CN102199643A - Preparation method of citicoline - Google Patents
Preparation method of citicoline Download PDFInfo
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- CN102199643A CN102199643A CN2011100526032A CN201110052603A CN102199643A CN 102199643 A CN102199643 A CN 102199643A CN 2011100526032 A CN2011100526032 A CN 2011100526032A CN 201110052603 A CN201110052603 A CN 201110052603A CN 102199643 A CN102199643 A CN 102199643A
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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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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention discloses a preparation method of citicoline. The method comprises the following steps: the culture of a single gene engineering bacterium or the treatment material of the culture is used as enzyme source to catalyze the reaction of a substrate comprising ammonium chloride, orotic acid and phosphocholine and ensure that citicoline is generated and accumulated in the reaction solution, and citicoline is extracted from the reaction solution. The invention has the following beneficial effects: (1) the single microorganism is utilized to catalyze the reaction, reaction conditions are easy to control; (2) the cost of the substrate is lower so that the production cost of citicoline is lower; and (3) the reaction is fast and the conversion rate is higher. The method can be widely used in the preparation of citicoline.
Description
Technical field
The present invention relates to a kind of preparation method of cytidine diphosphate, belong to biological technical field.
Background technology
Cytidine diphosphate has another name called cytidine diphosphocholine, and Chinese Pharmacopoeia version in 2005 is named and is citicoline, English name: Citichaline, its chemical name: choline cytidine(C-5 '-bisphosphate.It is the biosynthetic precursor of Yelkin TTS, and when brain function descended, the lecithin content in the cerebral tissue significantly reduced.The supplemented with exogenous cytidine diphosphate can activate the biosynthesizing of Yelkin TTS, stimulates the excitement of reticular formation of brain stem, improves the threshold value of reviving, and recovers the nervous tissue function, improves brain metabolism and nerve conduction, improves patient's level of consciousness.
Find and determine molecular structure that Rossiter etc. illustrates its function after a while the fifties in last century by people such as doctors kennedy.At the beginning of the seventies, the research and development of Japanese military Tanabe Selyaku Co., Ltd also are used for the treatment of cerebral trauma, the clinical achieving success of the disturbance of consciousness that the brain operation is followed, its trade(brand)name: Nichdin, middle translated name: Ni Keling.China is from the mid-1970s; unit cooperations such as biochemical teaching and research room of East China Normal University and day kitchen Gourmet Powder Factory; at first adopt cereuisiae fermentum to do the enzymatic living beings fermentation; carried out the exploration of chemosynthesis after a while again, and carried out the clinical application of about two terms in brain surgery such as Shanghai Huashan Hospital, No.1 People's Hospital Shanghai City, hospital of Xinhua and Neurology Department.At present, cytidine diphosphate is as brain surgery, Neurology Department and dissimilar senile dementia medications.Therefore, to being in great demand of it, the domestic market has reached about in the of 60 tons at present, and export volume also grows with each passing day.
The synthetic method of cytidine diphosphate mainly contains methods such as chemical synthesis, enzyme process and microbe transformation method, and still, preceding two kinds of methods no longer adopt now because cost is too high.Method commonly used at present is a microbe transformation method, utilizes microbial cell enzyme system synthetic.Because cell has the complete multienzyme system of keeping its vital movement, various enzymes are keeping residing state of original life cell and specific position again, therefore can finish the multistep enzymic catalytic reaction quickly and effectively.Yet the production method of existing cytidine diphosphate need be participated in by multiple microorganism mostly, makes that the processing condition of producing are comparatively harsh, and processing step is also comparatively loaded down with trivial details; And the price of the employed substrate of production method of existing cytidine diphosphate is comparatively expensive mostly, makes the cost of producing cytidine diphosphate greatly improve.
Summary of the invention
In view of the defective that above-mentioned prior art exists, the objective of the invention is to propose a kind of economy, efficient, and the preparation method of the simple cytidine diphosphate of technology.
Purpose of the present invention will be achieved by the following technical programs:
The preparation method of cytidine diphosphate, use the culture of single genetic engineering bacterium or its handled thing as the enzyme source, catalysis comprises that the substrate of ammonium chloride, vitamin B13 and phosphorylcholine reacts, make to generate in the reaction solution and the savings cytidine diphosphate, from described reaction solution, extract described cytidine diphosphate.
