CN102586383B - Process for preparing cytidine diphosphate choline - Google Patents
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Abstract
The invention relates to a process for preparing cytidine diphosphate choline. According to the method, denovo synthesis of pyrimidine is utilized as a basic reaction and choline materials are subjected to a reaction at the reaction temperature ranging from 20 DEG C to 38 DEG C for 10 to 72 hours with pH controlled at 5 to 7.5 in the presence of culture solution of microorganism A or further in the presence of culture solution of microorganism B or treatment so as to accumulatively produce cytidine diphosphate choline. Simple starting materials of carbon and nitrogen sources are utilized, in culture solution containing the choline materials, precursor of the cytidine diphosphate choline, namely cytidine monophosphate is synthesized by means of metabolic process, and cytidine diphosphate choline is cumulatively produced in the reaction solution. According to the method, expensive cytidine monophosphate or the precursor materials are avoided so that the preparing cost of the cytidine diphosphate choline is greatly reduced.
Description
Technical field
The present invention relates to a kind of preparation method of citicoline, be specifically related to fermentation method and prepare citicoline, belong to biological pharmacy technical field.
Background technology
Citicoline (CDPC) is the precursor substance of phospholipid phosphatidylcholine, is the synthetic necessary coenzyme of Yelkin TTS.Citicoline is widely used in the treatment of the diseases such as cerebral ischemia, brain injury, alzheimer's disease, Parkinson's disease and senile dementia clinically.Citicoline has another name called cytidine diphosphate, cytidine diphosphocholine.The preparation method that citicoline is known has chemical synthesis, use the enzyme process of the microbial cells such as yeast, utilize enzyme process that genetically modified microorganism transforms etc.
Common ground in these preparation methods is that as raw material, to have used the pyrimidines such as expensive cytidine monophosphate (CMP), cytidine diphosphate(CDP) (CDP), cytidine triphosphate(CTP) (CTP), cytosine(Cyt) be Nucleotide and precursor vitamin B13, uridylic etc., makes the preparation cost of citicoline higher.
Using the enzyme process of the microbial cells such as yeast to manufacture citicoline, is to using cytidylic acid and phosphorylcholine as raw material, utilizes microorganism that a series of enzymes such as the required choline phosphate cytidylyltransferase of reaction (CCT) are provided, and a step catalysis completes the preparation of citicoline.
The price of the initiator cytidylic acid in this process is very expensive, makes preparation cost high.
CN1074938 discloses the preparation method of cytidine diphosphocholine, and the method is that a series of enzymes such as the required UTP-ammonia ligase of reaction, choline phosphate cytidylyltransferase (CCT) are provided with microorganism, and a step catalysis completes the preparation of citicoline.CN1653188 discloses the preparation method of cytidine 5 '-diphosphate choline, and the method adopts choline, phosphorylcholine or their salt and uridylic to contact with biological catalyst in water-soluble medium, accumulates generation CDP-C.CN102199643A discloses a kind of preparation method of cytidine diphosphate, use the culture of single genetic engineering bacterium or its handled thing as enzyme source, catalysis comprises that the substrate of ammonium chloride, vitamin B13 and phosphorylcholine reacts, make to generate and savings cytidine diphosphate in reaction solution, from described reaction solution, extract described cytidine diphosphate.CN101538598A provides a kind of preparation method of cytidine diphosphate, it is characterized in that take that choline chloride 60, phosphate anion and cytidine monophosphate or its precursor are substrate, take glucose, fructose, sucrose or maltose as energy donor, add small molecules chemical effect material, utilize the yeast cell whole-cell catalytic of having property to prepare cytidine diphosphate; Wherein, described cytidine monophosphate precursor is vitamin B13 or cytidine; Described small molecules chemical effect material is any one or two kinds in magnesium ion, potassium ion, with any one or a few the composition in N.F,USP MANNITOL, halfcystine and spermine.
