Bio-control bacillus subtilis surfactin separation and purification method

A technology of Bacillus subtilis and surfactin, applied in chemical instruments and methods, analytical materials, material analysis by electromagnetic means, etc., can solve the problems of decreased antifungal activity of mutant strains, loss of antifungal activity of mutant strains, reduction, etc. , to achieve the effect of broad antibacterial spectrum, good thermal stability, high research and application value

Inactive Publication Date: 2015-04-15
JIANGSU ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A Bacillus subtilis strain with excellent antagonistic properties can usually synthesize and secrete surfactin, iturin and ubiquitin at the same time; the loss of any one of the lipopeptide antibiotic synthesis ability will lead to different degrees of antifungal activity of the mutant strains decline; when two or three of the lipopeptide antibiotics are all missing, it will greatly reduce or lead to the complete loss of antifungal activity of the mutant strain

Method used

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  • Bio-control bacillus subtilis surfactin separation and purification method
  • Bio-control bacillus subtilis surfactin separation and purification method
  • Bio-control bacillus subtilis surfactin separation and purification method

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Embodiment 1

[0030] Example 1 Construction of Bacitracin L and Ubiquitin Synthetic Gene Mutants

[0031]The neomycin resistance gene fragment was excised from the plasmid vector pBEST501 by enzyme digestion, cloned into the pUC19 plasmid vector to construct the recombinant plasmid vector pUC19-neo; amplified from the genomic DNA of Bacillus subtilis Bs916 by polymerase chain reaction (PCR) The partial gene fragment IBacD synthesized by bacitracin L was cloned into the pUC19-neo plasmid vector after enzyme digestion and enzyme ligation to construct the single exchange knockout plasmid vector pUC19-neo-bac; the polymerase chain reaction (PCR) method was used to obtain the The partial fragment IFenA of the ubiquitin synthesis gene was amplified from the genomic DNA of Bacillus subtilis Bs916, and the amplified fragment was cloned into the pSG1164 plasmid vector after digestion and enzyme ligation to construct the single crossover knockout plasmid vector pSG1164-Fen; the constructed single cros...

Embodiment 2

[0032] The preparation of embodiment 2 surfactin crude extracts

[0033] The double mutant strain BBFM of biocontrol Bacillus subtilis Bs-916 is transferred in the LB nutrient solution (the LB nutrient solution is tryptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L, pH7 .0, cultured with shaking at 28°C and 180r / min for 72h. The fermentation broth was centrifuged at 4°C and 5000r / min for 30min to remove the bacteria, and the supernatant was adjusted to pH 2.0 with concentrated hydrochloric acid to precipitate overnight. Centrifuged at 8000r / min After 25 minutes, the precipitate was obtained, and the precipitate was extracted with methanol, and the extract was subjected to 0.22 μmol L -1 After the microporous membrane, the crude surfactin sample was prepared.

Embodiment 3

[0034] The purification of embodiment 3 surfactin

[0035] The crude extract was diluted with deionized water into a 30% methanol aqueous solution, the pH value was adjusted to 7.0, and then loaded on NH 2 The solid phase extraction column is eluted with 50% methanol aqueous solution, 100% methanol, 0.5% formic acid methanol solution, 1% formic acid methanol solution, 2% formic acid methanol solution gradient elution, and the 1% formic acid methanol elution solution contains the target compound.

[0036] Adjust the pH value of the methanol eluting solution containing 1% formic acid to 7.0, dry and concentrate with nitrogen, dilute with deionized water to 30% methanol aqueous solution, and then load the sample on a C18 solid phase extraction column; use 40% of 3 times the column volume , 50%, 60%, 70%, 90% methanol aqueous solution gradient elution, wherein the pure product of surfactin was obtained in the eluent containing 90% methanol water, the purity was 95%, a total of 10...

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Abstract

The invention relates to a bio-control bacillus subtilis Bs916 extracellular antibiotic lipopeptide surfactin and a separation and purification method thereof. The molecular weight of surfactin is 1007.6, 1021.7 and 1035.7, and belongs to homologues with difference of one methylene. The separation and purification method comprises the following steps: constructing a mutant strain BBFM without a performance of synthesizing other lipopeptide antibiotics of Bs916 through a gene knock-out technology; then extracting coarse product of surfactin from the fermentation broth of BBFM through steps of acid precipitation and methanol extraction, and finally separating and purifying surfactin through NH2 and C18 column chromatography. Through experiments of polyacrylamide gel electrophoresis and high performance liquid chromatography (HPLC) analysis, the experiment results show that the prepared surfactin reaches the electrophoresis purity grade and the chromatography purity grade. The purified surfactin has high a research and application value for development of antifungal and antibacterial drugs, and can also be used as a standard substance in biochemistry.

Description

Technical field: [0001] The invention relates to a method for separating and purifying the extracellular lipopeptide antibiotic surfactant of a strain of biocontrol Bacillus subtilis Bs916. Background technique [0002] Bacillus subtilis is a dominant biological population in plant and soil ecology, and has high resistance to stress and antibacterial and disease-preventing effects. Because Bacillus subtilis can produce rich antibacterial metabolites outside the cell, and because it has fast growth rate, long survival time, strong tolerance to acid, alkali, high temperature, ultraviolet rays and electromagnetic radiation and other adverse environments, and simple nutritional requirements, It is safe and non-toxic, so it is widely used in the prevention and treatment of plant fungal diseases and bacterial diseases. [0003] There are three main categories of antibacterial metabolites secreted by Bacillus subtilis, one is small molecule compounds of polypeptides synthesized th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C12P21/04G01N27/447
CPCC07K7/06
Inventor 罗楚平周华飞陈志谊王晓宇刘永锋刘邮洲张荣胜
Owner JIANGSU ACAD OF AGRI SCI
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