Further, but described genetic engineering bacterium has the gene of abduction delivering choline phosphate cytidylyltransferase, orotidylic acid pyrophosphorylase, orotidylic decarboxylase, the sweet acid kinase of urine, nucleoside diphosphokinase and cytidine triphosphate(CTP) ligase enzyme.
Further, but the gene of described choline phosphate cytidylyltransferase of abduction delivering and cytidine triphosphate(CTP) ligase enzyme be positioned on the plasmid of described genetic engineering bacterium.
Further, but the gene of the described orotidylic acid pyrophosphorylase of abduction delivering, orotidylic decarboxylase, the sweet acid kinase of urine and nucleoside diphosphokinase be positioned on the karyomit(e) of described genetic engineering bacterium.
Further, but the gene nucleotide fragment of the described choline phosphate cytidylyltransferase of abduction delivering derives from yeast saccharomyces cerevisiae.
Further, but the gene fragment of the described cytidine triphosphate(CTP) ligase enzyme of abduction delivering, orotidylic acid pyrophosphorylase, orotidylic decarboxylase, the sweet acid kinase of urine and nucleoside diphosphokinase derives from intestinal bacteria.
Further, the preparation method of described genetic engineering bacterium comprises the steps:
But but the gene nucleotide fragment that a) will derive from the described choline phosphate cytidylyltransferase of abduction delivering of yeast saccharomyces cerevisiae is connected to form recombinant plasmid with plasmid vector with the gene nucleotide fragment that derives from colibacillary abduction delivering cytidine triphosphate(CTP) ligase enzyme after enzyme is cut;
B) but be inserted on the karyomit(e) of host cell by the gene fragment that homologous recombination will derive from the described orotidylic acid pyrophosphorylase of colibacillary abduction delivering, orotidylic decarboxylase, the sweet acid kinase of urine and nucleoside diphosphokinase;
C) described recombinant plasmid is transferred in the described host cell.
Further, described plasmid vector is a plasmid pUC18.
Further, described host cell is a prokaryotic cell prokaryocyte.
Further, described host cell is a Bacillus coli cells.
Compared with prior art, beneficial effect of the present invention is: (1) adopts single microorganism catalysis reaction, and reaction conditions is easy to control; (2) the substrate cost is lower, thereby makes that the production cost of cytidine diphosphate is lower; (3) be swift in response and transformation efficiency higher.
Following constipation closes the embodiment accompanying drawing, the specific embodiment of the present invention is described in further detail, so that technical solution of the present invention is easier to understand, grasp.
Description of drawings
Fig. 1 is the schema that the portion gene of the genetic engineering bacterium among the embodiment makes up.
Embodiment
The preparation method of cytidine diphosphate of the present invention is to use the culture of single genetic engineering bacterium or its handled thing as the enzyme source, catalysis comprises that the substrate of ammonium chloride, vitamin B13 and phosphorylcholine reacts, make to generate in the reaction solution and the savings cytidine diphosphate, from described reaction solution, extract described cytidine diphosphate.
[embodiment]
Employed genetic engineering bacterium is single bacterial strain in the present embodiment, simultaneously orotidylic acid pyrophosphorylase (pyrE), the orotidylic decarboxylase (pyrF) in abduction delivering zymic choline phosphate cytidylyltransferase (CCT) and the intestinal bacteria pyrimidine metabolic approach, urinate sweet acid kinase (pyrH), nucleoside diphosphokinase (NDK) and cytidine triphosphate(CTP) ligase enzyme (pyrG).
According to cct gene order (the genebank accession number is M36827) the design primer of known yeast saccharomyces cerevisiae, be that template amplification goes out the cct full length gene with the yeast chromosomal.PyrE gene, pyrF gene, pyrH gene, pyrG gene and ndk gene are that (the genebank accession number: ECK3632) design primer is that template amplification goes out these full length genes with the e. coli dna according to the intestinal bacteria whole genome sequence.
The preparation method of described genetic engineering bacterium specifically comprises the steps:
1, cct gene and pyrG expression vector make up
According to the pyrG gene nucleic acid sequence of known e. coli k12 MG1655 and the cct gene nucleic acid sequence of yeast saccharomyces cerevisiae, respectively with the karyomit(e) of intestinal bacteria and yeast saccharomyces cerevisiae as template, amplify total length cct gene and pyrG gene.
