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Extracellular antiseptic protein for Bacillus subtilis and its separation and purification method

A Bacillus subtilis, separation and purification technology is applied in the field of extracellular antibacterial protein of Bacillus subtilis and its separation and purification, which can solve the problems such as unseen and in-depth research on extracellular antibacterial protein, achieve wide application prospects and protect crops. Natural quality, the effect of reducing pesticide residues

Inactive Publication Date: 2009-02-25
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the large-scale application of Bacillus subtilis Bs-916 to control rice sheath blight has achieved relatively ideal results, but there is no in-depth study on the extracellular antibacterial protein of Bacillus subtilis Bs-916, and there are no related literatures to report

Method used

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  • Extracellular antiseptic protein for Bacillus subtilis and its separation and purification method
  • Extracellular antiseptic protein for Bacillus subtilis and its separation and purification method
  • Extracellular antiseptic protein for Bacillus subtilis and its separation and purification method

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Experimental program
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Effect test

Embodiment 1

[0037] Transfer Bacillus subtilis Bs-916 into YPG culture solution (the YPG culture solution is tryptone 5g, glucose 5g, yeast extract 5g), shake culture at 120r / min at 28°C for 48h, centrifuge at 10000rpm at 4°C for 10min, filter out The thalline is vacuum filtered with a bacterial filter to obtain the sterile filtrate of Bacillus subtilis Bs-916, and then ammonium sulfate is added to the sterile filtrate of Bacillus subtilis Bs-916, the saturation of ammonium sulfate is 100%, 4 After overnight at ℃, centrifuge at 12000rpm for 25min, remove the supernatant, wash the precipitate with 0.067mol / L phosphate buffer, put it in a dialysis bag (molecular weight cut-off is 8-10KD), dialyze at 4℃ for 48h, and obtain the Substance, that is, antibacterial crude protein.

[0038] The crude extracellular protein prepared above was passed through a DEAE Sepharose Fast Flow (manufactured by Amersh-am) chromatographic column with a column volume of 25 ml. DEAE Sepharose Fast Flow column chro...

Embodiment 2

[0041] The antibacterial protein bacteriostatic spectrum was determined by the filter paper method. The pathogens were Rhizoctonia solani, Magnaporthe grisease, Sclerotinia sclerotiorum, Alternaria oleracea, A. brassicae , Botrytis cinerea. Cultivate the above-mentioned pathogenic bacteria on PDA medium (200g of potatoes, 20g of glucose, 1000ml of water). In the center of the PDA medium plate, after mycelium grows, a sterile filter paper (65 mm in diameter) is placed diagonally at a distance of 25 mm from the mycelium, and isolated antibacterial protein is added to one side, and 0.067M phosphate buffer is added to the other side as control. Each treatment was repeated 3 times. When the hyphae grew to the control place, the antibacterial activity of the antibacterial protein against different pathogenic bacteria was observed. Discovery of antimicrobial proteins against Basidiomycotina, such as Rhizoctonia solani (see Figure 5 ), to Ascomycota subphylum, such as rice blast...

Embodiment 3

[0048] Assay method for lectin activity: rabbit erythrocytes were obtained by centrifugation, and the rabbit erythrocytes were prepared into a 2% suspension with PBS buffer (pH 7.2). The antibacterial protein was double-diluted. At 20°C, take 50 μl of each concentration and 50 μl of 2% rabbit erythrocyte PBS suspension and mix them evenly in U-Plates. phosphate buffer as a control. After standing at 20°C for about 1 hour, wait until the control erythrocytes are all precipitated, and observe the agglutination status of the erythrocytes in the treatment. The definition of the hemagglutination titer: The hemagglutination titer still exhibits lectin activity at the highest dilution factor. This concentration is one lectin unit (one titer) of the protein. The lectin activity of the protein is determined by how many per mg Expressed in units of lectin activity. The above method was used to determine, and the result showed that the protein had lectin activity.

[0049] Determinati...

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Abstract

The present invention relates to one kind of extracellular antiseptic protein of Bacillus subtilis and its separation and purification process. The extracellular antiseptic protein has molecular weight of 41.9 kD, 26 mass-to-charge ratio peaks, and 5 peptide amino acid sequences including VTIVDEKGR, FSFSDVHNR, VYIADSTNFK, ELPISENLASVNMR and EAEWAYMITGK. Its separation and purification process includes the following steps: separating coarse protein from Bacillus subtilis Bs-916 via DEAE Sepharose F.F. separation, separating its B peak drips via Phenyl Sepharose 6 F.F. separation, separating the E peak drips via with hydroxyapatite column and obtaining the G peak drips as the purified extracellular antiseptic protein. The extracellular antiseptic protein has broad spectrum antiseptic activity, ribonuclease activity and agglutinin activity.

Description

technical field [0001] The invention relates to an extracellular antibacterial protein of Bacillus subtilis and a separation and purification method thereof. Background technique [0002] It is impossible for any kind of organism to exist in isolation in nature. All kinds of organisms can survive because of their own defense mechanisms. Plants, microorganisms, etc. can synthesize some small molecule antibiotic substances, such as phenols (Phenols), melanin (Melanins), tannins (Tannins) or plant lectins (Phytoalexins) and other substances. Secondly, antibacterial proteins can also be synthesized. Antibacterial proteins are biological macromolecules encoded by genes. Antibacterial proteins can be divided into inducible expression antibacterial proteins and constitutive expression antibacterial proteins. The role of antibacterial proteins is mainly to resist the invasion of pathogenic bacteria, improve the disease resistance of the organism itself, and maintain the continuatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/32C07K1/14C12N9/88C12N9/22C12R1/125
Inventor 刘永锋陈志谊
Owner JIANGSU ACAD OF AGRI SCI
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