The prices such as the starting raw material vitamin B13 of above method or uridylic or cytidylic acid are all more expensive, and cost is also higher.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of and take microbe fermentation method that pyrimidine de novo synthesis is basic reaction and prepare the method for citicoline.
Summary of the invention
It is that Nucleotide or its precursor are as expensive raw materials such as vitamin B13, uridylics that method of the present invention is not used pyrimidine, and use industrial raw material (as choline chloride 60, phosphorylcholine etc.) that be easy to obtain, cheap, that choline can be provided to undergo microbial fermentation, prepare citicoline.
Method of the present invention be take pyrimidine de novo synthesis as basic reaction, by simple starting raw material carbon and nitrogen sources, started, utilize metabolic pathway of synthesizing in some specified microorganisms bodies, the simple materials such as synthetic ribose phosphoric acid, amino acid and one carbon unit and carbonic acid gas, then the synthetic precursor cytosine(Cyt) of preparing citicoline is Nucleotide, contain can provide the raw material of choline as the nutrient solution of phosphorylcholine or choline chloride 60 etc. in, under the participation of the choline phosphate cytidylyltransferase being provided by microorganism, accumulation generates citicoline.
The raw material of the de novo synthesis of pyrimidine nucleotide is aspartic acid, glutamine, CO
2deng, these amino acid are from the amino acid anabolism in organism.Key enzyme in reaction process is different in different organisms, in bacterium, the biosynthesizing of pyrimidine nucleotide, the point that is subject to end product feedback control has three: first regulatory enzyme of route of synthesis is carbamylphosphate synthetase, and it is subject to the feedback inhibition of uridylate (UMP); Another two regulatory enzymes are aspartate carbamoyltransferase and cytidine triphosphate(CTP) (CTP) synthetic enzyme, and they are all subject to the feedback inhibition of cytidine triphosphate(CTP) (CTP).The former is suppressed will affect the synthetic of uridylic acid and cytidylic acid, and suppressed of the latter affects the synthetic of cytidylic acid.
It is Nucleotide that a lot of microorganism fermentations can produce cytosine(Cyt), if remove the feedback inhibition of UMP, CTP, the branched metabolic pathway of the initial amino acid aspartic acid in cut-off pyrimidine route of synthesis, and select to produce cytosine(Cyt) be that to enliven thalline or the culture in period be the enzyme source of Nucleotide as producing cytosine(Cyt) to Nucleotide, can metabolism generation cytosine(Cyt) faster be Nucleotide, under the participation of the choline phosphate cytidylyltransferase providing in this microorganism or other microorganism, accumulation generates citicoline.Thereby avoiding using expensive pyrimidine is that Nucleotide is precursor, further reduces preparation cost.
The present invention, as starting point, has selected the bacterial strain of suitable the method, by microorganism, is fermented and is prepared citicoline.
Detailed Description Of The Invention
Technical scheme of the present invention is as follows:
A kind of preparation method of citicoline, take pyrimidine de novo synthesis as basic reaction, the method comprises: in the situation that there is the nutrient solution of microorganism A to exist, or also have in the nutrient solution of microorganism B or the situation of handled thing existence, choline raw material is reacted 10～72 hours 20～38 ℃ of temperature of reaction, control pH 5～7.5, accumulation generates citicoline.
Described choline raw material is selected from choline, choline chloride 60, phosphorylcholine, choline iodide or choline bromide.
