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Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli

An arginine deiminase, recombinant Escherichia coli technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of poor temperature tolerance (transformation temperature can only be controlled around 37 ℃, unfavorable separation and purification, wet The problem of high amount of bacterial cells can shorten the transformation time, shorten the production cycle and reduce the production cost.

Inactive Publication Date: 2015-06-24
WUHAN GRAND HOYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is following shortcoming in this invention: the enzyme activity of this escherichia coli genetically engineered bacterium is relatively low, causes the wet thalline consumption needed when converting arginine to be higher (need to add 1% wet thalline), and the conversion time is longer (conversion 10% arginine substrate needs 4h); the temperature resistance of the enzyme is poor (the conversion temperature can only be controlled at about 37°C); the conversion needs to be carried out under the acetate buffer system, which is not conducive to the later separation and purification

Method used

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  • Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli
  • Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli
  • Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli

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Experimental program
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Embodiment 1

[0037] The acquisition of embodiment 1 bacillus cereus mutant strain

[0038] 1. Preparation of Type-Based Strains

[0039] Take a strain of Bacillus cereus obtained from the soil as the starting strain, insert it into a medium containing 100ml, cultivate it at 30-37°C, 100-200r / min for 5-30h, then take 1ml of the bacterial solution and add it to 9ml of sterile water, mix well, dilute the concentration to 10 times, continue to dilute 10 times in this way -2 , 10 -3 and 10 -4 times, and the bacterial solution is set aside.

[0040] The composition of the culture medium is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium chloride, the balance is deionized water, and the pH is 6.0-7.5.

[0041] 2. N ion beam mutagenesis

[0042] Take 0.5ml of the bacterial solution prepared in step 1, apply it to a sterile petri dish and dry it, and use a TITAN ion beam implanter to perform N ion beam implantation on the bacteria with an energy of 30keV and an injection dose of 1-5×10 ...

Embodiment 2

[0052] Example 2 Genome Extraction, Target Fragment Amplification and Genetic Engineering Bacteria Construction

[0053] 1. Reagents and materials: Escherichia coli DH5α, BL21, pMD19-T simple vector, and pET28a vectors are all preserved by our laboratory.

[0054] Experimental reagents: DNA polymerase (KOD DNA Polymerase) was purchased from TOYOBO; restriction enzymes (EcoRI and NotI), T4DNA Ligase, dNTPs, DNA marker DL2,000, plasmid mini-extraction kit, and DNA purification kit were purchased from From Takara Corporation. Bacterial genome extraction kits were purchased from Tiangen Company, ampicillin, kanamycin sulfate, and IPTG were purchased from Biosharp Company, and other chemical reagents were of analytical grade.

[0055] 2. Extraction of Bacillus cereus genomic DNA

[0056] Select a single colony of Bacillus cereus 93-11 with the preservation number CCTCC NO: M 2015047 and inoculate it into 1-4ml of LB liquid medium for overnight culture; take 1-4ml of the overnight...

Embodiment 3

[0069] The fermentation culture of embodiment 3 recombinant bacteria

[0070] 1. Seed culture:

[0071] Put the engineered bacteria preserved in glycerin into a 250ml eggplant bottle LB solid slope and cultivate for 16-24h, then wash it down with sterile water, and take about 25ml and put it into the seed medium.

[0072] Seed medium: corn steep liquor 2-10%, monosodium glutamate 1-10%, MgSO 4 0.01-0.1%, KH 2 PO 4 0.01-0.1%, pH 4.0-7.5, prepared with deionized water;

[0073] Seed culture conditions: the strain is cultured in the seed medium for 5-30 hours at 30-37°C and a shaking speed of 100-300rpm to obtain seed bacteria;

[0074] 2. Fermentation culture

[0075] Fermentation medium: corn steep liquor 3-15%, monosodium glutamate 2-12%, MgSO 4 0.01-0.1%, KH 2 PO 4 0.01-0.1%, pH 4.0-7.5, prepared with deionized water;

[0076] Fermentation conditions: 1-10% inoculum size, 30-37°C, 150-450rpm rotation speed, culture in the fermentation medium for 0.5-4h, add lactose w...

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Abstract

The invention discloses recombinant Escherichia coli for producing arginine deiminase at high yield. The recombinant Escherichia coli is collected with the serial number of CCTCC (China Center for Type Culture Collection) NO:M2015048 in CCTCC; an arginine deiminase gene is from Bacillus cereus which is bred after being subjected to induced mutation of N<+> ion beams; and then, a mutant strain for producing arginine deiminase at high yield is obtained through screening, wherein the collection number of the mutant strain is CCTCC NO:M2015047. The invention also discloses application of the recombinant Escherichia coli to production of L-citrulline and a method for producing L-citrulline. By using the recombinant Escherichia coli disclosed by the invention, more than 99% of arginine can be converted into L-citrulline, and the purity of a product can be up to more than 99%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an arginine deiminase gene and a recombinant Escherichia coli expressing the gene, and also relates to the application of the recombinant Escherichia coli in the production of L-citrulline and a production method The method of L-citrulline. Background technique [0002] L-citrulline has multiple functions such as protecting the liver, relaxing blood vessels, treating sexual dysfunction, and improving brain power. At present, the production methods of L-citrulline mainly include plant extraction method, chemical synthesis method, fermentation method and enzyme conversion method. Among them, the plant extraction method has low content and high extraction cost, which is not suitable for large-scale production; the chemical organic synthesis method pollutes the environment and may leave toxic substances, and there are potential safety risks; the concentration of the product ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N1/21C12P13/10C12R1/19
Inventor 邢盼盼王君英曹新华苏海霞王炯宋盟军梅雪臣
Owner WUHAN GRAND HOYO
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