Adopt conventional bacterium and yeast DNA extraction method to extract respectively available from the original coli strain DH5 α of Germany and the genomic dna of Wine brewing yeast strain ATCC 26786/26787 (DSM 4266/4267).
What amplification was used is fidelity pfu enzyme preferably, is connected in pUC19-T vector after adding the A tail, and the picking positive colony is delivered order-checking company.
(1) structure of plasmid pUCG
The pyrG gene enzyme from the carrier that is connected on the pUC19-T vector is scaled off, the restriction enzyme that uses is Sma I and BamH I, while carrier pUC18 uses restriction enzyme Nru I and BamH I to carry out enzyme and cuts, after the carrier enzyme cut product and reclaim, carry out enzyme with the pyrG gene fragment that reclaims and connect reaction, by the digestion of Restriction Enzymes such as Sma I and BamH I, analyze, confirm fragment successful connection.The final plasmid pUCG that obtains, the pyrG gene is at this moment by lacZ promotor abduction delivering.
(2) structure of plasmid pUC-CCT
The cct gene enzyme from the carrier that is connected on the pUC19-T vector is scaled off, the flat end limit restriction endonuclease that uses is Dra I, equally, that pUC18 uses is flat terminal enzyme Sma I, after the carrier enzyme is cut back to close, carries out enzyme with the cct gene and connects reaction, elder generation's pcr amplification checking, by the digestion of Restriction Enzymes such as Sma I, analyze again, confirm fragment successful connection.
(3) structure of plasmid pUCG-CCT
By pcr amplification, the cct gene that will have promotor earlier is connected on the pUC19-T vector, and sequence is confirmed in order-checking, downcut the purpose fragment with Nde I enzyme, carry out agarose electrophoresis, reclaim DNA according to reclaiming the test kit operation instructions, be dissolved among the 20 μ L TE-20 ℃ of preservations in centrifuge tube.With being connected of dephosphorization carrier pUCG/Nde I, get a 0.2mL PCR pipe, add 7.5 μ L exogenous dna fragments, 1 μ LpUCG dephosphorylation carrier, 1 μ L, 10 * T4 Ligation Buffer, 0.5 μ L T4 ligase enzyme is positioned over 16 ℃ of water-baths and spends the night behind the mixing.The enzyme co-product is converted among the coli strain DH5 α picking positive colony on the flat board of the Amp that contains 100mg/L.Enzyme cut and pcr amplification with the checking positive colony exactness.
2, the structure of pyrE, pyrF, pyrH, ndk manipulator on the escherichia coli chromosome
From e. coli k12 MG1655 karyomit(e), amplify pyrE, pyrF, pyrH, ndk gene respectively, after the order-checking, the pyrE fragment is connected on the plasmid pUC18, obtain plasmid pUCpyrE by EcoRI, SacI restriction enzyme site; By SacI, SmaI site the pyrF fragment is connected on the plasmid pUCpyrE again, obtains plasmid pUC-pyrEF; Then the pyrH fragment is connected on the pUC-pyrEF via SmaI and XbaI site, obtains plasmid pUCpyrEFH; By XbaI and HindIII site the ndk fragment is connected on the pUCpyrEFH at last, but the plasmid pUCpyrEFHk of acquisition manipulator of abduction delivering pyrE, pyrF, pyrH, ndk gene after the lac promotor specifically makes up flow process as shown in Figure 1.
3, have pyrE, pyrF, the pyrH of KRT-Km-FRT element, the structure of ndk manipulator plasmid
Design has the XhoI site on the ndk gene primer, pUCpyrEFHk is cut with XhoI and ScaI enzyme, is that the Kan gene fragment that has the FRT site that template amplification goes out links to each other with what have XhoI and ScaI restriction enzyme site equally with plasmid pKD13, is built into plasmid pUCpyrEFHkKmFRT, as shown in Figure 1.
