca gcctgggcct gaagattgtt ctgtggaccg aagatgataa actgttctat 660

gtggatgaga atctgaaacg cctgctgggc atgttcgaag ttgttattgc caatgatgtt 720

gaacgcatgg tttcctatct gtcctccctg ggcattcgc 759