Described microorganism A is selected from or 2 above bacterial strains that belong to that belong in Corynebacterium (Corynebacterium), brevibacterium sp (Brevibacterium), bacillus (Bacillus), Rhodopseudomonas (Rseudomonas), yeast saccharomyces cerevisiae genus (Saccharomyces cerevisiae).Bacterial strain uses therefor possesses following characteristics:
(1) can metabolism generation cytosine(Cyt) be nucleosides product;
(2) there is pyrimidine architecture analogue resistance, can remove the feedback inhibition of UMP, CTP product;
(3) cytidine deaminase in metabolic enzyme system disappearance, the cytosine(Cyt) of blocking-up synthesized is that Nucleotide deamination recovers to be converted into uridylic is Nucleotide;
(4) can end homoserine dehydrogenase action pathway, the precursor aspartic acid of accumulation pyrimidine de novo synthesis;
(5) there is choline phosphate cytidylyltransferase activity, can be product by the cytidylic acid of synthesized be converted into citicoline as cytidylic acid, cytidine triphosphate(CTP) etc., the transformant that this enzyme can obtain from the DNA of natural microbial or transduttant coding phosphorylcholine dysuria due to the pressure of the fetus glycosides acyl enzyme.
Described microorganism B is selected from the 1. industrial cereuisiae fermentum thalline of beer fermentation, or 2. contain one of intestinal bacteria, Corynebacterium glutamicum of choline phosphate cytidylyltransferase, or 3. contain the intestinal bacteria of choline phosphate cytidylyltransferase, the transformant of Corynebacterium glutamicum, nutrient solution or extract.
Preferred according to the present invention, microorganism A is subtilis (Bacillus subtilis) QL28001 preserving number CGMCC No.5849, corynebacterium glutamicum preserving number ATCC No.13032, pseudomonas cepacia preserving number ATCCNo.25416, Arthrobacter citreus preserving number ATCC No.11624, one of yeast saccharomyces cerevisiae preserving number ATCC No.40075.
The most preferred microorganism A of the present invention is subtilis (Bacillus subtilis) QL28001, that subtilis (Bacillus subtilis) seed selection that the present invention is ATCC No.6051 by culture presevation number first comes, in (address: preservation Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences), preserving number is CGMCC No.5849, China Committee for Culture Collection of Microorganisms common micro-organisms center March 6 in 2012.
It is that all enzymes of Nucleotide are that the subtilis QL28001 preserving number CGMCC No.5849 being obtained for the subtilis seed selection of ATCC No.6051 by culture presevation number of the present invention contains synthesizing cytimidine, and can remove the feedback inhibition of UMP, CTP, ended the branched metabolic pathway of the initial amino acid aspartic acid in pyrimidine route of synthesis.Its bacterial characteristics is as follows:
1.1 morphology
1.1.1 shape and size: bacillus brevis (0.7-0.8 * 2.5-3 micron)
1.1.2 polymorphic: monotype, dimorphism is rare
1.1.3 mobility: nothing
1.1.4 sporulation: have
1.1.5 spore shape: oval
1.1.6 spore position: near central authorities
1.1.7 gramstaining: the positive
1.2 growth conditions
1.2.1 meat extract agar plate culture: irregular and divergent contour, surface irregularity and flat, opaque, light brown.
1.2.2 litmus milk: peptonize hypopigmentation
1.2.3 meat extract liquid culture: surface forms mycoderm, not muddy
1.3 physiological property
1.3.1 nitrate reduction :+
1.3.2 Starch Hydrolysis:
1.3.3v-p test: positive+
1.3.4 the utilization of propionic acid :-
1.3.5 the utilization of citric acid :+
1.3.6 the utilization of ammonium salt :+
1.3.7 urase: faint
1.3.8 catalase: exist
1.3.9 the requirement to oxygen: aerobic
1.3.10 resistance to acid: can grow during ph5.7
1.3.11 anti-sodium-chlor: can grow during concentration 7%
1.3.12 the 5 FU 5 fluorouracil of anti-pyrimidine analogue: 0.5ug/ml can be grown
1.3.13 cytidine deaminase activity is lower than 0.01 unit/mg albumen
According to the preparation method of above-mentioned citicoline, one of preferred version, step is as follows:
(1) subtilis QL28001 is inoculated in the nutrient solution that contains 0.1-5% glucose, 0.5-5% extractum carnis, 1-10% peptone, 0.5-5% yeast powder, 0.5-1% sodium-chlor composition, 32 ℃ when being cultured to sugar and exhausting, obtain the culture of microorganism A.