4, the reorganization of Red homologous recombination method pyrE, pyrF, pyrH, ndk gene
(1) preparation of linear dsdna target practice molecule and processing
The pcr amplification primer that is used for the foreign DNA of gene knockout, they are made up of 20bp homologous region on the kalamycin resistance gene homologous region of template plasmid pKD13 of pectinose manipulator homologous region and 3 ' end 20bps of 5 ' end 38bps or the plasmid pUCpyrEFHkKmFRT respectively.
Set up following amplification reaction system:
The pcr amplification reaction condition:
Use high-fidelity DNA polymerase (Pfu archaeal dna polymerase), synthesized linear dsdna target practice molecule (5 ' homology arm+selection markers+3 ' homology arm, the homology part marks with underscore) through PCR.The plasmid template that exists in the PCR product can cause the false positiveization of recon, acts on the PCR product with Dpn I enzyme, methylated template plasmid can be decomposed, for there not being the then not effect of methylated PCR product.Promptly can be used for the Red homologous recombination after PCR product after the Dpn I effect reclaimed with ethanol precipitation, take directly to cut the method that glue reclaims and also can reach same effect.
Dpn I handles: set up following system:
Enzyme is cut 2-3h under 37 ℃ of water-baths.
Recycling step reference reagent box is operated.
(2) preparation of the abduction delivering of recombinase gene and competent cell
Plasmid pKD46 CaCl with coding Red recombination system
2Method is converted in the intestinal bacteria, and host bacterium 30 ℃ of overnight shakings in containing the LB substratum of penbritin are cultivated, and inoculation 1mL bacterium liquid is in the LB liquid nutrient medium that contains Amp of 50mL, and 30 ℃ of shaking culture 3-4h are to OD600 ≈ 0.6.1h adds L-arabinose before cultivating termination, and making it final concentration is 0.1%, and Exo, Bet and three albumen of Gam on the plasmid pKD46 are given full expression to.Precooling 10min on ice, centrifugal collection thalline with 10% glycerine or ultrapure washing 3-4 time, is resuspended in thalline in 500 cold aseptic 10% glycerine at last, gets 50 μ L and is used for electric shock and transforms, and all the other can deposit in-80 ℃ of refrigerators standby.Above operation steps all will be carried out on ice.
(3) electric shock transforms
Get 2 μ L (20-100ng) PCR products and mix with the competence that 50 μ l have just prepared, adopt the BioRad Micro-Pulser of company electroporation apparatus to carry out, the 0.2cm electricity transforms cup, and the conversion parameter is 2.5kV, 5.8ms.Add LB substratum 1ml rapidly, place 37 ℃ of shaking tables to cultivate 1-2h, recovery uses the LB plate screening that contains selection markers that the positive colony of homologous recombination has taken place, and obtains bacterial strain reorganization bacterium K1.
(4) checking design of primers and amplification condition
When reorganization bacterium K1 was identified, the rule of design of primers was: a design of primers is peripheral in the district of practicing shooting, and other one then is positioned at linear target practice sequence inside.
Set up following amplification reaction system:
The pcr amplification reaction condition:
Confirmed the position that recombination event takes place by order-checking, the bacterial strain of acquisition promptly can be used for next step resistance and removes.
(5) removal of kalamycin resistance gene
Plasmid pCP20 expresses the FLP recombinase, acts on the recombinant chou karyomit(e) on two FRT target sites, and kalamycin resistance gene is lost.The plasmid pCP20 that will have amicillin resistance utilizes CaCl
2Method transforms and enters in the positive strain with Kan resistance, obtains positive transformant on the flat board that is containing Amp and Kan under 30 ℃ of culture condition.Being transferred to does not then have in the antibiotic LB substratum, cultivates 5h, 37 ℃ of plate loop method for 42 ℃.Resulting single bacterium colony is carried out Amp and the responsive detection of Kan, and the phenotype bacterial strain of last Amp and Kan sensitivity is exactly a recon of getting rid of the Kan gene.This eliminates the primer antagonism and verifies that condition is the same to use Kan simultaneously.Obtain removing the recon K1-E of resistance.
5, the acquisition of genetic engineering bacterium
The described recon K1-E that obtains in the step 4 is prepared as competent cell, import wherein making up the described plasmid pUCG-CCT that finishes in the step 1 again, can obtain having the genetic engineering bacterium K1-E/pUCG-CCT of the synthetic cytidine two phosphorus choline abilities of catalysis vitamin B13, phosphorylcholine and ammonium chloride.
