CN103849666B - A kind of method of immobilized enzyme catalysis production CITICOLINE SODIUM - Google Patents
A kind of method of immobilized enzyme catalysis production CITICOLINE SODIUM Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
A method for immobilized enzyme catalysis production CITICOLINE SODIUM, comprises the steps: to utilize the colibacillus engineering fermentation of molecular cloning cytidine phosphates transferase gene for cytidine phosphates transferring enzyme, and synthesis cytidine phosphates transferring enzyme liquid; Cytidine phosphates transfer enzyme immobilizatio; With phosphorylcholine and Cytidine Disodium Triphosphate for raw material, generate citicoline by the catalysis of immobilization cytidine phosphates transferring enzyme.Production technique of the present invention is simple, and with short production cycle, production cost is low, and the industrialization that can be widely used in citicoline is produced.
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 is also known as 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 that Yelkin TTS synthesizes necessary coenzyme, finds in 1954, synthesis in 1956,1973 domestic trial-produces successfully.
The pharmacological action of CITICOLINE SODIUM mainly contains: the structure can recovering cytolemma after brain cell injury, strengthens the function of cytolemma, improves neuronic function; Reduce cerebral vascular resistance, increase cerebral blood flow (CBF), promote brain metabolism, improve cerebral circulation, improve brain function; Increase the secretion comprising the nerve conduction medium of vagusstoff and Dopamine HCL, strengthen the reticular formation of brain stem function relevant with consciousness, after improving injury of head or the state of consciousness of the brain Post operation disturbance of consciousness and electroencephalogram, promote the recovery of the upper extremity exercise function of Patients with Hemiplegia, promotion brain function is recovered, promotes to revive have certain effect; Promote Yelkin TTS biosynthesizing and anti-phospholipase A effect, thus accelerate the heavily absorption of cerebral edema.Promote dopaminergic movable, 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 brain Post operation disturbance of consciousness 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, microbe fermentation method and enzymic synthesis three kinds of methods.Chemical synthesis, with cytidylic acid (CMP), phosphorylcholine (CP) for substrate, be condensing agent with Tosyl chloride, CITICOLINE SODIUM is condensed under dinethylformamide (DMF) exists, it is low to there is reaction conversion ratio in chemosynthesis, the problems such as by product is many, and production cost is high, and environmental pollution is serious; Microbe fermentation method utilizes glucose with the microorganism such as yeast or Brevibacterium ammoniagenes, and extract fermentation generation CITICOLINE SODIUM before adding CMP, CP etc., the problems such as it is low that microbe fermentation method exists production concentration, and productive rate is unstable; Enzymic synthesis once had to carry with Liver of Guinea Pig cell homogenates takes out cytidine phosphates transferring enzyme, and catalysis CTP and phosphorylcholine generate the Preliminary report of CITICOLINE SODIUM, but carrys out source problem because of enzyme, and enzymic synthesis cannot realize.
Summary of the invention:
The object of this invention is to provide one to 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, the method for the Enzyme catalyzed synthesis CITICOLINE SODIUM that the industrialization that can be widely used in CITICOLINE SODIUM is produced.
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 cultivates 26 ~ 32 hours, add that volume is substratum system volume 5% ~ 10% of the seed liquor of the colibacillus engineering of described molecular cloning cytidine phosphates transferase gene; Period supplements appropriate glucose, peptone and yeast extract, and fermented secondary fermentation liquid collected by centrifugation thalline.
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get above-mentioned thalline to be suspended in phosphoric acid buffer with ultrasonic wave or Syrup-homogenizing instrument fragmentation, obtain broken liquid, in above-mentioned broken liquid, add ammonium sulfate saltout, obtaining saturation ratio is 25% ~ 45% to saltout liquid, be that the purpose ceramic-film filter of 100 ~ 500nm is by above-mentioned solid and liquid separation of saltouing again with aperture, 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:
Add fixation support in above-mentioned cytidine phosphates transferring enzyme clear liquid, stir solidification, obtain immobilization cytidine phosphates transferring enzyme, the add-on of described fixation support and the ratio of cytidine phosphates transferring enzyme clear liquid are 100 ~ 200 grams/L.
