Method for catalytically producing citicoline sodium with immobilized enzyme

A technology of citicoline sodium and immobilized enzyme, which is applied in the field of immobilized enzyme catalyzed production of citicoline sodium, which can solve the problems of enzymatic synthesis, low reaction conversion rate, and high production cost, and reduce the synthesis Process time, environmental friendliness, and environmental protection effects

Active Publication Date: 2014-06-11
KAIPING GENUINE BIOCHEM PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical synthesis method, using cytidylic acid (CMP) and phosphorylcholine (CP) as substrates, using p-toluenesulfonyl chloride as a condensation agent, and condensing citicoline in the presence of N-dimethylformamide (DMF) Sodium, chemical synthesis has problems such as low reaction conversion rate, many by-products, high production cost, and serious environmental pollution; the microbial fermentation method uses microorganisms such as yeast or Brevibacterium ammoniagenes to utilize glucose, and add CMP, CP and other premise materials to ferment To produce citicoline sodium, the microbial fermentation method has problems such as low product concentration and unstable yield; the enzymatic synthesis method used to extract cytidine phosphotransferase from guinea pig liver cell homogenate, and catalyze the formation of CTP and phosphorylcholine A preliminary report on citicoline sodium, but due to the source of the enzyme, the enzymatic synthesis method cannot be realized

Method used

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  • Method for catalytically producing citicoline sodium with immobilized enzyme
  • Method for catalytically producing citicoline sodium with immobilized enzyme
  • Method for catalytically producing citicoline sodium with immobilized enzyme

Examples

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Effect test

Embodiment 1

[0041] A method for producing citicoline sodium catalyzed by immobilized enzymes, comprising the steps of:

[0042] (1) Engineering bacteria fermentation:

[0043] Inoculate 150ml seed liquid of Escherichia coli engineering bacteria with molecularly cloned cytidine phosphotransferase gene into a 3L culture medium system for 26 hours. The culture medium system contains peptone 10g / L, yeast extract 10g / L, glucose 5g / L L, inorganic salt 8g / L, supplemented with 150g glucose, 15g peptone and 15g yeast extract during the dissolution period in purified water; after the fermentation was completed, the fermentation broth was centrifuged to collect 152g of bacterial cells.

[0044] (2) Preparation of cytidine phosphotransferase serum:

[0045] Take 30g of bacteria, mix and suspend it with 180ml phosphate buffer solution, and use ultrasonic crushing for 30 minutes to obtain a broken liquid. Add ammonium sulfate to the broken liquid to 30% saturation for salting out, and then use 200nm mic...

Embodiment 2

[0053] A method for producing citicoline sodium catalyzed by immobilized enzymes, comprising the steps of:

[0054] (1) Engineering bacteria fermentation:

[0055] Inoculate 200ml of Escherichia coli engineered bacteria seed solution with molecularly cloned cytidine phosphotransferase gene into a 3L medium system (the medium system contains 15g / L peptone, 15g / L yeast extract, 8g / L glucose, and 13.5g inorganic salt / L, dissolved in purified water) for 28 hours, supplemented with 288g glucose, 30g peptone and 30g yeast extract during this period. After the fermentation was completed, 165 g of bacterial cells were collected by centrifugation of the fermented liquid.

[0056] (2) Preparation of cytidine phosphotransferase serum:

[0057] Take 20g of bacteria, mix and suspend it with 200ml phosphate buffer solution, and then crush it with ultrasonic wave for 30 minutes to obtain a crushed liquid. Add ammonium sulfate to the crushed liquid to 45% saturation, and then use 500nm mic...

Embodiment 3

[0065] The preparation of citicoline sodium comprises the following steps:

[0066] (1) Engineering bacteria fermentation:

[0067] Inoculate 300ml of Escherichia coli engineered bacteria seed solution with molecularly cloned cytidine phosphotransferase gene into a 3L medium system (the medium system contains 20g / L of peptone, 20g / L of yeast extract, 8g / L of glucose, 15g / L of inorganic salt L, the balance is dissolved in purified water) for 32 hours, during which 360g glucose, 48g peptone and 48g yeast extract were supplemented. After the fermentation was completed, 187 g of bacterial cells were collected by centrifugation of the fermented liquid.

[0068] (2) Preparation of cytidine phosphotransferase serum:

[0069] Take 30g of bacterial cells, mix and suspend them with 120ml phosphate buffer solution, crush the bacterial solution by ultrasonic wave for 30 minutes to obtain a broken liquid, add ammonium sulfate to the broken liquid to a saturation of 25%, and then use 500n...

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Abstract

The invention provides a method for catalytically producing citicoline sodium with an immobilized enzyme. The method comprises the following steps: by utilizing engineered escherichia coli of a molecularly cloned cytidine phosphotransferase gene for fermentation, preparing cytidine phosphotransferase, and preparing cytidine phosphotransferase liquid through purification; immobilizing cytidine phosphotransferase; by using phosphorylcholine and cytidine disodium triphosphate as raw materials, catalytically generating citicoline with immobilized cytidine phosphotransferase. The method is simple in production process, short in production cycle and low in production cost and can be widely applied to industrial production of citicoline.

Description

Technical field: [0001] The invention relates to the field of pharmacy, in particular to a method for catalyzing the production of citicoline sodium by immobilized enzymes. Background technique: [0002] Citicoline sodium, also known as citicoline sodium, the chemical name is the monosodium salt of choline cytosine nucleoside diphosphate, a brain metabolism activator, which is a nucleoside derivative and is a derivative of phosphatidylcholine. The precursor substance is a coenzyme necessary for the synthesis of lecithin. It was discovered in 1954, synthesized in 1956, and successfully produced in China in 1973. [0003] The pharmacological effects of citicoline sodium mainly include: it can restore the structure of the cell membrane after brain cell injury, enhance the function of the cell membrane, improve the function of neurons; reduce cerebrovascular resistance, increase cerebral blood flow, promote brain metabolism, improve cerebral circulation, Improve brain function;...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N9/12C12N11/12C12N11/10C12N11/08C12R1/19
Inventor 刘桂祯黄炯威周华润莫世艺劳瑞雄
Owner KAIPING GENUINE BIOCHEM PHARMA
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