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A cold-adapted peroxide reductase and its encoding gene and application

A technology of peroxide and reductase, which is used in the fields of cosmetics and food, biotechnology and medicine, and can solve problems such as poor stability

Active Publication Date: 2022-08-02
HARBIN INST OF TECH AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Peroxide reductase extracted from animals and plants is easily restricted by season, climate and region, and has poor stability, so it is difficult to be directly applied to related fields such as industrial production

Method used

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  • A cold-adapted peroxide reductase and its encoding gene and application
  • A cold-adapted peroxide reductase and its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Cloning and sequencing analysis of peroxidoreductase gene.

[0021] Antarctic Microbes Psychrobactor sp.ANT206 was activated in 2216E liquid medium and cultured to the middle and late logarithmic growth phase (about 4 days), and the total gene DNA of the strain was extracted by combining CTAB method and phenol-chloroform extraction method. PCR was performed using degenerate primers using the extracted total DNA as a template.

[0022] Upstream primer: 5'-AMTCAGTGACADTCAG ATGGCGTCT-3'

[0023] Downstream primer: 5'-GSAACTGGKGCATATGTTAGATTTT-3'

[0024] The amplification conditions were as follows: denaturation at 94 °C for 1 min, annealing at 58.2 °C for 1 min, and extension at 72 °C for 90 s, with 30 cycles. Then, it is detected by agarose gel electrophoresis to find the target gene. Ps Prx bands and sequenced. Analyzing the sequencing results, a gene with a full-length reading frame of 567 bp was obtained. The nucleotide sequence is shown in SEQ ID No....

Embodiment 2

[0025] Example 2: Expression and purification of peroxidoreductase gene

[0026] The primers containing the restriction sites were redesigned according to the determined full-length sequence of Prx.

[0027] Upstream primer: 5'-ATA GGATCC ATGGCGTCTATCATCA -3'

[0028] Downstream primer: 5'-CGC AAGCTT CGATTTTACCTACTAG -3’

[0029] The lines are respectively Bam HI, Hin dIII restriction site.

[0030] The Prx gene and pET-28a(+) double-enzyme digested gel recovery products were ligated by T4 ligase in proportion to construct the recombinant expression vector pET-Prx. Transform the recombinant expression vector into competent cells E.coli In BL21 (DE3), positive clones were screened and verified by enzyme digestion.

[0031] The recombinant strains obtained by screening were induced to express by IPTG. The recombinant bacteria were inoculated into LB medium and cultured to OD at 32-40°C 600 IPTG was added to the medium to a final concentration of 0.5-1.5 mM, and t...

Embodiment 3

[0032] Example 3: FeCl 3 Catalytic oxidative supercoiled DNA protection assay

[0033] A 15 μL reaction contains 15-18 mM FeCl 3 , 15-18 mM DTT and a certain amount of purified PsPrx in a water bath at 20-30 °C for 2-3 h, followed by adding 750-1250 ng pUC19 supercoiled DNA, and in a water bath at 20-30 °C for 2-3 h. After the reaction, the degradation results were detected by agarose gel electrophoresis. The results show that the cold-adapted peroxidoreductase PsPrx can protect supercoiled DNA from damage by metal-catalyzed oxidation systems. The result is as figure 2 As shown, 1, pUC19 plasmid; 2, pUC19 plasmid + FeCl; 3, pUC19 plasmid + DTT; 4, pUC19 plasmid + FeCl 3 +DTT+BSA; 5, pUC19 plasmid+FeCl 3 +DTT+PsPrx (5 μg / mL); 6, pUC19 plasmid+FeCl 3 +DTT+PsPrx (10 μg / mL); 7, pUC19 plasmid+FeCl 3 +DTT+PsPrx (15 μg / mL); 8, pUC19 plasmid+FeCl 3 +DTT+PsPrx (20 μg / mL); NF, nicked plasmid DNA; SF, supercoiled plasmid DNA. This experiment proves that the cold-adapted superox...

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Abstract

The invention discloses a cold-adapting peroxidase, its encoding gene and application, and aims to provide a cold-adapting peroxidase, its encoding gene and application. The present invention is first from the Antarctic sea ice microorganism psychrophilic bacillus ( Psychrobacter sp.) cloned the peroxide reductase gene PsPrx, the amino acid residue sequence of the peroxide reductase is shown in SEQ ID No.2. Its expression method is to construct a gene containing peroxide reductase Ps Prx recombinant expression vector, and the constructed recombinant expression vector is introduced into host cells ( Escherichia coli ), which induced the expression of the peroxidoreductase PsPrx gene. The expression product PsPrx of the present invention has outstanding cold adaptability, good stability, and good ability to protect supercoiled DNA from oxidative damage, and can be applied to related fields such as biomedicine, cosmetics and food.

Description

technical field [0001] The invention belongs to the fields of biotechnology, medicine, cosmetics, food and the like, and particularly relates to a cold-adapting peroxide reductase and its encoding gene and application. Background technique [0002] Peroxiredoxin (Prx) is a class of important thiol-dependent antioxidant enzymes widely present in eukaryotes and prokaryotes, which can efficiently degrade H 2 O 2 , peroxynitrate and various organic peroxides. Prx in microorganisms is mainly divided into three categories, namely 1-Cys Prx, typical 2-Cys Prx and atypical 2-Cys Prx. Typical 2-Cys Prx has been shown to be involved in oxidative signaling mechanisms that control cell apoptosis, differentiation and proliferation, and may be involved in immune responses, and can be used as biomarkers for tumor and cancer diagnosis. In addition, based on the antioxidant capacity of Prx, it can also be used as a food antioxidant to extend the shelf life of food. Therefore, typical 2-C...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0065C12Y111/01015
Inventor 侯艳华王全富王一帆王亚彤徐一凤
Owner HARBIN INST OF TECH AT WEIHAI
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