By the inoculum size of 5wt%, the culture of microorganism A is inoculated in nutrient solution, described nutrient solution is the aqueous solution that contains carbon source, nitrogenous source and trace element, this nutrient solution carbon source concentration is that 8-20wt%, nitrogen concentration are that 3-6wt%, microelement concentration are 0.2-5wt%, and add the phosphoric acid salt of 0.1-3%, cultivate 5～25h for 30～35 ℃; The nutrient solution that must contain microorganism A, or medium centrifugal is collected to thalline;
(2) in the nutrient solution that contains microorganism A of step (1), add choline raw material, choline raw material dosage is the 0.5-3wt% of nutrient solution weight, 20～38 ℃ of temperature of reaction, react 10～72 hours, control reaction accumulation under pH 5～7.5 conditions and generate citicoline.
Or,
The nutrient solution that contains microorganism A of step (1) is inoculated in the nutrient solution that contains carbon source, nitrogenous source, trace element, choline raw material and microorganism B according to the inoculum size of 5-30wt%, 20～38 ℃ of temperature of reaction, react 10～72 hours, control pH 5～7.5, in this nutrient solution, reaction accumulation generates citicoline.In this nutrient solution, carbon source concentration is 8-20wt%, and nitrogen concentration is 3-6wt%, and microelement concentration is 0.2-5wt%, and microorganism B concentration is 5-30wt%, and choline raw material dosage is the 0.5-3wt% of nutrient solution weight, cultivates 5～25h for 30～35 ℃.
Further preferably, in the nutrient solution of above-mentioned steps (2), be also added with the tensio-active agent of 0.005-0.05wt%.
According to the preparation method of above-mentioned citicoline, two of preferred version, step is as follows:
1) microorganism A is inoculated in the nutrient solution that contains 0.5% glucose, 0.5% extractum carnis, 1% peptone, 0.5% yeast powder, 0.5% sodium-chlor composition, 32 ℃ when being cultured to sugar and exhausting, separating thallus obtains the culture of microorganism A.
2) by step 1) culture of microorganism A is inoculated in the nutrient solution that contains carbon source, nitrogenous source, trace element according to the inoculum size of 5-20wt%, 20～38 ℃ of temperature of reaction, reacts 10～30 hours separating thallus; In described nutrient solution, carbon source concentration is 8-20wt%, and nitrogen concentration is 3-6wt%, and microelement concentration is 0.2-5wt%;
3) by step 2) thalline adds in the nutrient solution that contains carbon source, nitrogenous source, trace element, choline raw material, tensio-active agent and microorganism B according to 5-30wt%, 20～38 ℃ of temperature of reaction, react 20～72 hours, control pH 5～7.5, in this nutrient solution, reaction generates accumulation citicoline.In described nutrient solution, carbon source concentration is 8-20wt%, and nitrogen concentration is 3-6wt%, and microelement concentration is 0.2-5wt%, and microorganism B concentration is 5-30wt%, and choline raw material dosage is the 0.5-3wt% of nutrient solution weight.Or,
By step 2) thalline according to 5-30wt%, add in the nutrient solution that contains carbon source, nitrogenous source, trace element, choline raw material, 20～38 ℃ of temperature of reaction, react 20～72 hours, control pH 5～7.5, in this nutrient solution, reaction generates accumulation citicoline.In described nutrient solution, carbon source concentration is 8-20wt%, and nitrogen concentration is 3-6wt%, and microelement concentration is 0.2-5wt%, and choline raw material dosage is the 0.5-3wt% of nutrient solution weight.
Further preferably, in nutrient solution above-mentioned steps 3), be also added with the tensio-active agent of 0.005-0.05wt%.
Further preferably, above-mentioned steps 2), 3) in nutrient solution, be also added with the phosphoric acid salt of 0.1-3%.
