CN101768617A - New technology for full-cell biosynthesis of deoxynucleoside triphosphate - Google Patents
New technology for full-cell biosynthesis of deoxynucleoside triphosphate Download PDFInfo
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- CN101768617A CN101768617A CN201019026027A CN201019026027A CN101768617A CN 101768617 A CN101768617 A CN 101768617A CN 201019026027 A CN201019026027 A CN 201019026027A CN 201019026027 A CN201019026027 A CN 201019026027A CN 101768617 A CN101768617 A CN 101768617A
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- Prior art keywords
- triphosphate
- deoxynucleoside
- whole
- synthetic
- new technology
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
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- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a new technology for synthesizing deoxynucleoside triphosphate by whole-cell biosynthesis, which takes deoxynucleoside monophosphate (dNMP) and phosphate ions as substrates, sugar as an energy donor and permeable yeast cells to react in the presence of chemical effect substances to prepare the deoxynucleoside triphosphate. The invention utilizes the principles of whole-cell catalysis and metabolic engineering, adopts chemical effect substances to regulate and control metabolic flow, establishes an efficient energy self-coupling and regeneration system, and finally realizes the efficient preparation of the deoxyribonucleoside triphosphate. The invention overcomes the defects of the traditional production process of the deoxyribonucleoside triphosphate, and has the characteristics of simple process, high conversion rate, low cost, small pollution and the like.
Description
Technical field
The present invention relates to a kind of method by the synthetic deoxynucleoside triphosphate dNTP of deoxynucleoside one phosphoric acid dNMP.
Background technology
Deoxynucleoside triphosphate is artificial-synthetic DNA's an indispensable precursor raw material, and artificial-synthetic DNA's fragment is widely used in aspects such as genetically engineered, molecular biology, life science, genomic medicine.Deoxy-ribonucleoside triphosphate is the substrate of the short reaction of various archaeal dna polymerases, is that dna sequence analysis, genetic analysis, rite-directed mutagenesis, PCR (Polymerase ChainReaction), RT-PCR, reverse transcription and dna marker react indispensable lower molecular weight biological organic molecule.And being modern molecular biology, biological chemistry and modern biomedical, dna sequence analysis, rite-directed mutagenesis, PCR and DNA chip technology study indispensable technology.Especially round pcr has characteristics such as be widely used simple to operate, and it obtains application more and more widely in fields such as gene molecule clone, protein engineering, biological medicine research and development, heredity and communicable disease diagnosis, legal medical expert's evaluation, parent-offspring's calibratings.
Along with DNA synthetic PCR in biotechnology research and the industrialization uses, the demand of dNTP is stably increasing.Have only enterprise of several family can produce deoxy-ribonucleoside triphosphate at present in the world, the method that is adopted mainly is to utilize the enzymatic or the chemical phosphorylation of thymus nucleic acid hydrolysate, the acquisition of end product requires polystep reaction, complex production process, and product yield is low.
Commercially mainly produce dNTP by chemical process, reaction is to be substrate with corresponding tributyl ammonium salt of dNMP and ortho-phosphoric acid, is catalyzer with dicyclohexylcarbodiimide (DCC), carries out in pyridine or dimethylformamide organic solvents such as (DMF).The productive rate that chemical method production obtains different dNTP is 40～80%.Yet, each dNTP component in the purification reaction mixture, its process is very complicated and cost is very big, and it need separate inactive dNMP, dNDP, DCC and ortho-phosphoric acid and by product such as dezyribonucleoside four phosphoric acid or five phosphoric acid etc.In addition, because the principle of off gas treatment is very strict, pyridine or DMF solvent must be reduced, and separate, and reclaim then.Therefore, chemical method has very big environmental pollution, and very low by its yield behind reaction and the purifying, and cost is also higher relatively.
