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CN-122011139-A - Microbacterium MccY mutant resisting thermolysin and application thereof

CN122011139ACN 122011139 ACN122011139 ACN 122011139ACN-122011139-A

Abstract

The application belongs to the technical field of biological detection, and discloses a microzyme MccY mutant of antipyromonas protease and application thereof, wherein the microzyme MccY mutant of antipyromonas protease is obtained by mutating the 11 th amino acid of MccY from S to H, N or G and/or mutating the 16 th amino acid of MccY from Q to A, and the MccY mutant provided by the application can maintain structural and functional integrity for a long time under severe protease challenges, and solves the core problem of insufficient stability of natural peptide.

Inventors

  • SU BO
  • LIU YUTONG
  • SHAO WENLIN
  • FENG SAIXIANG
  • HUANG YIFENG
  • ZHENG HAN
  • LI JUNJIE

Assignees

  • 华南农业大学

Dates

Publication Date
20260512
Application Date
20260108

Claims (7)

  1. 1. A mutant of a thermolysin resistant micro-fungus MccY, characterized in that amino acid 11 of MccY is mutated from S to H, N or G; and/or mutation of amino acid MccY at position 16 from Q to A.
  2. 2. The mutant of claim 1, wherein the amino acid at position 11 of MccY is mutated from S to N.
  3. 3. The mutant of claim 1, wherein the amino acid at position 16 of MccY is mutated from Q to a.
  4. 4. The use of a mutant of a thermolysin MccY according to any one of claims 1 to 3 for the preparation of a thermolysin resistant to degradation by thermolysin.
  5. 5. A plasmid carrying the gene of the thermolysin MccY mutant of any one of claims 1-3.
  6. 6. A genetically engineered bacterium is characterized in that, a gene capable of expressing the thermolysin resistant mutant of claim 1-3.
  7. 7. A method for constructing the genetically engineered bacterium of claim 6, characterized in that the plasmid of claim 5 is transformed into competent cells to obtain the genetically engineered bacterium.

Description

Microbacterium MccY mutant resisting thermolysin and application thereof Technical Field The application relates to the technical field of biology, in particular to a mutant of a colicin MccY resistant to thermolysin and application thereof. Background With the vigorous development of the global intensive farming industry, food-borne gastrointestinal infections caused by Enterobacteriaceae (Enterobacteriaceae) pathogens such as E.coli (ESCHERICHIA COLI) and Salmonella (Salmonella) have become a significant challenge in restricting the development of the livestock industry and threatening public health safety. The long term abuse of antibiotics with sub-therapeutic dose use directly leads to the emergence of multi-drug resistant (MDR) or even broadly resistant (XDR) strains, leading to an increasing decay in the clinical efficacy of traditional antibiotics. Therefore, it has become urgent to develop antibiotic substitutes which have a novel mechanism of action and are not easy to induce drug resistance. Lasso peptides (Lasso peptides) are a class of natural antimicrobial peptides synthesized from ribosomes and subjected to complex post-translational modifications, whose unique "Lasso" like topology (consisting of an N-terminal ring and a C-terminal tail through the ring stabilized by disulfide bonds) confers excellent physicochemical stability, including significant resistance to thermal, acid and proteolytic hydrolysis. The microelement mycin MccY (Microcin Y) is used as I-type lasso peptide coded by escherichia coli AY25 plasmid, shows nanomolar (nM) -level powerful antibacterial activity on various enterobacteriaceae pathogenic bacteria, and has huge application potential. However, natural MccY, like many polypeptide drugs, is susceptible to degradation by proteases in complex application environments, which severely limits its practical use. Thermophilic protease (Thermolysin) is a zinc ion-dependent metalloprotease derived from bacillus thermophilus (Bacillus thermoproteolyticus), whose potent hydrolytic activity is a common challenge model for assessing polypeptide stability. The scheme needs to solve the problem of how to provide the micromycin resistant to the decomposition of thermophilic protease. Disclosure of Invention The application aims to provide a microzyme which is resistant to thermoprotease decomposition, and MccY mutant with good resistance to thermoprotease is obtained by carrying out single mutation and double mutation tests on a plurality of sites in MccY genes and screening. In order to achieve the aim, the application discloses a mutant of the colicin MccY resisting thermolysin, which is characterized in that the 11 th amino acid of MccY is mutated from S to H, N or G; And/or mutating amino acid number 16 of MccY from Q to A. Preferably, amino acid number 11 of MccY is mutated from S to N. Preferably, amino acid number 16 of MccY is mutated from Q to a. In addition, the application also discloses the effect of the mutant of the microzyme MccY of the thermophilic bacteria protease to prepare the microzyme of the thermophilic bacteria protease degradation resistance. In addition, the application also discloses a plasmid carrying the gene of the colistin MccY mutant of the antipyromonas protease. In addition, the application also discloses a genetic engineering bacterium which can express the gene of the colicin MccY mutant of the antipyrosis protease. In addition, the application also discloses a construction method for constructing the genetic engineering bacteria, and the plasmid is transformed into competent cells to obtain the genetic engineering bacteria. The beneficial effects of the application are as follows: The application firstly relates to a high-stability variant represented by MccY-S11N, which can maintain structural and functional integrity for a long time under the severe protease challenge, solves the core problem of insufficient stability of natural peptide, and secondly relates to a remarkable MccY-Q16A variant, the antibacterial efficacy of which is obviously superior to that of natural MccY, and the optimization and exceeding of functions are realized. This finding suggests that our engineering strategy not only avoids the drawbacks, but also actively enhances its intrinsic bioactivity. Drawings FIG. 1 is a plasmid map of MccY engineering strains; FIG. 2 is a schematic representation of the zone of inhibition of MccY prior to incubation with a thermophilic protease; FIG. 3 is a schematic representation of the zone of inhibition of MccY following incubation with a thermophilic protease; FIG. 4 is a diagram of liquid-phase secondary mass spectrometry of MccY before cleavage; FIG. 5 is a diagram of liquid-phase secondary mass spectrometry analysis of MccY after digestion; FIG. 6 is a chart of a test experiment of the inhibition zone of 21 mutants against Salmonella typhimurium; FIG. 7 is a schematic diagram of the time gradient bacteriostasis of natural MccY at diffe