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CN-122012465-A - Bacteriophage-derived antibacterial peptide lysozyme protein mutant of staphylococcus aureus with high thermal stability as well as preparation method and application thereof

CN122012465ACN 122012465 ACN122012465 ACN 122012465ACN-122012465-A

Abstract

The invention relates to the technical field of biology, and particularly discloses a bacteriophage-derived staphylococcus aureus antibacterial peptide lysozyme protein mutant with high thermal stability, and a preparation method and application thereof. The mutant carries out stability design on wild type staphylococcus aureus antibacterial peptide lysozyme protein (antimicrobial peptidase lysostaphin, APL) through PROSS on-line prediction tools to obtain 9 mutation schemes (design 1-design 9), wherein the thermal stability of the mutant APL (design 9) is obviously improved, the optimal temperature is improved to 55 ℃, and half lives of the mutant APL (design 9) at 70 ℃ and 80 ℃ respectively reach 57+/-4 min and 21+/-1 min which are 1.4 times and 2.1 times that of the wild type. The preparation method comprises the steps of gene synthesis, vector construction, recombinant expression and purification. The mutant can be used for preventing and treating staphylococcus aureus infection, antibiotic substitutes for animals, food preservatives and compound disinfectants, is suitable for high-temperature processing environments, and has industrial advantages.

Inventors

  • WEN JUNFAN
  • CHEN WEN
  • LIU TANG
  • DONG XIEPING

Assignees

  • 中山自然说生物科技有限公司
  • 小白鲸(中山)生物医药有限公司

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. A bacteriophage-derived antibacterial peptide lysozyme mutant of staphylococcus aureus with high heat stability is characterized in that the mutant is obtained by designing wild type APL through PROSS stability prediction tools, the heat stability of the mutant is higher than that of the wild type APL, and the half life of the mutant is not lower than 40 min at 70 ℃.
  2. 2. The mutant of claim 1, wherein the mutant has an optimum temperature of 55 ℃, a half-life of 57±4 min at 70 ℃ and a half-life of 21±1 min at 80 ℃.
  3. 3. The mutant according to claim 1 or claim 2, wherein the amino acid sequence of the mutant is as shown in SEQ ID No. 11.
  4. 4. A nucleic acid molecule encoding the mutant according to any one of claims 1 to 3, which sequence has been obtained by site-directed mutagenesis based on the wild-type APL gene.
  5. 5. A recombinant expression vector comprising the nucleic acid molecule of claim 4, wherein said vector is pET21a (+), and has a histidine tag.
  6. 6. A host cell which is a prokaryotic cell, preferably E.coli BL21 (DE 3), transformed with the vector of claim 5.
  7. 7. A method of preparing the mutant of any one of claims 1-3, comprising the steps of: (1) Synthesizing mutant genes and cloning the mutant genes to an expression vector; (2) Transforming the vector into a host cell for mutagenesis; (3) Cells were disrupted and the protein purified using Ni-NTA affinity chromatography.
  8. 8. The method of claim 7, wherein inducing expression is performed overnight at 16 ℃ using 0.1 mM IPTG and purifying is performed using a buffer containing 250 mM imidazole.
  9. 9. Use of a mutant according to any one of claims 1 to 3 for the preparation of an antibacterial, food preservative or disinfectant for inhibiting or killing staphylococcus aureus.
  10. 10. The use according to claim 9, comprising the use of a combination with vancomycin or gentamicin to synergistically enhance the antibacterial effect.

