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Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate from polyphosphate

A technology of deoxynucleoside triphosphate and polyphosphate, which is applied in the biological field, can solve the problems of activity difference, etc., and achieve the effects of low cost, raw material cost saving, and safe production

Pending Publication Date: 2021-07-16
ANHUI GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, through a large number of experimental studies, it has been found that the regenerated enzyme polyphosphate kinase EC 2.7.4.1 (PPK, which catalyzes the reaction of ADP and polyphosphoric acid or its salts thereof) added in the reaction can not only catalyze the reaction of ADP, but also catalyze ( d) CDP, (d)GDP and other (deoxy) nucleoside diphosphates react with different activities

Method used

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  • Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate from polyphosphate
  • Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate from polyphosphate
  • Method for preparing nucleoside triphosphate and deoxynucleoside triphosphate from polyphosphate

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Embodiment 1

[0034] The preparation of embodiment 1 enzyme

[0035] All the enzymes in the method of the present invention can be obtained commercially, or are artificially modified enzymes with the same catalytic function.

[0036] The enzyme preparation process is as follows:

[0037] All enzymes involved in the preparation method of this application are EC 2.7.4.25, EC 2.7.4.9, EC 2.7.4.22, EC 2.7.4.8, EC 2.7.4.1 and EC 2.7.4.6 respectively.

[0038] Primers were designed according to the gene sequences of the enzymes used in the reaction, the gene fragments were amplified by PCR, and connected to the corresponding vectors (commercially available, single expression vector or multiple expression vectors could be selected), after the sequencing was correct, transfection Enter E.coliBL21 (DE3) strain (commercially available).

[0039]Insert the transformed E.coli BL21(DE3) monoclonal into LB medium, culture to the logarithmic phase, add 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for ...

Embodiment 2

[0045] Add 2.0g CMP, 0.6g Tris, 0.38g potassium chloride, 0.26g ammonium sulfate, 2.0g polyphosphate and 0.6g magnesium chloride hexahydrate to the 100ml reaction system, adjust the pH value to 7.0, add 500U EC 2.7. 4.1 Enzyme and 500U EC2.7.4.25 Enzyme to start the reaction. During the reaction, the pH value was controlled to be 7.0, and the temperature was 37°C.

[0046] After reacting for 2 hours, the amount of CTP produced by high performance liquid chromatography (HPLC) was detected to be about 2.5 g, and the conversion rate of CMP into CTP was over 83%. figure 2 and image 3 Respectively, the HPLC detection chromatograms of reaction 0 hour and reaction 2 hours.

Embodiment 3

[0048] Add 2.0g GMP, 0.38g potassium chloride, 0.22g ammonium chloride, 2.0g polyphosphate and 0.6g magnesium chloride hexahydrate to the 100ml reaction system, adjust the pH value to 7.5, add 800U EC 2.7.4.1 enzyme and 400U EC 2.7.4.8 Enzyme started the reaction. During the reaction, the pH value was controlled to be 7.5, and the temperature was 40°C.

[0049] After reacting for 4 hours, the amount of GTP detected by high performance liquid chromatography (HPLC) was 2.3 g, and the conversion rate exceeded 80%.

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Abstract

The invention discloses a method for preparing nucleoside triphosphate and deoxynucleoside triphosphate from polyphosphate. The method comprises the following steps: (1) preparing nucleoside monophosphate kinase and PPK enzyme required by reaction; (2) reacting to generate NTP and dNTP: adding polyphosphate into a reaction system taking NMP and dNMP as substrates, and reacting the NMP, dNMP and polyphosphate under the combined action of nucleotide monophosphate kinase and PPK enzyme to generate dNTP and NTP; and (3) separating a target product. The high-efficiency and specificity of the biological enzyme of the invention enables the whole synthesis process to be simple, stable and rapid, and the whole process is free of addition of pollutants such as organic solvents, the whole process is green and environment-friendly, the production is safe, the period is short and the cost is low. ATP does not need to be added as an energy source in the reaction, and the raw material cost is saved by 50% or above; there are few by-products generated in the reaction, and it is easy for later purification without the interference of ATP, AMP and ADP; the enzyme dosage is small, and the reaction is rapid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing nucleoside triphosphate and deoxynucleoside triphosphate by using polyphosphate. Background technique [0002] Nucleosides are glycosides formed by the condensation of nitrogenous bases and sugar components. The nitrogenous bases mainly include pyrimidine bases and purine bases, while the sugar components mainly include ribose and deoxyribose. Ribonucleosides and tripolyphosphate form lipids to form nucleoside triphosphates (NTP); while deoxyribonucleosides and tripolyphosphates form lipids to form deoxynucleoside triphosphates (dNTP), which are all present in organisms high-energy phosphate compounds. According to different base types, nucleoside triphosphate (NTP) mainly includes adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP), etc.; Glycoside triphosphate (dNTP) mainly includes deoxyade...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12P19/32
CPCC12P19/305C12P19/32
Inventor 尹延明秦远超秦永发刘珊珊周稳文乔春鑫
Owner ANHUI GSH BIO TECH CO LTD
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