CN111349672A - Biosynthesis process for preparing deoxyribonucleoside triphosphate - Google Patents
Biosynthesis process for preparing deoxyribonucleoside triphosphate Download PDFInfo
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- CN111349672A CN111349672A CN202010287470.6A CN202010287470A CN111349672A CN 111349672 A CN111349672 A CN 111349672A CN 202010287470 A CN202010287470 A CN 202010287470A CN 111349672 A CN111349672 A CN 111349672A
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- dntp
- phosphodiesterase
- sperm dna
- dna
- active yeast
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- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 8
- 239000001226 triphosphate Substances 0.000 title claims description 9
- 235000011178 triphosphate Nutrition 0.000 title claims description 9
- 239000005549 deoxyribonucleoside Substances 0.000 title claims description 8
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 title claims description 7
- 238000004519 manufacturing process Methods 0.000 title description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000000203 mixture Substances 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 claims abstract description 13
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 claims abstract description 13
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 241000269821 Scombridae Species 0.000 claims abstract description 9
- 230000007062 hydrolysis Effects 0.000 claims abstract description 9
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 9
- 235000020640 mackerel Nutrition 0.000 claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 238000004237 preparative chromatography Methods 0.000 claims abstract description 7
- 238000013329 compounding Methods 0.000 claims abstract description 6
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 6
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 6
- 239000000178 monomer Substances 0.000 claims abstract description 5
- 239000002994 raw material Substances 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract 2
- 239000000758 substrate Substances 0.000 claims description 9
- 241000228153 Penicillium citrinum Species 0.000 claims description 6
- 241000736084 Scomber japonicus Species 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims description 3
- 238000011097 chromatography purification Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 150000002926 oxygen Chemical class 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 239000012264 purified product Substances 0.000 claims 1
- 238000012216 screening Methods 0.000 claims 1
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract 2
- 239000000126 substance Substances 0.000 abstract 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 18
- 239000011259 mixed solution Substances 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 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 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 239000003957 anion exchange resin Substances 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- DENRZWYUOJLTMF-UHFFFAOYSA-N diethyl sulfate Chemical compound CCOS(=O)(=O)OCC DENRZWYUOJLTMF-UHFFFAOYSA-N 0.000 description 2
- 229940008406 diethyl sulfate Drugs 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- KHWCHTKSEGGWEX-UHFFFAOYSA-N deoxyadenylic acid Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(O)=O)O1 KHWCHTKSEGGWEX-UHFFFAOYSA-N 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/305—Pyrimidine nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a preparation method of necessary raw materials for artificially synthesizing DNA, belonging to the technical field of biosynthesis and biocatalysis of important chemicals. The method comprises the steps of adopting a brand new process of enzyme hydrolysis and enzyme catalysis, selecting mackerel sperm DNA as a raw material, hydrolyzing the mackerel sperm DNA with phosphodiesterase to generate a dNMP mixture, separating and purifying through ion exchange chromatography, synthesizing the dNTP mixture through whole-cell catalysis of active yeast, separating and purifying through preparative chromatography to obtain four high-purity monomer compounds, and compounding into equimolar dNTP biochemical reagents. The process has the advantages of green and environment-friendly reaction, high efficiency, high product purity of over 99.9 percent, low cost, industrial production and strong competitiveness.
Description
Technical Field
The invention relates to a brand new biosynthesis method utilizing enzymatic hydrolysis and yeast catalysis, which has the advantages of easily obtained and cheap substrate, green, environment-friendly and efficient process reaction, high product purity of over 99.9 percent, low whole process cost, industrial production and strong competitiveness.
Background
dNTP is the abbreviation of deoxyribonucleoside triphosphate, including four products of dATP, dGTP, dTTP and dCTP, is an indispensable biochemical reagent for PCR amplification, DNA sequencing, molecular marking and cDNA synthesis, and the demand is continuously expanded. The dNTP reagent is required to have high purity and no nuclease pollution, and most of the raw materials of the dNTP reagent at present come from foreign countries and are expensive. The technological process includes hydrolyzing pneumatophorus japonicus sperm DNA as material with phosphodiesterase to produce dNMP mixture, ion exchange chromatographic separation and purification, yeast whole cell catalyzed synthesis of dNTP mixture, chromatographic separation to obtain four kinds of high purity monomeric compounds, and compounding into equimolar dNTP biochemical reagent. The enzymatic synthesis process adopted by the process has the advantages of cheap and easily obtained substrate, green and environment-friendly reaction, high efficiency, adoption of a preparative chromatographic separation technology, high product purity of over 99.9 percent, low production cost and high competitiveness.
Disclosure of Invention
The method for preparing the deoxyribonucleoside triphosphate by the new biosynthesis process comprises the following steps:
the phosphodiesterase is produced by fermentation of penicillium citrinum. Penicillium citrinum M71 strain is purchased from the market, and is optimized to obtain phosphodiesterase, the enzyme activity of which taking DNA as a substrate is more than 200u/ml, and the amount of secreted protein is more than 1 g/L.
