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Method for separating and purifying citicoline in issatchenkia orientalis biotransformation liquid

A technology for separation and purification of citicoline, applied in the field of bioseparation, which can solve the problems of severe loss, complicated process, and failure to recycle cytidylic acid

Active Publication Date: 2014-05-28
无锡泰仑达化机设备有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The reaction solution for preparing citicoline by biosynthesis contains proteins, pigments, incompletely reacted phosphorylcholine calcium, cytidylic acid, phosphate, magnesium sulfate, by-products of glucose metabolism and cytidine, etc. These impurities Bring many difficulties to the separation and purification of citicoline
The extraction of citicoline from the above reaction solution requires the removal of bacteria and protein, 769 activated carbon adsorption, ion exchange resin adsorption, step-by-step elution, carbon powder decolorization and other steps (Huang Ying, Chinese Journal of Pharmaceutical Industry, 1985.1: 001.), the process is complex, the yield is only 45%, the loss is serious, the remaining cytidylic acid has not been recycled, and it is not suitable for industrial large-scale production

Method used

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  • Method for separating and purifying citicoline in issatchenkia orientalis biotransformation liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1～ Embodiment 3

[0022] The preparation method of the I. orientalis citicoline biotransformation liquid in the following examples 1 to 3 is as follows:

[0023] Glucose is used as the energy donor, on the one hand to provide the energy demand of the bacteria, on the other hand to provide ATP for the citicoline synthetase system; to change the metabolic flow direction by adding one or a combination of potassium ions, magnesium ions and manganese ions , improve the regeneration efficiency of ATP, so as to match the rate of citicoline enzyme system, and realize the efficient preparation of citicoline. (Refer to the following patent documents for the specific preparation method: Zhang Liang, Shi Guiyang, You Cuiping, etc. A strain of Issaccharomyces orientalis and its whole cell transformation method to produce citicoline: China, CN102286386A[P].2011- 12-21.), among which I. orientalis Z1 was purchased from China Culture Collection Center, and the strain number is: CCTCC NO: 2011272.

[0024] The...

Embodiment 1

[0026] 300mL I. orientalis citicoline biotransformation solution, containing 1350mg of CDP-c, 650mg of cytidylic acid, 3750mg of phosphate, 500mg of calcium phosphocholine, and 900mg of sulfate.

[0027] (1) Pass the above citicoline biotransformation solution through 717HCO 3 - Type anion exchange resin column, the column size is 20×300mm, the column volume is 60mL, the flow rate of the upper column is 0.5mL / min, and the total amount of the column is 13.5% (mass ratio) of the total exchanged amount of the column.

[0028] (2) After loading the column, rinse the exchange column with 300mL deionized water to wash away unadsorbed substances until the effluent is completely water.

[0029] (3) Wash the column with 0.03M sodium chloride solution until the effluent at the outlet of the column no longer contains CDP-c, and collect 200 mL of the eluate to obtain a high-purity CDP-c solution.

[0030] (4) Wash the column with 0.3M sodium chloride solution until the effluent at the o...

Embodiment 2

[0033] 1000mL I. orientalis citicoline biotransformation solution, containing citicoline 4500mg, cytidylic acid 2039mg, phosphate 11380mg, phosphorylcholine calcium 1520mg, sulfate 2750mg.

[0034] (1) Pass the above citicoline biotransformation solution through 717HCO 3 -Type anion exchange resin column, the column size is 30×500mm, the column volume is 200mL, and the column flow rate is 1.0mL / min. The total amount on the column is 12.8% (mass ratio) of the total amount exchanged by the column.

[0035] (2) After loading the column, rinse the exchange column with 1000mL deionized water to wash away unadsorbed substances until the effluent is completely water.

[0036] (3) Wash the column with 0.04M sodium chloride solution until the effluent at the column outlet no longer contains CDP-c, and collect 800 mL of the eluent to obtain a high-purity CDP-c solution.

[0037] (4) Wash the column with 0.2M sodium chloride solution until the effluent at the outlet of the column no l...

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Abstract

The invention discloses a method for separating and purifying citicoline in an issatchenkia orientalis biotransformation liquid. The method comprises the following steps: the issatchenkia orientalis CDP-c biotransformation liquid is passed through an anion exchange resin column, deionized water is used for leaching an unadsorbed substance, a salt solution of 0.01-0.50M is employed for eluting CDP-C and the salting liquid of 0.10-1.00M is employed for eluting cytidylic acid, so that CDP-c with high purity (greater than 99%) and high yield (greater than 88%) can be obtained by one time, and the cytidylic acid without complete reaction can be recovered. The method for separating and purifying citicoline in the issatchenkia orientalis biotransformation liquid greatly simplify a separating process, the operation is simple, the cost is low, and the environment is protected, and is adapted to large-scale industrial production.

Description

technical field [0001] The invention relates to a method for separating and purifying citicoline, in particular to a method for separating and purifying citicoline from a biotransformation liquid of Issa orientalis, and belongs to the technical field of bioseparation. Background technique [0002] Citicoline, also known as cytidine-5'-diphosphate choline (Cytidine-5'-diphosphate choline, CDP-c, CDP-choline), is an important precursor of phospholipid metabolism and the synthesis of phosphatidylcholine The intermediate is an essential coenzyme for lecithin biosynthesis, and it has a significant effect on central nervous system damage models and encephalopathy (Sugino, Biochimica et Biophysica Acta, 1960.40:425-434). Studies have shown that CDP-choline can promote biological metabolism, especially the biosynthesis of phospholipids (Roe J.H., Journal of Biological Chemistry, 1954.210(2):703-707.). In the 1950s, Dr. Kennedy (Kennedy E.P., Journal of the American Chemical Society...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/10C07H1/06
Inventor 张梁石贵阳宋丽芳尤翠萍李赢顾正华丁重阳
Owner 无锡泰仑达化机设备有限公司
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