Engineering bacteria K1-E/pUCG-CCT bacterial strain on the solid medium is inserted 5-150ml contain in the seed culture fluid of penbritin (100 μ g/ml), 25-37 ℃ with 150-280rpm shaking culture 8-16 hour.By the inoculum size of 0.1-10% this nutrient solution is forwarded in the 1L Erlenmeyer flask of seed LB liquid medium that 10-500ml contains penbritin (100 μ g/ml), at 25-37 ℃ with 150-280rpm shaking culture 2-8 hour, the inductor isopropyl-that adds final concentration then and be 0.1-1mM is induced, continue to cultivate 4-12 hour, culture temperature is suitably reduced.
With refrigerated centrifuge with fermented liquid under 4-20 ℃ with the centrifugal 5-30min of the speed of 1000-5000rpm, abandon supernatant, use 25-150ml 0.05M, the resuspended somatic cells of the Tris-HC1 of pH 8.0.
The resuspended 4g Bacillus coli cells that obtains is like this added in the triangular flask, and, add distilled water again and be settled to 100mL to wherein adding 10g glucose, 0.6g vitamin B13,0.8g ammonium chloride, 1g dipotassium hydrogen phosphate, 1g potassium primary phosphate, 1.5g phosphorylcholine, 1mg ferrous sulfate, 0.8mg manganous sulfate, 40mg sal epsom and 0.4mL dimethylbenzene.This mixed solution is packed in the triangular flask of 2L capacity, under 30 ℃, the condition of 100rpm, react.In the reaction, adding KOH so that keep pH value of reaction system in suitable process is 7.2.React after 24 hours, the growing amount of measuring cytidine diphosphocholine with the HPLC method is 6.9g/L.
Originally be in the example, as carrier, as long as in host microorganism, can duplicate; As host microorganism,, be used for cytidine diphosphate production and get final product as long as can express recombinant DNA.
Cultivate the substratum of genetic engineering bacterium in the present embodiment, genetic engineering bacterium can be used for metabolic carbon source, nitrogenous source, inorganic salts etc., can cultivate microorganism of the present invention effectively in the present embodiment as long as contain, no matter be natural medium, or synthetic medium all can.Wherein:
Carbon source is so long as genetic engineering bacterium can be used for metabolizer and gets final product in the present embodiment, as glucose, fructose, sucrose; The carbohydrate such as molasses, starch or starch hydrolysate that contain above-mentioned sugar; Organic acid such as acetic acid, propionic acid; Alcohols such as ethanol and propyl alcohol.
Nitrogenous source can be used nitrogenous compounds such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; And peptone, meat extract, yeast extract, corn impregnation liquid, casein hydrolysate, soybean meal and soybean meal hydrolyzate, various fermentation thalline and digestion product thereof etc.
Inorganic salt can be used potassium primary phosphate, dipotassium hydrogen phosphate, trimagnesium phosphate, sal epsom, sodium-chlor, ferrous sulfate, manganous sulfate, copper sulfate and lime carbonate etc.
The optimum temperuture of cultivating genetic engineering bacterium in the present embodiment is 25-37 ℃, and the pH value of substratum preferably maintains neutral range in the cultivation, and incubation time is 6-24 hour.
The beneficial effect of present embodiment is: (1) adopts single microorganism catalysis reaction, and reaction conditions is easy to control; (2) the substrate cost is lower, thereby makes that the production cost of cytidine diphosphate is lower; (3) be swift in response and transformation efficiency higher.
The present invention still has numerous embodiments, and all employing equivalents or equivalent transformation and all technical schemes of forming all drop within protection scope of the present invention.
Claims (10)
1. the preparation method of cytidine diphosphate, it is characterized in that: use the culture of single genetic engineering bacterium or its handled thing as the enzyme source, catalysis comprises that the substrate of ammonium chloride, vitamin B13 and phosphorylcholine reacts, make to generate in the reaction solution and the savings cytidine diphosphate, from described reaction solution, extract described cytidine diphosphate.
2. the preparation method of cytidine diphosphate according to claim 1 is characterized in that: but described genetic engineering bacterium has the gene of abduction delivering choline phosphate cytidylyltransferase, orotidylic acid pyrophosphorylase, orotidylic decarboxylase, the sweet acid kinase of urine, nucleoside diphosphokinase and cytidine triphosphate(CTP) ligase enzyme.
