A strain of Issakia orientalis and its whole cell transformation method for producing citicoline

A technology of whole-cell transformation and ESZ yeast, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems of low conversion rate and yield, achieve high-efficiency preparation, and improve the effect of regeneration rate

Inactive Publication Date: 2011-12-21
江苏华晟知识产权运营有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used method is microbial transformation method, but there are problems of low conversion rate and yield.

Method used

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  • A strain of Issakia orientalis and its whole cell transformation method for producing citicoline
  • A strain of Issakia orientalis and its whole cell transformation method for producing citicoline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] For the cultivation of Issa orientalis, the yeast medium used is: glucose 50 g / L, urea 2.0 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, zinc sulfate heptahydrate 4.0 mg / L, ferric sulfate heptahydrate 3.0 mg / L, manganese sulfate tetrahydrate 0.3 mg / L, anhydrous calcium chloride 1.0 mg / L, biotin 0.05 mg / L, distilled water to volume. The inoculum amount of Issaccharomyces orientalis was 10%, cultured on a shaker at 30oC for 24 hours, then centrifuged at 4500r / min for 10min, and the supernatant was discarded to obtain yeast sludge, which could be used to prepare citicoline.

[0023] In the reactor, prepare 2000 g of yeast sludge and water obtained from 25 μmol / mL phosphorylcholine, 10 μmol / mL 5'-cytidylic acid, 200 μmol / mL glucose, 5 μmol / mL magnesium sulfate heptahydrate and centrifugation of the above-mentioned culture medium and water. The reaction solution was 10L, adjusted to pH 6.0 with sodium hydroxide, and converted at 32oC f...

Embodiment 2

[0025] In the reactor, prepare 25 μmol / mL phosphorylcholine, 10 μmol / mL 5'-cytidylic acid, 200 μmol / mL glucose, 5 μmol / mL magnesium sulfate heptahydrate and the method for cultivating Issahia orientalis as described in Example 1 Cultivate 10L of reaction solution consisting of 2000g of yeast sludge obtained by centrifugation and water, adjust the pH to 6.0 with sodium hydroxide, add 0.1mL / L of toluene, and transform at 32oC for 10h. After the reaction, measure the content of citicoline in the reaction solution. The yield was 2.4g / L, and the conversion rate reached 74.2%.

Embodiment 3

[0027] In the reactor, prepare 30 μmol / mL phosphorylcholine, 20 μmol / mL 5'-cytidylic acid, 300 μmol / mL glucose, 5 μmol / mL magnesium sulfate heptahydrate, 10 μmol / mL potassium dihydrogen phosphate and according to Example 1. The method of cultivating Issa orientalis is to cultivate 10L of reaction solution consisting of 5000g of yeast sludge obtained by centrifugation and water, adjust the pH to 5.0 with sodium hydroxide, add 0.5mL / L of toluene, and transform at 32oC for 10h. After the reaction, measure the reaction The output of citicoline in the solution was 5.1g / L, and the conversion rate reached 78.9%.

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Abstract

The invention relates to Issatchenkia orientalis and a method for producing citicoline by whole cell conversion of Issatchenkia orientalis, belonging to the technical field of biological pharmacy. In the method provided by the invention, the whole cells of Issatchenkia orientalis Z1, namely CCTCC (China Center for Type Culture Collection) NO: M2011272 are utilized to prepare citicoline, choline phosphate and 5'-cytidylic acid are used as substrates, glucose is used as an energy donor, and the ATP (adenosine triphosphate) regeneration efficiency is improved by adding inorganic ions; glucose is used as the energy donor, so that the energy requirement of the strain is provided and ATP is provided for a citicoline synthesis enzyme system; one or more of potassium ion, magnesium ion and manganese ion are added to change the metabolism flow direction and improve the ATP regeneration rate, so that the ATP regeneration rate is matched with the rate of the citicoline enzyme synthesis system and the high-efficiency preparation of citicoline is achieved; and the whole cells of Issatchenkia orientalis are used, and toluene is added to an aqueous solution during the preparation process so as to improve the cell permeability, so that the rate of the citicoline enzyme synthesis system is improved.

Description

technical field [0001] The invention discloses a strain of Issaccharomyces orientalis and a method for transforming whole cells thereof to produce citicoline, relates to the preparation of citicoline, and belongs to the technical field of biopharmaceuticals. Background technique [0002] Citicoline, also known as citicoline, the chemical name is choline cytosine-5'-diphosphate monosodium salt, which is the precursor of lecithin biosynthesis. 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. In addition, citicoline is also an important nucleic acid drug, which can regulate the tension of cerebrovascular movement and improve the metabolic function of the brain. The target organs of citicoline are the liver and brain tiss...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12P19/30C12R1/645
Inventor 张梁石贵阳尤翠萍丁重阳顾正华
Owner 江苏华晟知识产权运营有限公司
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