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Production method for enzymatic reaction by utilizing adenosine to replace ATP

A technology of enzymatic reaction and production method, which is applied in the biological field to achieve the effects of low price, wide source and simple operation

Active Publication Date: 2019-01-04
BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still needs to add a certain amount of ATP to carry out the enzymatic reaction

Method used

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  • Production method for enzymatic reaction by utilizing adenosine to replace ATP
  • Production method for enzymatic reaction by utilizing adenosine to replace ATP
  • Production method for enzymatic reaction by utilizing adenosine to replace ATP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The preparation of embodiment 1 crude enzyme

[0051] The ATP regenerating enzyme and AK enzyme in the method of the present invention can be obtained commercially, or are artificially modified enzymes having the same catalytic function.

[0052] The enzyme preparation process is as follows:

[0053] Primers were designed according to the gene sequences of PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme, and the gene fragments were respectively amplified by PCR and connected to the pET22b vector (commercially available). After the sequencing was correct, they were respectively transferred into E .coli BL21(DE3) strain (commercially available).

[0054] Insert the transformed E.coli BL21(DE3) monoclonal into LB medium, culture to the logarithmic phase, add 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) for induction, induce The cells were collected after 5 hours, and high-expression strains were screened by sodium dodecylsulfonate-polyacrylamide gel electrophoresis (S...

Embodiment 2

[0059] Embodiment 2 enzymatic method produces creatine phosphate

[0060] figure 2 A flow diagram of the reaction process using free enzymes for the present invention. Such as figure 2 Shown, the operation steps of enzymatic preparation creatine phosphate are as follows:

[0061] (1) The reaction of synthesizing creatine phosphate in the reaction tank:

[0062] In the reaction tank, the 100L reaction system is a substrate containing 2.0kg creatine, 0.2kg adenosine, 1.8kg sodium polyphosphate, 0.4kg potassium chloride, 0.5kg magnesium chloride hexahydrate and 0.3kg disodium hydrogen phosphate When preparing the solution, stir evenly to prevent precipitation. Adjust the pH value to 7.0, add creatine kinase 1000U / L, PPK enzyme 500U / L, ADK enzyme 500U / L and AK enzyme 500U / L to the reaction system to start the reaction. During the reaction, the pH value was controlled to be 7.0, and the temperature was 35°C.

[0063] Figure 4 It is a diagram of the remaining amount of cre...

Embodiment 3

[0067] Embodiment 3 enzymatic method produces 1,6-diphosphate fructose (immobilized enzyme)

[0068] image 3 Process flow diagram for the reaction of the method of the present invention using immobilized enzymes. Such as image 3 As shown, the operation steps for preparing 1,6-diphosphate fructose by the immobilized enzyme method are as follows:

[0069] (1) Immobilization of catalytic enzyme, ATP regeneration enzyme and AK enzyme:

[0070] Catalytic enzymes fructokinase (FK) and phosphofructokinase (PFK) were obtained commercially, and together with ADK enzyme, PAP enzyme and AK enzyme initially purified in Example 1, were immobilized on the commercial epoxy immobilized carrier LX1000EP.

[0071] Mix FK, PFK, ADK, PAP and AK enzymes according to the activity ratio of 2:2:1:1:1 to make a mixed enzyme solution, and the AK enzyme activity in the enzyme solution is 2000U / L. Add 2 kg of LX1000EP wet carrier to the constant temperature stirring tank to mix with the above enzym...

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Abstract

The invention discloses a production method for enzymatic reaction by utilizing adenosine to replace ATP and application thereof. The method comprises the following steps that (1) in an enzymatic reaction system, ATP regeneration enzyme, AK enzyme and the adenosine are added in proportion for enzymatic reaction; (2) immobilized ATP regeneration enzyme and AK enzyme are directly separated in a reaction tank, and free ATP regeneration enzyme and AK enzyme are subjected to ultrafiltration membrane separation in a filter; (3) percolate in step (2) is separated and purified to obtain a product. Theproduction method for enzymatic reaction by utilizing the adenosine to replace the ATP and the application thereof have the advantages that a large amount of cost is saved for industrial production by utilizing the adenosine to replace the ATP or AMP; a stable enzyme recovery system is established, and the energy conservation and environment protection are achieved; a small quantity of generatedby-products ATP, ADP and AMP are directly used for cyclic reaction, or used for producing the ATP, or purified intensively through filtration, ion exchange and other methods, and the operation is simple.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a production method using adenosine instead of ATP for enzymatic reaction. Background technique [0002] Adenosine triphosphate (ATP) consists of one adenosine 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 converter and storage of biological energy, and plays an irreplaceable and important role in the enzymatic reaction of utilizing and converting energy. [0003] Due to the high cost of ATP at present, the direct use of ATP for enzymatic reactions in industrial production has little benefit. Therefore, it is the current research direction to use ATP for industrial production to put a small amount of ATP into the reaction production and to recycle ATP in the reaction process by establishing a stable and effective ATP regeneration system. [0004] A common method for ATP regeneration in industry is to use the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12P13/12C12P19/02C12P17/10C12P13/04C12N9/00C12N15/70
CPCC12P19/02C12N11/18C12N11/089C12N9/1229C12N9/1205C12Y207/04003C12Y207/04C12Y207/0102C12Y207/01011C12Y207/01004
Inventor 刘珊珊刘辉周稳文秦永发
Owner BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
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