CN103849666A - Method for catalytically producing citicoline sodium with immobilized enzyme - Google Patents
Method for catalytically producing citicoline sodium with immobilized enzyme Download PDFInfo
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- CN103849666A CN103849666A CN201310167034.5A CN201310167034A CN103849666A CN 103849666 A CN103849666 A CN 103849666A CN 201310167034 A CN201310167034 A CN 201310167034A CN 103849666 A CN103849666 A CN 103849666A
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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:
The present invention relates to pharmacy field, specifically refer to a kind of method of immobilized enzyme catalysis production CITICOLINE SODIUM.
Background technology:
CITICOLINE SODIUM claims again citicoline sodium, single sodium salt of chemical name choline Cytidine diphosphate ester, the agent of a kind of brain metabolic activation, for nucleoside derivates, being the precursor substance of phosphatldylcholine, is the synthetic necessary coenzyme of Yelkin TTS, finds in 1954,1956 synthetic, domestic trial-produceing successfully in 1973.
The pharmacological action of CITICOLINE SODIUM mainly contains: can recover the structure of the rear cytolemma of brain cell damage, strengthen the function of cytolemma, improve neuronic function; Reduce cerebral vascular resistance, increase cerebral blood flow (CBF), promote brain metabolism, improve cerebral circulation, improve brain function; Increase the secretion of the nerve conduction medium including vagusstoff and Dopamine HCL, strengthen the reticular formation of brain stem function relevant with consciousness, state of consciousness and the electroencephalogram of the disturbance of consciousness after improving injury of head or after brain operation, promote the recovery of the upper extremity exercise function of Patients with Hemiplegia, to promoting brain function to recover, promoting to revive have certain effect; Promote Yelkin TTS biosynthesizing and anti-phospholipase A effect, thereby accelerate the heavily absorption of cerebral edema.Promote dopaminergic activity, the physiological function of regulation and control extrapyramidal system.Share with proteolysis enzyme inhibitors, can protect and repairing pancreas tissue.
CITICOLINE SODIUM is mainly used in Acute Brain Injury and the rear disturbance of consciousness of brain operation clinically, treatment Parkinsonism (Parkinson ' s disease); Neural heariing loss; To senile dementia (Alzheimer ' s disease), dysthymia disorders, delay senility, improving results of learning and memory etc. has certain curative effect.
The production method of CITICOLINE SODIUM comprises chemical synthesis, three kinds of methods of microbe fermentation method and enzymic synthesis.Chemical synthesis, take cytidylic acid (CMP), phosphorylcholine (CP) as substrate, with Tosyl chloride be condensing agent, under existing, dinethylformamide (DMF) is condensed into CITICOLINE SODIUM, chemosynthesis exists reaction conversion ratio low, the problems such as by product is many, and production cost is high, and environmental pollution is serious; Microbe fermentation method is to utilize glucose with the microorganism such as yeast or Brevibacterium ammoniagenes, and adds the problems such as the front extract fermentation such as CMP, CP generates CITICOLINE SODIUM, and microbe fermentation method exists production concentration low, and productive rate is unstable; Enzymic synthesis once had with Liver of Guinea Pig cell homogenates to be carried and takes out cytidine phosphates transferring enzyme, and catalysis CTP and phosphorylcholine generate the Preliminary report of CITICOLINE SODIUM, but because of the source problem that comes of enzyme, enzymic synthesis cannot realize.
Summary of the invention:
The object of this invention is to provide one and be intended to solve chemical synthesis, the many disadvantages of microbe fermentation method and enzymic synthesis production CITICOLINE SODIUM, production technique is simple, with short production cycle, production cost is low, can be widely used in the method for the synthetic CITICOLINE SODIUM of enzyme catalysis of the industrialization production of CITICOLINE SODIUM.
