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Method for preparing adenosine triphosphate with immobilized enzyme method

A technique for the reaction of adenosine triphosphate and adenosine triphosphate, which is applied in the direction of biochemical equipment and methods, fixed on/in organic carriers, enzymes, etc., can solve problems such as ineffective recycling, affecting application value, and rapid decline in enzyme activity, and achieves The effect of stable product quality, easy reaction and easy purification

Active Publication Date: 2016-06-08
BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In practical applications, the cost of enzymes is expensive. Using free enzymes to produce ATP, the enzyme activity declines rapidly and cannot be effectively recycled, which will inevitably lead to high production costs and affect the actual application value.

Method used

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  • Method for preparing adenosine triphosphate with immobilized enzyme method
  • Method for preparing adenosine triphosphate with immobilized enzyme method
  • Method for preparing adenosine triphosphate with immobilized enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation of embodiment 1Ppk, Adk and Pap enzyme

[0042] The Ppk, Adk and Pap enzymes in the method of the present invention can be obtained commercially, or are artificially modified enzymes having the same catalytic function.

[0043] The preparation process of Ppk, Adk and Pap enzyme is as follows:

[0044] According to the sequence of the following genes, three pairs of amplification primers were designed and synthesized by Zhongmei Taihe Biotechnology Co., Ltd. The primer sequences are as follows:

[0045] ppk sense primer: 5'-CCATATGGGTCAGGAAAAGCTATACATCG-3';

[0046] ppk antisense primer: 5'-CGGATCCTTTATTCAGGTTGTTCGAGTGATT-3';

[0047] adk sense primer: 5'-CCATATGCGTATCATTCTGCTTGGCGCTCCGG-3';

[0048] ADK antisense primer: 5'-CGGATCCTTAGCCGAGGATTTTTTTCCAGATC-3';

[0049] PAP sense primer: 5'-GCCATGGATACAGAAACGATCGCCAGTGCAG-3'; and

[0050] pap antisense primer: 5'-CGGATCCTTAATCCGTGTCGCGATCCGCTT-3';

[0051] Extract the DNA of Escherichia coli (Escher...

Embodiment 2

[0056] Example 2 Preparation of adenosine triphosphate by immobilized ATP production enzyme

[0057] Highly expressing Ppk and Pap strains were respectively prepared according to Example 1, and the bacterial cells were collected by centrifugation after the fermentation was completed. Take 2.0 kg of Pap-containing bacteria and 2.0 kg of Ppk-containing bacteria, mix and suspend them with 20 L of 0.1M pH7.5 Tris hydrochloric acid buffer, crush the bacteria with a high-pressure homogenizer, and collect the supernatant by centrifugation to obtain the ATP-producing enzyme.

[0058] Add 5 kg of amino-containing synthetic polymer carrier LX1000HA and 20 L of the above-mentioned ATP production enzyme into a constant temperature stirring reaction tank (the mass ratio of immobilized carrier to enzyme is 25:1), and stir at 150 rpm for 12 hours at 20°C. The carrier was collected by filtration, and washed twice with 0.02M pH 8.0 potassium phosphate buffer solution to obtain the immobilized ...

Embodiment 3

[0061] Example 3 Preparation of adenosine triphosphate by immobilized ATP production enzyme

[0062] Highly expressing Pap and Adk strains were respectively prepared according to Example 1, and the bacterial cells were collected by centrifugation after the fermentation was completed. Take 1.6 kg of Pap-containing bacteria and 0.4 g of Adk-containing bacteria, mix and suspend them in 20 L of phosphate buffer, crush the bacteria with a high-pressure homogenizer, and collect the supernatant by centrifugation to obtain an ATP-producing enzyme solution.

[0063] Add 4 kg of epoxy-containing polyacrylic acid carrier and 16 L of the above-mentioned ATP production enzyme solution into a constant temperature stirring reaction tank and mix (the ratio of immobilized carrier to enzyme protein is 40:1). Stir at 150 rpm for 5 h at 25°C. The carrier was collected by filtration and washed twice with 0.01 M pH 8.0 potassium phosphate buffer. The immobilized ATP-producing enzyme was placed in...

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Abstract

The invention discloses a method for preparing adenosine triphosphate (ATP) with an immobilized enzyme method. The method comprises the following steps: (1) fixing an ATP production enzyme on an immobilized carrier to prepare an immobilized ATP production enzyme; (2) catalyzing by using the immobilized ATP production enzyme to prepare an adenosine triphosphate reaction solution; (3) separating the product adenosine triphosphate. According to the method disclosed by the invention, three types of novel ATP production enzymes including Ppk, Adk and Pap are adopted, and the ATP can be synthesized through only two steps of enzymatic reaction; a reaction process is simpler, a reaction is more easily controlled and the product quality is more stable; the ATP is prepared by adopting an immobilized enzyme catalysis method, and an immobilized enzyme can he continuously and repeatedly used for a plurality of times, so that the production cost is greatly reduced. Meanwhile, impurities including a lot of proteins, pigments and the like, which are introduced by utilizing yeasts, are avoided, and the product is more easily purified; an intermittent stirring reaction and enzyme reaction column continuous reaction system which is applicable to large-scale production of the ATP is established.

Description

technical field [0001] The invention relates to a method for preparing adenosine triphosphate, in particular to a method for preparing adenosine triphosphate by an immobilized enzyme method. Background technique [0002] Adenosine triphosphate (ATP) is a high-energy compound composed of one adenine, one ribose sugar and three phosphate groups, with the molecular formula C 10 h 16 N 5 o 13 P 3 , the molecular weight is 507. It is an energy converter and storage device in a living organism, 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 fat, protein, sugar and nucleic acid in the living body. In clinical application, ATP has been used as a therapeutic agent, and has good therapeutic and adjuvant therapeutic effects on progressive muscular atrophy, stroke sequelae, myocardial infarction, myocarditis, coronary arteriosclerosis, hepatitis and other diseases. In recent yea...

Claims

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Application Information

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IPC IPC(8): C12P19/32C12N11/08C12N11/12C12N11/10
CPCC12N9/1229C12N11/08C12N11/10C12N11/12C12P19/32C12Y207/04001C12Y207/04003Y02P20/50
Inventor 于铁妹刘珊珊黄庆军秦永发
Owner BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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