CN112143766A - Biosynthesis method for efficiently preparing nucleoside triphosphate - Google Patents
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- -1 nucleoside triphosphate Chemical class 0.000 title claims abstract description 58
- 239000001226 triphosphate Substances 0.000 title claims abstract description 44
- 235000011178 triphosphate Nutrition 0.000 title claims abstract description 44
- 239000002777 nucleoside Substances 0.000 title claims abstract description 43
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000000203 mixture Substances 0.000 claims abstract description 57
- 239000000243 solution Substances 0.000 claims abstract description 30
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 12
- 239000003637 basic solution Substances 0.000 claims abstract description 12
- 235000015097 nutrients Nutrition 0.000 claims abstract description 12
- 239000010452 phosphate Substances 0.000 claims abstract description 12
- 238000010438 heat treatment Methods 0.000 claims abstract description 11
- 238000003756 stirring Methods 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 6
- 239000002994 raw material Substances 0.000 claims abstract description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 30
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 15
- 239000003960 organic solvent Substances 0.000 claims description 15
- 210000005253 yeast cell Anatomy 0.000 claims description 15
- 229910021645 metal ion Inorganic materials 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 5
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 5
- 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 description 5
- 239000004367 Lipase Substances 0.000 claims description 5
- 102000004882 Lipase Human genes 0.000 claims description 5
- 108090001060 Lipase Proteins 0.000 claims description 5
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 5
- 229930195725 Mannitol Natural products 0.000 claims description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims description 5
- 235000015278 beef Nutrition 0.000 claims description 5
- 229910001424 calcium ion Inorganic materials 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 claims description 5
- 230000002925 chemical effect Effects 0.000 claims description 5
- 238000013375 chromatographic separation Methods 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 229910052742 iron Inorganic materials 0.000 claims description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 5
- 235000019421 lipase Nutrition 0.000 claims description 5
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 5
- 239000000594 mannitol Substances 0.000 claims description 5
- 235000010355 mannitol Nutrition 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 230000035699 permeability Effects 0.000 claims description 5
- 239000011574 phosphorus Substances 0.000 claims description 5
- 229910052698 phosphorus Inorganic materials 0.000 claims description 5
- 238000000053 physical method Methods 0.000 claims description 5
- 229910001414 potassium ion Inorganic materials 0.000 claims description 5
- 229910001415 sodium ion Inorganic materials 0.000 claims description 5
- 239000000600 sorbitol Substances 0.000 claims description 5
- 235000010356 sorbitol Nutrition 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 239000011593 sulfur Substances 0.000 claims description 5
- 229910052717 sulfur Inorganic materials 0.000 claims description 5
- QXJQHYBHAIHNGG-UHFFFAOYSA-N trimethylolethane Chemical compound OCC(C)(CO)CO QXJQHYBHAIHNGG-UHFFFAOYSA-N 0.000 claims description 5
- 235000013343 vitamin Nutrition 0.000 claims description 5
- 239000011782 vitamin Substances 0.000 claims description 5
- 229940088594 vitamin Drugs 0.000 claims description 5
- 229930003231 vitamin Natural products 0.000 claims description 5
- 239000008096 xylene Substances 0.000 claims description 5
- 239000000811 xylitol Substances 0.000 claims description 5
- 235000010447 xylitol Nutrition 0.000 claims description 5
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 5
- 229960002675 xylitol Drugs 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 238000001308 synthesis method Methods 0.000 abstract description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- PCDQPRRSZKQHHS-CCXZUQQUSA-N Cytarabine Triphosphate 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)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-CCXZUQQUSA-N 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- RZCIEJXAILMSQK-JXOAFFINSA-N TTP Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 RZCIEJXAILMSQK-JXOAFFINSA-N 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 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
- 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 2
- 230000007547 defect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- PGAVKCOVUIYSFO-UHFFFAOYSA-N [[5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 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
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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)
Abstract
The invention provides a biosynthesis method for efficiently preparing nucleoside triphosphate, and relates to the field of nucleoside triphosphate synthesis. The biosynthesis method for efficiently preparing the nucleoside triphosphate comprises the following steps: s1, DNMP and phosphate radical are used as raw materials, then the DNMP and the phosphate radical are dissolved in 2-3 times of aqueous solution to obtain a mixture, and hydrochloric acid solution is added into the mixture to adjust the PH of the mixture to 4-5 to obtain a basic solution; s2, adding 1.5-1 time of nutrient into the basic solution obtained in the step S1 for energy supply, then heating and uniformly stirring the solution to fully mix the solution to obtain a mixture, and then placing the mixture in a container for storage for later use. Through the design of the synthesis method, the method has the advantage of simple operation during synthesis, and the substances obtained in the synthesis process have high purity and low production cost, thereby further promoting the production.
