Bacteriophage lysin chimera or variant thereof and use thereof

Abstract

The present invention relates to the field of anti-infection treatment and prevention. The present invention relates to a bacteriophage lysin chimera or a variant thereof and a use thereof. The bacteriophage lysin chimera or the variant thereof of the present invention comprises a catalytic domain, a binding domain and a linker. The lysin and the chimera thereof of the present invention have improved biophysical properties, have excellent applicability, and are suitable for cosmetics, medical devices, disinfectants and / or therapeutic applications.

Description

A phage lysin chimera or a variant thereof and its applications Technical Field

[0001] This invention belongs to the field of anti-infective treatment and prevention. Specifically, this invention relates to a phage lyase chimera or a variant thereof and its use in the preparation of medicaments for treating and / or preventing infections caused by Gram-positive bacterial pathogens. Background Technology

[0002] This invention specifically relates to the treatment and / or prevention of infections caused by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus ludensundus, Staphylococcus hemolyticus, Staphylococcus saprophyticus, Staphylococcus Schwetze, Staphylococcus capitulata, Staphylococcus hominis, Staphylococcus wortii, Staphylococcus pettenkoferi, Staphylococcus mimicus, Staphylococcus coliformis, Staphylococcus intermedius, pseudo-intermedius, Staphylococcus hemolyticus, and other staphylococci. Most preferably, this disclosure relates to the treatment and / or prevention of Staphylococcus aureus. This invention relates to engineered chimeric phage lysin peptides and the use of such modified peptides in killing Gram-positive bacteria, particularly in the treatment of infections, for prevention, in medical devices, in cosmetics, as skin products, in disinfection, and in diagnostic products. The lysin can be formulated for topical, oral, or non-gastrointestinal administration, and can be used alone or in combination with other antimicrobial agents. This invention also relates to the use of the disclosed reagents as components in cosmetic and / or medical device formulations. This invention also extends to downstream effects on the health of the treated person or animal, where such downstream effects are only indirectly related to the mechanism of action, such as health benefits resulting from microbiome alterations. This invention addresses the need for effective treatment of Gram-positive bacterial infections, particularly those caused by antibiotic-resistant bacteria.

[0003] Antimicrobial resistance (AMR) among bacterial pathogens is a growing global problem. A recent systematic literature review identified AMR as a contributing factor to 4.95 million deaths in 2019, with 1.27 million of those deaths potentially attributable directly to AMR. The World Health Organization (WHO) currently lists methicillin-resistant Staphylococcus aureus (MRSA) as a high-risk bacterium requiring urgent new treatments. This bacterium often colonizes the anterior nasal cavity and skin of healthy individuals, but becomes an opportunistic bacterium if it breaches the outer skin barrier. Staphylococcus aureus can cause a variety of invasive infections, ranging from skin and soft tissue infections to severe bacteremia, endocarditis, pneumonia, and osteomyelitis. Staphylococcus aureus's infectious capacity is attributed to its ability to express a large number of virulence factors, including many toxins that play a key role in the establishment, progression, and severity of infection. Staphylococcus aureus has also evolved mechanisms for forming biofilms, complex extracellular polymeric structures that protect bacterial cells from the host immune system and various antimicrobial agents, often leading to treatment failure. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) clones has made Staphylococcus aureus extremely difficult to treat. Despite recent advancements in antibiotics such as daptomycin and linezolid, mortality rates associated with invasive infections remain high, even with optimal treatment regimens. There is an urgent need for new treatment options targeting the causative agents, including Gram-positive bacteria, common staphylococci, and Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA).

[0004] Phage lyases are produced by bacteriophages in the later stages of infection and are used to degrade the peptidoglycan layer of the host bacterial cell wall, leading to rapid bacterial lysis. Peptidoglycan is a large molecule that encapsulates the entire bacterial cell, providing structural support and protection against environmental stresses. It consists of repeating units of N-acetylglucosamine and N-acetylmuramic acid, which cross-link to form a highly stable structure. Lyases are classified according to their specific enzymatic activities: glucosidases and muramamidinases cleave the peptidoglycan sugar backbone, N-acetylmuramyl-L-alanine amidases cleave the amide bonds connecting the sugar backbone and the peptide moiety, and endopeptidases cleave within the peptide moiety of peptidoglycan (stem peptides and / or cross-bridges). In nature, lyases are produced within phage-infected cells and pass through the cell membrane to reach the cell wall peptidoglycan via pores formed by phage-encoded cavitary proteins. Recent studies have shown that lyases can be recombinantly expressed, purified, and used externally to kill target pathogens, thus possessing potential therapeutic applications.