To achieve these goals, the present invention adopts following technical scheme:
A method for immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps:
(1), engineering bacterium fermentation:
The seed liquor of the colibacillus engineering of molecular cloning cytidine phosphates transferase gene is inoculated in substratum system and is cultivated 26～32 hours, and the volume that adds of the seed liquor of the colibacillus engineering of described molecular cloning cytidine phosphates transferase gene is 5%～10% of substratum system volume; Supplement during this time appropriate glucose, peptone and yeast extract, the centrifugal collection thalline of the secondary fermentation liquid that fermented.
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Getting above-mentioned thalline is suspended in phosphoric acid buffer with ultrasonic wave or Syrup-homogenizing instrument fragmentation, obtain broken liquid, in above-mentioned broken liquid, add ammonium sulfate to saltout, obtaining saturation ratio is 25%～45% liquid of saltouing, the purpose ceramic-film filter that is 100～500nm with aperture again separates the above-mentioned solid and liquid of saltouing, obtain cytidine phosphates transferring enzyme clear liquid, wherein, the suspension ratio of described thalline and described phosphoric acid buffer is 1:4～1:10.
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
In above-mentioned cytidine phosphates transferring enzyme clear liquid, add fixation support, stir and solidify, obtain immobilization cytidine phosphates transferring enzyme, the ratio of the add-on of described fixation support and cytidine phosphates transferring enzyme clear liquid is 100～200 grams/L.
(4), CITICOLINE SODIUM is synthetic:
To be mixed with synthesis reaction solution containing the aqueous solution of Cytidine Disodium Triphosphate 30～80mmol/L, phosphorylcholine 60～400mmol/L and magnesium acetate 10～50mmol/L, regulate pH value to 6.0～9.0 of synthesis reaction solution, add immobilization cytidine phosphates transferring enzyme, and under the bath temperature of 10～50 ℃ stirring reaction 4～10 hours, the clear liquid obtaining after filtration is CITICOLINE SODIUM liquid, and the add-on of described immobilization cytidine phosphates transferring enzyme and the ratio of synthesis reaction solution are 10～50g/L.
(5), extract purifying
Above-mentioned CITICOLINE SODIUM liquid, through chromatographic separation crystallizing and drying, is obtained to the dry product of CITICOLINE SODIUM.
Wherein, the Classification And Nomenclature of the colibacillus engineering of described molecular cloning cytidine phosphates transferase gene is that colon bacillus (Escherichia coli) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation centre address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica's deposit number is CGMCC NO:4428.
Further, in described substratum system, contain peptone 10～20g/L, yeast extract 10～20g/L, glucose 5～8g/L, inorganic salt 8～15g/L, surplus is purified water, in engineering bacterium fermentation fermenting process, during this time the amount of supplementary glucose be the glucose that contains in substratum system amount 10-15 doubly, the 50-80% of the amount that the amount of supplementary peptone is the peptone that contains in substratum system, the 50-80% of the amount that the amount of supplementary yeast extract is the yeast extract that contains in substratum system.
Further, described fixation support is selected from any of Mierocrystalline cellulose, glucose gel, agarose, polyacrylamide gel, polyamino acid, polystyrene, nylon or porous glass matrix.
Further, in described fixation support, contain at least one group in fragrant amido or hydroxyl or carboxyl or carboxymethyl or epoxy group(ing) or amino functional group.
Preferably, in the synthetic step of CITICOLINE SODIUM, immobilization cytidine phosphates transferring enzyme 10～50g/L, Cytidine Disodium Triphosphate is 30～80mmol/L; Phosphorylcholine 60～400mmol/L; Magnesium acetate 10～50mmol/L; 25～40 ℃ of temperature of reaction; PH value is 6.0～9.0.
Preferably, in the synthetic step of CITICOLINE SODIUM, immobilization cytidine phosphates transferring enzyme content is 20～30g/L, and Cytidine Disodium Triphosphate is 40～60mmol/L; Phosphorylcholine 120～240mmol/L; Magnesium acetate 20～30mmol/L; 30～40 ℃ of temperature of reaction; PH value is 6.5～8.0.
