CN101792786A - Method for synthesizing cytidine phosphoryl compound by directional catalysis - Google Patents
Method for synthesizing cytidine phosphoryl compound by directional catalysis Download PDFInfo
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- CN101792786A CN101792786A CN201010118203.2A CN201010118203A CN101792786A CN 101792786 A CN101792786 A CN 101792786A CN 201010118203 A CN201010118203 A CN 201010118203A CN 101792786 A CN101792786 A CN 101792786A
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- 238000000034 method Methods 0.000 title claims abstract description 46
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 title claims abstract description 35
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 title claims abstract description 35
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 22
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 17
- -1 cytidine phosphoryl compound Chemical class 0.000 title claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 49
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 14
- 239000008103 glucose Substances 0.000 claims abstract description 14
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 12
- 235000019743 Choline chloride Nutrition 0.000 claims abstract description 12
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 12
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 12
- 229960003178 choline chloride Drugs 0.000 claims abstract description 12
- 239000010452 phosphate Substances 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 9
- RZZPDXZPRHQOCG-OJAKKHQRSA-M CDP-choline(1-) Chemical compound O[C@@H]1[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OCC[N+](C)(C)C)O[C@H]1N1C(=O)N=C(N)C=C1 RZZPDXZPRHQOCG-OJAKKHQRSA-M 0.000 claims abstract 2
- ZWIADYZPOWUWEW-XVFCMESISA-N CDP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O1 ZWIADYZPOWUWEW-XVFCMESISA-N 0.000 claims description 32
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims description 32
- 150000001875 compounds Chemical class 0.000 claims description 27
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- AZRZBCASYOBNKQ-UHFFFAOYSA-N 6-chloro-3,5-diaminopyrazine-3-carboxamide Chemical group CN(C)C(N)=NC(=O)C1=NC(Cl)=C(N)N=C1N AZRZBCASYOBNKQ-UHFFFAOYSA-N 0.000 claims 2
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- 102000002745 Choline Kinase Human genes 0.000 description 4
- 108010018888 Choline kinase Proteins 0.000 description 4
- 102000015083 Choline-Phosphate Cytidylyltransferase Human genes 0.000 description 4
- 108010078853 Choline-Phosphate Cytidylyltransferase Proteins 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 4
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- 238000011069 regeneration method Methods 0.000 description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 3
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- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical compound [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 description 2
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- OTEKOJQFKOIXMU-UHFFFAOYSA-N 1,4-bis(trichloromethyl)benzene Chemical compound ClC(Cl)(Cl)C1=CC=C(C(Cl)(Cl)Cl)C=C1 OTEKOJQFKOIXMU-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for synthesizing cytidine phosphoryl compounds by directional catalysis, which takes CMP, choline chloride and phosphate radical ions as substrates, takes glucose as an energy donor, utilizes permeable microbial cells as an enzyme source, and promotes a reaction system to synthesize cytidine phosphoryl compounds by directional biological catalysis by taking the change of reaction temperature as a means. The invention can produce any one of three substances according to the requirement, has simple reaction system, no toxicity, low production cost and simple and easy control means, and greatly facilitates the production control in industrialization. When CMP is used as substrate, the yield of CDP, CTP and CDP-choline can reach 80%, 91.8% and 93.3% respectively.
Description
Technical field
The invention belongs to the biocatalysis technology field, be specifically related to utilize the control technique method of synthesizing cytidine phosphinylidyne compounds through oriented catalysis respectively.
Background technology
Cytidine diphosphate(CDP) (CDP) is a kind of important biochemical reagents as the derivative of cytosine(Cyt).It and bisphosphate inosine prepare Polyinosinic-polycytidylic acid (Poly I:C) under the effect of nucleotide phosphodiesterase enzyme.Polyinosinic-polycytidylic acid is a kind of interferon inducers efficiently, has broad-spectrum antiviral and immunosuppressive action, is widely used in multiple treatment of diseases.