[0005] Using recombinantly expressed lyases as therapeutic agents offers several important advantages: they are extremely potent, generating osmotic pressure within minutes to lyse and kill target bacteria. Furthermore, given the billions of years of selective evolutionary pressures on lyases, the cell wall targets they attack are highly conserved and unlikely to be easily altered by bacteria. This characteristic makes resistance to lyases extremely unlikely, making them suitable for long-term use. To date, no bacterial resistance to bacteriophage lyases has been discovered. Moreover, with a few exceptions, each lyase targets bacteria belonging to a single genus or a limited number of species, allowing lyases to eliminate target pathogens while preserving beneficial microbiota. Lyases targeting Gram-positive bacteria typically consist of one or more N-terminal catalytic domains (CAT) and one or more C-terminal binding domains (BD), usually linked by flexible linkers. While some natural lyases can be used directly as therapeutic agents, modern approaches include engineered lyases that often offer superior properties. These engineered lyases may incorporate catalytic and binding domains from diverse sources, resulting in lyase chimeras with properties superior to their parent enzymes.

[0006] ClyO is a chimeric phage lyase highly active against Staphylococcus bacteria, including Staphylococcus aureus (US2018 / 0291357A1). ClyO consists of an N-terminal catalytic domain derived from the Staphylococcus aureus lyase Ply187 and a peptidoglycan-binding domain derived from the Streptococcus suis lyase PlySs2. ClyO exhibits high activity and a broad spectrum of activity against Staphylococcus species, killing Staphylococcus aureus (including MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus equorum, Staphylococcus capitulata, Staphylococcus albus, Staphylococcus xylose, Staphylococcus aureus, Staphylococcus hemolyticus, and Staphylococcus chromogenicus. It kills Staphylococcus aureus very rapidly through osmotic lysis, as clearly seen in a visibly reduced optical density of the culture. Colony-forming units also decrease rapidly, typically killing several log units within minutes. In addition, ClyO has strong anti-biofilm activity and can protect animals when attacked by infectious doses of Staphylococcus aureus.

[0007] ClyF is a lyase very similar to ClyO. Like ClyO, it consists of an N-terminal catalytic domain derived from the Staphylococcus aureus lyase Ply187 and a peptidoglycan-binding domain derived from the Streptococcus suis lyase PlyS2. Like ClyO, ClyF is active against a variety of Staphylococcus species and biofilms, and can protect animals from Staphylococcus aureus infection.

[0008] Despite the high catalytic activity of ClyO, the commercial use of lyases requires additional properties, such as product stability, which has not yet been selected in the natural environment but is absolutely crucial for the successful market acceptance of such products. Summary of the Invention

[0009] In response to the above situation, we surprisingly discovered that other chimeric lyases with specific range of linkers combine the Ply187 catalytic domain and the PlySs2 binding domain to produce a protein with appropriate stability for commercial applications, which outperforms ClyO and ClyF.

[0010] Therefore, the present invention provides a phage lysin chimera or a variant thereof, the lysin chimera comprising a catalytic domain, a binding domain, and a linker polypeptide, wherein,

[0011] The catalytic domain has the amino acid sequence shown in SEQ ID NO:3;

[0012] The binding domain has the amino acid sequence shown in SEQ ID NO:4;

[0013] The linker polypeptide includes a linker polypeptide with one or more catalytic domains or a linker polypeptide with one or more binding domains or a combination thereof.

[0014] The linker of the catalytic domain is selected from any linker in the amino acid sequence shown in SEQ ID NO:113 to SEQ ID NO:121;

[0015] The linker for the binding domain is selected from any linker in the amino acid sequences shown in SEQ ID NO:125 to SEQ ID NO:137, including G, AG, LAG, or the amino acid sequences shown in SEQ ID NO:125 to SEQ ID NO:137.