According to the preparation method of citicoline of the present invention, from the accumulation citicoline reaction solution of above gained, further extract described citicoline.By prior art.
In the preparation method of above-mentioned citicoline, described carbon source is selected from one of following three classes:
A, carbohydrate, be specifically selected from glucose, fructose, sucrose, maltose, seminose, sorbose, gluconic acid, molasses, glycerine or starch hydrolyzates.
B, organic acid, be specifically selected from pyruvic acid, lactic acid, citric acid or pyruvic acid.
C, natural organic carbon source, be specifically selected from corn steep liquor or bagasse.
Above-mentioned citicoline preparation method, described nitrogenous source be selected from one of corn steep liquor, yeast powder, yeast extract, ammonium sulfate, ammoniacal liquor, ammonia, ammonium acetate, glutamine, urea or combination;
Above-mentioned citicoline preparation method, described trace element is selected from a kind of in magnesium salts, manganese salt, cobalt salt, mantoquita, sylvite, Bio or combination, further a kind of the or combination in preferably sulfuric acid magnesium, potassium primary phosphate, Bio.
Above-mentioned citicoline preparation method, preferred reaction conditions is pH 6～7,30～35 ℃ of temperature of reaction, the reaction times is controlled at 30～48 hours.
Preparation method of the present invention, in order to promote the synthetic of citicoline, also can add suitable tensio-active agent in reaction system, and preferred, tensio-active agent is tween-80.
In preparation method of the present invention, do not itemize by prior art.Adopt above listed condition to prepare the method for citicoline, having adopted cheap phosphorylcholine, choline chloride 60 etc. is raw material, and preparation cost is reduced greatly.Preparation level by the method cytidine diphosphate can reach 11.2g/L reaction solution.
Embodiment
Further illustrate by the following examples the present invention, these embodiment should be as restriction of the present invention.If no special instructions, in embodiment, the consumption of each composition is mass percent.
Microbe-derived as follows in embodiment:
Industry cereuisiae fermentum wet thallus is purchased from Jinan Beer factory.
Bio is purchased from Shanghai Sheng Gong biotechnology company limited.
Corynebacterium glutamicum preserving number ATCC No.13032, pseudomonas cepacia preserving number ATCC No.25416, Arthrobacter citreus preserving number ATCC No.11624, Corynebacterium glutamicum preserving number ATCC No.13287, intestinal bacteria preserving number ATCCNo.21148 etc. are purchased from the biological product of USS collecting center.The center preservation of subtilis (Bacillus subtilis) QL28001 ，You China Committee for Culture Collection of Microorganisms's common micro-organisms, culture presevation numbering CGMCC No.5849.
The culture of described microorganism, handled thing except have special instruction all by prior art, obtain.
Embodiment 1,
A, subtilis QL28001 is inoculated in the nutrient solution that contains 0.5% glucose, 0.5% extractum carnis, 1% peptone, 0.5% yeast powder, 0.5% sodium-chlor composition, 32 ℃ when being cultured to sugar and exhausting, obtains the culture of subtilis;
B, the inoculum size by 5%, be inoculated in the culture of gained subtilis QL28001 in the nutrient solution that contains 20% glucose, 5% yeast powder, 0.5% sal epsom, 1% potassium primary phosphate composition, cultivates 6h for 32 ℃; Obtain the nutrient solution of subtilis QL28001;
The nutrient solution Continuous Flow of C, gained subtilis QL28001 adds 1% phosphorylcholine, continues reaction 30h, in process, with strong aqua, regulates pH 6.8, contains citicoline 5.7g/L in the nutrient solution of acquisition.
Embodiment 2, by the steps A of embodiment 1, make the culture of subtilis; By 5% inoculum size, the culture of subtilis is inoculated in the nutrient solution that contains 8% glucose, 2% yeast powder, 1% urea, 0.5% sal epsom, 1% potassium primary phosphate, 0.01%D-vitamin H, cultivate for 35 ℃ and exhaust to sugar for approximately 20 hours.