Major technique advantageous characteristic of the present invention is as follows:
1. adopt the colibacillus engineering high density fermentation of molecular cloning cytidine phosphates transferase gene to prepare cytidine phosphates transferring enzyme, enzyme source is stable, and the workshop condition of avoiding the at present domestic cereuisiae fermentum reaction system generally adopting to bring pollutes and a large amount of waste yeast slag such as need process at the shortcoming.
2. adopt immobilization cytidine phosphates transferring enzyme catalytic synthesis, can repeated multiple timesly use, reaction conditions is gentle easily to be controlled, and reduces the synthesis procedure time, and transformation efficiency reaches more than 90%, effectively reduces production costs.
3. compared with traditional technology, technique of the present invention is environmentally friendly, aspect energy-saving consumption-reducing, protection of the environment, is having a clear superiority in.
Accompanying drawing explanation
Accompanying drawing 1 is immobilized enzyme catalysis production born of the same parents alkali choline sodium schema;
Accompanying drawing 2 is the IR collection of illustrative plates of CITICOLINE SODIUM standard substance;
The IR collection of illustrative plates that accompanying drawing 3 is the CITICOLINE SODIUM that makes;
Accompanying drawing 4 is CITICOLINE SODIUM standard substance
1hNMR collection of illustrative plates;
Accompanying drawing 5 is the CITICOLINE SODIUM that makes
1hNMR collection of illustrative plates;
Accompanying drawing 6 is CITICOLINE SODIUM standard substance
13cNMR collection of illustrative plates;
Accompanying drawing 7 is the CITICOLINE SODIUM that makes
13cNMR collection of illustrative plates;
Accompanying drawing 8 is the mass spectrum of CITICOLINE SODIUM standard substance;
The mass spectrum that accompanying drawing 9 is the CITICOLINE SODIUM that makes.
Embodiment
Below by embodiments of the invention, the present invention is described in detail:
With reference to accompanying drawing 1, a kind of method of immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps: to utilize the colibacillus engineering fermentation of molecular cloning cytidine phosphates transferase gene to prepare cytidine phosphates transferring enzyme, and synthesis cytidine phosphates transferring enzyme liquid, the immobilization of cytidine phosphates transferring enzyme, take phosphorylcholine and Cytidine Disodium Triphosphate as raw material, generate citicoline by the catalysis of immobilization cytidine phosphates transferring enzyme, the Classification And Nomenclature of the colibacillus engineering of the molecular cloning cytidine phosphates transferase gene of wherein using in the present invention is that colon bacillus (Escherichia coli) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation centre address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica's deposit number is CGMCC NO:4428, in following examples, all adopt the colibacillus engineering of this molecular cloning cytidine phosphates transferase gene.
Embodiment mono-
A method for immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps:
(1), engineering bacterium fermentation:
The seed liquor of the colibacillus engineering of 150ml molecular cloning cytidine phosphates transferase gene is inoculated in 3L substratum system and is cultivated 26 hours, in this substratum system, contain peptone 10g/L, yeast extract 10g/L, glucose 5g/L, inorganic salt 8g/L, supplements 150g glucose, 15g peptone and 15g yeast extract between purified water breaking-in period; The centrifugal collection thalline of secondary fermentation liquid 152g has fermented.
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Get thalline 30g, within 30 minutes, obtain broken liquid with the miscible suspension of 180ml phosphoric acid buffer and with ultrasonic disruption, in broken liquid, add ammonium sulfate to 30% saturation ratio to saltout, then use 200nm mocromembrane solids removed by filtration material, obtain cytidine phosphates transferring enzyme clear liquid 205ml.
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
Get above-mentioned cytidine phosphates transferring enzyme clear liquid 205ml, add 41g to contain the polyacrylamide gel fixation support of epoxy group(ing), being fixed of stirring, obtains 59g immobilization cytidine phosphates transferring enzyme, amounts to every gram of fixation support fixing cytidine phosphates transferase protein 11mg.