[0016] The linker optionally includes one, two, or more glycine residues (G).

[0017] Furthermore, the chimera does not include the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2.

[0018] In this invention, the adaptor polypeptide may be an adaptor polypeptide with one or more catalytic domains, an adaptor polypeptide with one or more binding domains, or a combination of both.

[0019] In this invention, as one embodiment, the lyase chimera includes a catalytic domain of the amino acid sequence shown in SEQ ID NO:3, a binding domain of the amino acid sequence shown in SEQ ID NO:4, and a linker selected from GG or any of the amino acid sequences shown in SEQ ID NO:77 to SEQ ID NO:108.

[0020] In this invention, as one embodiment, the lyase chimera includes an amino acid sequence as shown in any one of SEQ ID NO:5 to SEQ ID NO:37.

[0021] In this invention, as one embodiment, the lyase chimera includes an amino acid sequence as shown in any one of SEQ ID NO:5 to SEQ ID NO:18 or SEQ ID NO:26 to SEQ ID NO:29; more preferably, the lyase chimera is an amino acid sequence as shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:14; preferably, it is an amino acid sequence as shown in SEQ ID NO:11.

[0022] In this invention, as one embodiment, the lyase chimera includes any nucleotide sequence shown in SEQ ID NO:42 to SEQ ID NO:74; preferably, the lyase chimera includes any nucleotide sequence shown in SEQ ID NO:42 to SEQ ID NO:55 or SEQ ID NO:63 to SEQ ID NO:66; more preferably, the lyase chimera includes any nucleotide sequence shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:51; more preferably, it is the nucleotide sequence shown in SEQ ID NO:48.

[0023] The present invention also provides a variant of a lyase chimera, the variant comprising the lyase chimera of the present invention modified by adding a positively charged amino acid or a polypeptide composed of one or more positively charged amino acids to the C-terminus, wherein the positively charged amino acid is selected from lysine (K), arginine (R), or histidine (H), or more preferably from lysine (K) and arginine (R); preferably, the added polypeptide is selected from K, KK, KKK, R, RR, RRR, KR, RK, KRR, KRK, KKR, RKK, RRK, RKR; more preferably, the added polypeptide is selected from K, KK, KKK, R, RR, RRR;

[0024] In this invention, as one embodiment, the variant of the lyase chimera includes the amino acid sequence shown in any one of SEQ ID NO:109–SEQ ID NO:112, SEQ ID NO:122–SEQ ID NO:124, or SEQ ID NO:138–SEQ ID NO:340; preferably, the variant of the lyase chimera includes the amino acid sequence shown in any one of SEQ ID NO:138–SEQ ID NO:299; preferably, the variant of the lyase chimera includes SEQ ID NO:138–143, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:198–SEQ ID NO:203, SEQ ID NO:204 ... The amino acid sequence is any one of SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:276, SEQ ID NO:277, or SEQ ID NO:281; more preferably, the variant of the lyase chimera includes any one of SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:198 to SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:235, SEQ ID NO:277, or SEQ ID NO:281; further preferably, the variant of the lyase chimera is SEQ ID NO:187, SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:276, SEQ ID NO:277, or SEQ ID NO:281; NO:199 or SEQ ID NO:205; most preferably, the variant of the lyase chimera has the amino acid sequence shown in SEQ ID NO:187.

[0025] In this invention, as one embodiment, the lyase chimera or its variant further includes an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more preferably 100% identity.

[0026] In this invention, as one embodiment, the lyase chimera is modified by fusing it with other amino acids or peptides at the N-terminus or C-terminus. The peptide includes a lyase-binding domain, a lyase-catalytic domain, a functional protein that promotes purification, enhances activity or stability, or a functional protein that imparts other desirable properties to the peptide.

[0027] In this invention, as one embodiment, the polypeptide is modified and located between the catalytic domain and the binding domain. The modification may include deleting amino acids derived from the polypeptide of this invention, introducing other amino acids into the linker, replacing amino acids in the linker with different amino acids, and / or introducing a linker region or fragment thereof, other lyases, or other proteins having a predetermined linker domain. Such added linker domain may be a naturally occurring and / or synthetic peptide.

