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Method for preparing citicoline through biological enzyme catalysis

A technology for citicoline and catalyzed preparation is applied in the field of preparation of citicoline, and can solve the problems of high cost, expensive substrate, difficult to control and the like

Inactive Publication Date: 2016-04-06
苏州正济药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current citicoline production methods mostly involve the participation of various microorganisms or the substrates used are expensive, costly, and difficult to control. The present invention uses a single strain for fermentation, and the purpose is to provide citicoline as a drug. A More Efficient and Economical Manufacturing Method for Alkalis

Method used

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  • Method for preparing citicoline through biological enzyme catalysis
  • Method for preparing citicoline through biological enzyme catalysis
  • Method for preparing citicoline through biological enzyme catalysis

Examples

Experimental program
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Embodiment 1

[0022] Construction of cct and pyrG gene expression vectors

[0023] According to the known nucleic acid sequence of the Escherichia coli K12MG1655pyrG gene and the cct gene nucleic acid sequence of Saccharomyces cerevisiae, the full-length cct gene and pyrG gene were amplified by using the chromosomes of Escherichia coli and Saccharomyces cerevisiae as templates respectively.

[0024] Genomic DNA of original Escherichia coli strain DH5α and Saccharomyces cerevisiae strain ATCC26786 / 26787 (DSM4266 / 4267) purchased from Germany were extracted by conventional bacterial and yeast DNA extraction methods.

[0025] The pfu enzyme with better fidelity was used for amplification, and after adding A tail, it was connected to pUC19-Tvector, and the positive clones were picked and sent to the sequencing company.

[0026] ① Construction of plasmid pUCG

[0027] The pyrG gene connected to pUC19-Tvector was digested from the vector, and the restriction enzymes used were SmaI and BamHI. At t...

Embodiment 3

[0062] The recombinant K1-E obtained in Example 2 was prepared as a competent cell, and then the plasmid pUCG-CCT constructed in Example 1 was introduced into it to obtain a cytidine diphosphate compound with the ability to catalyze orotate, phosphorylcholine and ammonium chloride to synthesize cytidine diphosphate Choline-capable engineered bacteria K1-E / pUCG-CCT.

[0063] Insert the engineering bacteria K1-E / pUCG-CCT strain on the solid medium into 5-150 ml seed culture solution containing ampicillin (100 μg / ml), and vibrate at 150-280 rpm for 8-16 hours at 25-37°C. According to the inoculum size of 0.1-10%, transfer this culture solution to 1L Erlenmeyer flask containing ampicillin (100μg / ml) seed LB medium solution in 10-500ml, shake and cultivate with 150-280rpm at 25-37℃ for 2 -8 hours, then add the inducer isopropyl-β-D-thiogalactopyranoside with a final concentration of 0.1-1mM for induction, continue to cultivate for 4-12 hours, and lower the culture temperature appro...

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Abstract

The invention discloses a method for preparing citicoline through biological enzyme catalysis. The method is characterized by comprising the steps that a single artificially-transformed gene engineering strain is used as an enzyme source, an ammonium chloride catalyst, orotic acid, choline phosphate and other chemical substances are subjected to a reaction to generate citicoline, and then the citicoline is extracted from the reaction solution. Compared with the prior art, single strain is adopted for fermentation, the production technology is simple, the production period is short, the production cost is low, and the method for preparing citicoline through biological enzyme catalysis can be widely applied to industrial production of citicoline.

Description

technical field [0001] The invention relates to the fields of biotechnology and pharmacy, including genetic engineering and enzyme engineering, and discloses a method for preparing citicoline by using the bacterial strain. Background technique [0002] Citicoline, also known as cytidine diphosphate choline, was named citicoline in the Chinese Pharmacopoeia 2005 edition, English name: Citichaline, and its chemical name: choline cytosine nucleoside-5'-diphosphate. It is a precursor for the biosynthesis of lecithin, and when brain function declines, the lecithin content in brain tissue is significantly reduced. Supplementing exogenous citicoline can activate the biosynthesis of lecithin, stimulate the excitement of the brainstem reticular structure, increase the threshold of awakening, restore the function of nerve tissue, improve brain metabolism and nerve conduction, and improve the patient's consciousness level. [0003] In the 1950s, Dr. Kennedy and others discovered and d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N1/21C12R1/19
Inventor 罗勇薛程良朱江姚青钱超时花
Owner 苏州正济药业有限公司
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