Description

Bacteriophage-derived antibacterial peptide lysozyme protein mutant of staphylococcus aureus with high thermal stability as well as preparation method and application thereof Technical Field The invention relates to the technical field of biology, in particular to a bacteriophage-derived antibacterial peptide lysozyme protein mutant of staphylococcus aureus with high thermal stability, and a preparation method and application thereof. Background Staphylococcus aureus (Staphylococcus aureus) is a common zoonotic primordium, and can cause various diseases such as skin soft tissue infection, pneumonia, septicemia and the like. With the spread of drug-resistant strains such as methicillin-resistant staphylococcus aureus (MRSA), the clinical treatment difficulty is increasing. The antibacterial peptide lysozyme protein (antimicrobial peptidase lysostaphin, APL) derived from staphylococcus aureus (Staphylococcus simulans) is a bacteriophage-derived endolysin, can specifically identify and hydrolyze peptidoglycan cross-linked structures in the cell wall of staphylococcus aureus, has the advantages of high sterilization efficiency, strong targeting, difficult induction of bacterial drug resistance and the like, and is considered as a potential candidate molecule for replacing traditional antibiotics. However, wild-type APL has poor thermal stability, and its optimal reaction temperature is only 37 ℃ and its enzyme activity rapidly decreases in an environment above 40 ℃ and is almost completely inactivated at 70 ℃ and above. For example, wild-type APL has a half-life of about 41±3 min at 70 ℃ and only 10±1 min at 80 ℃. This drawback severely limits its application in situations where high temperature treatment is required (e.g. food pasteurization, medical instrument heat sterilization, high temperature pelletization of feed, etc.). In the prior art, the transformation of the stability of endolysins is dependent on directed evolution or random mutation, and the problems of large screening workload, long period and low success rate exist, so that stable mutants meeting the requirements of industrial high-temperature processing are difficult to obtain efficiently. Aiming at the problems, an APL mutant with high thermal stability is necessary to be developed through a rational design strategy so as to expand the application value of the APL mutant in a high-temperature environment and reduce the cold chain dependence in the production, storage and transportation processes. Disclosure of Invention The invention firstly provides a bacteriophage-derived antibacterial peptide lysozyme protein mutant of staphylococcus aureus with high thermal stability, which is obtained by designing a wild type APL (the sequence is shown as SEQ ID NO: 1) through PROSS stability prediction tools, wherein the thermal stability of the mutant is higher than that of the wild type APL, and the half life of the mutant is not lower than 40 min at 70 ℃. In certain embodiments, the mutant is APL (design 9) with an optimum temperature of 55 ℃, a half-life of 57±4 min at 70 ℃, and a half-life of 21±1 min at 80 ℃. In certain embodiments, the mutant amino acid sequence is set forth in SEQ ID NO. 11. The invention also provides a nucleic acid molecule for coding the mutant, and the sequence of the nucleic acid molecule is obtained by site-directed mutagenesis based on a wild APL gene. The invention also provides a recombinant expression vector which comprises the nucleic acid molecule, wherein the vector is pET21a (+), and is provided with a histidine tag. The invention also provides a host cell which is a prokaryotic cell, preferably E.coli BL21 (DE 3), transformed with the vector. The invention also provides a method for preparing the APL mutant, which comprises the following steps: (1) Synthesizing mutant genes and cloning the mutant genes to an expression vector; (2) Transforming the vector into a host cell for mutagenesis; (3) Cells were disrupted and the protein purified using Ni-NTA affinity chromatography. In certain embodiments, the induced expression is performed overnight at 16 ℃ using 0.1 mM IPTG and the purification is eluted using a buffer containing 250 mM imidazole. The invention finally provides application of the APL mutant in preparing antibacterial drugs, food preservatives or disinfectants for inhibiting or killing staphylococcus aureus. In certain embodiments, the use includes a combination with vancomycin or gentamicin to synergistically enhance the antibacterial effect. Compared with the prior art, the invention has at least the following beneficial effects: (1) The heat stability is obviously improved, the optimal temperature of the mutant APL (design 9) is improved to 55 ℃, and the half life of the mutant APL (design 9) is prolonged to 1.4 times and 2.1 times of that of the wild type at 70 ℃ and 80 ℃, so that the mutant APL (design 9) is suitable for a high-temperature environment. (2) The method has the advantages of improving