Consider that from economy and environmental factors occurred the synthetic deoxynucleoside triphosphate of enzyme process in recent years in the world, this method has more advantage than chemical process.The reaction of two enzyme phosphorylations of synthetic needs of dNTP generates dNDP and second step generation dNTP from dNMP.Ladner and Whitesides are by the synthetic dNTP[Ladner W of isolating deoxynucleoside one phosphoric acid (dNMP) mixture from herring sperm dna, Whitesides G.Enzymatic synthesis of deoxyATP using DNAas starting material[J] .J Org Chem, 1985,50:1076-1079].In recent years, Jie Bao, people such as the Dewey D.Y.Ryu four kinds of kinase whose genes of '-deoxynucleoside monophosphate of successfully will encoding are separated from the genome of Saccharomyces cerevisiae ATCC2610 bacterial strain, be respectively the ADK1 of coding deoxydenylate kinase (AK), the GUK1 of coding deoxyguanylate kinase (GK), the URA6 of coding Deoxyribose cytidine acid kinase (CK), and the CDC8 gene of coding deoxythymidine acid kinase (TK).They arrive these gene clones in E.coli BL21 (DE3) bacterial strain, and make AK, GK, CK, TK overexpression.The conversion of deoxynucleotide kinase realization from the '-deoxynucleoside monophosphate to the deoxynucleoside diphosphate with purifying.Second step added conversion [the Bao J of pyruvate kinase (PK) realization from dNDP to dNTP that extracts from tame rabbit muscle, Ryu DDY.Total Biosynthesis of Deoxynucleoside Triphosphates UsingDeoxynucleoside Monophosphate Kinases for PCR Application[J] .Biotechnol Bioeng.2007,98 (1): 1-11].However, the productive rate of dNTP is still lower, have only about 25% [Bao J, Bruque GA, RyuDDY.Biosynthesis of deoxynucleoside triphosphates, dCTP and dTTP:Reaction mechanismand kinetics[J] .Enzyme Microb Technol.2005,36:350-356; Bao J, Ryu DDY.Biosynthesisreaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP[J] .Biotechnol Bioeng.2005,89:485-491].In addition, the man research institution of existing in the world number is is researching and developing ribonucleotide reductase, and the shortcomings such as complicated condition and enzyme stability difference but these ribonucleotide reductases that are in development mostly respond are not suitable for industrialization, are of limited application.
At present both at home and abroad the dNTP yield general not high, cost is high and a major reason of complex production process is that energy regeneration and coupling efficiency are low.Need to consume lot of energy (ATP) in the building-up process of dNTP, therefore needing two enzyme systems is the regeneration system of ATP and the synthetase series of dNTP.The regeneration system of ATP is substrate with sugar, and (EMP) realizes by glycolytic pathway, and this approach is one of most economical approach of energy regeneration, and donor ATP in the dNTP building-up process as phosphodonor and energy and exist.Therefore dNTP synthetic key just is how to set up an energy regeneration efficiently and from coupling system.Adopt yeast whole-cell catalytic technology, can overcome that substrate utilization efficient is low in the building-up process, efficiency of pcr product is low and problem such as production cost height.Simultaneously, compare with enzyme process, because use is full cell, the stability of enzyme is better, and the adaptability of organic solvent-resistant is stronger, the in-situ regeneration of easier realization energy and coenzyme.In the prior art, sugar is very low by the efficient that EMP Embden Meyerbof Parnas pathway generates ATP, can only keep the general life metabolism of yeast, strengthen the flux of EMP Embden Meyerbof Parnas pathway, overexpression substrate phosphorylation level, have only method to realize, adopt the latter more convenient, be easy to realize by genetic engineering technique or employing chemical effect substance change metabolism stream.Under the adjusting of chemical effect material, can make the metabolic flux of EMP Embden Meyerbof Parnas pathway take place obviously to change, ATP regenerated speed also is greatly improved, and when the speed of its speed and dNTP synthetic system is complementary, promptly realizes efficiently synthesizing of dNTP.