Gained nutrient solution is inoculated in the nutrient solution that contains 12% glucose, 1% sal epsom, 1% potassium primary phosphate, 1% phosphorylcholine, 0.01% tween-80,10% industrial cereuisiae fermentum wet thallus according to 30% inoculum size, continue reaction 30h, in process, with strong aqua, regulate pH to remain on 6.8, in the nutrient solution of acquisition, contain citicoline 7.6g/L.
Embodiment 3, by the steps A of embodiment 1, make the culture of subtilis; Inoculum size by 5%, the culture of subtilis is inoculated in the nutrient solution that contains 8% glucose, 2% yeast powder, 1% urea, 0.5% sal epsom, 1% potassium primary phosphate, 0.01%D-vitamin H, 35 ℃ are cultured to 24 hours, centrifugal collection thalline, according to the inoculum size of wet thallus 10%, be inoculated in the nutrient solution that contains 12% glucose, 1% sal epsom, 1% potassium primary phosphate, 1% phosphorylcholine, 0.01% tween-80, reaction 30h, in process, with strong aqua, regulate pH to remain on 6.8, in the nutrient solution of acquisition, contain citicoline 8.2g/L.
Embodiment 4, by the steps A of embodiment 1, make the culture of subtilis, inoculum size by 5%, the culture of subtilis is inoculated in and contains 12% glucose, 2% yeast powder, 1% urea, 2% corn steep liquor, 0.5% sal epsom, 1% potassium primary phosphate, in the nutrient solution of 0.01%D-vitamin H, 35 ℃ are cultured to 24 hours, centrifugal collection thalline, according to the inoculum size of wet thallus 10%, be inoculated in and contain 10% glucose, 1% sal epsom, 4% potassium primary phosphate, 1% phosphorylcholine, 0.01% tween-80, in the nutrient solution of 10% industrial cereuisiae fermentum wet thallus, 24 ℃ of reaction 46h, in process, with strong aqua, regulate pH to remain on 6.5, in the nutrient solution obtaining, contain citicoline 11.2g/L.
Embodiment 5, by the steps A of embodiment 1, make the culture of subtilis; Inoculum size by 5%, the culture of subtilis is inoculated in the nutrient solution of the industrial cereuisiae fermentum wet thallus that contains 13% glucose, 2% yeast powder, 1% urea, 0.5% sal epsom, 1% potassium primary phosphate, 0.01%D-vitamin H, 10% freeze thawing, 2% phosphorylcholine, cultivate 48 hours for 30 ℃, in process, with strong aqua, regulate pH to remain on 6.5, in the nutrient solution of acquisition, contain citicoline 8.7g/L.
Embodiment 6, by the steps A of embodiment 1, make the culture of subtilis; Inoculum size by 5%, culture is inoculated in the nutrient solution that contains 10% glucose, 2% yeast powder, 2% urea, 0.8% sal epsom, 1% potassium primary phosphate, 0.01%D-vitamin H, cultivate after 24 hours for 30 ℃, add the phosphorylcholine of 15% industrial cereuisiae fermentum wet thallus and 1.5%, continue to cultivate 48h, in process, with strong aqua, regulate pH to remain on 6.8, in the nutrient solution of acquisition, contain citicoline 7.2g/L.
Embodiment 7
Corynebacterium glutamicum preserving number ATCC No.13032 is inoculated in the nutrient solution that contains 0.5% glucose, 0.5% extractum carnis, 1% peptone, 0.5% yeast powder, 0.5% sodium-chlor composition, 31 ℃ are cultured to sugar and exhaust, and obtain the culture of corynebacterium glutamicum ATCC 13032.Inoculum size by 5%, gained culture is inoculated in and contains 8% glucose, 2% yeast powder, 1% urea, 0.5% sal epsom, 1% potassium primary phosphate, in the nutrient solution of 0.01%D-vitamin H, 37 ℃ when being cultured to sugar and exhausting, nutrient solution is inoculated in and contains 13% glucose according to 30% inoculum size, 0.6% sal epsom, 1.5% potassium primary phosphate, 1.5% phosphorylcholine, 0.01% tween-80, in the nutrient solution of 10% industrial cereuisiae fermentum wet thallus, continue reaction 27h, in process, with strong aqua, regulate pH to remain on 6.8, in the nutrient solution obtaining, contain citicoline 4.8g/L.