(4), the synthesis of CITICOLINE SODIUM:
Aqueous solution containing Cytidine Disodium Triphosphate 30 ~ 80mmol/L, phosphorylcholine 60 ~ 400mmol/L and magnesium acetate 10 ~ 50mmol/L is mixed with synthesis reaction solution, 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 DEG C stirring reaction 4 ~ 10 hours, the clear liquid obtained after filtration is CITICOLINE SODIUM liquid, and the described add-on of immobilization cytidine phosphates transferring enzyme and the ratio of synthesis reaction solution are 10 ~ 50g/L.
(5), extraction purification
By above-mentioned CITICOLINE SODIUM liquid through chromatographic separation crystallizing and drying, obtain 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, containing peptone 10 ~ 20g/L in described substratum system, yeast extract 10 ~ 20g/L, glucose 5 ~ 8g/L, inorganic salt 8 ~ 15g/L, surplus is purified water, in engineering bacterium fermentation fermenting process, the amount of the glucose that period supplements is 10-15 times of the amount of the glucose contained in substratum system, the amount of the peptone supplemented is the 50-80% of the amount for the peptone contained in substratum system, and the amount of supplementary yeast extract is the 50-80% of the amount for the yeast extract contained in substratum system.
Further, described fixation support is selected from any one of Mierocrystalline cellulose, glucose gel, agarose, polyacrylamide gel, polyamino acid, polystyrene, nylon or porous glass matrix.
Further, in described fixation support containing at least one group in fragrant amido or hydroxyl or carboxyl or carboxymethyl or epoxy group(ing) or amino-functional group.
Preferably, in the step of the synthesis 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; Temperature of reaction 25 ~ 40 DEG C; PH value is 6.0 ~ 9.0.
Preferably, in the step of the synthesis 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; Temperature of reaction 30 ~ 40 DEG C; 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 stablized, and the workshop condition of avoiding the domestic cereuisiae fermentum reaction system generally adopted at present to bring pollutes and a large amount of waste yeast slag such as need to 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 reduce the synthesis procedure time, transformation efficiency reaches more than 90%, effectively reduces production cost.
3. compared with traditional technology, present invention process is environmentally friendly, has a clear superiority in energy-saving consumption-reducing, protection of the environment.
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;
Accompanying drawing 3 is the IR collection of illustrative plates of obtained CITICOLINE SODIUM;
Accompanying drawing 4 is CITICOLINE SODIUM standard substance
1hNMR collection of illustrative plates;
Accompanying drawing 5 is the CITICOLINE SODIUM obtained
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 obtained
13cNMR collection of illustrative plates;
Accompanying drawing 8 is the mass spectrum of CITICOLINE SODIUM standard substance;
Accompanying drawing 9 is the mass spectrum of obtained CITICOLINE SODIUM.
Embodiment
Below by way of 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 for cytidine phosphates transferring enzyme, and synthesis cytidine phosphates transferring enzyme liquid, cytidine phosphates transfer enzyme immobilizatio, with phosphorylcholine and Cytidine Disodium Triphosphate for raw material, citicoline is generated 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 wherein 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, the colibacillus engineering of this molecular cloning cytidine phosphates transferase gene is all adopted in following examples.
Embodiment one
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 cultivates 26 hours, containing peptone 10g/L in this substratum system, 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; Fermented secondary fermentation liquid collected by centrifugation thalline 152g.
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get thalline 30g, within 30 minutes, broken liquid is obtained with ultrasonic disruption with the miscible suspension of 180ml phosphoric acid buffer, in broken liquid, add ammonium sulfate to 30% saturation ratio 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 the polyacrylamide gel fixation support of 41g containing epoxy group(ing), being fixed of stirring, obtains 59g immobilization cytidine phosphates transferring enzyme, amounts to every gram of fixation support and fix cytidine phosphates transferase protein 11mg.
(4), the synthesis of CITICOLINE SODIUM:
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 the pH value regulating reaction solution is 7.5, adds immobilization cytidine phosphates transferring enzyme 50g, water-bath 40 DEG C of stirring reactions 10 hours, and obtain CITICOLINE SODIUM liquid 960ml after filtering, the transformation efficiency of this Cytidine Disodium Triphosphate reaches 88%.
