Method for separating antibacterial proteins from bacillus pumilus E14

A technology of Bacillus pumilus and E14, which is applied to the preparation method of peptides, methods based on microorganisms, chemical instruments and methods, etc., can solve the problems of large losses and high frequency of disease occurrence, and achieve the effect of convenient operation and simple and reasonable design

Active Publication Date: 2014-04-16
HUAIHAI INST OF TECH
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, with the continuous development of the aquaculture industry and the continuous expansion of the breeding scale, the frequency of disease occurrence is getting higher and higher, and the losses caused are also increasing.

Method used

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  • Method for separating antibacterial proteins from bacillus pumilus E14
  • Method for separating antibacterial proteins from bacillus pumilus E14
  • Method for separating antibacterial proteins from bacillus pumilus E14

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1, a kind of method for isolating antibacterial protein from Bacillus pumilus E14, its steps are as follows:

[0031] (1) Fermentation production: Bacillus pumilus E14 ( Bacillus pumilus ) Inoculate the Bacillus pumilus E14 seed liquid into 2216E liquid medium, the inoculum amount is 5%, 180rpm, 28°C for 24h; obtain the fermentation liquid;

[0032] (2) Preparation of supernatant: ferment the bacteria liquid at 1200rpm, centrifuge at 4°C for 30min, centrifuge the supernatant again, repeat 2-3 times; obtain the supernatant;

[0033] (3) Ammonium sulfate precipitation: Dry and grind the ammonium sulfate, weigh different aliquots respectively according to the preparation table of saturated ammonium sulfate solution, add to the supernatant while stirring on ice, and obtain ammonium sulfate with a mass volume percentage of 40% solution, then stand on ice for 20 min, centrifuge at 4°C, 8000rpm for 5 min; take the supernatant, continue to add ammonium sulfate while...

Embodiment 2

[0047] Embodiment 2, with reference to figure 1 -5. Experiment on the method of isolating antibacterial proteins from Bacillus pumilus E14:

[0048] 1. Experimental method

[0049] 1.1 Detection of Bacillus pumilus E14 antagonistic activity

[0050] Bacillus pumilus E14 is preserved in CGMCC, General Microorganism Center of China Committee for Culture Collection of Microorganisms, with preservation number CGMCC No.6682.

[0051] In this study, the zone of inhibition method of filter paper was used. Inoculate Bacillus pumilus E14 in 2216E liquid medium, culture at 28°C for 20 h, draw 0.1 mL of culture solution and spread it evenly on the 2216E solid medium plate. Place the sterile filter paper in a petri dish, then pipette 50 μL of the antagonistic bacteria to be tested dropwise onto the filter paper, incubate at 28°C, observe the inhibition zone and measure the size of the inhibition zone after 16 hours.

[0052] 1.2 Determination of the location of the active substance o...

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Abstract

The invention discloses a method for separating antibacterial proteins from bacillus pumilus E14. The method comprises the following steps: preparing a zymophyte solution through fermentation production, and centrifuging to prepare supernatant; precipitating by using ammonium sulfate, resuspending the obtained precipitate by using a buffer solution to obtain resuspension; separating through an ion exchange method, collecting an elution peak, concentrating, dialyzing, and obtaining an active ingredient separation peak; and measuring the molecular weight of the antibacterial proteins separated from the bacillus pumilus E14 to be between 97.2 and 116.0kD through antibacterial activity detection and protein electrophoresis detection. The method is simple and reasonable in design and convenient to operate, and proteins with an effect of resisting Vibrio harveyi ((i) Vibrio( / i) (i) harveyi( / i) can be separated according to the method.

Description

technical field [0001] The invention relates to a method for preparing antibacterial protein, in particular to a method for isolating antibacterial protein from bacillus pumilus E14. Background technique [0002] In the past 30 years, my country's fishery has made great progress. The output of aquatic products has ranked first in the world for more than ten consecutive years. It is the only country in the world where the output of aquaculture exceeds that of fishing. The export of aquatic products has also accounted for more than 50% of the net income of agricultural product exports. . Fishery has become one of the fastest-growing industries in my country's large-scale agriculture. It has made important contributions to promoting the adjustment of rural industrial structure, increasing farmers' income, ensuring food safety, optimizing the national dietary structure, and safeguarding national marine rights and interests. However, with the continuous development of the aquacu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K14/32C07K1/36C07K1/30C07K1/18A61P31/04C12R1/07
Inventor 安贤惠李联泰阎斌伦朱明
Owner HUAIHAI INST OF TECH
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