[0028] In this invention, as one embodiment, the lyase chimera or its variant is modified in one or more amino acids using local mutagenesis or gene synthesis methods to improve enzyme expression, yield or activity.

[0029] The present invention provides a nucleic acid molecule encoding the above-mentioned chimera, wherein the nucleic acid molecule is DNA or RNA.

[0030] The present invention provides an expression vector containing the above-mentioned nucleic acid molecules; the expression vector includes, but is not limited to, plasmids, bacteriophages, viruses or artificial chromosomes; as one embodiment; plasmids are preferred, and pET matrix particles or pBAD matrix particles are more preferred.

[0031] In this invention, as one embodiment, the expression vector further includes a promoter, preferably having constant expression or inducible expression characteristics; more preferably, an arabinose-induced promoter, a lactose-induced promoter, or an isopropyl β-D-1-thiogalactoside (IPTG)-induced promoter; and even more preferably, an IPTG-induced promoter.

[0032] The present invention provides a host cell containing the above-mentioned expression vector, characterized in that the host cell is a microbial cell, preferably a bacterial cell, and most preferably an Escherichia coli cell.

[0033] The present invention provides a method for producing the above-mentioned lyase chimera or variant thereof, the method comprising culturing the above-mentioned host cell population under conditions expressing the lyase or chimera thereof, and isolating therefrom.

[0034] The present invention provides a purification method comprising the above-described lyase chimera or a variant thereof.

[0035] The present invention provides a pharmaceutical composition comprising the above-described lyase chimera or a variant thereof, and a pharmaceutically acceptable carrier.

[0036] The present invention discloses a dosage form containing the above-mentioned pharmaceutical composition, the dosage form including injections, inhalers, topical preparations, surgical cleansers, sprays, mouthwashes, nasal sprays, direct nasal applications, solid preparations, or dosage forms coated on clips, patches, implantable devices, sutures, wound dressings, or other objects.

[0037] In this invention, as one embodiment, the injectable dosage form includes a liquid injection preparation or a lyophilized injection preparation, and the topical preparation includes a gel, ointment, spray, cream, or powder.

[0038] The present invention provides the use of the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell or pharmaceutical composition in the preparation of a medicament for treating or preventing pathogen colonization and / or infection-related diseases.

[0039] In this invention, as one embodiment, the pathogen is a Gram-positive bacterial pathogen; preferably, the pathogen is selected from one or more of the genus Staphylococcus; more preferably, the pathogen is one of the following: Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus - MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus equorum, Staphylococcus capitulata, Staphylococcus albus, Staphylococcus xylose, Staphylococcus aureus, Staphylococcus pyogenes, or Staphylococcus chromogenic; most preferably, the pathogen is Staphylococcus aureus (including MRSA).

[0040] In this invention, as one embodiment, the pathogen has drug resistance, multidrug resistance (MDR), or extensive drug resistance (XDR).

[0041] In this invention, as one embodiment, the infection is a multi-microbial infection.

[0042] In this invention, as one embodiment, the lyase chimera or a variant thereof is co-formulated or co-administered with another antibacterial agent, antifungal agent, preservative or disinfectant, including co-formulation with standard care antibiotics.

[0043] This invention provides the use of the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or dosage form in the preparation of a medicament for treating or preventing infectious diseases, respiratory infections, gastrointestinal infections, blood infections, urinary tract infections, reproductive tract infections, skin and soft tissue infections, nervous system and brain infections, structural system and skeletal infections, device-related infections, immunodeficiency infections, or diseases caused by bacterial infections; wherein,

[0044] The urinary tract infection includes urinary tract infection, and further includes cystitis, pyelonephritis, prostatitis or urethritis;

[0045] The diseases caused by the gastrointestinal infections include gastroenteritis, enteritis, enterocolitis, food poisoning, typhoid fever, cholera, or Helicobacter pylori infection;

[0046] Diseases caused by the bacterial infection include pneumonia, bronchitis, sinusitis, pharyngitis, tonsillitis, tuberculosis, meningitis, encephalitis, osteomyelitis, suppurative arthritis, peritonitis, pericarditis, abscess, botulism, tetanus, diphtheria, or pertussis, as well as skin and soft tissue infections, further including cellulitis, impetigo, folliculitis, furunculosis, carbuncle, erysipelas, necrotizing fasciitis, myonecrosis, or wound infection;

[0047] The diseases caused by blood infections include sepsis, bacteremia, endocarditis, and septicemia;

[0048] The diseases caused by the reproductive tract infections include gonorrhea, chlamydia, syphilis, chancroid, and lymphogranuloma venereum.

