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CN-121991938-A - Lyase composition and application thereof

CN121991938ACN 121991938 ACN121991938 ACN 121991938ACN-121991938-A

Abstract

The present invention relates to the field of biological medicine, and more specifically, to a lyase and compositions comprising the same, and their related uses for broad-spectrum antibacterial, disease treatment.

Inventors

  • WANG MENG
  • HUANG XIAOBO
  • WANG SIQIN
  • JIN LEI
  • WANG JIANFENG
  • SHEN YUN
  • LI XIYUE
  • AN HUIFANG
  • RUAN YANTING
  • Fei Keke
  • LIN XIA
  • LIU KAI

Assignees

  • 上海赛增医疗科技有限公司

Dates

Publication Date
20260508
Application Date
20251103
Priority Date
20241104

Claims (17)

  1. 1. An isolated and purified lyase having activity in hydrolyzing peptidoglycan, the amino acid sequence of the lyase comprising, but not limited to, any of SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, or an amino acid sequence having at least 90% sequence identity with any of the amino acid sequences shown as SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and having the same activity.
  2. 2. A fusion protein comprising the lyase of claim 1, and one or more cleavage site sequences; preferably, the fusion protein comprises the lyase of claim 1, and a cleavage site sequence for a 3C enzyme; Preferably, the fusion protein comprises the lyase of claim 1, and a cleavage site sequence for a 3C enzyme, said cleavage site sequence being linked to the N-terminus of the lyase; Preferably, the amino acid sequence of the enzyme cleavage site of the 3C enzyme is shown as SEQ ID NO. 4, Preferably, the nucleotide sequence of the enzyme cutting site of the 3C enzyme is shown as SEQ ID NO. 3.
  3. 3. An isolated nucleic acid molecule encoding the lyase of claim 1 or the fusion protein of claim 2; Preferably, the sequence of the nucleic acid molecule is a natural nucleotide sequence or a codon-optimized nucleotide sequence, e.g.E.coli codon-optimized nucleotide sequence Preferably, the nucleic acid molecule has a sequence as shown in any one of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9, or a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence as shown in any one of SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9.
  4. 4. A vector comprising the isolated nucleic acid molecule of claim 3, preferably said vector is a cloning vector or an expression vector.
  5. 5. A host cell comprising the isolated nucleic acid molecule of claim 3 or the vector of claim 4, Preferably, the host cell is a genetically engineered strain, Preferably, the strain comprises a bacterium or a fungus; preferably, the strain is selected from the group consisting of genetically engineered E.coli, bacillus, corynebacterium, yeast or Streptomyces.
  6. 6. A method of preparing the lyase according to claim 1, comprising the steps of: culturing the host cell of claim 5 under conditions permitting expression of the protein, and recovering the lyase from the cultured host cell culture.
  7. 7. A conjugate comprising the lyase of claim 1 and a coupling moiety; preferably, the coupling moiety is selected from a protein tag, such as a purification tag, a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin, a therapeutic agent, such as an antibacterial agent, or an additional biologically active polypeptide.
  8. 8. A composition comprising the lyase of claim 1, the fusion protein of claim 2, or the conjugate of claim 7.
  9. 9. The composition of claim 8, further comprising one or more additional bacterial inhibiting or killing agents, such as antibiotics, sulfonamides, imidazoles, nitroimidazoles, quinolones; Preferably, the active agent is chlorhexidine, fluoride, povidone iodine, cetylpyridinium chloride, or a quaternary ammonium salt.
  10. 10. The composition of claim 8 or 9, further comprising one or more pharmaceutically acceptable excipients.
  11. 11. The composition of any one of claims 8-10, which is any one of the following: 1) A pharmaceutical composition; 2) An oral cleaning composition selected from the group consisting of toothpaste, mouthwash product, oral cleaner, tooth whitener, or gel; 3) A food composition selected from chewing gum, soft candy, beverage or health functional food.
  12. 12. Use of the lyase of claim 1, the fusion protein of claim 2, the conjugate of claim 7 or the composition of any of claims 8-11 in: (1) Inhibit or kill bacteria, and/or (2) Hydrolyzing peptidoglycan, preferably peptidoglycan in the cell wall of a bacterium; preferably, the bacterium is a gram-negative bacterium; preferably, the content of peptidoglycan in the cell wall of the bacterium is not less than 80%; Preferably, the bacterium is a bacterium of the genus Fusobacterium, Preferably, the bacteria are selected from one or more of the group consisting of fusobacterium nucleatum, fusobacterium necroseum, fusobacterium microbiogenes, fusobacterium mortiferum, fusobacterium navigatum, fusobacterium lansium, fusobacterium variabilis and fusobacterium ulcerans.
  13. 13. Use of the lyase of claim 1, the fusion protein of claim 2, the conjugate of claim 7 or the composition of any one of claims 8-11 for the manufacture of a medicament for preventing and/or treating a disease caused by a bacterial infection of the genus fusobacterium in a subject; Such diseases include oral diseases such as gingivitis, periodontitis; Systemic infections, such as sepsis and brain abscess; respiratory infections, such as lung abscess, aspiration pneumonia, gastrointestinal diseases, such as colon cancer, genital infections, gynecological infections, meningitis; preferably, the subject is a mammal, such as a human.
  14. 14. A method of inhibiting or killing bacteria, preferably bacteria of the genus fusobacterium, in vitro comprising the step of administering an effective amount of a lyase according to claim 1, a fusion protein according to claim 2, a conjugate according to claim 7 or a composition according to any of claims 8-10.
  15. 15. A method of hydrolyzing peptidoglycan in vitro comprising the step of administering an effective amount of the lyase of claim 1, the fusion protein of claim 2, the conjugate of claim 7, or the composition of any of claims 8-10.
  16. 16. The use according to any one of claims 12-13 or the method according to any one of claims 14-15, wherein the lyase, fusion protein, conjugate, composition is administered orally, by painting or by intravenous injection.
  17. 17. The use according to any one of claims 12-13 or the method according to any one of claims 14-15, wherein the amount of active ingredient used is from 0.001g to 20g.

