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 negat

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

Example Embodiment

[0047] Example 1: The active adsorption component Q1 was chromatographed again using a quaternary ammonium salt (Capto Q) strong anion exchange column

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

Example Embodiment

[0049] Example 2: Active component 2 runs small molecule protein glue

[0050] The separated active components 2 were run on small molecule protein electrophoresis. After silver nitrate staining, the electrophoretic staining pattern obtained component 2 as a single band, and the molecular weight of the protein was about 4.6kDa ( figure 2 ), named as MD antibacterial protein.

Example Embodiment

[0051] Example 3: The inhibitory effect of the isolated product MD antibacterial protein on Staphylococcus aureus and Listeria monocytogenes

[0052] The specific steps of using agar plate diffusion method to test the antibacterial activity are as follows: Use a sterilized puncher (10mm in diameter) to punch holes on the mixed plate of Staphylococcus aureus and Listeria, and use a sterilized toothpick to pick out and culture Base block, add 50μL of separation component 2 (MD antibacterial protein) to each well, blow-dry it in an ultraclean table, and place it in a 37℃ 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
CPCC07K14/32C12N1/205C12R2001/07G01N30/02G01N2030/027
Inventor 陈新华吴雅萍郭文斌
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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