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Bifunctional glutathione synthetase expression cassette derived from lactobacillus as well as construction and application thereof

A glutathione and lactobacillus technology, applied in the direction of enzymes, bacteria, ligases, etc., can solve the problems of low yield and complex glutathione synthesis methods, and achieves improved synthesis, improved GSH synthesis ability, and improved ability Effect

Inactive Publication Date: 2018-08-28
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a bifunctional glutathione synthetase expression cassette derived from Lactobacillus and its construction and application. The bifunctional glutathione synthase derived from Lactobacillus The peptide synthetase expression cassette and its construction and application need to solve the technical problems of complex glutathione synthesis methods and low yields in the prior art

Method used

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  • Bifunctional glutathione synthetase expression cassette derived from lactobacillus as well as construction and application thereof
  • Bifunctional glutathione synthetase expression cassette derived from lactobacillus as well as construction and application thereof
  • Bifunctional glutathione synthetase expression cassette derived from lactobacillus as well as construction and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning of the gshF gene in Lactobacillus

[0029] Using the Lactobacillus plantarum WCFS1 genome (NCBI accession number: NC_004567.2, https: / / www.ncbi.nlm.nih.gov / nuccore / NC_004567.2) as a template, use gshF-Lp-F / gshF-Lp-R This pair of primers amplifies the gshF gene in Lactobacillus plantarum and is named gshF-lp (SEQ ID NO.6).

[0030] Using the Lactobacillus casei LC2W genome (NCBI accession number: NC_017473.1, https: / / www.ncbi.nlm.nih.gov / nuccore / NC_017473.1) as a template, use gshF-Lc-F / gshF-Lc-R This pair of primers amplifies the gshF gene in Lactobacillus casei and is named gshF-lc (SEQ ID NO.5).

[0031] From figure 1 and figure 2 It can be seen that the gshF-lp fragment amplified with the gshF-Lp-F / gshF-Lp-R primer and the gshF-lc fragment amplified with the gshF-Lc-F / gshF-Lc-R primer are respectively at about 2300bp A clear band can be seen at 200bp and 200bp, which is consistent with the expected result.

Embodiment 2

[0032] Embodiment 2: the construction of lactobacillus gshF expression cassette and its vector

[0033] The PCR products of the above two Lactobacillus gshF genes were recovered using a gel recovery kit or a PCR clean recovery kit, and then double-digested with restriction endonucleases NdeI and XhoI, and then used a PCR clean recovery kit The digestion product of the lactobacillus gshF gene is recovered. The pET series expression vectors (such as pET24a) were also double-digested with restriction endonucleases NdeI and XhoI, and the digested products of the vector were recovered with a PCR cleaning and recovery kit.

[0034] The gshF gene digestion product of Lactobacillus and the vector digestion product were ligated by T4 DNA ligase, and transformed into Escherichia coli Top10, and cultured at 37° C. for 16 hours. Take a single colony on the plate for liquid culture, use the bacterial liquid as a template, and use primers gshF-Lc-F / gshF-Lc-R or gshF-Lc-F / gshF-Lc-R for PCR ...

Embodiment 3

[0036] Embodiment 3: Lactobacillus gshF expression cassette plasmid induces expression in Escherichia coli

[0037] Add the Lactobacillus gshF expression cassette plasmid and the pET24a expression vector (control) to Escherichia coli BL21(DE3) competent cells, mix gently and place on ice for 30 minutes, then quickly transfer them to a water bath at 42°C for 90 seconds. Perform heat shock treatment, then quickly transfer to ice, and keep for 2-5min; then add 0.9mL LB liquid medium (yeast extract 5.0g / L, tryptone 10.0g / L, sodium chloride 10.0g / L), After culturing at a low speed of 150 r / min on a shaker at 37°C for 1 h, spread it on an LB solid medium plate containing 50 mg / L kanamycin, and culture overnight at 37°C. Single colonies were cultured overnight, inoculated into LB solid medium containing 50 mg / L kanamycin at 1% (v / v), cultured at 37°C until OD600=0.6, and induced by adding 0.1-1mM IPTG for 6 hours. Centrifuge the fermentation broth at 10,000rpm at 4°C for 10min, disc...

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Abstract

The invention provides a bifunctional glutathione synthetase expression cassette derived from lactobacillus. The bifunctional glutathione synthetase expression cassette includes cloning a gshF gene derived from the lactobacillus. The invention further provides a preparation method of the bifunctional glutathione synthetase expression cassette derived from the lactobacillus. The preparation methodcomprises the following steps: designing a primer for amplifying the gshF gene by taking a lactobacillus genome as a template; performing enzyme digestion with Ndel and Xhol; connecting to a polyclonal site of an escherichia coli pET expression vector through DNA ligase in order to construct the expression cassette expressing the lactobacillus gshF. The invention further provides a lactobacillus-containing gshF expression cassette escherichia coli genetically engineered bacterium. The invention further provides application of the lactobacillus-containing gshF expression cassette escherichia coli genetically engineered bacterium to preparation of glutathione. After the bifunctional glutathione synthetase expression cassette is applied to GSH synthesis, the GSH yield is up to 103.1mu m, theescherichia coli GSH yield is increased by 11.5 times or more, and the GSH synthesis ability of the escherichia coli is significantly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene expression cassette, in particular to a lactobacillus-derived bifunctional glutathione synthetase expression cassette and its construction and application. Background technique [0002] Glutathione (γ-L-glutamy-L-cysteiny-glycine, GSH) is composed of glutamic acid, cysteine ​​and glycine, and contains γ-amide bonds and sulfhydryl groups. As an active tripeptide, GSH exists widely in most animals, plants and microorganisms. It is the most important non-protein sulfhydryl compound in organisms. It has unique anti-oxidation and anti-aging properties and can be used as food. Additives, health care products and drugs, the market demand for GSH has been increasing year by year in recent years. [0003] In eukaryotes and most prokaryotes, glutathione (GSH) is synthesized by a two-step enzyme-catalyzed reaction involving γ-glutamylcysteine ​​synthetase (γ-GCS) and Glutathione synthase...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/70C12N1/21C12P21/02
CPCC12N9/93C12N15/70C12P21/02C12Y603/02003
Inventor 艾连中熊智强王光强夏永军张汇
Owner UNIV OF SHANGHAI FOR SCI & TECH
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