[0049] The indwelling devices include catheters, intravenous catheters, ventilators, pacemakers, implantable cardioverter defibrillators, central venous catheters, gastrostomy tubes, tracheostomy tubes, nerve stimulators, artificial heart valves, vascular access devices, tympanostomy tubes, hemodialysis catheters, feeding tubes, or drainage tubes.

[0050] The present invention provides the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or dosage form, including but not limited to its use as a food preservative, disinfectant, bactericide, medical device or cosmetic.

[0051] The present invention provides the application of the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or dosage form in the treatment or prevention of bacterial biofilm formation.

[0052] The present invention provides an application of the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or dosage form for treating or preventing the formation of bacterial persistent cells or metabolically dormant cells in the presence or absence of a biofilm.

[0053] The present invention provides an application of the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or dosage form to decolonize target bacteria in individual form.

[0054] This invention provides an application of the above-mentioned lyase chimera or variant thereof, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or dosage form to alter an individual's microbiome by removing unwanted bacteria while retaining desired bacteria.

[0055] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to treat multiple microbial infections. For example, the lyase chimera or a variant thereof is used as a single agent against multiple Staphylococcus species or other species with lyase activity, or in combination with another antimicrobial agent to treat multiple microbial infections, wherein each microbial species in the infection is sensitive to at least one component of the combined formulation.

[0056] In this invention, as one embodiment, the bacterial infection refers to a respiratory tract infection, including pneumonia.

[0057] In this invention, as one embodiment, the infectious disease being treated includes urinary tract infection.

[0058] In this invention, as one of the embodiments, the disease being treated is gastrointestinal infection.

[0059] In this invention, as one embodiment, the bacterial infection being treated is a skin and soft tissue infection.

[0060] In this invention, as one embodiment, the infection being treated is a blood infection with or without endocarditis.

[0061] In this invention, as one of the implementation schemes, the infection being treated is a sexually transmitted infection or a reproductive tract infection.

[0062] In this invention, as one of the embodiments, the infection refers to infection of the brain or meninges, or infection of the nervous system.

[0063] In this invention, as one embodiment, the infection refers to an infection related to the indwelling device.

[0064] In this invention, as one embodiment, the infection refers to an infection related to bone or joint infection, including osteomyelitis, in the presence or absence of artificial devices or implants related to bone or joint.

[0065] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to treat, control, or prevent bacterial infection or colonization in patients whose risk of bacterial infection is increased due to impaired immune system.

[0066] In this invention, as one embodiment, the infection refers to an infection related to deep tissue abscess.

[0067] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to prevent infection (or reinfection) of high-risk individuals with Gram-positive pathogens in patients with increased infection risk (e.g., patients with cystic fibrosis).

[0068] In this invention, as one embodiment, the lyase chimera or a variant thereof is used in combination therapy with another antimicrobial agent or antiseptic. This includes co-formulation with standard care antibiotics to prevent the development of resistance to these antibiotics and / or to re-sensitize resistant bacteria to standard care antibiotics.

[0069] In this invention, as one embodiment, the lysin chimera or a variant thereof is administered together with a reagent that enhances bacterial membrane permeability.

[0070] In this invention, as one embodiment, the lyase chimera or a variant thereof is administered together with an antibacterial agent effective against the target bacteria.

[0071] In this invention, as one embodiment, the lyase chimera or its variant is administered together with an antibacterial agent. When administered alone, the organism develops resistance to the antibacterial agent; however, the combination of the antibacterial agent and the lyase is effective.

[0072] In this invention, as one embodiment, the lyase chimera or a variant thereof is used in combination with an antibacterial disinfectant.

[0073] In this invention, as one embodiment, the lyase chimera or a variant thereof is used as an antibacterial disinfectant or rinsing solution.