Description
Technical Field
The invention relates to the technical field of nucleoside triphosphate synthesis, in particular to a biosynthesis method for efficiently preparing nucleoside triphosphate.
Background
Nucleoside Triphosphate (NTP) is a nucleotide containing three phosphate groups. Common types in nature include Adenosine Triphosphate (ATP), Guanosine Triphosphate (GTP), Cytidine Triphosphate (CTP), Thymidine Triphosphate (TTP), and Uridine Triphosphate (UTP). These molecules contain a ribose, and if ribose is substituted for deoxyribose, the nucleoside triphosphate is converted to deoxynucleoside triphosphate, which is written as dNTP, such as deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), and the like.
Nucleoside triphosphate has important significance in biology, so the demand for nucleoside triphosphate is increasing day by day at present, but the nucleoside triphosphate has the problem of low purity no matter imported from foreign countries or independently synthesized in China at present, and the nucleoside triphosphate preparation in the past has the defects of complex process, high cost and difficult synthesis.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a biosynthesis method for efficiently preparing nucleoside triphosphate, which solves the problems of low purity of the existing nucleoside triphosphate synthesis and complex process, high cost and difficult synthesis of the existing nucleoside triphosphate preparation.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a biosynthesis method for efficiently preparing nucleoside triphosphate comprises the following steps:
s1, DNMP and phosphate radical are used as raw materials, then the DNMP and the phosphate radical are dissolved in 2-3 times of aqueous solution to obtain a mixture, and hydrochloric acid solution is added into the mixture to adjust the PH of the mixture to 4-5 to obtain a basic solution;
s2, adding 1.5-1 time of nutrients into the basic solution obtained in the step S1 for energy supply, then heating and uniformly stirring the solution to fully mix the solution to obtain a mixture, and then storing the mixture in a container for later use;
s3, adding 10-15% of metal ions and 1-2 times of organic solvent into the mixture obtained in the S2, and mixing and stirring the mixture to obtain a secondary mixture;
s4, adding yeast cells with permeability and sodium hydroxide into the mixture in the S3 according to the ratio of 1:1, adjusting the pH to 6-8, performing the mixing at the temperature of 30-45 ℃ to generate chemical effect so as to prepare nucleoside triphosphate solution, and performing chromatographic separation on the nucleoside triphosphate solution to obtain 93-98% nucleoside triphosphate solution
Preferably, the concentration of the hydrochloric acid in the S1 is 1-1.5 mol/L.
Preferably, the nutrients in the S2 are peptone 1-2 parts, glucose 5-10 parts, sucrose 0.5-1 part, vitamins 1-2 parts and beef extract 3-4 parts, and the heating treatment temperature is 40-55 ℃.
Preferably, the metal ions in S3 are one or more of potassium ions, sodium ions, iron ions, magnesium ions, calcium ions, phosphorus ions, and sulfur ions.
Preferably, the organic solvent in S3 is at least one of sorbitol, mannitol, xylitol, trimethylolethane, ethyl acetate, toluene, xylene, and lipase, and the concentration of the organic solvent is 10-20 ml/L.
Preferably, the yeast cells permeable in S4 are cells with modified yeast cell membranes by chemical or physical methods.
Preferably, the concentration of the sodium hydroxide in the S4 is 1-3 mol/L.
(III) advantageous effects
The invention provides a biosynthesis method for efficiently preparing nucleoside triphosphate. The method has the following beneficial effects:
1. through the design of the synthesis method, the method has the advantage of simple operation during synthesis, and the substances obtained in the synthesis process have high purity and low production cost, thereby further promoting the production.
2. Through the intervention of nutrient substances, the efficient reaction in the synthesis process is ensured, and the synthesis rate can be increased.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows:
the embodiment of the invention provides a biosynthesis method for efficiently preparing nucleoside triphosphate, which comprises the following steps:
s1, DNMP and phosphate radical are used as raw materials, then the DNMP and the phosphate radical are dissolved in 2-3 times of aqueous solution to obtain a mixture, and hydrochloric acid solution is added into the mixture to adjust the PH of the mixture to 4-5 to obtain a basic solution;
s2, adding 1.5-1 time of nutrients into the basic solution obtained in the step S1 for energy supply, then heating and uniformly stirring the solution to fully mix the solution to obtain a mixture, and then storing the mixture in a container for later use;
s3, adding 10-15% of metal ions and 1-2 times of organic solvent into the mixture obtained in the S2, and mixing and stirring the mixture to obtain a secondary mixture;
s4, adding yeast cells with permeability and sodium hydroxide into the mixture in the S3 according to the ratio of 1:1, adjusting the pH value to 6-8, performing the mixing at the temperature of 30-45 ℃ to generate a chemical effect so as to prepare a nucleoside triphosphate solution, and performing chromatographic separation on the nucleoside triphosphate solution to obtain 93-98% nucleoside triphosphate.