3. the preparation method of cytidine diphosphate according to claim 2 is characterized in that: but the gene of described choline phosphate cytidylyltransferase of abduction delivering and cytidine triphosphate(CTP) ligase enzyme is positioned on the plasmid of described genetic engineering bacterium.
4. the preparation method of cytidine diphosphate according to claim 3 is characterized in that: but the gene of the described orotidylic acid pyrophosphorylase of abduction delivering, orotidylic decarboxylase, the sweet acid kinase of urine and nucleoside diphosphokinase is positioned on the karyomit(e) of described genetic engineering bacterium.
5. the preparation method of cytidine diphosphate according to claim 4 is characterized in that: but the gene nucleotide fragment of the described choline phosphate cytidylyltransferase of abduction delivering derives from yeast saccharomyces cerevisiae.
6. the preparation method of cytidine diphosphate according to claim 5 is characterized in that: but the gene fragment of the described cytidine triphosphate(CTP) ligase enzyme of abduction delivering, orotidylic acid pyrophosphorylase, orotidylic decarboxylase, the sweet acid kinase of urine and nucleoside diphosphokinase derives from intestinal bacteria.
7. according to the preparation method of any described cytidine diphosphate among the claim 1-6, it is characterized in that: the preparation method of described genetic engineering bacterium comprises the steps:
But but the gene nucleotide fragment that a) will derive from the described choline phosphate cytidylyltransferase of abduction delivering of yeast saccharomyces cerevisiae is connected to form recombinant plasmid with plasmid vector with the gene nucleotide fragment that derives from colibacillary abduction delivering cytidine triphosphate(CTP) ligase enzyme after enzyme is cut;
B) but be inserted on the karyomit(e) of host cell by the gene fragment that homologous recombination will derive from the described orotidylic acid pyrophosphorylase of colibacillary abduction delivering, orotidylic decarboxylase, the sweet acid kinase of urine and nucleoside diphosphokinase;
C) described recombinant plasmid is transferred in the described host cell.
8. the preparation method of cytidine diphosphate according to claim 7, it is characterized in that: described plasmid vector is a plasmid pUC18.
9. the preparation method of cytidine diphosphate according to claim 8, it is characterized in that: described host cell is a prokaryotic cell prokaryocyte.
10. the preparation method of cytidine diphosphate according to claim 9, it is characterized in that: described host cell is a Bacillus coli cells.
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| CN102586383A (en) * | 2012-03-15 | 2012-07-18 | 齐鲁制药有限公司 | Process for preparing cytidine diphosphate choline |
| CN105463042A (en) * | 2015-10-23 | 2016-04-06 | 苏州天马精细化学品股份有限公司 | Method for preparing citicoline through biological enzyme catalysis |
| CN109207415A (en) * | 2017-07-07 | 2019-01-15 | 苏州华赛生物工程技术有限公司 | A method of producing the recombinant microorganism and production citicoline of citicoline |
| CN110699373A (en) * | 2019-10-16 | 2020-01-17 | 中国药科大学 | Uridine diphosphate glucose high-producing strain and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102586383A (en) * | 2012-03-15 | 2012-07-18 | 齐鲁制药有限公司 | Process for preparing cytidine diphosphate choline |
| CN102586383B (en) * | 2012-03-15 | 2014-01-22 | 齐鲁制药有限公司 | Process for preparing cytidine diphosphate choline |
| CN105463042A (en) * | 2015-10-23 | 2016-04-06 | 苏州天马精细化学品股份有限公司 | Method for preparing citicoline through biological enzyme catalysis |
| CN109207415A (en) * | 2017-07-07 | 2019-01-15 | 苏州华赛生物工程技术有限公司 | A method of producing the recombinant microorganism and production citicoline of citicoline |
| CN109207415B (en) * | 2017-07-07 | 2022-09-16 | 苏州华赛生物工程技术有限公司 | Recombinant microorganism for producing citicoline and method for producing citicoline |
| CN110699373A (en) * | 2019-10-16 | 2020-01-17 | 中国药科大学 | Uridine diphosphate glucose high-producing strain and application thereof |
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Application publication date: 20110928 |