Embodiment 8
Pseudomonas cepacia preserving number ATCC No.25416 is inoculated in the nutrient solution that contains 1% glucose, 0.5% peptone, yeast extract paste 0.1%, Sodium phosphate dibasic 0.05% composition, cultivates 19h for 28 ℃, obtain the culture of pseudomonas cepacia ATCC 25416.Inoculum size by 5%, gained culture is inoculated at the nutrient solution that contains 5% glucose, 1.5% corn steep liquor, 1% soybean meal hydrolysate, maltose 2%, 0.5% sal epsom, 1% potassium primary phosphate composition and is supported in base, cultivate after 10h for 26 ℃, Continuous Flow adds 1% phosphorylcholine, continue reaction 40h, in process, with strong aqua, regulate pH 6.8, in the nutrient solution of acquisition, contain citicoline 4.7g/L.
Embodiment 9
Arthrobacter citreus preserving number ATCC No.11624 is inoculated in the nutrient solution that contains 1% glucose, 0.5% yeast extract paste, 1% peptone, 0.1% Sodium phosphate dibasic composition, and 25 ℃ are cultured to sugar and exhaust, and obtain the culture of Arthrobacter citreus ATCC 11624.Inoculum size by 5%, culture is inoculated in the nutrient solution that contains 8% glucose, 1% corn steep liquor, 1% soybean meal hydrolysate, maltose 2%, 0.5% sal epsom, 1% potassium primary phosphate, 0.01%D-vitamin H, 25 ℃ are cultured to 36 hours, centrifugal collection thalline, according to the inoculum size of wet thallus 10%, be inoculated in the nutrient solution that contains 12% glucose, 1% sal epsom, 1% potassium primary phosphate, 1% phosphorylcholine, 0.01% tween-80, reaction 30h, in process, with strong aqua, regulate pH to remain on 6.8, in the nutrient solution of acquisition, contain citicoline 3.4g/L.
Embodiment 10
Yeast saccharomyces cerevisiae preserving number ATCC No.40075 is inoculated in the nutrient solution that contains 0.5% glucose, 0.5% extractum carnis, 1% peptone, 0.5% yeast powder, 0.5% sodium-chlor composition, 32 ℃ are cultured to sugar and exhaust, and obtain the culture of yeast saccharomyces cerevisiae ATCC 40075.Inoculum size by 5%, culture is inoculated in and contains 12% glucose, 2% yeast powder, 1% urea, 2% corn steep liquor, 0.5% sal epsom, 1% potassium primary phosphate, in the nutrient solution of 0.01%D-vitamin H, 35 ℃ are cultured to 24 hours, centrifugal collection thalline, according to the inoculum size of wet thallus 10%, be inoculated in and contain 10% glucose, 1% sal epsom, 4% potassium primary phosphate, 1% phosphorylcholine, 0.01% tween-80, in the nutrient solution of 25% intestinal bacteria ATCC 21148 dry myceliums, 24 ℃ of reaction 40h, in process, with strong aqua, regulate pH to remain on 6.7, in the nutrient solution obtaining, contain citicoline 4.2g/L.