Wherein the concentration of hydrochloric acid in S1 is 1-1.5 mol/L.
Wherein the nutrients in the S2 comprise 1 part of peptone, 5 parts of glucose, 0.5 part of sucrose, 1 part of vitamins and 3 parts of beef extract, and the heating treatment temperature is 40-55 ℃.
Wherein the metal ions in S3 are one or more of potassium ions, sodium ions, iron ions, magnesium ions, calcium ions, phosphorus ions and sulfur ions.
Wherein the organic solvent in S3 is at least one of sorbitol, mannitol, xylitol, trimethylolethane, ethyl acetate, toluene, xylene, and lipase, and the concentration of the organic solvent is 10-20 ml/L.
Wherein the yeast cells permeable in S4 are cells with modified yeast cell membranes by chemical or physical methods.
Wherein the concentration of the sodium hydroxide in the S4 is 1-3 mol/L.
Example two:
the embodiment of the invention provides a biosynthesis method for efficiently preparing nucleoside triphosphate, which comprises the following steps:
s1, DNMP and phosphate radical are used as raw materials, then the DNMP and the phosphate radical are dissolved in 2-3 times of aqueous solution to obtain a mixture, and hydrochloric acid solution is added into the mixture to adjust the PH of the mixture to 4-5 to obtain a basic solution;
s2, adding 1.5-1 time of nutrients into the basic solution obtained in the step S1 for energy supply, then heating and uniformly stirring the solution to fully mix the solution to obtain a mixture, and then storing the mixture in a container for later use;
s3, adding 10-15% of metal ions and 1-2 times of organic solvent into the mixture obtained in the S2, and mixing and stirring the mixture to obtain a secondary mixture;
s4, adding yeast cells with permeability and sodium hydroxide into the mixture in the S3 according to the ratio of 1:1, adjusting the pH value to 6-8, performing the mixing at the temperature of 30-45 ℃ to generate a chemical effect so as to prepare a nucleoside triphosphate solution, and performing chromatographic separation on the nucleoside triphosphate solution to obtain 93-98% nucleoside triphosphate.
Wherein the concentration of hydrochloric acid in S1 is 1-1.5 mol/L.
Wherein the nutrients in the S2 comprise 1.5 parts of peptone, 7 parts of glucose, 1.8 parts of sucrose, 1.5 parts of vitamins and 3.6 parts of beef extract, and the heating treatment temperature is 40-55 ℃.
Wherein the metal ions in S3 are one or more of potassium ions, sodium ions, iron ions, magnesium ions, calcium ions, phosphorus ions and sulfur ions.
Wherein the organic solvent in S3 is at least one of sorbitol, mannitol, xylitol, trimethylolethane, ethyl acetate, toluene, xylene, and lipase, and the concentration of the organic solvent is 10-20 ml/L.
Wherein the yeast cells permeable in S4 are cells with modified yeast cell membranes by chemical or physical methods.
Wherein the concentration of the sodium hydroxide in the S4 is 1-3 mol/L.
Example three:
the embodiment of the invention provides a biosynthesis method for efficiently preparing nucleoside triphosphate, which comprises the following steps:
s1, DNMP and phosphate radical are used as raw materials, then the DNMP and the phosphate radical are dissolved in 2-3 times of aqueous solution to obtain a mixture, and hydrochloric acid solution is added into the mixture to adjust the PH of the mixture to 4-5 to obtain a basic solution;
s2, adding 1.5-1 time of nutrients into the basic solution obtained in the step S1 for energy supply, then heating and uniformly stirring the solution to fully mix the solution to obtain a mixture, and then storing the mixture in a container for later use;
s3, adding 10-15% of metal ions and 1-2 times of organic solvent into the mixture obtained in the S2, and mixing and stirring the mixture to obtain a secondary mixture;
s4, adding yeast cells with permeability and sodium hydroxide into the mixture in the S3 according to the ratio of 1:1, adjusting the pH value to 6-8, performing the mixing at the temperature of 30-45 ℃ to generate a chemical effect so as to prepare a nucleoside triphosphate solution, and performing chromatographic separation on the nucleoside triphosphate solution to obtain 93-98% nucleoside triphosphate.
