CN112143766A - Biosynthesis method for efficiently preparing nucleoside triphosphate - Google Patents
Biosynthesis method for efficiently preparing nucleoside triphosphate Download PDFInfo
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- CN112143766A CN112143766A CN202011015996.5A CN202011015996A CN112143766A CN 112143766 A CN112143766 A CN 112143766A CN 202011015996 A CN202011015996 A CN 202011015996A CN 112143766 A CN112143766 A CN 112143766A
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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 |
|---|---|---|---|---|
| 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 |
-
2020
- 2020-09-24 CN CN202011015996.5A patent/CN112143766A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 由洋: "利用固定化啤酒酵母合成5’-核苷三磷酸", 《工业微生物》 * |
| 邱蔚然等: "《核酸类产品的生物合成》", 31 January 2006, 华东理工大学出版社 * |
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Application publication date: 20201229 |