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Method for regenerating ATP(adenosine triphosphate) through enzyme method

A polyphosphate kinase and bacillus technology, which is applied in biochemical equipment and methods, enzymes, transferases, etc., can solve the problems that the enzyme coupling reaction cannot be carried out at room temperature, cannot widely meet the needs of industrial production, and the degree of polyphosphate polymerization is high. , to reduce costs

Inactive Publication Date: 2016-04-06
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the polyphosphokinase TePpk derived from Thermotogamaritima is a thermophilic enzyme. Its optimum temperature is 70 degrees, which is suitable for participating in reactions with higher temperatures. The enzyme activity is low, and it cannot be coupled with room temperature enzymes, so it cannot widely meet the needs of industrial production
There are also some polyphosphate kinases. Although the optimum temperature is about 30 degrees, the polyphosphate used has a high degree of polymerization. This polyphosphate with a high degree of polymerization is not easy to obtain in the market and is expensive. Increased production difficulty and production cost
On the other hand, with regard to industrial mass production of ATP, some polyphosphate kinases can be inhibited by polyphosphate, so the method of feeding polyphosphate has to be adopted in large-scale production to reduce inhibition and make the production process easier. cumbersome

Method used

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  • Method for regenerating ATP(adenosine triphosphate) through enzyme method
  • Method for regenerating ATP(adenosine triphosphate) through enzyme method
  • Method for regenerating ATP(adenosine triphosphate) through enzyme method

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Experimental program
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Effect test

Embodiment 1

[0031] 1.1 Cloning of ppk polyphosphate kinase gene in Corynebacterium glutamicum strain and construction of recombinant vector

[0032] The ppk polyphosphokinase gene in the Corynebacterium glutamicum strain was synthesized by genome amplification method. Using the whole genome of the strain as a template, primers were designed according to the GenBank sequence, and corresponding restriction sites (NcoI and EcoRI) and protective bases were added.

[0033] The primers used for PCR amplification of polyphosphate kinase gene ppk are as follows:

[0034] Upstream primer: 5'-GCATATGATGACCGCCACCGATTC-3'

[0035] Downstream primer: 5'-GAAGCTTCCGGTTGGTAGCTGTGAC-3'

[0036] The PCR system reaction system contains 0.2 μL of genomic DNA, 0.5 μL of upstream and downstream primers, 10 μL of Extaq mixture, and added double distilled water to make up to 20 μL. The reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, exten...

Embodiment 2

[0040] Transfer the constructed expression vector pET28a-ppk plasmid into BL21(DE3) Escherichia coli, use LB medium after picking the bacteria, grow at 37°C until the OD600 is about 0.4-1.0, and induce with 0.1mM-1mM IPTG , for intracellular expression at 30°C. The vector pET28a was transformed into BL21(DE3) Escherichia coli, and the expression was induced and expressed under the same conditions as above, and the expressed crude enzyme solution was subjected to protein electrophoresis to verify the expression result. The protein electrophoresis was as follows: image 3 As shown, the protein content measured by the Coomassie brilliant blue method was 2.3 g / L.

Embodiment 3

[0042] Three experimental groups were set up in reaction system 1. The phosphoric acid donors in the experimental groups were tripolyphosphate, tetrapolyphosphate and hexametaphosphate respectively. 1mMADP, 30mMMgCl 2 , 2mM polyphosphate, 250ul PPK crude enzyme solution (containing 0.547mg of protein), add 3M pH8.0 Tris-HCl10ul to adjust the pH to 8.0, system 1 reacted at 37 degrees for 5 minutes, placed in a 60 degrees water bath and heated for 20 minutes to inactivate the PPK enzyme . Take the supernatant and add it to NADPH reaction system 2. System 2 includes 1mM glucose, 1mM NADP, 100ul hexokinase (1U), 100ul 6-phosphate glucose dehydrogenase (1U). System 2 measures NADPH at 340nm after reacting at 30°C for 30min According to the absorbance of the molar absorptivity, the amount of NADPH generated was calculated. The PPK enzyme activities measured with the three phosphate substrates were 0.162umol / min / ml, 0.176umol / min / ml, and 0.193umol / min / ml, respectively.

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Abstract

The invention relates to a method for regenerating ATP(adenosine triphosphate). A heterogeneous source is used for expressing polyphosphate kinase, and under catalyzing of polyphosphate kinase, polyphosphates with the low polymerization degree are used as phosphoric acid donors for regenerating ATP. Compared with an existing ATP regeneration method, adopted polyphosphate kinase is a normal-temperature enzyme, can be combined with multiple enzymes for promoting reaction coupling, and adopts a tripolyphosphate, a tetrapolyphosphate and a hexametaphosphate as the phosphoric acid donors, these polyphosphates are low in polymerization degree, common, easy to obtain and low in price, on the basis of simplifying the ATP regeneration process, feasibility of ATP regeneration is improved, and the ATP regeneration cost is reduced; activity of a PPK enzyme is not inhibited by polyphosphate concentration, and possibility is provided for industrialized large-scale production.

Description

technical field [0001] The invention belongs to the field of biochemical industry, in particular to a method for enzymatically regenerating ATP. Background technique [0002] ATP is the most important high-energy phosphate compound in the body, and it is widely used in medicine as a treatment or an important auxiliary treatment drug for muscle atrophy, myocardial infarction, hepatitis and various emergency diseases. There are also many biosynthetic processes of important bioactive substances or biochemical drugs in industry, which are also enzymatic reactions that require ATP to participate. Therefore, the production of ATP and the recycling of ATP in industry have become an important issue in the development of enzyme engineering, and are the key factors to determine whether industrial production is economical. [0003] In recent years, the research on ATP regeneration is mainly based on the method catalyzed by polyphosphate kinase, that is, ADP and polyphosphate are used ...

Claims

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Application Information

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IPC IPC(8): C12P19/32C12N9/12
CPCC12P19/32C12N9/1229C12Y207/04001
Inventor 刘珞李成成王峥
Owner BEIJING UNIV OF CHEM TECH
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