[0074] In this invention, as one embodiment, the lysin is formulated into a topical preparation for use on the skin or mucous membrane surface.

[0075] In this invention, as one embodiment, the lyase chimera or a variant thereof is embedded in a wound dressing material.

[0076] In this invention, as one embodiment, the lyase chimera or a variant thereof is coated onto an implantable device.

[0077] In this invention, as one embodiment, the lyase chimera or variant thereof may be used in combination or in combination, or one or more lyases disclosed in this patent may be used in combination with additional lyases from different sources, or any of the above combinations may be used except for another antimicrobial agent or preservative.

[0078] In this invention, as one embodiment, the lyase chimera or its variants encapsulate or modify the lyase in a manner that improves its pharmacokinetic properties.

[0079] In this invention, as one embodiment, the lyase chimera or its variant lyase is used as a food preservative to prevent or eliminate contamination by target bacteria.

[0080] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to combat bacterial biofilms or persistent bacterial cells.

[0081] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to alter the composition of bacterial communities inside or on the surface of humans and / or animals and / or inanimate objects.

[0082] Detailed description of the invention:

[0083] In this invention, as one embodiment, the invention includes the lyase chimera or a variant thereof or a functional portion thereof.

[0084] In this invention, as one embodiment, the lyase chimera or its variants or functional portions thereof may be fused with other peptides at the N-terminus or C-terminus, and may include additional lyase-binding domains, lyase-catalytic domains, functional tags that promote purification, enhance activity or stability, or impart other desired properties to the peptide.

[0085] In this invention, as one embodiment, a chimeric lyase of the present invention or a variant thereof or a functional portion thereof is included, thereby modifying the linker region between the catalytic domain and the binding domain. Linker modification may include deleting amino acids derived from the polypeptide of the present invention, introducing additional amino acids into the linker, replacing amino acids in the linker with different amino acids, and / or introducing a linker region or fragment thereof, other lyases, or other proteins having an established linker domain. Such added linker domain may be a naturally occurring and / or synthetic peptide.

[0086] In this invention, as one embodiment, a chimeric lyase of the present invention or a variant thereof or a functional portion thereof has been modified in one or more amino acids using methods known to those skilled in the art (such as site-directed mutagenesis or gene synthesis) to improve enzyme expression, yield, or activity. Such mutant forms of the lyase are also included within the scope of this invention.

[0087] In this invention, as one embodiment, a lyase polypeptide or its functional portion is included, wherein the polypeptide has been modified by adding a C-terminal positively charged amino acid or a polypeptide composed of one or more positively charged amino acids, wherein the positively charged amino acid is selected from lysine (K), arginine (R), or histidine (H), or more preferably from lysine and arginine. Optionally, the positively charged polypeptide may contain other non-positively charged amino acids, as long as the total charge of the polypeptide is positive. Optionally, the positively charged polypeptide can be separated from the lyase chimera or other variant polypeptide by a linker. Preferably, the added positively charged amino acid or polypeptide is selected from K, KK, KKK, R, RR, RRR, KR, RK, KRR, KRK, KKR, RKK, RRK, RKR. More preferably, the added amino acid or polypeptide is selected from K, KK, KKK, R, RR, RRR.

[0088] Most preferably, the modified lyase chimera is selected from the amino acid sequences shown in any one of SEQ ID NO:109 to SEQ ID NO:112, SEQ ID NO:122 to SEQ ID NO:124 or SEQ ID NO:138 to SEQ ID NO:340;

[0089] Preferably, the variants of the lyase chimera include the amino acid sequences shown in any one of SEQ ID NO:138–143, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:198–SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:276, SEQ ID NO:277, or SEQ ID NO:281;

[0090] More preferably, the variants of the lyase chimera include the amino acid sequences shown in any one of SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:198 to SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:235, SEQ ID NO:277, or SEQ ID NO:281; even more preferably, the variants of the lyase chimera are the amino acid sequences shown in SEQ ID NO:187, SEQ ID NO:199, or SEQ ID NO:205; most preferably, the variants of the lyase chimera are the amino acid sequences shown in SEQ ID NO:187.

