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Microcin C7 heptapeptide and prepared strain thereof

An adenosine heptapeptide and strain technology, applied in the directions of peptides, bacteria, biochemical equipment and methods, etc., can solve the problems of inability to genetically modify Lactobacillus johnsonii, inability to achieve large-scale industrialization, poor biological stability, etc. Low, achieve large-scale industrialized production, high stability

Active Publication Date: 2020-04-17
CHONGQING ACAD OF ANIMAL SCI
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AI Technical Summary

Problems solved by technology

The adenosine heptapeptide secreted by wild-type Lactobacillus johnsonii has poor tolerance to pepsin, trypsin and heat treatment, and the secretion amount is small, so the production of adenosine heptapeptide by wild-type Lactobacillus johnsonii is safe, but the biological stability is poor. And the production cost is high, and large-scale industrialization cannot be realized
[0005] The traditional method of screening target strains is heavy and time-consuming. Patent CN 109400685 A adopts the Quickchange method to transform adenosine heptapeptide. The host needs to be Escherichia coli, and only strains resistant to trypsin can be screened, and it cannot be used for John's milk. Bacteria genes for rapid modification

Method used

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  • Microcin C7 heptapeptide and prepared strain thereof
  • Microcin C7 heptapeptide and prepared strain thereof
  • Microcin C7 heptapeptide and prepared strain thereof

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Experimental program
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Embodiment Construction

[0036] specific implementation plan

[0037] 1. Establish a strain library

[0038] The wild-type Lactobacillus johnsonii was cultured in MRS broth medium for 10 hours to reach the middle stage of logarithmic growth, irradiated with ultraviolet light for 120s, and then treated with 4mg / L nitrosoguanidine (NTG) for 30min for combined mutagenesis to construct the strain library.

[0039] 2. High-throughput screening: the screening process is as follows: figure 1 shown.

[0040] 1. Obtain the culture supernatant of a single bacterial strain: first spread the culture of the bacterial strain library on MRS agar, so that about 100 colonies grow on each plate, pick a single colony with a sterilized toothpick and inoculate it in a 96-well plate (each well contains 100 μL The culture medium, whose medium composition is MRS without Tween and diammonium hydrogen citrate) or a 48-well plate or a 24-well plate, after culturing for 24 hours, centrifuged, and sucked 100ul supernatant to a...

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Abstract

The invention relates to an microcin C7 heptapeptide with an amino acid sequence of MATINAN. The heptapeptide has stronger biological characteristics of heat resistance, pepsin resistance, trypsin resistance and chymotrypsin resistance, high stability and strong bacteriostatic activity. The invention further provides a high-throughput screening method for obtaining a heat-resistant and protease-resistant strain. A lactobacillus johnsonii strain capable of producing the heptapeptide is screened out; the lactobacillus johnsonii strain is deposited in China General Microbiological Culture Collection Center on October 16th, 2019, and has the accession number CGMCC No.18695. The lactobacillus johnsonii strain has advantages of good stress resistance and high secretion amount and is low in cost.An important foundation is laid for industrial large-scale preparation of microcin C7 heptapeptide in the fields of animal husbandry feed, cosmetics, health care products and the like.

Description

technical field [0001] The invention relates to an adenosine heptapeptide and its producing strain, belonging to the field of biotechnology. [0002] technical background [0003] Lactobacillus johnsonii is a safe and harmless probiotic, which can be used to ferment feed, food, and as a probiotic to regulate intestinal health and improve animal or human immunity. Antimicrobial peptides are small molecular polypeptides produced by organisms that are related to host defense and innate immunity, generally consisting of 5-50 amino acids. Antimicrobial peptides have the functions of inhibiting and killing pathogens, promoting immunity, promoting wound healing and mucosal defense. [0004] Adenosine heptapeptide (also known as bacitracin heptapeptide, microcin C7 peptide) is a bacteriocin antibacterial peptide secreted by Enterobacter, Lactobacillus and other microorganisms. The combination of phosphoramide bond and 5'-adenosine phosphate (AMP) forms a small molecular weight anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N1/20C12N1/02C12P21/02C12R1/225
CPCC07K7/06C12N1/20C12N1/02C12R2001/225C12N1/205
Inventor 周晓容杨飞云肖融刘作华
Owner CHONGQING ACAD OF ANIMAL SCI
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