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AMP phosphotransferase mutant, coding gene thereof and application of AMP phosphotransferase mutant in ATP synthesis

A phosphotransferase and mutant technology, applied in the field of genetic engineering, can solve the problems of difficult process control, many types of enzymes, low enzyme activity, etc., and achieve the effects of cost reduction, less impurities and pigments, and simple production process

Pending Publication Date: 2022-08-09
美邦美和生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a mutant of AMP phosphotransferase, its coding gene and its application in ATP synthesis, so as to solve the problems of low enzyme activity, many types of enzymes, high cost and difficult process control in the existing ATP synthesis technology. question

Method used

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  • AMP phosphotransferase mutant, coding gene thereof and application of AMP phosphotransferase mutant in ATP synthesis
  • AMP phosphotransferase mutant, coding gene thereof and application of AMP phosphotransferase mutant in ATP synthesis
  • AMP phosphotransferase mutant, coding gene thereof and application of AMP phosphotransferase mutant in ATP synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Obtaining method of AMP phosphotransferase mutant

[0043] Random mutation was introduced by random mutation PCR kit, and the difference in ATP synthesis ability between mutant and wild type was screened by HPLC method. Select forward mutant monoclonal strains, extract plasmids and send them for sequencing to obtain mutation points. A total of 4 single point mutations were obtained, see mutants 1-4. In the later stage, mutants 5-10 were constructed by introducing site-directed mutagenesis through primer design and tested for activity.

[0044] A total of ten strains with high vigor and high stability were obtained by the viability screening method under the same conditions, which were named as mutants 1-10, which contained mutations at positions 124 (A124L), 312 (G312M) and 393 (F393K). Body 9 (named AMP-PPTM enzyme) can increase about 80 times in ATP synthesis activity, about 20 times in temperature stability, and the most significant improvement in activity...

Embodiment 2

[0047] Example 2: Determination of AMP-PPTM enzyme properties

[0048] The plasmids containing AMP phosphotransferase and its mutant were respectively transformed into Escherichia coli BL231 (DE)) to obtain mutants, the strains were inoculated in LB medium, and the OD of the bacteria was treated. 600 When the value reached 0.6, the final concentration of 0.1 mM IPTG was added to induce the expression of AMP phosphotransferase and its mutants, overnight induction, and the induction condition was 20°C. The amount of cells collected horizontally in shake flasks was 5 g / L. The bacterial cells were crushed to prepare a crude enzyme solution.

[0049] According to the activity value of AMP-PPTM enzyme, high-quality strains were determined. Enzyme activity test 1mL reaction system: substrate adenosine (13.5mM / L), ATP disodium salt (5mM / L), magnesium ion (50mM / L) and sodium hexametaphosphate (50mM / L), pH7.0, The reaction was performed at 37°C for 30 min, and the amount of ATP gener...

Embodiment 3

[0050] Example 3: Determination of the detection method of each substance in the catalytic reaction

[0051] High performance liquid chromatography was used to detect the remaining substrate of the catalytic reaction and the formation of the product. Chromatographic conditions: C18 reversed-phase column, mobile phase phosphate buffered saline-methanol, flow rate 1.2 mL / min, column temperature 30 °C, wavelength 254 nm. Test the 100mL reaction system, react for 30min, and detect the conditions of each substance in the system, such as figure 1 : The ATP generation rate is 33%, and a little AMP and ADP are generated; the reaction is 2h, and the ATP generation is detected, such as figure 2 : The generation rate of ATP is 81%, followed by AMP; after 3 hours of reaction, the amount of ATP generated is detected, such as image 3 , the substrate was basically consumed completely, and the ATP generation rate was 97%.

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Abstract

The invention discloses an AMP (adenosine monophosphate) phosphotransferase mutant, a coding gene thereof and application of the AMP phosphotransferase mutant in ATP (adenosine triphosphate) synthesis. The amino acid sequence of the mutant is obtained by carrying out one or more mutations on a sequence shown as SEQ: ID: NO: 1. The mutation site of the gene comprises one or more mutation sites in the 124 site (A124L), the 312 site (G312M) and the 393 site (F393K). The invention further provides a composite biocatalyst containing adenosine kinase and the AMP phosphotransferase mutant and application of the composite biocatalyst in ATP synthesis. The mutant is higher in enzyme activity, higher in stability and higher in ATP generation ratio, the repeated batch after immobilization is larger than 100, the cost for producing ATP through a whole enzyme method can be further reduced, the whole adenosine phosphorylation ATP synthesis reaction can be completed by reducing the number of enzyme types to 2, and large-scale industrial application is facilitated.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an AMP phosphotransferase mutant, its encoding gene and its application in ATP synthesis. Background technique [0002] Adenosine triphosphate is abbreviated as ATP, which is an unstable high-energy compound. It is interconverted with ADP in cells to achieve energy storage and release, thus ensuring the energy supply of various life activities of cells. ATP plays an important role in human energy metabolism. As a metabolic intermediate and coenzyme, it participates in the metabolism of carbohydrates, proteins, nucleic acids and fats in vivo. In clinical application, it has good therapeutic and adjuvant therapeutic effects on muscle atrophy, stroke sequelae, myocardial infarction, coronary sclerosis, hepatitis and other diseases. [0003] The synthesis of ATP mainly includes chemical method and biological method, and biological method includes microbial fermentation m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12P19/32
CPCC12N9/1229C12N9/1205C12P19/32C12Y207/0102C12Y207/04003
Inventor 秦浩杰任丽梅杨梦茜张蕴之米雅萱王素霞刘东高文杲
Owner 美邦美和生物科技有限公司
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