Summary of the invention
Technical problem to be solved by this invention provides the preparation method of a kind of simply and efficiently deoxynucleoside triphosphate dNTP, to overcome shortcomings such as dNTP traditional preparation process complexity, the high yield of cost is low, pollution is big.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological, with deoxynucleoside one phosphoric acid and phosphate anion is substrate, with sugar is energy donor, the yeast cell of having property of utilization is set up efficiently energy self coupling connection and regeneration system rapidly and is prepared deoxynucleoside triphosphate in the presence of the chemical effect material.
Wherein, described deoxynucleoside triphosphate dNTP is meant any one among deoxyadenosine triphosphate dATP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP and the deoxythymidine triphosphate dTTP, and its structural formula is as follows:
Wherein, described deoxynucleoside one phosphoric acid dNMP is meant any one among deoxyadenosine monophosphate dAMP, deoxyguanosine monophosphate dGMP, deoxycytidine monophosphate dCMP and the deoxythymidine monophosphate dTMP, and its structural formula is as follows:
Wherein, the initial action concentration of described deoxynucleoside one phosphoric acid is 1～100mM; Described phosphate anion initial action concentration is 0.01～1.0M; The initial action concentration of sugar is 0.1～1.0M; Described phosphate ion can be enumerated Tripyrophosphoric acid such as ortho-phosphoric acid, tetra-sodium, tripolyphosphate, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, inorganic phosphates such as Sodium phosphate dibasic; Described sugar is glucose, fructose, sucrose or maltose.
Wherein, described chemical effect material is meant the combination of metal ion and organic compound; Described metal ion is Mg
2+, K
+And Na
+In any one or a few; Described organic compound is the combination of any one or two kinds in acetaldehyde and the polyvalent alcohol; Mg
2+Can be inorganic magnesium salts such as sal epsom, magnesium nitrate, magnesium chloride, initial action concentration be 1～100mM; K
+Can be inorganic potassium salts such as potassium primary phosphate, dipotassium hydrogen phosphate, vitriolate of tartar, saltpetre, Repone K, initial action concentration be 1～100mM; Acetaldehyde initial action concentration is 10～80mL/L, and polyvalent alcohol can be ethylene glycol, glycerol, sorbyl alcohol or N.F,USP MANNITOL, preferred glycerol, and polyvalent alcohol initial action concentration is 5～100mL/L.
Wherein, described yeast cell is any one during yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to, preferred example can be lifted the microorganism yeast saccharomyces cerevisiae that belongs to yeast belong, bread yeast etc.; The microorganism Candida parapsilosis that belongs to mycocandida; The Ao Molieshi pichia spp that belongs to Pichia; The microorganism white torulopsis that belongs to torulopsis; The spherical Dbaly yeast of class that belongs to Debaryomyces; Belong to the Lu Shi zygosaccharomyces that zygosaccharomyces belongs to; The kluyveromyces marxianus that belongs to genus kluyveromyces; The outstanding fourth debaryomyces hansenii that belongs to Hansenula; Belong to the different Brettanomyces of Brettanomyces genus etc.
The usage quantity of yeast cell is for to press wet thallus 100～800g/L, and preferred 200～600g/L promptly is the reaction solution of 1L for cumulative volume, needs to add 100～800g wet thallus, preferably adds 200～600g wet thallus.
Zymic utilizes form to be the dry thing of yeast cell, the centrifugal cell that obtains of culture of isolated, immobilized cell, the lyophilized products of cell, commercially available yeast powder, air-dry yeast or waste yeast mud by fermentation.
Wherein, the yeast cell of having property is meant the yeast cell that the permeability changes of the cytolemma of handling by chemistry, physics or biological method is crossed, and described chemistry, physics or biological method comprise 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, or anion surfactant Sarkosyl L salt, the tensio-active agent usage quantity is 0.1～50g/L, and preferred 1～20g/L is promptly during surfactant method process for producing bacterial strain, tensio-active agent is directly added reaction solution, for cumulative volume is the reaction solution of 1L, adds 0.1～50g, preferably adds 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, organic solvent concentration is 0.1～50mL/L, preferably with 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.