(5), extraction purification:
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 two
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 (containing peptone 15g/L in substratum system, yeast extract 15g/L, glucose 8g/L, inorganic salt 13.5g/L, purified water dissolve) in cultivate 28 hours, period supplements 288g glucose, 30g peptone and 30g yeast extract.Fermented secondary fermentation liquid collected by centrifugation thalline 165g.
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get thalline 20g, use ultrasonic disruption 30 minutes with 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 and fix cytidine phosphates transferase protein 17mg.
(4), the synthesis of CITICOLINE SODIUM:
(contain Cytidine Disodium Triphosphate in synthesis reaction solution is 30mmol/L to preparation 1L synthesis reaction solution, phosphorylcholine 60mmol/L, magnesium acetate 10mmol/L, surplus is water), regulate the pH value to 9.0 of reaction solution, add immobilization cytidine phosphates transferring enzyme 10g, water-bath 25 DEG C of stirring reactions 9 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 90%, clear liquid (CITICOLINE SODIUM liquid) 973ml obtained after filtering.
(5), extraction purification:
Above-mentioned CITICOLINE SODIUM liquid, after chromatographic separation crystallizing and drying, obtains the dry product 11g of CITICOLINE SODIUM, product content 99.7%.
Embodiment three
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 (containing peptone 20g/L in substratum system, yeast extract 20g/L, glucose 8g/L, inorganic salt 15g/L, surplus be purified water dissolve) in cultivate 32 hours, period supplements 360g glucose, 48g peptone and 48g yeast extract.Fermented secondary fermentation liquid collected by centrifugation thalline 187g.
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get thalline 30g, with the miscible suspension of 120ml phosphoric acid buffer, bacterium liquid 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 and fix cytidine phosphates transferase protein 20mg.
(4), the synthesis of CITICOLINE SODIUM:
(contain Cytidine Disodium Triphosphate in synthesis reaction solution is 50mmol/L to preparation 1L synthesis reaction solution; Phosphorylcholine 180mmol/L; Magnesium acetate 20mmol/L), regulate the pH to 8.0 of synthesis reaction solution, add immobilization cytidine phosphates transferring enzyme 30g, water-bath 30 DEG C of stirring reactions 4 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 91%, obtains CITICOLINE SODIUM liquid 966ml after filtering.
(5), extraction purification:
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 four
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) in cultivate 28 hours, period supplements 2880g glucose, 320g peptone and 320g yeast extract.Fermented secondary fermentation liquid collected by centrifugation thalline 1670g.
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get thalline 300g, with the miscible suspension of 2400ml phosphoric acid buffer, the clarifixator fragmentation 3 times of bacterium liquid, adds ammonium sulfate to 35% saturation ratio in broken bacterium liquid, then the clear clear enzyme solution of 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 containing amino agarose fixation support, stir being fixed, obtain 580g immobilization cytidine phosphates transferring enzyme, amount to every gram of fixation support and fix cytidine phosphates transferase protein 15mg.
(4), the synthesis of CITICOLINE SODIUM:
(contain Cytidine Disodium Triphosphate in synthesis reaction solution is 60mmol/L to preparation 10L synthesis reaction solution; Phosphorylcholine 200mmol/L; Magnesium acetate 25mmol/L, surplus is water), regulate the pH value to 8.0 of synthesis reaction solution, add immobilization cytidine phosphates transferring enzyme 300g, water-bath 30 DEG C of stirring reactions 5 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 91%.CITICOLINE SODIUM liquid 10.4L is obtained after filtering.
(5), extraction purification:
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, the dry product infrared absorption pattern of comparative sample and the CITICOLINE SODIUM obtained, nuclear magnetic resonance map and mass spectrum are shown in known all consistent with standard substance.
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 (containing peptone 15g/L in substratum system, yeast extract 15g/L, glucose 8g/L, inorganic salt 13.5g/L, purified water dissolve) in cultivate 30 hours, period supplements 576g glucose, 60g peptone and 60g yeast extract.Fermented secondary fermentation liquid collected by centrifugation thalline 330g.
