Method for synthesizing choline phosphate through whole-cell biocatalysis

A technology of phosphorylcholine and biocatalysis, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effects of maintaining integrity, environmental protection in the preparation process, and reducing difficulty

Inactive Publication Date: 2010-07-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for synthesizing choline phosphate through whole-cell biocatalysis
  • Method for synthesizing choline phosphate through whole-cell biocatalysis

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Experimental program
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Embodiment 1

[0032] Yeast medium: glucose 40g / L, urea 2.0g / L, potassium dihydrogen phosphate 1.5g / L, zinc sulfate heptahydrate 4.0×10 -3 g / L, ferrous sulfate heptahydrate 3.0×10 -3 g / L, manganese chloride tetrahydrate 0.3×10 -3 g / L, anhydrous calcium chloride 1.0×10 -3 g / L, biotin 0.05×10 -3 g / L.

[0033] The inoculum amount of yeast was 10%, cultured on a shaker at 120 rpm at 30°C for 24 hours, and centrifuged at 4000 rpm for 20 minutes. Take the yeast paste and store it at -7°C for later use.

Embodiment 2

[0035] In a reaction tank with a capacity of 15L, the Saccharomyces cerevisiae mud cultivated by choline chloride 100mmol, glucose 1mol, zinc sulfate 10mmol, embodiment 1 method was repeatedly frozen and thawed 5 times 1000g, cysteine ​​10mol, sodium dihydrogen phosphate 1mol The reaction solution composed of water and water was 10L, the pH was adjusted to 5.0 with sodium hydroxide, and the temperature was 20°C. After 4 hours of reaction, the reaction was terminated, and the product was precipitated with perchloric acid, and the product was quantitatively analyzed by HPLC. The phosphorylcholine content was 5.6mmol / L, the yield was 56%.

Embodiment 3

[0037] In a reaction tank with a capacity of 15L, the wet thallus of Brevibacterium ammoniagenes cultivated by 5000mmol of choline chloride, 10mol of glucose, 2mol of zinc chloride, and 8000g of Brevibacterium ammoniagenes, 0.5mol of cysteine, and phosphoric acid were prepared after ultrasonic treatment for 30min. The reaction solution composed of 20 mol of potassium dihydrogen and water was 10L, the pH was adjusted to 10.0 with sodium hydroxide, and the temperature was 40°C. After 30 hours of reaction, the reaction was terminated, and the product was precipitated with perchloric acid, and the product was quantitatively analyzed by HPLC. Phosphorylcholine The content is 251.3mmol / L, and the yield is 50.3%.

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Abstract

The invention discloses a method for synthesizing choline phosphate through whole-cell biocatalysis, which comprises the following steps of: using choline chloride and phosphate ions as substrates and glucose as an energy donor; adding a micro-molecule chemical effect substance into the substrates; and synthesizing the choline phosphate by using microorganism whole-cell biocatalysis having permeability. The method for synthesizing the choline phosphate by adopting the whole-cell biocatalysis overcomes the defects that a chemical synthesis method has high cost, low conversion rate, high toxicity of a solvent and the like. In the method, the whole-cell catalysis has high stability, and the micro-molecule chemical effect substance is adopted to adjust and control the metabolic flow so as to improve the energy situ regeneration and coupling efficiencies; besides, the product concentration is greatly improved and the conversion rate is also improved.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for synthesizing phosphorylcholine by whole cell biocatalysis. Background technique [0002] Phosphorylcholine is an important intermediate in the biosynthesis of lecithin in eukaryotic cells. It is formed by condensation of choline and ATP under the catalysis of choline kinase existing in animals and yeast. It has the strongest binding ability to CRP (C-reactive protein) among phosphate monoesters, and can enhance the body's defense against bacteria and fungi in the presence of calcium ions (European Journal of Internet Medicine, 2002, 13: 412-422). Phosphorylcholine can be used to prepare photoelectrochemical cells to generate photovoltage. The biochemical properties of the phosphorylcholine bilayer modified by glow discharge are similar to the antithrombotic surface of human red blood cells, which greatly inhibits the surface connection of platelets ...

Claims

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Application Information

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IPC IPC(8): C12P9/00C12R1/01C12R1/185C12R1/265C12R1/13C12R1/125C12R1/72C12R1/84C12R1/78C12R1/645C12R1/865
Inventor 应汉杰汤佳鹏陈勇熊健柏建新陈晓春
Owner NANJING UNIV OF TECH
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