(4), CITICOLINE SODIUM is synthetic:
Preparation 1L synthesis reaction solution, in synthesis reaction solution, Cytidine Disodium Triphosphate is 80mmol/L; Phosphorylcholine 400mmol/L; Magnesium acetate 50mmol/L, surplus is water, and regulating the pH value of reaction solution is 7.5, adds immobilization cytidine phosphates transferring enzyme 50g, and 40 ℃ of stirring reactions of water-bath 10 hours obtain CITICOLINE SODIUM liquid 960ml after filtering, and the transformation efficiency of this Cytidine Disodium Triphosphate reaches 88%.
(5), extract purifying:
Above-mentioned by above-mentioned CITICOLINE SODIUM liquid through chromatographic separation crystallizing and drying, obtain the dry product 27g of CITICOLINE SODIUM, product content 99.8%.
Embodiment bis-
A method for immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps:
(1), engineering bacterium fermentation:
The colibacillus engineering seed liquor of 200ml molecular cloning cytidine phosphates transferase gene is inoculated into 3L substratum system and (in substratum system, contains peptone 15g/L, yeast extract 15g/L, glucose 8g/L, inorganic salt 13.5g/L, purified water is dissolved) middle cultivation 28 hours, supplement during this time 288g glucose, 30g peptone and 30g yeast extract.The centrifugal collection thalline of secondary fermentation liquid 165g has fermented.
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Get thalline 20g, with using ultrasonic disruption 30 minutes after the miscible suspension of 200ml phosphoric acid buffer, obtain broken liquid, in broken liquid, add ammonium sulfate to 45% saturation ratio, then use 500nm mocromembrane solids removed by filtration material, obtain cytidine phosphates transferring enzyme clear liquid 218ml.
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
Get cytidine phosphates transferring enzyme clear liquid 200ml, add the polystyrene fixation support of 20g hydroxyl, stir being fixed, obtain 28g immobilization cytidine phosphates transferring enzyme, amount to every gram of fixation support fixing cytidine phosphates transferase protein 17mg.
(4), CITICOLINE SODIUM is synthetic:
Preparation 1L synthesis reaction solution (is 30mmol/L containing Cytidine Disodium Triphosphate in synthesis reaction solution, phosphorylcholine 60mmol/L, magnesium acetate 10mmol/L, surplus is water), the pH value to 9.0 that regulates reaction solution, adds immobilization cytidine phosphates transferring enzyme 10g, 25 ℃ of stirring reactions of water-bath 9 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 90%, clear liquid (CITICOLINE SODIUM liquid) 973ml obtaining after filtering.
(5), extract purifying:
Above-mentioned CITICOLINE SODIUM liquid, after chromatographic separation crystallizing and drying, obtains the dry product 11g of CITICOLINE SODIUM, product content 99.7%.
Embodiment tri-
The preparation of CITICOLINE SODIUM comprises the steps:
(1), engineering bacterium fermentation:
The colibacillus engineering seed liquor of 300ml molecular cloning cytidine phosphates transferase gene is inoculated into 3L substratum system and (in substratum system, contains peptone 20g/L, yeast extract 20g/L, glucose 8g/L, inorganic salt 15g/L, surplus is that purified water is dissolved) middle cultivation 32 hours, supplement during this time 360g glucose, 48g peptone and 48g yeast extract.The centrifugal collection thalline of secondary fermentation liquid 187g has fermented.
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Get thalline 30g, with the miscible suspension of 120ml phosphoric acid buffer, bacterium liquid is used ultrasonic disruption 30 minutes, obtain broken liquid, in broken liquid, add the saturation ratio of ammonium sulfate to 25%, then use 500nm mocromembrane solids removed by filtration material, obtain cytidine phosphates transferring enzyme clear liquid 144ml.