Cytidine triphosphate(CTP) (CTP) is that termolecular phosphoric acid is combined in the Nucleotide on cytidine ribose 5 '-OH base, is a kind of normal composition in the body cell, and is widely distributed, is the direct precursor of RNA synthetic.It participates in nucleic acid and phospholipid (Yelkin TTS in vivo, kephalin, serine phosphatide, sphingophospholipid etc.) biosynthetic process, phosphatide is the important component of nervous tissue, also be a kind of natural fat emulsifier, have the brain of perfecting and nervous tissue, increase intelligence and memory and the transportation that promotes body inner cholesterol, fat is arranged, improve metabolism of fat, thereby prevent the effect of piling up.CTP is widely used in the multiple nerve of treatment, vascular disease as biochemical drug.
Cytidine diphosphate (CDPC) claims CITICOLINE SODIUM again, is the agent of brain metabolic activation, can promote the synthetic of neuron membrane Yelkin TTS, has the reparation brain injury, and anti-hypoxia improves memory, the effect that increases intelligence, and clinical application is wider.So the synthetic technology of cytidine diphosphate is the research topic that people relatively are concerned about.
CDP, CTP and CDPC all belong to the category of cytidine phosphinylidyne compounds.
At present, the above CDP preparation method of document has two kinds of chemical synthesis and biological synthesis process.Chemical synthesis is to be reactant with CMP, with hexichol phosphoryl chloride phosphorylation, re-refine CDP.This method is used multiple volatile toxic reagent, severe operational environment, and exist yield low, the problem that cost is high (Biochim.Biophys.Acta., 1964,91 (1): 1-13).Biological synthesis process was divided into for two steps, with the synthetic CTP of CMP, again the CTP degraded was generated CDP earlier, can obtain highly purified CDP, and yield is in (chemistry and biotechnology, 2005,22 (7): 52-54) more than 70%.
CTP can utilize microorganism (based on cereuisiae fermentum) cellular enzymes system synthetic.The source of this technology required yeasts is wide, and cost is low, is subject to seasonal restrictions few; And productive rate is higher, has to excavate to improve potentiality.It is one of main mode of production of present cytidine.According to reports, Kilajima etc. utilize yeast, and from the synthetic CTP of CMP, the reaction yield is 80% (HakkoKogaku Zasshi.1970,48 (12): 753-762).
CDPC synthetic method at present commonly used is a microbe transformation method, utilizes microbial cell enzyme system synthetic.In yeast cell, CMP can synthesize CDP under the catalysis of nucleoside monophosphate kinase; CDP can generate CTP under the effect of nucleoside diphosphokinase, choline can be under the catalysis of choline kinase synthesizing choline phosphate.CTP and phosphorylcholine can generate CDPC and tetra-sodium through choline phosphate cytidylyltransferase catalysis then.But because cellular enzymes system is complicated, have the feedback regulation effect, transformation efficiency is generally on the low side, and fermentation period is oversize, causes transformation efficiency, product concentration lower, has a large amount of by products in the reaction solution, comprises CMP, CDP and CTP.The at present domestic relevant report of also not utilizing same reaction system by control reaction temperature and the synthetic cytidine phosphinylidyne compounds of time orientation catalysis.
Summary of the invention
Technical problem to be solved by this invention provides the method for the synthetic above-mentioned three kinds of cytidine phosphinylidyne compounds of a kind of microorganism cells directional catalyzing.
For solving the problems of the technologies described above, thinking of the present invention is:
In the preparation process in view of CDPC, need to consume lot of energy (ATP), therefore needing two enzyme systems in the preparation process of cytidine phosphinylidyne compounds is regeneration system and the cytidine phosphinylidyne compounds synthetase series of ATP.The regeneration system of ATP is a substrate with the glucose of cheapness, and (EMP) realizes by glycolytic pathway, and this approach is one of most economical approach of energy regeneration; The cytidine phosphinylidyne compounds synthetase series is made of nucleoside monophosphate kinase, nucleoside diphosphokinase, choline kinase and choline phosphate cytidylyltransferase.
