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Enzymatic reaction composition, method for increasing amount of adenosine triphosphate (ATP) in enzymatic reaction and application of method

A technology of adenosine triphosphate and adenosine monophosphate, applied in the field of enzymatic reaction composition and increasing the content of adenosine triphosphate in enzymatic reaction, can solve the problems that adenosine triphosphate cannot be widely used, and achieve the goal of increasing the pressure of purification, reducing production cost and consuming production capacity Effect

Pending Publication Date: 2020-12-11
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current enzymatic process using adenosine triphosphate (ATP) cannot be widely used due to cost and other reasons

Method used

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  • Enzymatic reaction composition, method for increasing amount of adenosine triphosphate (ATP) in enzymatic reaction and application of method
  • Enzymatic reaction composition, method for increasing amount of adenosine triphosphate (ATP) in enzymatic reaction and application of method
  • Enzymatic reaction composition, method for increasing amount of adenosine triphosphate (ATP) in enzymatic reaction and application of method

Examples

Experimental program
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Effect test

example 1

[0103] Example 1: Preparation of Creatine Kinase (CK)

[0104] Creatine kinase PCR primer sequence design according to Chinese patent application publication number CN102808006, specifically:

[0105] Upstream primer CPK1:

[0106] 5'-ctgacc ggatcc atgccgttcggtaacacccacaac-3' (SEQ NO.1)

[0107] Wherein, the BamHI restriction site is underlined.

[0108] Downstream primer CPK2:

[0109] 5'-tat gcg gaattc ttacttctgggcggggatcatgtc-3' (SEQ NO.2)

[0110] Among them, the EcoRI restriction site is underlined.

[0111] According to the method described in Chinese Patent Application Publication No. CN102808006, the total RNA of mouse skeletal muscle was extracted, and cDNA was prepared by reverse transcription. Using mouse skeletal muscle cDNA as a model, the creatine kinase gene was amplified by PCR with the above primers, and connected to pGEX-2T (purchased from GE Healthcare, USA) to obtain pGEX-2T(+)-CK (SEQ NO.15), which was transformed into Escherichia coli BL21(DE3) ...

example 2

[0113] Example 2: Preparation of polyphosphate: AMP transferase (PAP)

[0114] According to polyphosphoric acid: AMP phosphotransferase sequence design PCR primers, specifically:

[0115] Upstream primer PAP1:

[0116] 5'-ctgacc ggatccatgttcgaatccgcggaagttggc-3 (SEQ NO. 3)

[0117] Wherein, the BamHI restriction site is underlined.

[0118] Downstream primer PAP2:

[0119] 5'-tatgcg aagctt ttacttgtccttcttgtacgccgcctc-3' (SEQ NO.4)

[0120] Among them, the EcoRI restriction site is underlined.

[0121] With Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1-VE13AGY71676DNA as substrate, carry out PCR amplification polyphosphoric acid with above-mentioned primer: AMP transferase gene, utilize restriction endonuclease BamH I and EcoRI to process PCR product and connect it to pGEX-2T (purchased from GE Healthcare, USA) to obtain pGEX-2T(+)-PAP (SEQ NO.16). The recombinant expression vector was transformed into Escherichia coli HB101 to obtain polyphosphate: AMP phosp...

example 3

[0123] Example 3: Preparation of adenylate kinase (AK)

[0124] Design PCR primers based on the sequence of adenylate kinase, specifically:

[0125] Upstream primer AK1:

[0126] 5'-ctgacc ggatcc atggcagtcgattcctccaactcg-3 (SEQ NO. 5)

[0127] Wherein, the BamHI restriction site is underlined.

[0128] Downstream primer AK2:

[0129] 5'-tat gcg gaattc ttaacacggaagtgaagtgaagct-3' (SEQ NO. 6)

[0130] Among them, the EcoRI restriction site is underlined.

[0131] Using Salmonella enterica subsp.enterica serovarATCC700720 (ATCC, USA) DNA as a substrate, adenylate kinase gene was amplified by PCR with the above primers, and the PCR product was treated with restriction endonucleases BamHI and EcoRI and converted to Ligated into pGEX-2T (purchased from GE Healthcare, USA) to obtain pGEX-2T(+)-ADK (SEQ NO.17). The recombinant expression vector was transformed into Escherichia coli HB101 to obtain adenylate kinase recombinant expression strain.

[0132] Select a single co...

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Abstract

The invention provides an enzymatic reaction composition, a method of increasing the amount of adenosine triphosphate (ATP) in an enzymatic reaction and a method for synthesizing an amino acid or a derivative, polypeptide, enzyme or protein thereof by utilizing the adenosine triphosphate (ATP). According to the method, a first enzyme or an enzyme group and adenosine for producing adenosine monophosphate (AMP) are added during the enzymatic reaction so as to increase the amount of the adenosine triphosphate (ATP). The method and composition provided by the invention can increase the amount of the adenosine triphosphate (ATP), thereby improving the enzymatic reaction efficiency and reducing the cost.

Description

technical field [0001] The present invention relates to the field of biochemistry, in particular, to a biochemical reaction utilizing adenosine triphosphate (ATP), particularly an enzymatic reaction utilizing adenosine triphosphate, an enzymatic reaction composition and a method for increasing the content of adenosine triphosphate (ATP) in an enzymatic reaction Background technique [0002] Adenosine triphosphate (ATP) is a high-energy compound composed of three linked phosphate groups, ribose and adenine. Adenosine triphosphate (ATP) is the most important coenzyme in organisms, providing energy for metabolism and providing phosphate groups and adenosine groups. The important components of adenosine triphosphate (ATP) are three connected α, β and γ-phosphate groups; the α-phosphate group is connected to adenosine, and the β and γ phosphate groups are high-energy phosphate bonds. The compound containing only α-phosphate group is adenosine monophosphate (AMP); the compound co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12N9/12
CPCC12P19/32C12N9/1205C12N9/1229C12N9/1223C12N9/1217C12N9/12C12Y207/0102C12Y207/04003C12Y207/04001C12Y207/03002C12Y207/02008C12N15/70
Inventor 曾实现潘永强萧游龙卢锦春王骏
Owner BIORIGHT WORLDWIDE
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