Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for enzymatically preparing adenosine triphosphate

An enzymatic preparation technology of adenosine triphosphate, which is applied in the field of enzymatic preparation of adenosine triphosphate, can solve the problems of unclear classification, etc., and achieve the effects of easy control, low pollution and cost reduction

Active Publication Date: 2019-04-16
BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

"ATP-producing enzymes" used include polyphosphate kinase (EC 2.7.4.1, Ppk), adenylate kinase (EC 2.7.4.3, Adk) and polyphosphate-adenylate phosphotransferase (EC 2.7.4.- , Pap, it has been reported in the literature that this enzyme also belongs to the class of Ppk enzymes, but the classification of this enzyme by IUBMB is not very clear)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for enzymatically preparing adenosine triphosphate
  • A method for enzymatically preparing adenosine triphosphate
  • A method for enzymatically preparing adenosine triphosphate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 AK, Ppk, Adk and Pap enzyme

[0047] The AK, Ppk (Ppk1 and / or Ppk2), Adk and Pap enzymes in the method of the present invention can be obtained commercially, or are enzymes with the same catalytic function after artificial modification.

[0048] The preparation process of AK, Ppk1, Ppk2, Adk and Pap enzymes is as follows:

[0049] According to the sequences of the five enzyme genes, five pairs of amplification primers were designed. Extract Saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain genomic DNA, use it as a template, PCR amplify ado1 (AK enzyme gene) fragment, and connect it to pET22b carrier (purchased from Novagene Company); Extract Escherichia coli (Escherichia coli) K12 bacterial strain ( Purchased from Tiangen Biochemical Technology Co., Ltd.) genomic DNA, using it as a template, PCR amplified ppk1 and adk gene fragments, and connected them to the pET 22b vector (purchased from Novagene); extract Pseudomonas...

Embodiment 2

[0057] Embodiment 2 uses free enzyme to prepare ATP

[0058] image 3 Process flow diagram for the production of ATP using free enzymes in the method of the present invention. see image 3 , the operation steps of using free enzyme to prepare ATP are as follows:

[0059] (1) The reaction of synthesizing ATP in the reaction tank:

[0060] In the reaction tank, the reaction system of 100L sterile water contains substrate 2.5kg adenosine, and 0.1kg ATP, 0.5kg sodium dihydrogen phosphate, 0.3kg sodium chloride, 0.3kg ammonium sulfate, 1.0kg magnesium chloride hexahydrate and 2.0kg sodium hexametaphosphate solution, stir evenly during preparation to prevent precipitation. The pH value was adjusted to 7.5, and 500U / L Pap enzyme, 800U / LPpk2 enzyme and 800U / L Pap enzyme were added to the reaction system to start the reaction. During the reaction, the pH value was controlled to be 7.5, and the temperature was 45°C.

[0061] Figure 5 , Image 6 Respectively, the HPLC detection ...

Embodiment 3

[0068] Embodiment 3 uses free enzyme to prepare ATP

[0069] see image 3 , the operation steps of using free enzyme to prepare ATP are as follows:

[0070] (1) The reaction of synthesizing ATP in the reaction tank:

[0071] In the reaction tank, the reaction system of 100L sterile water contains substrate 3.0kg adenosine, and 0.1kg AMP, 0.5kg disodium hydrogen phosphate, 0.3kg potassium chloride, 0.25kg ammonium chloride, 2.5kg heptahydrate sulfuric acid The solution of magnesium and 3.0kg tetrapolyphosphoric acid should be uniformly stirred during preparation to prevent precipitation. Adjust the pH value to 7.0, add 500U / LAK enzyme, 500U / LPpk2 enzyme, 300U / LAdk enzyme and 500U / L Pap enzyme to the reaction system to start the reaction. During the reaction, the pH value was controlled to be 7.0, and the temperature was 40°C.

[0072] After 8 hours of reaction, the amount of ATP produced was about 50 g / L, and the conversion rate of adenosine reached 85%. The HPLC detection...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for preparing triphosadenine by an enzymic method. The method comprises the following steps of (1) preparing ATP production enzymes: obtaining the ATP production enzymes in gene engineering transformation, fermentation, purification or direct extraction modes; (2) preparing ATP in reaction liquid; adding adenosine, the ATP production enzymes and polyphosphoric acid or salts of the polyphosphoric acid for ATP preparation; (3) separating products and ATP production enzymes; directly separating immobilized ATP production enzymes in a reaction tank, and separating free ATP production enzymes through an ultrafiltration membrane in a filtering device; (4) preparing a finished product: preparing the reaction liquid subjected to the enzyme separation into finished product ATP through the steps of ion-exchange column chromatography, concentration, crystallization; drying and the like. The method has the advantages that the novel ATP production enzymes are used; the production process is simple; the control is easy; the adenosine is used as a substrate; the cost is reduced; an APT production enzyme recovery system is built; the method is applicable to industrial production; the content of impurities such as impurity protein and pigment in the enzymic method preparation reaction liquid is low; the subsequent purification is easy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for enzymatically preparing adenosine triphosphate. Background technique [0002] Adenosine triphosphate (ATP) consists of one adenosine (A) and three phosphate groups, with a molecular weight of 507 and molecular formula C 10 h 16 N 5 o 13 P 3 . It is the energy converter and storage device in the living body, and plays an important role in the energy metabolism of the human body. As a metabolic intermediate and coenzyme, it participates in the metabolism of carbohydrates, proteins, nucleic acids and fats in the living body. In clinical application, ATP has a wide range of functions, and has good therapeutic and adjuvant therapeutic effects on progressive muscular atrophy, stroke sequelae, myocardial infarction, myocarditis, coronary arteriosclerosis, hepatitis and other diseases. [0003] The synthesis of ATP mainly includes chemical synthesis, biological enzyme cata...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/32
CPCC12P19/32
Inventor 刘珊珊刘辉于铁妹秦永发
Owner BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com