In the whole process of CDPC synthetic, the cytidine phosphinylidyne compounds synthetase series plays keying action by nucleoside monophosphate kinase, nucleoside diphosphokinase, choline kinase and choline phosphate cytidylyltransferase.These enzymes in the cereuisiae fermentum have different optimum temperutures respectively.Four kinds of enzyme optimum temperutures are respectively cytidylate kinase, 25 ℃; Nucleoside diphosphokinase, 37 ℃; Choline kinase, 30 ℃; Choline phosphate cytidylyltransferase, 30 ℃.We can produce any one product among CDP, CTP and the CDPC by control reaction temperature and time.
Key of the present invention is:
1) the present invention has adopted the whole-cell catalytic technology, and the own enzyme that has directly utilized cell is the synthetic three kinds of cytidine phosphinylidyne compounds of catalysis CMP, can produce any one of three kinds of materials according to demand, and reaction system is simple, and is nontoxic, low production cost.
2) the present invention as means, influences the activity of relevant enzyme in the cytidine phosphinylidyne compounds building-up process by control reaction temperature.Under differing temps, the enzymic activity that can help synthetic a kind of material is strengthened, and the enzymic activity that helps generating other materials simultaneously then weakens, and the active selectivity of this kind of enzyme has determined the final product that reacts different.Control device is simple, has greatly made things convenient for the production control in the industrialization.
Concrete technical scheme of the present invention is as follows:
A kind of method of synthesizing cytidine phosphinylidyne compounds through oriented catalysis; with CMP, choline chloride 60 and phosphate anion is substrate; with glucose as energy donor; the microorganism cells of having property of utilization is the enzyme source; by changing temperature of reaction is means, impels the reaction system biocatalysis to synthesize cytidine phosphinylidyne compounds.
Wherein, described cytidine phosphinylidyne compounds is cytidine diphosphate(CDP) (CDP), cytidine triphosphate(CTP) (CTP) or CDP-choline (CDPC), and its structural formula is as follows:
Wherein, cytidine monophosphate, i.e. CMP, its structural formula is as follows:
The method of above-mentioned synthesizing cytidine phosphinylidyne compounds through oriented catalysis is: carry out one of following three kinds of reactions in the aqueous solution of pH5～10, directed synthetic cytidine phosphinylidyne compounds:
(a) under 28～40 ℃, reacted 3 hours, primary product is CTP;
(b) under 28～40 ℃, reacted 3 hours; Adjust temperature to 28～34 ℃ again, reacted 9～15 hours, primary product is CDPC;
(c) under 28～40 ℃, reacted 3 hours; Adjust temperature to 28～34 ℃ again, reacted 9～15 hours; Adjust temperature to 20～27 ℃ again, reacted 6～10 hours, primary product is CDP.
Wherein, the initial action concentration of substrate CMP is 10～100mmol/L, preferred 20～50mmol/L; The initial action concentration of choline chloride 60 is 10～300mmol/L, preferred 20～150mmol/L; The initial action concentration of phosphate anion is 0.1～2mol/L, preferred 0.2～0.5mol/L; The initial action concentration of glucose is 0.1～1mol/L, preferred 0.2～0.5mol/L; The add-on of microorganism cells is by wet thallus 100～800g/L, preferred 200～600g/L.Phosphate ion can be selected from Tripyrophosphoric acid such as ortho-phosphoric acid, tetra-sodium, tripolyphosphate, inorganic phosphates such as potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic.
Wherein, reaction system also adds Mn
2+The composition of ion and dithiothreitol (DTT); Mn
2+Initial action concentration is 1～200mmol/L, preferred 10～100mmol/L; Dithiothreitol (DTT) initial action concentration is 1～200mmol/L, preferred 10～100mmol/L.
Wherein, described microorganism cells is meant the microorganism that can utilize the synthetic CDPC of CMP, comprises the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to.Preferred intestinal bacteria, subtilis, yeast saccharomyces cerevisiae or Torulopsis candida.
Wherein, the microorganism cells of described having property is meant the microorganism cells that the permeability changes of the cytolemma of handling by chemistry, physics or biological method is crossed, and concrete grammar comprises surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process.