[0091] This invention also relates to polynucleotide molecules encoding the lysin chimeras of the present invention disclosed herein. The nucleic acid molecule may be DNA or RNA. Here, the terms polynucleotide and polynucleotide molecule are used synonymously with the term nucleic acid molecule.

[0092] This invention also relates to vectors comprising the polynucleotide molecules described herein. The choice of vector will depend on the choice of host expression system. For example, the vector may be a plasmid.

[0093] In this invention, as one embodiment, the vector is an expression vector, including any suitable expression vector known in the art, such as pET-based, pBAD-based, or any other suitable vector known in the art. Furthermore, the vector also includes a promoter with constant or inducible expression function. Promoters known in the art with inducible expression function include arabinose-induced promoters, lactose or isopropyl β-D-1-thiogalactoside (IPTG)-induced promoters, etc. Preferably, a vector with an IPTG-induced promoter is used.

[0094] In this invention, as one embodiment, the nucleotide is the nucleotide sequence shown in SEQ ID NO:47.

[0095] In this invention, as one embodiment, the nucleotide is the nucleotide sequence shown in SEQ ID NO:48.

[0096] In this invention, as one embodiment, the nucleotide is the nucleotide sequence shown in SEQ ID NO:50.

[0097] In this invention, as one embodiment, the nucleotide is the nucleotide sequence shown in SEQ ID NO:51.

[0098] In this invention, as one embodiment, the nucleotide may be selected from any one of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 or SEQ ID NO:55.

[0099] In one embodiment of the present invention, the nucleotides of the present invention may be selected from any one of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.

[0100] The present invention also provides a host cell comprising the polynucleotide molecule of the present invention or the vector or plasmid of the present invention. The host cell may be selected from a range of possibilities known in the art, including mammalian cells, yeast or fungal cells, insect cells, microbial cells, etc.

[0101] In this invention, as one embodiment, the host cell is a microbial cell, such as a bacterial cell. Preferably, the host cell is non-pathogenic. Most preferably, the host cell is *Escherichia coli*. Therefore, one aspect of the invention relates to a bacterial host cell comprising the plasmid of the invention, preferably *Escherichia coli* cells.

[0102] The present invention also provides a method for producing the chimeric lyase of the present invention or a variant thereof, comprising culturing a host cell population containing the polynucleotide molecule of the present invention or the plasmid / vector of the present invention under conditions expressing the lyase, and isolating therefrom.

[0103] This invention includes the application of various vector expression systems for cloning and expressing lyase genes. Examples of suitable vector expression systems include, but are not limited to, plasmids, bacteriophages, viruses, and artificial chromosomes. These vector systems can be used to clone lyase genes into appropriate expression vectors, which are then transformed into suitable host cells for expression.

[0104] This invention also includes any modifications or alterations to the vector expression system known to those skilled in the art that result in improved enzyme expression, yield, or purity. Recombinant cloning of the lyase gene can be achieved using standard molecular biology techniques such as PCR, restriction enzyme digestion, and ligation.

[0105] This invention includes any methods known to those skilled in the art for recombinant cloning of lyase genes, whether now known or to be developed in the future.

[0106] This invention includes various recombinant expression systems for producing enzymes, including but not limited to bacterial systems such as *Escherichia coli*, *Bacillus subtilis*, and *Pseudomonas putida*; yeast systems such as *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Lactobacillus kluyveromyces*; plant systems such as *Arabidopsis thaliana*, tobacco, and *Alfalfa truncatula*; algal systems such as *Chlamydomonas reinhardtii*, *Phaeodactylum tricornutum*, and marine microchlorophyll; and mammalian systems such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, and mouse myeloma cells. These recombinant expression systems can be used to produce enzymes using molecular biology techniques known to those skilled in the art, such as DNA cloning, expression vector design, and gene transformation.

[0107] This invention includes any method by which a person skilled in the art produces enzymes using known recombinant expression systems, including methods currently known or those to be developed in the future. Furthermore, any modifications or alterations to the recombinant expression system known to a person skilled in the art that improve enzyme expression, yield, or purity are also included within the scope of this invention.

[0108] This invention also includes the application of cell-free expression systems for the production of lyases. Examples of suitable cell-free expression systems include, but are not limited to, E. coli S30 extract, wheat germ extract, and lysates of rabbit reticulocytes. These systems can be optimized for lyase expression using various modifications and additives, such as molecular chaperones, energy regeneration systems, and stabilizers.

