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Bacillus subtilis and application of bacillus subtilis to production of antimicrobial peptide

A Bacillus subtilis and antibacterial peptide technology is applied to a Bacillus subtilis strain and its application field in the production of antibacterial peptides to achieve the effects of easy popularization, reduced production costs and high product purity

Active Publication Date: 2013-07-10
LINZHOU SINAGRI YINGTAI BIOLOGICAL PEPTIDES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no production method of sublancin antibacterial peptide that can realize industrial production at home and abroad

Method used

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  • Bacillus subtilis and application of bacillus subtilis to production of antimicrobial peptide
  • Bacillus subtilis and application of bacillus subtilis to production of antimicrobial peptide
  • Bacillus subtilis and application of bacillus subtilis to production of antimicrobial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, acquisition and preservation of Bacillus subtilis BF168

[0028] 1. Acquisition of Bacillus subtilis BF168

[0029] 1. The carrier pBluescriptII SK (+) is used as a backbone carrier, and the expression operon (double-stranded DNA molecule shown in sequence 2 of the sequence table) of sumo protease is inserted forward between its EcoRI and BamHI restriction sites. Between the BamHI and SacI restriction sites, the fusion protein expression operon of Saccharomyces cerevisiae micromolecule ubiquitin-like protein and sublancin antimicrobial peptide is forwardly inserted (the double-stranded DNA molecule shown in sequence 3 of the sequence table, wherein the sublancin antimicrobial peptide gene is as follows Sequence 1 of the sequence listing), the recombinant plasmid D1778-1 was obtained.

[0030] 2. Synthesize the double-stranded DNA molecule (Bacillus subtilis p43 promoter) shown in sequence 4 of the sequence listing, insert it into vector pGJ103, and obtain...

Embodiment 2

[0047] Embodiment 2, application Bacillus subtilis BF168 produces Sublancin antibacterial peptide or antibacterial substance

[0048] Seed medium: 30g cornmeal, 18g soybean meal, 12.5g peptone, 15g glucose, 3g KH 2 PO 4 and 1.25g of ammonium sulfate were dissolved in water and made up to 1L with water.

[0049] Fermentation medium: 30kg cornmeal, 18kg soybean meal, 20kg peptone, 3kg glucose, 3kg KH 2 PO 4 and 5kg of ammonium sulfate were dissolved in water and the volume was adjusted to 1 cubic meter with water.

[0050] 1. Activation of bacteria

[0051] The freeze-dried Bacillus subtilis BF168 was taken out from the -70°C refrigerator, inoculated on LB slant medium, and cultured at 37°C for 30 hours.

[0052] 2. Bacteria expansion and cultivation

[0053] 1. Take colonies from the slant in step 1 and inoculate them into LB liquid medium (each 250ml Erlenmeyer flask contains 30ml LB liquid medium), shake at 37°C and 220rpm for 30 hours.

[0054] 2. Inoculate the bacter...

Embodiment 3

[0092] Embodiment 3, application Bacillus subtilis BF168 produces Sublancin antimicrobial peptide

[0093] Seed medium: 35g cornmeal, 20g soybean meal, 15g peptone, 20g glucose, 5g KH 2 PO 4 and 1.5g of ammonium sulfate were dissolved in water and made up to 1L with water.

[0094] Fermentation medium: 35kg cornmeal, 20kg soybean meal, 25kg peptone, 5kg glucose, 5kg KH 2 PO 4 and 8kg of ammonium sulfate were dissolved in water and the volume was adjusted to 1 cubic meter with water.

[0095] 1. Activation of bacteria

[0096] Same as Step 1 of Example 2.

[0097] 2. Bacteria expansion and cultivation

[0098] Same as Step 2 of Example 2.

[0099] 3. Seed tank cultivation

[0100] Same as Step 3 of Example 2.

[0101] 4. Fermentation tank culture

[0102] Same as Step 4 of Example 2.

[0103] Sublancin antimicrobial peptide concentration is 80mg / L.

[0104] 5. Purification of Sublancin Antimicrobial Peptides from Fermentation Broth

[0105] 1. Press filtration of f...

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Abstract

The invention discloses bacillus subtilis and an application of the bacillus subtilis to production of antimicrobial peptide. The preservation number of the bacillus subtilis BF168 provided by the invention is CGMCC No. 7200. The invention further provides the application of the bacillus subtilis BF168 to the production of sublancin antimicrobial peptide or antibiotic substance. The application of the bacillus subtilis has the following specific advantages that (1) cereal products, such as corn and soybean processed goods, are taken as the main raw materials to replace the raw materials of chemical reagent grade in a laboratory, so that the production cost is reduced; (2) the industrial production of the sublancin antimicrobial peptide or antibiotic substance can be realized; (3) the production efficiency of the sublancin antimicrobial peptide or antibiotic substance can be improved, and the content of the sublancin antimicrobial peptide in a fermentation liquid can be up to 80-150mg / L; and (4) the yield is high, the cost is low, the purity of product is high, the requirements on operators during production are low, the process is simple, and the popularization is easy.

Description

technical field [0001] The invention relates to a bacillus subtilis and its application in producing antibacterial peptides. Background technique [0002] Sublancin antimicrobial peptide is an endogenous polypeptide with antibacterial activity produced by Bacillus subtilis, which is mainly synthesized through the ribosomal pathway, and is mainly post-transcriptionally modified and transported through the leader sequence. [0003] The molecular weight of Sublancin antimicrobial peptide is 3877U, it is colorless crystal and white crystalline powder, easily soluble in water. Sublancin antimicrobial peptides contain disulfide bonds and have good stability. [0004] Sublancin antimicrobial peptide has a strong inhibitory and killing effect on Gram-positive bacteria, and can be applied to food preservation, anti-infection and environmental disinfection. At present, there is no production method of sublancin antibacterial peptide that can realize industrial production at home and...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/00C12R1/125
Inventor 谯仕彦
Owner LINZHOU SINAGRI YINGTAI BIOLOGICAL PEPTIDES CO LTD
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