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
Get cytidine phosphates transferring enzyme clear liquid 144ml, add the polystyrene fixation support of 28g hydroxyl, stir being fixed, obtain 40g immobilization cytidine phosphates transferring enzyme, amount to every gram of fixation support fixing cytidine phosphates transferase protein 20mg.
(4), CITICOLINE SODIUM is synthetic:
Preparation 1L synthesis reaction solution (is 50mmol/L containing Cytidine Disodium Triphosphate in synthesis reaction solution; Phosphorylcholine 180mmol/L; Magnesium acetate 20mmol/L), the pH to 8.0 of adjusting synthesis reaction solution, adds immobilization cytidine phosphates transferring enzyme 30g, 30 ℃ of stirring reactions of water-bath 4 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 91%, obtains CITICOLINE SODIUM liquid 966ml after filtering.
(5), extract purifying:
Above-mentioned CITICOLINE SODIUM liquid, after chromatographic separation crystallizing and drying, obtains the dry product 18.5g of CITICOLINE SODIUM, product content 99.9%.
Embodiment tetra-
The preparation of cytidine phosphates transferring enzyme liquid comprises the steps:
(1), engineering bacterium fermentation:
The colibacillus engineering of 2.5L molecular cloning cytidine phosphates transferase gene is inoculated into 30L substratum system (containing peptone 15g/L, yeast extract 15g/L, glucose 8 g/L, inorganic salt 13.5g/L, surplus is purified water) middle cultivation 28 hours, supplement during this time 2880g glucose, 320g peptone and 320g yeast extract.The centrifugal collection thalline of secondary fermentation liquid 1670g has fermented.
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Get thalline 300g, with the miscible suspension of 2400ml phosphoric acid buffer, clarifixator fragmentation 3 times for bacterium liquid adds ammonium sulfate to 35% saturation ratio in broken bacterium liquid, then the clear clear enzyme solution of the purpose ceramic-film filter of general aperture 500nm, obtains cytidine phosphates transferring enzyme clear liquid 2610ml.
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
Get cytidine phosphates transferring enzyme clear liquid 2610ml, add 400g to contain amino agarose fixation support, stir being fixed, obtain 580g immobilization cytidine phosphates transferring enzyme, amount to every gram of fixation support fixing cytidine phosphates transferase protein 15mg.
(4), CITICOLINE SODIUM is synthetic:
Preparation 10L synthesis reaction solution (is 60mmol/L containing Cytidine Disodium Triphosphate in synthesis reaction solution; Phosphorylcholine 200mmol/L; Magnesium acetate 25mmol/L, surplus is water), the pH value to 8.0 of adjusting synthesis reaction solution, adds immobilization cytidine phosphates transferring enzyme 300g, 30 ℃ of stirring reactions of water-bath 5 hours, and the transformation efficiency of Cytidine Disodium Triphosphate reaches 91%.After filtering, obtain CITICOLINE SODIUM liquid 10.4L.
(5), extract purifying:
Above-mentioned CITICOLINE SODIUM liquid, after chromatographic separation crystallizing and drying, obtains the dry product 203g of CITICOLINE SODIUM, product content 99.7%.
With reference to accompanying drawing 2-accompanying drawing 9, comparative sample is shown in known all consistent with standard substance with dry product infrared absorption pattern, nuclear magnetic resonance map and the mass spectrum of the CITICOLINE SODIUM making.
Embodiment five
A method for immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps:
(1), engineering bacterium fermentation:
The colibacillus engineering seed liquor of 400ml molecular cloning cytidine phosphates transferase gene is inoculated into 6L substratum system and (in substratum system, contains peptone 15g/L, yeast extract 15g/L, glucose 8g/L, inorganic salt 13.5g/L, purified water is dissolved) middle cultivation 30 hours, supplement during this time 576g glucose, 60g peptone and 60g yeast extract.The centrifugal collection thalline of secondary fermentation liquid 330g has fermented.