Four kinds of desorbed nucleotide mixtures (dMTP) are produced by hydrolyzing mackerel sperm DNA with phosphodiesterase. The phosphodiesterase prepared by the method can hydrolyze the mackerel sperm DNA, the concentration of the hydrolyzed mackerel sperm DNA taking the DNA as a substrate is 2.0-4.0%, the enzymolysis temperature is 55-60 ℃, the pH value is 6.5-7.0, the enzymolysis time is 2-4 hours, and the hydrolysis rate is more than 99%.
Purifying the hydrolyzed mixture by ion exchange chromatography, separating and purifying by ion exchange chromatography, and then adopting 201 × 8 anion exchange resin in the separation process, wherein the specific conditions are that the height of a column bed is 105mm, the diameter of the column bed is 45mm, the sample concentration is 213mg/mL, the elution flow rate is 0.5 mL/(cm-2. min), the impurity removal rate is more than 90%, and the purified dMTP mixed solution is obtained.
Active yeast cells having a capability of converting oxynucleotides into deoxyribonucleoside triphosphates (dNTPs) are removed. Taking saccharomyces cerevisiae to culture active yeast, wherein the optimal reaction conditions of the yeast are as follows: dNMP 1.40 g/L, glucose 40.98g/L, MgCl2 & 6H2O4.00 g/L, KCl 2.00 g/L, NaH2 PO431.20 g/L, yeast 300.00 g/L, ammonium chloride 0.67 g/L, acetaldehyde 11.7 mL/L, pH 7.0, temperature 29.6 ℃. Under the condition, the concentration of dNTP is 2.10 g/L, the yield reaches 93.7 percent, and the concentration of the product is four times of that of 0.52 g/L before optimization.
Active yeast whole cells catalyze the conversion of deoxyribonucleotides (dnmps) to deoxyribonucleoside triphosphates (dntps).
And (3) carrying out whole-cell catalysis on the purified dNMP mixed solution by using the prepared active yeast to convert the purified dNMP mixed solution into a dNTP mixture, wherein the concentration of a substrate for the conversion reaction of the active yeast is 0.5-1.0 g/L, the reaction temperature is 37 ℃, the pH value is 6.5-7.0, the reaction time is 8-10 hours, and the conversion rate of each compound is more than 70%.
The high-efficiency preparative chromatography purification dNTP mixture is monomer compound and then is compounded into equimolar dNTP biochemical reagent. The product is purified by adopting high-efficiency preparative chromatography, the purity of each component can be more than 99.9%, the process is simple and high, and the product purity is high.
The specific implementation mode is as follows:
a technique for producing phosphodiesterase by fermenting penicillium citrinum.
The method is characterized in that purchased penicillium citrinum ST817 is used as an initial strain, a high-yield strain P citrinum YB-1 of 5' -phosphodiesterase is obtained through Ultraviolet (UV), Nitrosoguanidine (NTG), high-temperature treatment and diethyl sulfate (DES) mutagenesis, the shake flask fermentation enzyme activity reaches 693U/mL and is improved by 2.5 times compared with the initial strain, and genetic stability experiments prove that the strain YB-1 has stable enzyme production performance. A single-factor test is adopted to optimize a fermentation medium and culture conditions of the strain P.citrinum YB-1, the optimal fermentation medium composition (g/L) is determined to be glucose 60, peptone 6, KH _2PO _ 40.4, K _2HP _ O40.6, MgSO 4 & 7H _2O 0.3, CaCl _ 20.2 and MnSO _ 40.15, and the determined optimal culture conditions are initial pH 5.5, culture temperature 30 ℃, liquid loading amount of a shake flask per 250mL of a triangular flask, rotating speed of a shaking table of 180r/min and fermentation time of 72H.
Technology for producing deoxynucleotide mixture (dMTP) by hydrolyzing pneumatophorus japonicus milt DNA with phosphodiesterase is provided.
The phosphodiesterase hydrolysis pneumatophorus japonicus milt DNA prepared by the method has the concentration of 2.0-4.0 percent by taking DNA as a substrate, the enzymolysis temperature of 55-60 ℃, the pH value of 6.5-7.0, the enzymolysis time of 2-4 hours and the hydrolysis rate of more than 99 percent, and after the mixture after hydrolysis is separated and purified by ion exchange chromatography, anion exchange resin of 201 × 8 is adopted in the separation, wherein the specific conditions are that the height of a column bed is 105mm, the diameter of the column bed is 45mm, the sample concentration is 213mg/mL, the elution flow rate is 0.5 mL/(cm-2. min), the impurity removal rate is more than 90 percent, and the purified dMTP mixed solution is obtained.
An active yeast culture technology with the capability of whole-cell catalyzing the conversion of dNMP into dNTP.