Description

Lyase composition and application thereof Cross Reference to Related Applications The application claims priority from a prior application filed by 11.04.2024 to China national intellectual property agency with patent application number 202411567608.2 and the name of "a lyase composition and application thereof". The entire contents of said prior application are incorporated by reference into the present application. Technical Field The invention relates to the technical field of lyase, in particular to a lyase composition and application thereof. Background Fusobacterium nucleatum (Fusobacterium nucleatum, F.n) is an obligate anaerobic gram-negative bacterium, with the bacterial body being long shuttle, which is one of the common oral microorganisms. Fusobacterium nucleatum has high detection rate in periodontitis, and the Fusobacterium nucleatum have strong correlation. Normally, the clostridium nucleatum and other floras of the oral cavity act together to maintain the microecology of the oral cavity health, but when the oral cavity ecological is unbalanced, the clostridium nucleatum can form dental plaque biomembrane by means of various adhesion copolymerization pathogenic bacteria (such as streptococcus, porphyromonas odontoid and the like), can adhere and invade epithelial cells, and activate host immune reaction by using virulence factors, metabolites and the like to destroy periodontal tissues, thereby promoting the occurrence and development of various oral diseases such as acute and chronic periodontitis, gingivitis and the like. According to the fourth national oral health epidemiological investigation result, the periodontal health rate of adults in China is only 9.1%, the prevalence rate of periodontal disease in the population of 35-44 years is as high as 90.9%, and severe periodontitis becomes the world sixth pandemic disease. At present, the oral disease is treated by combining mechanical scaling and auxiliary antibacterial treatment clinically, and the auxiliary antibacterial treatment medicament mainly comprises nitroimidazoles and other antibiotics such as azithromycin, tetracycline, doxycycline and the like. However, these therapeutic agents are not directed against specific periodontal pathogens such as fusobacterium nucleatum, which may cause problems such as dysbacteriosis and drug resistance, and dental plaque biofilm formed by fusobacterium nucleatum in combination with other pathogens has serious resistance to existing antibiotic drugs, resulting in unsatisfactory therapeutic effects of antibiotics. Therefore, the search and development of new antibacterial drugs against pathogenic bacteria such as fusobacterium nucleatum is still an urgent need for current therapeutic drugs. In addition to the important role of clostridium nucleatum in the development of oral diseases, in recent years, as the research on intestinal microorganisms is in progress and the abundance of clostridium nucleatum is significantly increased in colorectal cancer (colorectal cancer, CRC) patients, the research on the relationship between clostridium nucleatum and colorectal cancer has been receiving extensive attention from researchers. CRC has been the second largest cancer lethal factor worldwide, and is also the third largest cancer worldwide. Studies have shown that fusobacterium nucleatum is highly specifically enriched in CRC patients compared to normal colon tissue and is highly correlated with poor patient prognosis and recurrence of CRC after treatment. In addition, fusobacterium nucleatum is closely related to occurrence and development of CRC, is an important pathogenic risk factor of colorectal cancer, and can promote proliferation and transfer of CRC cells through various mechanisms (immune regulation, virulence factors, intestinal metabolites, DNA damage) and can promote chemoresistance of CRC through autophagy. In summary, fusobacterium nucleatum may be a potential target for CRC prevention. Therefore, with fusobacterium nucleatum as a target, intensive research and development of drug molecules capable of killing fusobacterium nucleatum to reduce abundance and activity thereof in the intestinal tract of a patient may provide a new direction for targeted treatment and prevention of CRC. Phage lyase is a bacterial cell wall hydrolase that the phage encodes for release in the late stages of infection of the host, also known as endolysin (Endolysin). Because the enzyme has the characteristics of good specificity, rapid sterilization, good safety, difficult generation of drug resistance and the like, the research of phage lyase enzyme attracts the attention of scientists and is also taken as an effective substitute of antibiotics. At present, a great deal of research data has demonstrated that phage lytic enzymes exhibit significant bactericidal activity and good biosafety both in vivo and in vitro, and that some lytic enzymes have been successfully entered into clinical laboratory stages and markets