The tensio-active agent that uses in the surfactant method is nonionic surface active agent polyethylene oxide amines or triton x-100, cationic surfactant hexadecyl trimethylamine bromide, perhaps anion surfactant Sarkosyl L salt, usage quantity is 0.1～50g/L, preferred 1～20g/L, when being surfactant method processing yeast cell, tensio-active agent directly being added reaction solution, is the reaction solution of 1L for cumulative volume, add 0.1～50g, preferably add 1～20g.
The organic solvent that uses in the organic solvent method is dimethylbenzene, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ethyl acetate, usage quantity is 0.1～50mL/L, preferred 1～20mL/L, when being organic solvent method process for producing bacterial strain, organic solvent is directly added reaction solution, for cumulative volume is the reaction solution of 1L, adds 0.1～50mL, preferably adds 1～20mL.
It can be the dry thing of yeast cell, the centrifugal cell that obtains of culture of isolated, the lyophilized products of cell, commercially available yeast powder, air-dry yeast or waste yeast mud by fermentation that above-mentioned zymic utilizes form.
Beneficial effect of the present invention is:
The invention provides a kind of method of synthesizing cytidine phosphinylidyne compounds through oriented catalysis; with CMP, choline chloride 60 and phosphate anion is substrate; with glucose as energy donor; the microorganism cells of having property of utilization is the enzyme source; by changing temperature of reaction is means, impels the synthetic cytidine phosphinylidyne compounds of the directed biocatalysis of reaction system.The present invention can produce any a kind of of three kinds of materials according to demand, and reaction system is simple, and is nontoxic, low production cost, and control device is simple, has greatly made things convenient for the production control in the industrialization.The yield of CDP, CTP and CDP-choline can reach 80%, 91.8%, 93.3% respectively when doing substrate with CMP.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
Yeast culture base: glucose 40g/L, urea 2.0g/L, potassium primary phosphate 1.5g/L, Zinc vitriol 4.0 * 10
-3G/L, ferrous sulfate 3.0 * 10
-3G/L, four hydration Manganous chloride tetrahydrates 0.3 * 10
-3G/L, Calcium Chloride Powder Anhydrous 1.0 * 10
-3G/L, vitamin H 0.05 * 10
-3G/L.
Yeast-inoculated amount 10% was cultivated centrifugal 4000rpm, 20 minutes 24 hours in 30 ℃ of following 120rpm shaking tables.Get yeast slurry ,-7 ℃ of preservations are standby.
Embodiment 2: utilize the directed CTP of production of CMP.
Modulation is by CMP 300mmol in the reactive tank of capacity 15L, choline chloride 60 100mmol, glucose 5mol, manganous sulfate 500mmol, dithiothreitol (DTT) 300mmol, press the subtilis 2400g of the method cultivation of embodiment 1, potassium primary phosphate 3mol, the reaction solution 10L that triton x-100 1g and water are formed, transferring pH with sodium hydroxide is 7.0, temperature is 35 ℃, react and finish reaction after 3 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid, and its content is 18.6mmol/L, and yield is 62.1%, this moment, CDP content was 2.5mmol/L, and CDPC content is 5.8mmol/L.
Embodiment 3: utilize the directed CTP of production of CMP.
Modulation is by CMP 300mmol in the reactive tank of capacity 15L, choline chloride 60 3000mmol, glucose 5mol, manganous nitrate 500mmol, dithiothreitol (DTT) 300mmol, press the yeast saccharomyces cerevisiae 2500g of the method cultivation of embodiment 1, potassium primary phosphate 3mol, the reaction solution 10L that toluene 10mL and water are formed, transferring pH with sodium hydroxide is 7.0, temperature is 40 ℃, react and finish reaction after 3 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid, its content is 27.5mmol/L, yield is 91.8%, this moment, CDP content was 0.7mmol/L, and CDPC content is 0.8mmol/L.
Embodiment 4: utilize the directed CDPC of production of CMP.
