Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof

A regeneration reaction and enzymatic technology, applied in the biological field, can solve the problems of expensive substrates and difficult to use in large quantities, and achieve the effects of simple and efficient regeneration process, reduced usage and high stability.

Inactive Publication Date: 2016-08-17
SHENZHEN GSH BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrates used by these enzymes are expensive, such as phosphoenolpyruvate used by pyruvate kinase, and the by-products generated have certain biological toxicity and pollution, such as the products of the reaction catalyzed by acetate kinase and ammonia kinase They are acetic acid and ammonia respectively, so it is difficult to use them in large quantities in industrial production

Method used

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  • Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof
  • Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof
  • Method for regenerating ATP (adenosine triphosphate) by enzyme process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of embodiment 1Ppk, Adk and Pap enzyme

[0055] The Ppk (including Ppk1 and Ppk2), Adk and Pap enzymes in the method of the present invention can be obtained commercially, or can be artificially modified to have the same catalytic function.

[0056] The preparation process of Ppk1, Ppk2, Adk and Pap enzymes is as follows:

[0057] According to the sequences of the four enzyme genes, four pairs of amplification primers were designed and synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The primer sequences are as follows:

[0058] ppk1 sense primer: 5'-CCATATGGGTCAGGAAAAGCTATACATCG-3';

[0059] ppk1 antisense primer: 5'-CGGATCCTTTATTCAGGTTGTTCGAGTGATT-3';

[0060] ppk2 sense primer: 5'-TACATATGGCCAGCCCGGCGCAGAAG-3';

[0061] ppk2 antisense primer: 5'-TGAATTCAGGCCGGGATATCCAGGTTC-3';

[0062] adk sense primer: 5'-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3';

[0063] ADK antisense primer: 5'-CGGATCCTTAGCCGAGGATTTTTTTCCAGATC-3';

[0064] PAP sense primer: 5...

Embodiment 2

[0073] Example 2 Enzymatic production of phosphocreatine-coupled ATP regeneration system

[0074] image 3 It is a process flow diagram of using free enzyme to carry out enzymatic reaction and coupling ATP regeneration system in the method of the present invention. see image 3 , Enzymatic preparation of creatine phosphate while coupling free ATP regeneration system steps are as follows:

[0075] (1) The reaction of synthesizing creatine phosphate and regenerating ATP in the reaction tank:

[0076] In the reaction tank, the reaction system of 100L sterile water contains substrate 1.5kg creatine, and 1.0kg ATP, 0.5kg disodium hydrogen phosphate, 0.38kg potassium chloride, 0.3kg sodium chloride, 0.2kg ammonium sulfate, The solution of 1.0kg magnesium chloride hexahydrate and 2.0kg sodium tetrapolyphosphate should be uniformly stirred to prevent precipitation during preparation. Adjust the pH value to about 7.5, add 500U / L creatine kinase, 500U / L Ppk2 enzyme, and 500U / L Adk e...

Embodiment 3

[0081] Example 3 Enzymatic production of 1,6-diphosphate fructose-coupled ATP regeneration system (immobilized enzyme)

[0082] Figure 4 It is a process flow chart of using immobilized enzyme to carry out enzymatic reaction to produce and coupling ATP regeneration system in the method of the present invention. see Figure 4 , the operation steps of the immobilized enzyme method to prepare 1,6-diphosphate fructose while coupling the ATP regeneration system are as follows:

[0083] (1) Immobilization of catalytic enzyme and ATP regeneration enzyme:

[0084] Catalytic enzymes fructokinase (FK) and phosphofructokinase (PFK) were obtained commercially, and together with the ATP regeneration enzyme Ppk2 enzyme and Pap enzyme initially purified in Example 1, they were immobilized on the commercial epoxy immobilized carrier LX1000EP.

[0085] Mix FK, PFK, Ppk2 and Pap enzymes according to the activity ratio of 1:1:1:1 to make 10L of mixed enzyme solution, and the activity of each ...

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Abstract

The invention discloses a method for regenerating ATP by enzymatic method, which comprises the following steps: (1) preparing and obtaining ATP regenerating enzyme; (2) regenerating ATP in reaction liquid; (3) separating products and ATP regenerating enzyme. According to the method for regenerating ATP of the present invention, it has the following beneficial effects: (1) adopt three kinds of ATP regenerating enzymes of novel Ppk, Adk and Pap, the ATP consumed in the enzymatic reaction can be recycled and regenerated, greatly reducing the amount of ATP used, regenerated The process is simple and efficient, the reaction is easy to control, and the stability is high. (2) The substrate polyphosphoric acid or its salt to be added to the reaction is cheap and less polluting. (3) The enzymatic reaction does not need to add expensive ATP to start the reaction, add ADP or cheap AMP. (4) ATP regenerating enzyme recovery system was established, which is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for enzymatically regenerating ATP and its application. Background technique [0002] Adenosine triphosphate (ATP) consists of one adenosine (A) and three phosphate groups (Pi), with a molecular weight of 507 and molecular formula C 10 h 16 N 5 o 13 P 3 . It is the converter and storage of biological energy, and plays an irreplaceable and important role in the enzymatic reactions involving energy. The ATP molecule has a structure of A-Pi~Pi~Pi, and the two phosphate bonds away from adenosine are represented by ~, which are high-energy phosphate bonds. During the reaction, the high-energy phosphate bond of the ATP molecule can be broken to release a large amount of energy: when a high-energy phosphate bond is broken, the ATP molecule is converted into adenosine diphosphate (ADP), releasing energy of 30.5kJ / mol; when the second high-energy phosphate When the bond is bro...

Claims

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Application Information

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IPC IPC(8): C12P19/32C12N9/12C12P13/00C12P19/02C12P9/00C12P19/36C12P19/30C12P21/02C12P13/14C12P13/04C12P17/10
CPCC12P19/32C12N9/1229C12P7/62C12P13/001C12P13/04C12P13/14C12P17/10C12P19/02C12P19/305C12P19/36C12P21/02C12Y207/04001C12Y207/04003
Inventor 刘珊珊于铁妹秦永发
Owner SHENZHEN GSH BIO TECH CO LTD
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