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Preparation method of high-purity glycerophosphate diester

A glycerophosphodiester, high-purity technology, applied in the field of genetic engineering of enzymes, can solve the problems of complex process, many steps, high cost, etc.

Active Publication Date: 2021-06-01
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the preparation method of glycerophosphodiester mainly contains 3 kinds: ① biological tissue direct extraction method, this method process is complicated, yield is low, and cost is high; ② Preparation from lecithin hydrolysis: This method has simple process and low cost, but the post-treatment is quite cumbersome, the product purity is low, and the most important thing is that it cannot solve environmental problems
3. Synthesis by chemical method: it is prepared from basic chemical raw materials, but the steps are numerous, the process is complicated, and the yield is low
But can produce glycerophosphodiester (such as GPC, GPE, GPI etc.) and lysophosphatidic acid (LPA) two kinds of products in the experiment of hydrolyzing lysophospholipid substrate, the application in the high-purity preparation of glycerophosphodiester is still not enough ideal

Method used

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  • Preparation method of high-purity glycerophosphate diester
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  • Preparation method of high-purity glycerophosphate diester

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1: Treatment of purified pfGDPD for hydrolysis of lysophospholipids

[0020] (1) Treatment of pfGDPD with a metal ion chelator: EDTA-2Na with a final concentration of 20 mM was added to the purified pfGDPD, and it was left to stand at 4° C. for 2 h.

[0021] (2) Hydrolyzed lysophospholipids of pfGDPD treated with EDTA-2Na: dissolve lysophosphatidylcholine LPC (16:0) with borate buffer (pH 8.0), add pfGDPD treated in step (1), and shake at constant temperature The reaction was carried out at 30 °C in the instrument at 300 r / min for 4 h.

[0022] Get the product after reaction and carry out nuclear magnetic resonance detection, the result shows ( figure 2 ) The ability of wild-type pfGDPD to catalyze the hydrolysis of lysophospholipid substrates after treatment with metal ion chelator was significantly enhanced. The reaction system only contained GPC, and the formation of LPA(16:0) and the residue of LPC(16:0) were not detected. Glycerophosphoric acid diester (...

Embodiment 2

[0025] Detection of GPC generation by hydrolyzing lysophospholipids after treating pfGDPD with different metal ion chelators: adding disodium EDTA, aminotriacetic acid, diethylenetriaminepentaacetic acid and Among its sodium salt, citric acid and its sodium salt, tartaric acid and its sodium salt, gluconic acid and its sodium salt, after standing at 4 °C for 2 hours, the treated pfGDPD was taken to hydrolyze lysophospholipids, respectively. The reaction system and reaction conditions Same as Example 1. The reaction product was taken for nuclear magnetic resonance detection. The results showed that there was no significant difference in the treatment effect of different metal ion chelating agents, and the conversion rate of glycerophosphoric acid diester (GPC) during the reaction process could reach 100%.

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Abstract

The invention belongs to the technical field of gene engineering of enzymes, and discloses a preparation method of high-purity glycerophosphodiesters, which comprises the following steps: hydrolyzing lysophosphatide in a water phase system by taking lysophosphatide as a raw material and pfGDPD treated by a metal ion chelating agent as a catalyst to obtain a glycerophosphodiester product; the amino acid sequence of the pfGDPD is shown as SEQ ID No: 1. Starting from lysophospholipid, high-purity glycerophosphodiesters (such as GPC, GPE, GPI and the like) can be prepared through one step of hydrolysis by a single enzyme method, and the conversion rate is close to 100%. The invention provides a new thought and method for the production of glycerophosphate diester.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of enzymes, in particular to a method for obtaining high-purity catalytic product glycerophosphoric acid diester by using an enzyme method. Background technique [0002] Diglycerol phosphate is a water-soluble small molecular substance that normally exists in the human body. Common ones are glycerol phosphatidyl choline (GPC), glycerol phosphatidyl ethanolamine (GPE), and glycerol phosphatidyl inositol (GPI). Diglycerol phosphate is a product of phospholipid metabolism and plays an important physiological function in the human body. Taking GPC as an example, in vivo, the most important physiological function of GPC is to cross the blood-brain barrier and provide the necessary choline source for the synthesis of acetylcholine and phospholipid (PC). Clinical studies have shown that: GPC has a significant effect in the treatment of cerebral ischemic stroke, Alzheimer's disease, and mult...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/64
CPCC12P7/6481
Inventor 王方华王永华闵雪珂杨博王卫飞
Owner SOUTH CHINA UNIV OF TECH
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