Taking saccharomyces cerevisiae to carry out optimized cultivation of active yeast, wherein the optimal reaction conditions of the yeast are as follows: dAMP 1.40 g/L, glucose 40.98g/L, MgCl 2.6H 2O 4.00.00 g/L, KCl 2.00 g/L, NaH2 PO431.20 g/L, yeast 300.00 g/L, ammonium chloride 0.67 g/L, acetaldehyde 11.7 mL/L, pH 7.0, temperature 29.6 ℃. Under the condition, the concentration of dATP is 2.10 g/L, the yield reaches 93.7 percent, and the concentration of the product is four times of that of the product which is 0.52 g/L before optimization. Obtaining the optimized yeast catalysis stock solution.
The technology of converting dNMP into dNTP by the whole cell catalysis of active yeast.
And (3) carrying out whole-cell catalysis on the purified dNMP mixed solution by using the prepared active yeast to convert the purified dNMP mixed solution into a dNTP mixture, wherein the concentration of a substrate for the conversion reaction of the active yeast is 0.5-1.0 g/L, the reaction temperature is 37 ℃, the pH value is 6.5-7.0, the reaction time is 8-10 hours, and the conversion rate of each compound is more than 70%.
And purifying the dNTP mixture into a monomer compound by high-efficiency preparative chromatography.
The purity of each component of the monomer compound, namely the dNTP mixture purified by the efficient preparative chromatography of a novel instrument is more than 99.9 percent, the process is simple, and the product purity is high.
Claims (2)
1. The invention relates to a new biosynthesis method for preparing deoxyribonucleoside triphosphate, which is realized by the following processes and methods:
(1) the mackerel sperm DNA is selected as a basic raw material, and multiple tests show that the mackerel sperm DNA is easy to obtain and low in price, and the hydrolysis rate of hydrolysis by phosphodiesterase reaches over 99 percent. The cost of the whole process is reduced;
(2) performing enzyme hydrolysis with phosphodiesterase, hydrolyzing pneumatophorus japonicus milt DNA to produce four kinds of desorbed nucleotide mixtures (dNMP), and purifying by ion exchange chromatography. The preparation method comprises the steps of producing phosphodiesterase by fermenting penicillium citrinum, wherein the enzyme activity is more than 200u/ml, the amount of secreted protein is more than 1g/L, hydrolyzing mackerel sperm DNA by using the phosphodiesterase, the concentration of the substrate DNA is 2.0-4.0%, the enzymolysis temperature is 55-60 ℃, the pH value is 6.5-7.0, the enzymolysis time is 2-4 hours, the hydrolysis rate is more than 99%, and after separation and purification of a hydrolyzed mixture through ion exchange chromatography, the impurity removal rate is more than 90% for enzyme catalysis in the next step;
(3) active yeast whole cell catalysis, active yeast whole cell catalysis divorced oxygen nucleotide mixture (dNMP) conversion
Deoxyriboside triphosphate (dNTP), and active yeast can convert four monophosphoric acid compounds into triphosphoric acid compounds at one time, the conversion rate of each compound is greater than 70%, and high-quality strain yeast is obtained by continuous screening. Then, the active yeast is used for whole-cell catalysis dNMP, the concentration of a conversion reaction substrate is 0.5-1.0 g/L, the reaction temperature is 37 ℃, the pH value is 6.5-7.0, the reaction time is 8-10 hours, and the conversion rate of each compound is more than 70%;
(4) purifying and compounding, namely compounding a high-efficiency preparative chromatography purified dNTP mixture into a monomer compound, and then compounding into an equimolar dNTP biochemical reagent, adopting a high-efficiency preparative chromatography purified product, having a simple process, and compounding into an equimolar dNTP biochemical reagent, wherein the purity of each component product is more than 99.9%.
2. The method of producing deoxyribonucleoside triphosphates according to claim 1, wherein: selecting a mackerel sperm DNA raw material, culturing active yeast and using a chromatographic purification instrument.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375796A (en) * | 2020-11-07 | 2021-02-19 | 潍坊华诺医药科技有限公司 | Preparation method of high-purity deoxyribonucleoside triphosphate |
| CN113842671A (en) * | 2021-09-24 | 2021-12-28 | 上海蔚之星生物科技有限公司 | NTP/dNTP chromatographic separation method and system based on intelligent control |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101633681A (en) * | 2008-07-25 | 2010-01-27 | 华东理工大学 | Method for separating and purifying deoxynucleoside triphosphate |
| CN101768617A (en) * | 2010-02-04 | 2010-07-07 | 南京工业大学 | New technology for full-cell biosynthesis of deoxynucleoside triphosphate |
-
2020
- 2020-04-13 CN CN202010287470.6A patent/CN111349672A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101633681A (en) * | 2008-07-25 | 2010-01-27 | 华东理工大学 | Method for separating and purifying deoxynucleoside triphosphate |
| CN101768617A (en) * | 2010-02-04 | 2010-07-07 | 南京工业大学 | New technology for full-cell biosynthesis of deoxynucleoside triphosphate |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112375796A (en) * | 2020-11-07 | 2021-02-19 | 潍坊华诺医药科技有限公司 | Preparation method of high-purity deoxyribonucleoside triphosphate |
| CN113842671A (en) * | 2021-09-24 | 2021-12-28 | 上海蔚之星生物科技有限公司 | NTP/dNTP chromatographic separation method and system based on intelligent control |
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