Wherein the concentration of hydrochloric acid in S1 is 1-1.5 mol/L.
Wherein the nutrients in the S2 comprise 2 parts of peptone, 10 parts of glucose, 1 part of sucrose, 2 parts of vitamins and 4 parts of beef extract, and the heating treatment temperature is 40-55 ℃.
Wherein the metal ions in S3 are one or more of potassium ions, sodium ions, iron ions, magnesium ions, calcium ions, phosphorus ions and sulfur ions.
Wherein the organic solvent in S3 is at least one of sorbitol, mannitol, xylitol, trimethylolethane, ethyl acetate, toluene, xylene, and lipase, and the concentration of the organic solvent is 10-20 ml/L.
Wherein the yeast cells permeable in S4 are cells with modified yeast cell membranes by chemical or physical methods.
Wherein the concentration of the sodium hydroxide in the S4 is 1-3 mol/L.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A biosynthesis method for efficiently preparing nucleoside triphosphate is characterized in that: the method comprises the following steps:
s1, DNMP and phosphate radical are used as raw materials, then the DNMP and the phosphate radical are dissolved in 2-3 times of aqueous solution to obtain a mixture, and hydrochloric acid solution is added into the mixture to adjust the PH of the mixture to 4-5 to obtain a basic solution;
s2, adding 1.5-1 time of nutrients into the basic solution obtained in the step S1 for energy supply, then heating and uniformly stirring the solution to fully mix the solution to obtain a mixture, and then storing the mixture in a container for later use;
s3, adding 10-15% of metal ions and 1-2 times of organic solvent into the mixture obtained in the S2, and mixing and stirring the mixture to obtain a secondary mixture;
s4, adding yeast cells with permeability and sodium hydroxide into the mixture in the S3 according to the ratio of 1:1, adjusting the pH value to 6-8, performing the mixing at the temperature of 30-45 ℃ to generate a chemical effect so as to prepare a nucleoside triphosphate solution, and performing chromatographic separation on the nucleoside triphosphate solution to obtain 93-98% nucleoside triphosphate.
2. The biosynthesis method for efficiently preparing nucleoside triphosphates according to claim 1, wherein: the concentration of the hydrochloric acid in the S1 is 1-1.5 mol/L.
3. The biosynthesis method for efficiently preparing nucleoside triphosphates according to claim 1, wherein: the nutrients in the S2 comprise 1-2 parts of peptone, 5-10 parts of glucose, 0.5-1 part of sucrose, 1-2 parts of vitamins and 3-4 parts of beef extract, and the heating treatment temperature is 40-55 ℃.
4. The biosynthesis method for efficiently preparing nucleoside triphosphates according to claim 1, wherein: the metal ions in the S3 are one or a mixture of more of potassium ions, sodium ions, iron ions, magnesium ions, calcium ions, phosphorus ions and sulfur ions.
5. The biosynthesis method for efficiently preparing nucleoside triphosphates according to claim 1, wherein: the organic solvent in the S3 is at least one of sorbitol, mannitol, xylitol, trimethylolethane, ethyl acetate, toluene, xylene and lipase, and the concentration of the organic solvent is 10-20 ml/L.
6. The biosynthesis method for efficiently preparing nucleoside triphosphates according to claim 1, wherein: the yeast cells permeable in S4 are cells with modified yeast cell membranes by chemical or physical methods.
7. The biosynthesis method for efficiently preparing nucleoside triphosphates according to claim 1, wherein: the concentration of the sodium hydroxide in the S4 is 1-3 mol/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN1896264A (en) * | 2006-06-13 | 2007-01-17 | 南京工业大学 | Preparation method of nucleoside triphosphate |
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| CN1861625A (en) * | 2006-06-12 | 2006-11-15 | 南京工业大学 | Crystallization method of 5' -nucleoside triphosphate sodium salt |
| CN1896264A (en) * | 2006-06-13 | 2007-01-17 | 南京工业大学 | Preparation method of nucleoside triphosphate |
| CN101705270A (en) * | 2009-10-15 | 2010-05-12 | 江苏华荣生物科技有限公司 | Method for synthesizing deoxycytidine triphosphate through biotechnology |
| CN101768617A (en) * | 2010-02-04 | 2010-07-07 | 南京工业大学 | New technology for full-cell biosynthesis of deoxynucleoside triphosphate |
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