A recombinant expression vector, recombinant expression host and method for synthesizing adenosine triphosphate

An expression vector and adenylate kinase technology, which is applied in the field of preparation of adenosine triphosphate, can solve the problems of large product batch quality differences, difficult control of the reaction process, complex reaction system, etc., to achieve industrial production and easy subsequent purification Industry, the effect of improving reaction efficiency

Active Publication Date: 2021-02-23
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current method for industrial production mainly uses adenosine or adenosine monophosphate (AMP) as a substrate to synthesize ATP by using enzymes in the glycolysis pathway of yeast, but this method has complex reaction systems and difficulties in separation and purification. , the reaction process is not easy to control, and the quality difference between product batches is large, etc.

Method used

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  • A recombinant expression vector, recombinant expression host and method for synthesizing adenosine triphosphate
  • A recombinant expression vector, recombinant expression host and method for synthesizing adenosine triphosphate

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The construction of embodiment 1 recombinant escherichia coli

[0035] Experimental materials and reagents:

[0036] Escherichia coli BL21(DE3) (E.coil BL21(DE3)): purchased from Invitrogen;

[0037] Escherichia coli DH5α (E.coil DH5α): purchased from Invitrogen;

[0038] Saccharomyces cerevisiae strain: China Center for Type Culture Collection, No. CCTCC AY 93175;

[0039] pET24a plasmid: used for expression plasmid, with T7 promoter, kanamycin resistance gene, purchased from NOVAGEN Company, Cat. No. 69749;

[0040] PCR primers for adenosine kinase, adenylate kinase and acetate kinase: synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.;

[0041] Pyrobest enzyme and 10×Pyrobest buffer: purchased from TaKaRa, product number R005;

[0042] Restriction endonuclease Nde I: purchased from TaKaRa, Cat. No. 1161A;

[0043] Restriction endonuclease BamH I: purchased from TaKaRa, Cat. No. 1010A;

[0044] Restriction endonuclease BglⅡ: purchased from TaKaRa, Cat. No. ...

Embodiment 2

[0077] Example 2 Recombinant Escherichia coli Whole Cell Catalytic Synthesis of ATP

[0078] In the reaction system (1L), add 10mmol / L adenosine, 50mmol / L borax, 50mmol / L ACP and 5mmol / L magnesium sulfate, add 10g / L thalline as above collected in embodiment 1 as enzyme source, adjust reaction The pH of the solution was 7.5, and the temperature was controlled at 35° C. during the reaction, and the reaction was carried out at a stirring speed of 150 r / min for 3 hours under mechanical stirring.

[0079] Collect the supernatant of the reaction solution, use high performance liquid chromatography (Agilent 1260) to analyze the concentration of residual adenosine by high performance liquid chromatography (HPLC) after filter sterilization; Diluted 10-fold, filter-sterilized and analyzed by HPLC.

[0080] HPLC conditions: Welch Ultimate XB-C18 chromatographic column (250mm×4.6mm I.D., 5μm particle size, purchased from Yuexu Technology (Shanghai) Co., Ltd., article number 00201-31043);...

Embodiment 3

[0083] Example 3 Recombinant Escherichia coli Whole Cell Catalytic Synthesis of ATP

[0084] Add 30mmol / L adenosine, 50mmol / L borax, 180mmol / L ACP and 10mmol / L magnesium sulfate in reaction system (1L), add 5g / L bacterium as enzyme source, adjust reaction solution pH to 7.5, during the reaction The temperature was controlled at 30° C., and the reaction was performed at a stirring speed of 150 r / min for 5 hours under mechanical stirring. ATP and adenosine concentrations were measured as described in Example 2. The generated ATP concentration was 15.1 g / L, and the conversion rate of adenosine was over 99% as calculated by the method described in Example 2.

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Abstract

The invention belongs to the field of bioengineering and relates to the preparation of adenosine triphosphate. The invention provides a recombinant expression vector and a recombinant expression host comprising the vector, the recombinant expression vector comprises polynucleotides encoding adenosine kinase, adenylate kinase and acetate kinase; the invention also provides the use of the A method for the synthesis of adenosine triphosphate by a recombinant expression host. The invention improves the adenosine conversion rate, ATP production rate and reaction efficiency by using the whole cell of the recombinant expression host to catalyze the synthesis of adenosine triphosphate, and meanwhile, the components of the reaction system are simple and the cost is low.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to the preparation of adenosine triphosphate. Background technique [0002] Adenosine triphosphate (ATP) is an important metabolite in organisms, with a molecular weight of 507 and a molecular formula of C 10 h 16 N 5 o 13 P 3 , as a metabolic intermediate, coenzyme and energy donor, participates in various biochemical reactions in organisms, and is a high-energy compound necessary for life activities and biochemical reactions; in vitro, ATP is often used as a cosubstrate for enzyme reactions in industrial products such as glutathione Glycopeptide production. Clinically, ATP is also commonly used in the adjuvant treatment of progressive muscle atrophy, myocardial infarction, myocarditis and other diseases. [0003] The methods for synthesizing ATP mainly include chemical synthesis, biological enzyme catalysis, and microbial enzyme fermentation. The current method for industrial pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P19/32
CPCC12N15/70C12P19/32
Inventor 杨勇江林林吴磊许海霞黄坚丽张永进徐期
Owner ZHEJIANG HISUN PHARMA CO LTD
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