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Marine-derived bacillus antimicrobial protein MD and preparation method thereof

A kind of marine bacillus, antibacterial protein technology, applied in the field of antibacterial protein

Pending Publication Date: 2016-08-17
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the massive use of preservatives, antibiotics, etc. to inhibit bacteria in food has caused a huge negative impact on human health, so antibacterial substances from natural sources have attracted widespread attention

Method used

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  • Marine-derived bacillus antimicrobial protein MD and preparation method thereof
  • Marine-derived bacillus antimicrobial protein MD and preparation method thereof
  • Marine-derived bacillus antimicrobial protein MD and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Active adsorption component Q1 is chromatographically separated again using a quaternary ammonium salt (Capto Q) strong anion exchange chromatographic column

[0048] Balance solution A: 50mM MES, pH 6, eluent B: 50mM MES+1M NaCl, pH 6. First use equilibration buffer A to equilibrate the Capto Q anion-exchange chromatography column, load the adsorbed component Q1 concentrated to about 700 μL on the Capto Q anion-exchange chromatography column, and use two column volumes of equilibration buffer A to wash the chromatography column. Obtain the non-adsorbed components, and then wash 50mL by linear elution until the eluent B is 20%, and the adsorbed components 1 and 2 are eluted ( figure 1 ).

Embodiment 2

[0049] Embodiment 2: active component 2 runs small molecule protein glue

[0050] Each active component 2 that is separated runs small molecular protein electrophoresis, obtains component 2 as a single band in the electrophoresis staining map after silver nitrate staining, and shows that the molecular weight of this protein is about 4.6kDa ( figure 2 ), named as MD antimicrobial protein.

Embodiment 3

[0051] Embodiment 3: the inhibitory effect of isolated product MD antimicrobial protein to Staphylococcus aureus, Listeria monocytogenes

[0052] The specific steps of using the agar plate diffusion method to test the antibacterial activity are as follows: use a sterilized puncher (10 mm in diameter) to punch holes on the mixed plate of Staphylococcus aureus and Listeria, and pick out the cultured bacteria with a sterilized toothpick. Add 50 μL of separation component 2 (MD antibacterial protein) to each well of the base block, dry it in an ultra-clean bench, place it in a 37°C incubator for 12 hours, and observe that it is a zone of inhibition ( image 3 , Figure 4 ).

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Abstract

The invention provides a marine-derived bacillus antimicrobial protein MD and a preparation method thereof and relates to a marine-derived bacillus antimicrobial protein. Supernatant is taken after bacillus culture, ammonium sulfate is added to reach the saturation of 60%, centrifugation is performed, a precipitate is taken and is dissolved in water again, dialysis, lyophilization and re-dissolution are performed to obtain a crude protein solution, then separation and purification are performed on an ethyl amino ethyl anion exchange chromatographic column, components are collected, and a staphylococcus aureus is used as a tested indicator bacterium to track active components after centrifugal ultra-filtration and concentration; the concentrated active components are separated and purified on a quaternary ammonium salt strong anion exchange column, elution components are collected, ultra-filtration and concentration are performed, the bacteriostatic activity is checked, and the component purity is judged through SDS-PAGE electrophoresis after the active components are determined; single strips on an SDS-PAGE electrophoretogram are cut to perform liquid chromatography-mass spectrometry combined detection and identification, and a whole genome encoding protein sequence is used as a database to perform comparison so as to obtain the antimicrobial protein sequence MD.

Description

technical field [0001] The invention relates to an antibacterial protein derived from microorganisms, in particular to a marine-derived bacillus antibacterial protein MD and a preparation method thereof. Background technique [0002] At present, the world is facing serious food safety problems, especially food contamination bacteria and pathogenic bacteria have a great impact on food safety. Food contamination bacteria mainly include Escherichia coli, Salmonella, Clostridium botulinum, Listeria, Staphylococcus and so on. No matter in the field of food or medicine, the demand for antibacterial drugs is constantly increasing. However, the extensive use of preservatives, antibiotics, etc. to inhibit bacteria in food has caused a huge negative impact on human health, so antibacterial substances from natural sources have attracted widespread attention. Among them, the antibacterial protein not only has a wide source, has a unique mechanism of action, but also basically does not...

Claims

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Application Information

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IPC IPC(8): C12N1/20C07K14/32C07K1/36C07K1/30C07K1/18C12P21/02G01N30/02C12R1/07
CPCG01N30/02C07K14/32G01N2030/027C12R2001/07C12N1/205Y02A50/30
Inventor 陈新华吴雅萍郭文斌
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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