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Antibacterial lipopeptide of endophytic Bacillus subtilis and separation and purification method

A technology of Bacillus subtilis and antibacterial lipopeptide, applied in the field of extracellular antibacterial lipopeptide and its separation and purification

Inactive Publication Date: 2010-06-09
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the extracellular antimicrobial lipopeptide produced by Bacillus subtilis Jaas ed1 has not been studied in depth, and there are no related reports

Method used

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  • Antibacterial lipopeptide of endophytic Bacillus subtilis and separation and purification method
  • Antibacterial lipopeptide of endophytic Bacillus subtilis and separation and purification method
  • Antibacterial lipopeptide of endophytic Bacillus subtilis and separation and purification method

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Embodiment 1

[0020] The acquisition of embodiment 1 antimicrobial lipopeptide crude extract

[0021] The endophytic Bacillus subtilis Jaas ed1 was transferred to the NB culture solution (the NB culture solution is beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, pH 6.8-7.0), in Shake culture at 30°C and 120r / min for 48h, 4°C, 10000r / min, centrifuge for 10min, remove the bacteria, collect the supernatant, then add ammonium sulfate solids in batches while stirring with a magnetic bar, statically at 4°C After standing overnight, centrifuge at 10000r / min for 18min, and dissolve the precipitate obtained in 0.05mol / L phosphate buffer (pH 7.0). According to the above method, carry out ammonium sulfate segmental salting-out on the supernatant, and collect the precipitates with ammonium sulfate saturation at 0-30%, 30-40%, 40-50%, 50-60%, and 60-70% respectively , dissolved in 0.05mol / L phosphate buffer (pH 7.0). The antibacterial activity of the above ammonium sulfate precipita...

Embodiment 2

[0022] The separation and purification of embodiment 2 antimicrobial lipopeptides

[0023]The antimicrobial lipopeptide crude extract is loaded on the Sephedex G-25 molecular sieve chromatography column, eluted with 0.05mol / L phosphate buffer (pH7.0), the flow rate is 1ml / min, and detected at OD 280nm of ultraviolet light, there is I , II two absorption peaks, collect each peak component, and detect the bacteriostasis activity to Verticillium dahliae of eggplant, wherein peak I has bacteriostasis activity. Concentrate the peak I collection solution obtained by molecular sieve chromatography, load the sample on Cellulose DEAE-52 anion exchange column, and use 0.05mol / L containing 0mol / L, 0.1mol / L, 0.4mol / L, 1.0mol / L NaCl respectively Gradient elution with L phosphate buffer (pH 7.0) at a flow rate of 1ml / min, detected at UV light OD 280nm, and eluted in 0.1mol / L, 0.4mol / L, 1.0mol / L NaCl phosphate buffer respectively Absorption peaks appeared at each concentration, and the peak...

Embodiment 3

[0024] Embodiment 3 Utilizes biological self-imaging technique to search for antimicrobial lipopeptide target band

[0025] The antibacterial activity of the antibacterial activity peak substance collection solution after the antibacterial lipopeptide crude extract was chromatographed by Sephedex G-25 molecular sieve chromatography column and Cellulose DEAE-52 anion exchange column was detected in situ by Tricine-SDS-PAGE gel, That is to use biological autoradiography to find the target band of antibacterial lipopeptide. In Tricine-SDS-PAGE, the separation gel concentration is 16.5%, the interlayer gel concentration is 10%, and the stacking gel concentration is 4%. The antibacterial lipopeptide crude extract is subjected to Sephedex G-25 molecular sieve chromatography column and CelluloseDEAE-52 anion exchange column After the bacteriostatic activity peak substance collection solution after chromatography was concentrated, the sample was loaded in parallel on two sample wells ...

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Abstract

The invention relates to an extracellular antibacterial lipopeptide of plant endophytic Bacillus subtilis Jaasedl and a separation and purification method. The distribution range of the molecular weight of the extracellular antibacterial lipopeptide is mainly between 1,000Da and 2,200Da. The extracellular antibacterial lipopeptide contains an Iturin homologue, a Fengycin homologue and a Surfactin-like Compound homologue. The extracellular antibacterial lipopeptide is a group of uncommon antibacterial lipopeptide mixture produced by a single bacterial strain. The separation and purification method of the extracellular antibacterial lipopeptide comprises the following steps of: after salting out to obtain the crude extract of the antibacterial lipopeptide by using ammonium sulfate from the fermentation liquor of the endophytic Bacillus subtilis Jaasedl, performing Sephedex G-25 molecular sieve chromatography, Cellulose DEAE-52 anion exchange chromatography and FPLC300SB-C18 column chromatography successively, wherein 34 to 37 min of collecting peak has bacteriostatic activity; detecting by Tricine-SDS-PAGE after concentrating; and achieving electrophoretically pure at only one strip to obtain pure extracellular antibacterial lipopeptide. The antibacterial lipopeptide has extremely high research and application value for the development of broad-spectrum antifungal medicaments, and has wide application prospect for natural quality protection of crops and pesticide residue reduction.

Description

Technical field: [0001] The invention relates to an extracellular antibacterial lipopeptide of plant endogenous Bacillus subtilis Jaas ed1 and a separation and purification method thereof. Background technique: [0002] Because endophytes live in the microenvironment of plants for a long time and co-evolve with host plants, they form a mutualistic relationship during the evolution process. Endophytes can not only participate in the synthesis of plant secondary components, or transform plant secondary metabolites, but also independently produce abundant secondary metabolites, which are important sources of natural products. Some endophytic bacteria can produce some antibacterial substances in plants, which can antagonize pathogenic bacteria. The antagonistic substances produced by Bacillus, except for a few exceptions, belong to a single group, that is, polypeptides, which mainly inhibit Gram-positive bacteria; some bacteria can inhibit Gram-negative bacteria and yeast; some...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K4/04C12P21/02C07K1/36C07K1/30C07K1/18C07K1/20C07K1/16A01P3/00C12R1/125
Inventor 林玲孙义乔勇升张昕周益军
Owner JIANGSU ACAD OF AGRI SCI
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