Other handles the method for cell permeability, as freeze-thaw method, ultrasonication method, aeration drying etc., after employing is handled strain cell earlier, the bacterial strain of handling well is added the mode of reaction solution again.
Being reflected in the aqueous solution of above-mentioned preparation deoxynucleoside triphosphate carried out, and reacts 2～24 hours under 5.0～9.0,20～50 ℃ of conditions of pH, preferably reacts 2～24 hours under 7.0～8.0,30 ℃ of conditions of pH.
Beneficial effect: the invention has the advantages that:
1, the present invention utilizes yeast cell to carry out catalyzed reaction, and it is abundant that yeast cell contains the enzyme class, has the potentiality of the multiple biochemical reaction of catalysis.Special yeast cell has coenzyme, and (ATP) refresh function can play a significant role aspect catalytic oxidation-reduction reaction, the phosphoric acid shift reaction for NADH, NADPH.Utilize the enzyme system of yeast cell carry out enzymic catalytic reaction produce desired product in extensive range, have good market outlook.
2, the present invention is based upon on the basis of whole-cell catalytic, its characteristics be to have overcome the additive method substrate conversion efficiency not high, be difficult to realize defectives such as energy and regenerating coenzyme.Compare with enzyme process, because cell has and keeps the complete multienzyme system of its vital movement, various enzymes are keeping residing state of original life cell and specific position again, reaction energy needed and cofactors do not need extraneous the supply, directly produce by cell, therefore can finish the multistep enzymic catalytic reaction quickly and effectively, have transformation efficiency height, cost low, and pollute little advantage.Compare with existing chemical process simultaneously, the present invention is a kind of bioconversion method that can obviously reduce environmental pollution.
3, the present invention has set up energy self coupling connection and regeneration system rapidly efficiently, it is glycolysis-(EMP) path enzyme system (hexokinase that reaction has utilized the intravital enzyme of microorganism, glucose phosphate isomerase, phosphofructokinase, zymohexase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydro-genase, phosphoglyceric kinase, phosphoglycerate phosphomutase, enolase, pyruvate kinase, pyruvic carboxylase, ethanol dehydrogenase) and dNTP synthetase series (nucleoside monophosphate kinase, nucleoside diphosphokinase) carries out catalyzed reaction, it is synthetic that the ATP that EMP Embden Meyerbof Parnas pathway produces can apply to dNTP, adding by chemical substance in addition, can accelerate the regeneration rate of ATP, form efficiently energy self coupling connection and regeneration system rapidly, make the dNTP excess accumulation.
4, the present invention utilizes the metabolic engineering principle, adds magnesium ion, potassium ion, and the composition of acetaldehyde and polyvalent alcohol, mainly plays following several respects effect:
1) regulate metabolic flux, make intrasystem pathways metabolism flow take place obviously to change, the metabolic flux that flows to glycerine significantly reduces.Metabolic flux is after regulating, and the approach of EMP make that EMP master's approach is strengthened, thereby improved energy utilization ratio, and the accumulation of dNTP is just being needed ATP energize and phosphate radical by strongly inhibited, dNTP and then be able to a large amount of accumulation.
2) accelerate NADH regeneration, keep NADH/NAD in the born of the same parents
+Ratio, the redox equilibrium of recovery cell.And the NADH oxidative pathway is turned to the ethanol fermentation approach, thereby strengthen the flux of glycolytic pathway, promote the conversion of dAMP to dATP.
3) add metal ions such as magnesium ion, potassium ion, make the cumulative speed of FDP obviously accelerate, stimulate the pyruvate kinase activity; Be because the decomposition of phosphoenolpyruvic acid on the other hand, make the regeneration of the NAD that former cause glycerolphos phate dehydrogenase catalysis phosphodihydroxyacetone causes, still take on by ethanol dehydrogenase.And acetaldehyde and polyvalent alcohol can guarantee and accelerate NADH regeneration effectively, and it is alive and stable to keep enzyme.Thus, metal ion and organism can promote mutually that both act synergistically efficiently in generation, make intrasystem pathways metabolism flow take place obviously to change, and the metabolic flux that flows to glycerine significantly reduces, and realization dNTP's is efficient synthetic.