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get thalline 40g, use ultrasonic disruption 30 minutes with 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 and fix cytidine phosphates transferase protein 17mg.
(4), the synthesis of CITICOLINE SODIUM:
(contain Cytidine Disodium Triphosphate in synthesis reaction solution is 70mmol/L to preparation 1L 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, water-bath 10 DEG C of stirring reactions 8 hours, the transformation efficiency of Cytidine Disodium Triphosphate reaches 90%.Clear liquid (CITICOLINE SODIUM liquid) 973ml obtained after filtering.
(5), extraction purification:
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 amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within claims 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 cultivates 26 ~ 32 hours, add that volume is substratum system volume 5% ~ 10% of the seed liquor of the colibacillus engineering of described molecular cloning cytidine phosphates transferase gene; Period supplements appropriate glucose, peptone and yeast extract, and fermented secondary fermentation liquid collected by centrifugation thalline;
(2) cytidine phosphates transferring enzyme clear liquid, is prepared:
Get above-mentioned thalline to be suspended in phosphoric acid buffer with ultrasonic wave or Syrup-homogenizing instrument fragmentation, obtain broken liquid, in above-mentioned broken liquid, add ammonium sulfate saltout, obtaining saturation ratio is 25% ~ 45% to saltout liquid, be that the purpose ceramic-film filter of 100 ~ 500nm is by above-mentioned solid and liquid separation of saltouing again with aperture, 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:
Add fixation support in above-mentioned cytidine phosphates transferring enzyme clear liquid, stir solidification, obtain immobilization cytidine phosphates transferring enzyme, the add-on of described fixation support and the ratio of cytidine phosphates transferring enzyme clear liquid are 100 ~ 200 grams/L;
(4), the synthesis of CITICOLINE SODIUM:
Aqueous solution containing Cytidine Disodium Triphosphate 30 ~ 80mmol/L, phosphorylcholine 60 ~ 400mmol/L and magnesium acetate 10 ~ 50mmol/L is mixed with synthesis reaction solution, 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 DEG C stirring reaction 4 ~ 10 hours, the clear liquid obtained after filtration is CITICOLINE SODIUM liquid, and the described add-on of immobilization cytidine phosphates transferring enzyme and the ratio of synthesis reaction solution are 10 ~ 50g/L;
(5), extraction purification:
By above-mentioned CITICOLINE SODIUM liquid through chromatographic separation crystallizing and drying, obtain the dry product of CITICOLINE SODIUM.
2. method according to claim 1, is characterized in that, containing peptone 10 ~ 20g/L, yeast extract 10 ~ 20g/L, glucose 5 ~ 8g/L, inorganic salt 8 ~ 15g/L in described substratum system, surplus is purified water.
3. method according to claim 2, it is characterized in that, in engineering bacterium fermentation fermenting process, the amount of the glucose that period supplements is 10-15 times of the amount of the glucose contained in substratum system, the amount of the peptone supplemented is the 50-80% of the amount for the peptone contained in substratum system, and the amount of supplementary yeast extract is the 50-80% of the amount for the yeast extract contained in substratum system.
4. method according to claim 3, is characterized in that, described fixation support is selected from any one of Mierocrystalline cellulose, glucose gel, agarose, polyacrylamide gel, polyamino acid, polystyrene, nylon or porous glass matrix.
5. method according to claim 4, is characterized in that, containing 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. method according to claim 5, is characterized in that, in the step of the synthesis of CITICOLINE SODIUM, and immobilization cytidine phosphates transferring enzyme 10 ~ 50g/L; Temperature of reaction 25 ~ 40 DEG C; PH value is 6.0 ~ 9.0.
7. method according to claim 6, is characterized in that, in the step of the synthesis of CITICOLINE SODIUM, immobilization cytidine phosphates transferring enzyme content is 20 ~ 30g/L, and Cytidine Disodium Triphosphate is 40 ~ 60mmol/T; Phosphorylcholine 120 ~ 240mmo1/T; Magnesium acetate 20 ~ 30mmol/L; Temperature of reaction 30 ~ 40 DEG C; 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 |
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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