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Get thalline 40g, with using ultrasonic disruption 30 minutes after the miscible suspension of 400ml phosphoric acid buffer, obtain broken liquid, in broken liquid, add ammonium sulfate to 40% saturation ratio, then use 100nm mocromembrane solids removed by filtration material, obtain cytidine phosphates transferring enzyme clear liquid 436ml.
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
Get cytidine phosphates transferring enzyme clear liquid 400ml, add the carboxylic cellulose fixed carrier of 40g, stir being fixed, obtain 56g immobilization cytidine phosphates transferring enzyme, amount to every gram of fixation support fixing cytidine phosphates transferase protein 17mg.
(4), CITICOLINE SODIUM is synthetic:
Preparation 1L synthesis reaction solution (is 70mmol/L containing Cytidine Disodium Triphosphate in synthesis reaction solution, phosphorylcholine 300mmol/L, magnesium acetate 40mmol/L, surplus is water), regulate the pH value to 6.0 of reaction solution, add immobilization cytidine phosphates transferring enzyme 10g, 10 ℃ of stirring reactions of water-bath 8 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 90%.Clear liquid (CITICOLINE SODIUM liquid) 973ml obtaining after filtering.
(5), extract purifying:
Above-mentioned CITICOLINE SODIUM liquid, after chromatographic separation crystallizing and drying, obtains the dry product 11g of CITICOLINE SODIUM, product content 99.8%.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any modification of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in claim protection domain of the present invention.
Claims (7)
1. a method for immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps:
(1), engineering bacterium fermentation:
The seed liquor of the colibacillus engineering of molecular cloning cytidine phosphates transferase gene is inoculated in substratum system and is cultivated 26～32 hours, and the volume that adds of the seed liquor of the colibacillus engineering of described molecular cloning cytidine phosphates transferase gene is 5%～10% of substratum system volume; Supplement during this time appropriate glucose, peptone and yeast extract, the centrifugal collection thalline of the secondary fermentation liquid that fermented;
(2), prepare cytidine phosphates transferring enzyme clear liquid:
Getting above-mentioned thalline is suspended in phosphoric acid buffer with ultrasonic wave or Syrup-homogenizing instrument fragmentation, obtain broken liquid, in above-mentioned broken liquid, add ammonium sulfate to saltout, obtaining saturation ratio is 25%～45% liquid of saltouing, the purpose ceramic-film filter that is 100～500nm with aperture again separates the above-mentioned solid and liquid of saltouing, obtain cytidine phosphates transferring enzyme clear liquid, wherein, the suspension ratio of described thalline and described phosphoric acid buffer is 1:4～1:10;
(3), the immobilization of cytidine phosphates transferring enzyme clear liquid:
In above-mentioned cytidine phosphates transferring enzyme clear liquid, add fixation support, stir and solidify, obtain immobilization cytidine phosphates transferring enzyme, the ratio of the add-on of described fixation support and cytidine phosphates transferring enzyme clear liquid is 100～200 grams/L;
(4), CITICOLINE SODIUM is synthetic:
To be mixed with synthesis reaction solution containing the aqueous solution of Cytidine Disodium Triphosphate 30～80mmol/L, phosphorylcholine 60～400mmol/L and magnesium acetate 10～50mmol/L, regulate pH value to 6.0～9.0 of synthesis reaction solution, add described immobilization cytidine phosphates transferring enzyme, and under the bath temperature of 10～50 ℃ stirring reaction 4～10 hours, the clear liquid obtaining after filtration is CITICOLINE SODIUM liquid, and the add-on of described immobilization cytidine phosphates transferring enzyme and the ratio of synthesis reaction solution are 10～50g/L;
(5), extract purifying:
Above-mentioned CITICOLINE SODIUM liquid, through chromatographic separation crystallizing and drying, is obtained to the dry product of CITICOLINE SODIUM.