4) enzyme of assurance yeast enzyme system is lived and is stable, helps improving the combined coefficient of dNTP.
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 (g/L): glucose 40, urea 2.0, potassium primary phosphate 1.5, bitter salt 0.5, Zinc vitriol 4.0 * 10
-3, ferrous sulfate 3.0 * 10
-3, four hydration Manganous chloride tetrahydrates 0.3 * 10
-3, Calcium Chloride Powder Anhydrous 1.0 * 10
-3, vitamin H 0.05 * 10
-3Yeast saccharomyces cerevisiae inoculum size 10% was cultivated centrifugal 4000rpm, 20 minutes 24 hours in 30 ℃ of following 120rpm shaking tables.Get yeast slurry ,-7 ℃ of preservations are standby.
The yeast that following examples are used all is to cultivate by above-mentioned training method.
Embodiment 2: utilize dAMP to prepare dATP.
Capacity be in the beaker of 500mL modulation by dAMP 1.49mmol, glucose 0.072mol, bread yeast mud 100g, sal epsom 14.96mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, glycerol 3mL, hexadecyl trimethylamine. the reaction solution 300mL that brometo de amonio 0.3g and water are formed, transfer pH to 7.0 with sodium hydroxide, the stirring at low speed reaction is 6 hours under 30 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dATP is carried out quantitative analysis, contain dATP 2.59g/L in the conversion fluid, the yield of dATP reaches 93.6% (mol meter).
Embodiment 3: utilize dAMP to prepare dATP.
Capacity be in the beaker of 500mL modulation by dAMP 1.66mmol, glucose 0.08mol, the bread yeast mud 110g after air-dry, Repone K 8.05mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, the reaction solution 300mL that acetaldehyde 3mL and water are formed transfers pH to 7.0 with sodium hydroxide, the stirring at low speed reaction is 6 hours under 30 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dATP is carried out quantitative analysis, contain dATP 2.85g/L in the conversion fluid, the yield of dATP reaches 92.4% (mol meter).
Embodiment 4: utilize dCMP to prepare dCTP.
Capacity be in the beaker of 500mL modulation by dCMP 1.50mmol, glucose 0.068mol, yeast saccharomyces cerevisiae 90g, air-dry processing, magnesium chloride 6.89mmol, Repone K 5.37mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, acetaldehyde 3mL, glycerol 3mL, the reaction solution 300mL that triton x-100 6g and water are formed transfers pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 18 hours under 37 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dCTP is carried out quantitative analysis, contain dCTP 0.71g/L in the conversion fluid, the yield of dCTP reaches 25.6% (mol meter).
Embodiment 5: utilize dCMP to prepare dCTP.
Capacity be in the beaker of 500ml modulation by dCMP 1.61mmol, glucose 0.076mol, Lu Shi zygosaccharomyces 100g, multigelation 3 times, Repone K 8.05mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, the reaction solution 300mL that glycerol 3mL and water are formed, transfer pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 18 hours under 37 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dCTP is carried out quantitative analysis, contain dCTP 0.80g/L in the conversion fluid, the yield of dCTP reaches 26.9% (mol meter).
Embodiment 6: utilize dGMP to prepare dGTP.
Capacity be in the beaker of 500ml modulation by dGMP 1.30mmol, glucose 0.065mol, Lu Shi zygosaccharomyces 90g, sal epsom 16.62mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, acetaldehyde 3mL, hexadecyl trimethylamine. the reaction solution 300mL that brometo de amonio 0.3g and water are formed, transfer pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 12 hours under 37 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dGTP is carried out quantitative analysis, contain dGTP 0.99g/L in the conversion fluid, the yield of dGTP reaches 45.0% (mol meter).