Modulation is by CMP 300mmol in the reactive tank of capacity 15L, choline chloride 60 3mol, glucose 5mol, manganous sulfate 500mmol, dithiothreitol (DTT) 300mmol, press the intestinal bacteria 2500g of the method cultivation of embodiment 1, potassium primary phosphate 3mol, the reaction solution 10L that acetone 10mL and water are formed, transferring pH with sodium hydroxide is 7.0, temperature is 35 ℃, react after 3 hours, temperature is adjusted into 28 ℃, continues reaction and finishes reaction in 9 hours, uses the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDPC in the conversion fluid, and its content is 28mmol/L, and yield is 93.3%, this moment, CDP content was 0.8mmol/L, and CTP content is 0.3mmol/L.
Embodiment 5: utilize the directed CDPC of production of CMP.
Modulation is by CMP 300mmol in the reactive tank of capacity 15L, choline chloride 60 3mol, glucose 5mol, manganous sulfate 500mmol, dithiothreitol (DTT) 300mmol, press the pichia spp 2500g of the method cultivation of embodiment 1, potassium primary phosphate 3mol, the reaction solution 10L that ethyl acetate 10mL and water are formed, transferring pH with sodium hydroxide is 7.0, temperature is 40 ℃, react after 3 hours, temperature is adjusted into 34 ℃, continue reaction and finished reaction in 15 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDPC in the conversion fluid, its content is 21mmol/L, yield is 70.0%, and this moment, CDP content was 1.2mmol/L, and CTP content is 0.7mmol/L.
Embodiment 6: utilize the directed CDP of production of CMP.
Modulation is by CMP 100mmol in the reactive tank of capacity 15L, choline chloride 60 100mmol, glucose 1mol, manganous sulfate 20mmol, dithiothreitol (DTT) 10mmol, press the intestinal bacteria 1000g that embodiment 1 method is cultivated, SODIUM PHOSPHATE, MONOBASIC 2mol, triton x-100 50g, the reaction solution 10L that toluene 50mL and water are formed, transferring pH with sodium hydroxide is 6.0, temperature is 35 ℃, react after 3 hours, temperature is adjusted into 28 ℃, continue reaction 9 hours, temperature is adjusted into 20 ℃ again, continue reaction and finished reaction in 6 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid, and its content is 8mmol/L, and yield is 80.0%, this moment, CTP content was 1mmol/L, and CDPC content is 0.8mmol/L.
Embodiment 7: utilize the directed CDP of production of CMP.
Modulation is by CMP 1000mmol in the reactive tank of capacity 15L, choline chloride 60 3000mmol, glucose 10mol, Manganous chloride tetrahydrate 500mmol, dithiothreitol (DTT) 2mol, the Torulopsis candida of pressing the cultivation of embodiment 1 method is through 3 8000g of multigelation, the reaction solution 10L that SODIUM PHOSPHATE, MONOBASIC 5mol and water are formed, transferring pH with sodium hydroxide is 9.0, temperature is 40 ℃, react after 3 hours, temperature is adjusted into 34 ℃, continue reaction 15 hours, temperature is adjusted into 27 ℃ again, continue reaction and finished reaction in 10 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is CDP in the conversion fluid, its content is 42mmol/L, yield is 42.0%, and this moment, CTP content was 0.5mmol/L, and CDPC content is 11mmol/L.
Claims (9)
1. the method for a synthesizing cytidine phosphinylidyne compounds through oriented catalysis; it is characterized in that with CMP, choline chloride 60 and phosphate anion be substrate; with glucose as energy donor; the microorganism cells of having property of utilization is the enzyme source; by changing temperature of reaction is means, impels the reaction system biocatalysis to synthesize cytidine phosphinylidyne compounds.