Embodiment 11, by the steps A of embodiment 1, make the culture of subtilis, inoculum size by 5%, culture is inoculated in and contains 12% glucose, 2% yeast powder, 1% urea, 2% corn steep liquor, 0.5% sal epsom, 1% potassium primary phosphate, in the nutrient solution of 0.01%D-vitamin H, 35 ℃ are cultured to 24 hours, centrifugal collection thalline, according to the inoculum size of wet thallus 10%, be inoculated in and contain 10% glucose, 1% sal epsom, 4% potassium primary phosphate, 1% phosphorylcholine, 0.01% tween-80, in the nutrient solution of 9% Corynebacterium glutamicum preserving number ATCC No.13287 dry mycelium, 24 ℃ of reaction 40h, in process, with strong aqua, regulate pH to remain on 6.7, in the nutrient solution obtaining, contain citicoline 6.9g/L.
Claims (1)
1. the preparation method of a citicoline, take pyrimidine de novo synthesis as basic reaction, and the method comprises: in the situation that there is the nutrient solution of microorganism A to exist, choline raw material is reacted 10～72 hours 20～38 ℃ of temperature of reaction, control pH 6～7, accumulation generates citicoline;
Described choline raw material is selected from choline chloride 60, phosphorylcholine, choline iodide or choline bromide;
Described microorganism A be selected from the subtilis that preserving number is CGMCC No.5849 (
bacillus subtilis), preserving number is the pseudomonas cepacia of ATCC No.25416, preserving number is one of Arthrobacter citreus of ATCC No.11624.
2
.the preparation method of citicoline as claimed in claim 1, is characterized in that step is as follows:
(1) by the subtilis of preserving number CGMCC No.5849 (
bacillus subtilis) be inoculated in the nutrient solution that contains 0.1-5% glucose, 0.5-5% extractum carnis, 1-10% peptone, 0.5-5% yeast powder, 0.5-1% sodium-chlor composition, 32 ℃ when being cultured to sugar and exhausting, obtain the culture of microorganism A;
By the inoculum size of 5wt%, the culture of microorganism A is inoculated in nutrient solution, described nutrient solution is the aqueous solution that contains carbon source, nitrogenous source and trace element, this nutrient solution carbon source concentration is that 8-20 wt %, nitrogen concentration are that 3-6 wt %, microelement concentration are 0.2-5 wt %, and add the phosphoric acid salt of 0.1-3%, cultivate 5～25h for 30～35 ℃; The nutrient solution that must contain microorganism A;
(2) in the nutrient solution that contains microorganism A of step (1), add choline raw material, choline raw material dosage is the 0.5-3 wt % of nutrient solution weight, 20～38 ℃ of temperature of reaction, react 10～72 hours, control reaction accumulation under pH 6～7 conditions and generate citicoline.
3
.the preparation method of citicoline as claimed in claim 2, is characterized in that being also added with in the nutrient solution of described step (2) tensio-active agent of 0.005-0.05 wt %, and described tensio-active agent is tween-80.
4
.the preparation method of citicoline as claimed in claim 2, is characterized in that, described carbon source is selected from one of glucose, maltose, corn steep liquor.
5
.the preparation method of citicoline as claimed in claim 2, is characterized in that described nitrogenous source is selected from one of yeast powder, yeast extract, corn steep liquor, ammoniacal liquor, urea or combination.
6
.the preparation method of citicoline as claimed in claim 2, is characterized in that described trace element is selected from the combination of sal epsom and potassium primary phosphate, or the combination of sal epsom, potassium primary phosphate and Bio.
7
.the preparation method of citicoline as claimed in claim 1, is characterized in that reaction conditions is pH 6～7,30～35 ℃ of temperature of reaction, and the reaction times is controlled at 30～48 hours.
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| CN101538598A (en) * | 2008-03-19 | 2009-09-23 | 南京工业大学 | Method for preparing citicoline |
| CN102199643A (en) * | 2011-03-04 | 2011-09-28 | 苏州天马医药集团天吉生物制药有限公司 | Preparation method of citicoline |
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| CN102199643A (en) * | 2011-03-04 | 2011-09-28 | 苏州天马医药集团天吉生物制药有限公司 | Preparation method of citicoline |
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