2. the method for a kind of immobilized enzyme catalysis production CITICOLINE SODIUM according to claim 1, is characterized in that, in described substratum system, contains peptone 10～20g/L, yeast extract 10～20g/L, glucose 5～8g/L, inorganic salt 8～15g/L, surplus is purified water.
3. the method for a kind of immobilized enzyme catalysis production CITICOLINE SODIUM according to claim 2, it is characterized in that, in engineering bacterium fermentation fermenting process, during this time the amount of supplementary glucose be the glucose that contains in substratum system amount 10-15 doubly, the 50-80% of the amount that the amount of supplementary peptone is the peptone that contains in substratum system, the 50-80% of the amount that the amount of supplementary yeast extract is the yeast extract that contains in substratum system.
4. the method for a kind of immobilized enzyme catalysis production CITICOLINE SODIUM according to claim 3, it is characterized in that, described fixation support is selected from any of Mierocrystalline cellulose, glucose gel, agarose, polyacrylamide gel, polyamino acid, polystyrene, nylon or porous glass matrix.
5. the method for a kind of immobilized enzyme catalysis production CITICOLINE SODIUM according to claim 4, is characterized in that, contains at least one group in fragrant amido or hydroxyl or carboxyl or carboxymethyl or epoxy group(ing) or amino functional group in described fixation support.
6. the method for a kind of immobilized enzyme catalysis production CITICOLINE SODIUM according to claim 5, is characterized in that, in the synthetic step of CITICOLINE SODIUM, and immobilization cytidine phosphates transferring enzyme 10～50g/L, Cytidine Disodium Triphosphate is 30～80mmol/L; Phosphorylcholine 60～400mmol/L; Magnesium acetate 10～50mmol/L; 25～40 ℃ of temperature of reaction; PH value is 6.0～9.0.
7. the method for a kind of immobilized enzyme catalysis production CITICOLINE SODIUM according to claim 6, it is characterized in that, in the synthetic step of CITICOLINE SODIUM, immobilization cytidine phosphates transferring enzyme content is 20～30g/L, and Cytidine Disodium Triphosphate is 40～60mmol/L; Phosphorylcholine 120～240mmol/L; Magnesium acetate 20～30mmol/L; 30～40 ℃ of temperature of reaction; PH value is 6.5～8.0.
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| CN110257362A (en) * | 2019-06-04 | 2019-09-20 | 开平牵牛生化制药有限公司 | Preparation method and application of cholic acid and derivative thereof surfactant modified metal organic framework nano composite catalyst |
| CN111808899A (en) * | 2020-08-31 | 2020-10-23 | 宁波酶赛生物工程有限公司 | Synthesis method of citicoline sodium |
| CN114262726A (en) * | 2022-01-11 | 2022-04-01 | 深圳华酶生物科技有限公司 | Method for synthesizing citicoline sodium by using cytidine enzymatic method |
| CN117025697A (en) * | 2023-10-10 | 2023-11-10 | 开平牵牛生化制药有限公司 | Method for producing adenosylmethionine by hydroxy resin immobilized enzyme method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257362A (en) * | 2019-06-04 | 2019-09-20 | 开平牵牛生化制药有限公司 | Preparation method and application of cholic acid and derivative thereof surfactant modified metal organic framework nano composite catalyst |
| CN111808899A (en) * | 2020-08-31 | 2020-10-23 | 宁波酶赛生物工程有限公司 | Synthesis method of citicoline sodium |
| CN114262726A (en) * | 2022-01-11 | 2022-04-01 | 深圳华酶生物科技有限公司 | Method for synthesizing citicoline sodium by using cytidine enzymatic method |
| CN117025697A (en) * | 2023-10-10 | 2023-11-10 | 开平牵牛生化制药有限公司 | Method for producing adenosylmethionine by hydroxy resin immobilized enzyme method |
| CN117025697B (en) * | 2023-10-10 | 2024-01-30 | 开平牵牛生化制药有限公司 | Method for producing adenosylmethionine by hydroxy resin immobilized enzyme method |
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