2. the method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis according to claim 1 is characterized in that described cytidine phosphinylidyne compounds is cytidine diphosphate(CDP), cytidine triphosphate(CTP) or CDP-choline, and its structural formula is as follows:
3. the method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis according to claim 2 is characterized in that
Carry out one of following three kinds of reactions in the aqueous solution of pH5～10, directed synthetic cytidine phosphinylidyne compounds:
(a) under 28～40 ℃, reacted 3 hours, primary product is CTP;
(b) under 28～40 ℃, reacted 3 hours; Adjust temperature to 28～34 ℃ again, reacted 9～15 hours, primary product is CDPC;
(c) under 28～40 ℃, reacted 3 hours; Adjust temperature to 28～34 ℃ again, reacted 9～15 hours; Adjust temperature to 20～27 ℃ again, reacted 6～10 hours, primary product is CDP.
4. according to the method for any described synthesizing cytidine phosphinylidyne compounds through oriented catalysis in the claim 1～3; the initial action concentration that it is characterized in that substrate CMP is 10～100mmol/L; the initial action concentration of choline chloride 60 is 10～300mmol/L; the initial action concentration of phosphate anion is 0.1～2mol/L; the initial action concentration of glucose is 0.1～1mol/L, and the add-on of microorganism cells is by wet thallus 100～800g/L.
5. according to the method for any described synthesizing cytidine phosphinylidyne compounds through oriented catalysis in the claim 1～3, it is characterized in that described reaction system also adds Mn
2+The composition of ion and dithiothreitol (DTT); Mn
2+Initial action concentration is 1～200mmol/L, and dithiothreitol (DTT) initial action concentration is 1～200mmol/L.
6. according to the method for any described synthesizing cytidine phosphinylidyne compounds through oriented catalysis in the claim 1～3, it is characterized in that described microorganism cells is meant the microorganism that can utilize the synthetic CDPC of CMP, comprises the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to.
7. according to the method for any described synthesizing cytidine phosphinylidyne compounds through oriented catalysis in the claim 1～3; the microorganism cells that it is characterized in that described having property is meant the microorganism cells that the permeability changes of the cytolemma of handling by chemistry, physics or biological method is crossed, and concrete grammar comprises surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process.
8. the method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis according to claim 7; it is characterized in that the tensio-active agent that uses in the surfactant method is nonionic surface active agent, cationic surfactant or anion surfactant, usage quantity is 0.1～50g/L.
9. the method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis according to claim 7 is characterized in that the organic solvent that uses in the organic solvent method is dimethylbenzene, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ethyl acetate, and usage quantity is 0.1～50mL/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102605025A (en) * | 2011-01-19 | 2012-07-25 | 中国科学院生物物理研究所 | Bioengineering method for synthesis of citicoline |
| CN103436455A (en) * | 2013-08-15 | 2013-12-11 | 南通秋之友生物科技有限公司 | S. cerevisiae strain for producing citicoline through bioconversion and application of S. cerevisiae strain |
| CN108486195A (en) * | 2018-02-26 | 2018-09-04 | 安徽翠鸟生物技术有限公司 | A method of preparing UDP with enzyme process |
| 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 |
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| EP0553821A1 (en) * | 1992-01-30 | 1993-08-04 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing cytidine diphosphate choline |
| CN101555509A (en) * | 2009-04-17 | 2009-10-14 | 南京工业大学 | Method for synthesizing uridine phosphoryl compound by directional catalysis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1055202A (en) * | 1990-12-30 | 1991-10-09 | 陕西省微生物研究所 | Technology by nucleic acid direct production nucleoside diphosphate |
| EP0553821A1 (en) * | 1992-01-30 | 1993-08-04 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing cytidine diphosphate choline |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102605025A (en) * | 2011-01-19 | 2012-07-25 | 中国科学院生物物理研究所 | Bioengineering method for synthesis of citicoline |
| CN102605025B (en) * | 2011-01-19 | 2014-07-02 | 中国科学院生物物理研究所 | Bioengineering method for synthesis of citicoline |
| CN103436455A (en) * | 2013-08-15 | 2013-12-11 | 南通秋之友生物科技有限公司 | S. cerevisiae strain for producing citicoline through bioconversion and application of S. cerevisiae strain |
| CN108486195A (en) * | 2018-02-26 | 2018-09-04 | 安徽翠鸟生物技术有限公司 | A method of preparing UDP with enzyme process |
| 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 |
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