Embodiment 7: utilize dGMP to prepare dGTP.
Capacity be in the beaker of 500ml modulation by dGMP 1.44mmol, glucose 0.068mol, yeast saccharomyces cerevisiae 90g, air-dry processing, sal epsom 14.96mmol, SODIUM PHOSPHATE, MONOBASIC 0.03mol, ethylene glycol 3mL, the reaction solution 300mL that triton x-100 6mL and water are formed transfers pH to 6.5 with sodium hydroxide, the stirring at low speed reaction is 12 hours under 37 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dGTP is carried out quantitative analysis, contain dGTP1.13g/L in the conversion fluid, the yield of dGTP reaches 46.4% (mol meter).
Embodiment 8: utilize dTMP to prepare dTTP.
Capacity be in the beaker of 500ml modulation by dTMP 1.50mmol, glucose 0.09mol, different Brettanomyces 150g, sal epsom 14.96mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, acetaldehyde 3mL, the reaction solution 300mL that toluene 3mL and water are formed, transfer pH to 8.0 with sodium hydroxide, the stirring at low speed reaction is 10 hours under 30 ℃ of conditions, after reaction finishes, use the perchloric acid precipitation, with HPLC dTTP is carried out quantitative analysis, contain dTTP 0.84g/L in the conversion fluid, the yield of dTTP reaches 34.8% (mol meter).
Embodiment 9: utilize dTMP to prepare dTTP.
Capacity be in the beaker of 500ml modulation by dTMP 1.64mmol, glucose 0.10mol, yeast saccharomyces cerevisiae 100g, air-dry processing, magnesium chloride 6.89mmol, Repone K 8.05mmol, SODIUM PHOSPHATE, MONOBASIC 0.036mol, acetaldehyde 3mL, glycerol 3mL, the reaction solution 300mL that acetone 6mL and water are formed transfers pH to 7.0 with sodium hydroxide, the stirring at low speed reaction is 10 hours under 30 ℃ of conditions, reaction is used the perchloric acid precipitation after finishing, and with HPLC dTTP is carried out quantitative analysis, contain dTTP 0.95g/L in the conversion fluid, the yield of dTTP reaches 36.0% (mol meter).
Claims (12)
1. the new technology of the synthetic deoxynucleoside triphosphate of a whole-cell biological, it is characterized in that with deoxynucleoside one phosphoric acid and phosphate anion be substrate, with sugar is energy donor, the yeast cell of having property of utilization is set up efficiently energy self coupling connection and regeneration system rapidly and is prepared deoxynucleoside triphosphate in the presence of the chemical effect material.
2. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, it is characterized in that described deoxynucleoside triphosphate is meant any one among deoxyadenosine triphosphate dATP, deoxyguanosine triphosphate dGTP, deoxycytidine triphosphate dCTP and the deoxythymidine triphosphate dTTP, its structural formula is as follows:
3. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, it is characterized in that described deoxynucleoside one phosphoric acid is meant any one among deoxyadenosine monophosphate dAMP, deoxyguanosine monophosphate dGMP, deoxycytidine monophosphate dCMP and the deoxythymidine monophosphate dTMP, its structural formula is as follows:
4. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, the initial action concentration that it is characterized in that described deoxynucleoside one phosphoric acid is 1～100mM; Described phosphate anion initial action concentration is 0.01～1.0M; The initial action concentration of sugar is 0.1～1.0M; Described sugar is glucose, fructose, sucrose or maltose.
5. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1 is characterized in that described chemical effect material is meant the combination of metal ion and organic compound; Described metal ion is Mg
2+And K
+In the combination of any one or two kinds; Described organic compound is the combination of any one or two kinds in acetaldehyde and the polyvalent alcohol; Mg
2+Initial action concentration is 1～100mM, K
+Initial action concentration is 1～100mM, Na
+Initial action concentration is 1～100mM, and acetaldehyde initial action concentration is 10～80mL/L, and polyvalent alcohol initial action concentration is 5～100mL/L.
6. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 5 is characterized in that described polyvalent alcohol is ethylene glycol, glycerol, sorbyl alcohol or N.F,USP MANNITOL.
7. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1, it is characterized in that described yeast cell is any one during yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to, the zymic usage quantity is by wet thallus 100～800g/L.
8. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 7 is characterized in that described yeast cell is yeast saccharomyces cerevisiae, Candida parapsilosis, bread yeast, Ao Molieshi pichia spp, white torulopsis, the spherical Dbaly yeast of class, Lu Shi zygosaccharomyces, kluyveromyces marxianus, outstanding fourth debaryomyces hansenii or different Brettanomyces.
9. according to the new technology of the synthetic deoxynucleoside triphosphate of claim 1,7 or 8 described whole-cell biologicals, the yeast cell that it is characterized in that described having property is meant the yeast cell that the permeability changes of the cytolemma of handling by chemistry, physics or biological method is crossed, and described chemistry, physics or biological method comprise surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze enzyme process.
10. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 9, it is characterized in that the tensio-active agent that uses in the described surfactant method is nonionic surface active agent, cationic surfactant or anion surfactant, working concentration is 0.1～50g/L.
11. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 9, it is characterized in that the organic solvent that uses in the described organic solvent method is dimethylbenzene, toluene, lipase, acetone or ethyl acetate, working concentration is 0.1～50ml/L.
12. the new technology of the synthetic deoxynucleoside triphosphate of whole-cell biological according to claim 1 is characterized in that being reflected in the aqueous solution of described preparation deoxynucleoside triphosphate carry out, and reacts 2～24 hours under 5.0～9.0,20～50 ℃ of conditions of pH.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102168123A (en) * | 2011-01-10 | 2011-08-31 | 吕朝阳 | Novel method for preparing deoxyribonucleoside triphosphate (dNMP) |
| CN102199644A (en) * | 2011-04-15 | 2011-09-28 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
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| CN111349672A (en) * | 2020-04-13 | 2020-06-30 | 南京曼蕊生物科技有限公司 | Biosynthesis process for preparing deoxyribonucleoside triphosphate |
| CN112143766A (en) * | 2020-09-24 | 2020-12-29 | 天津全和诚科技有限责任公司 | Biosynthesis method for efficiently preparing nucleoside triphosphate |
| CN114940985A (en) * | 2022-04-20 | 2022-08-26 | 苏州酶泰生物科技有限公司 | Protein with activity of deoxyadenosine diphosphate kinase and acetate kinase and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102168123A (en) * | 2011-01-10 | 2011-08-31 | 吕朝阳 | Novel method for preparing deoxyribonucleoside triphosphate (dNMP) |
| 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 |
| CN102199644A (en) * | 2011-04-15 | 2011-09-28 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
| CN102199644B (en) * | 2011-04-15 | 2014-12-17 | 江苏省中国科学院植物研究所 | Genetic engineering preparation method of cytidine triphosphate |
| CN111349672A (en) * | 2020-04-13 | 2020-06-30 | 南京曼蕊生物科技有限公司 | Biosynthesis process for preparing deoxyribonucleoside triphosphate |
| CN112143766A (en) * | 2020-09-24 | 2020-12-29 | 天津全和诚科技有限责任公司 | Biosynthesis method for efficiently preparing nucleoside triphosphate |
| CN114940985A (en) * | 2022-04-20 | 2022-08-26 | 苏州酶泰生物科技有限公司 | Protein with activity of deoxyadenosine diphosphate kinase and acetate kinase and application thereof |
| CN114940985B (en) * | 2022-04-20 | 2024-03-19 | 苏州酶泰生物科技有限公司 | Protein with deoxyadenosine diphosphate kinase and acetate kinase activities and application thereof |
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