[0109] This invention includes any modifications or alterations to cell-free expression systems known to those skilled in the art that result in improvements in enzyme expression, yield, or purity.

[0110] This invention includes any modifications or alterations to the lyase gene sequence known to those skilled in the art, resulting in increased expression, yield, or activity of the enzyme in a cell-free expression system. These modifications include, but are not limited to, codon optimization, signal sequence modification, and truncation of non-functional or harmful regions of the lyase protein.

[0111] The present invention also includes any methods known to those skilled in the art for formulating and optimizing reaction conditions for cell-free expression systems to improve the expression, yield, or purity of lysins, whether now known or to be developed in the future.

[0112] This invention also includes various methods known to those skilled in the art for purifying enzymes, such as the purification of lysins, including but not limited to ultrafiltration, dialysis, precipitation, chromatography, and centrifugation. Ultrafiltration can be used to remove low molecular weight impurities, while dialysis can be used for buffer exchange and desalting. Precipitation techniques, such as salting out or solvent precipitation, can be used to concentrate and purify enzymes. Chromatography, including affinity, ion exchange, size exclusion, and hydrophobic interaction chromatography, can be used to separate and purify enzymes from other proteins, contaminants, and impurities. Furthermore, centrifugation can be used to separate enzymes from cell debris and insoluble substances. Therefore, this invention includes any methods known to those skilled in the art for purifying enzymes, whether now known or developed in the future.

[0113] The lyase chimera of the present invention can be formulated into various pharmaceutical compositions suitable for different routes of administration, including but not limited to injection (liquid or lyophilized), inhalation, topical (gel, ointment, cream, spray, powder, lotion), mouthwash, nasal application (spray or gel), solid dosage forms, and sustained-release formulations. The composition may also include additional agents such as antibacterial agents, preservatives, excipients, stabilizers, and carriers to improve stability, bioavailability, and efficacy.

[0114] The following sections describe the various formulations and their respective delivery methods:

[0115] In one embodiment of this invention, an injectable formulation containing a phage lyase chimera or a variant thereof is provided. As one embodiment, the injectable formulation includes a liquid injection formulation. As an example, the lyase chimera or a variant thereof can be formulated as a solution or suspension and placed in a pharmaceutically acceptable carrier, such as saline, phosphate-buffered saline (PBS), or water for injection (WFI). The pH of the formulation is typically adjusted to physiological conditions (pH 7.0 to 7.4). This invention includes a wider pH range (pH 5.0–8.0). Preservatives such as benzyl alcohol or other suitable reagents can be used to stabilize the enzyme.

[0116] As one implementation method, the injectable formulation includes a lyophilized injectable formulation. As an example, to maintain stability during storage and transportation, the lyase chimera or a variant thereof can be prepared as a lyophilized powder and reconstituted with a suitable diluent (such as sterile water or physiological saline) before administration. Lyophilization may require the addition of stabilizers such as mannitol, sucrose, or trehalose to maintain lyase activity during the lyophilization process.

[0117] In one embodiment of this invention, an inhalation formulation containing a phage lyase chimera or a variant thereof is provided. As an example, the lyase chimera or variant thereof can be formulated for delivery via the pulmonary route, and can be a dry powder inhaler (DPI) or a nebulized liquid formulation. For DPI formulations, the lyase can be co-processed with lactose, mannitol, or other excipients that promote particle formation and pulmonary deposition. The nebulized formulation may contain the lyase chimera or variant thereof in a sterile buffer solution and may contain surfactants or other excipients to ensure stable nebulization.

[0118] In this invention, as one embodiment, the invention provides a topical preparation containing a phage lysin chimera or a variant thereof.

[0119] As one embodiment, the topical formulation includes a gel or ointment formulation. As an example, a phage lysin chimera or a variant thereof may be incorporated into a gel or ointment for topical application. The formulation includes a gelling agent, such as carbomer, hydroxyethyl cellulose, or sodium alginate, and an emulsifier to facilitate the delivery of the phage lysin chimera or a variant thereof to the target site.

[0120] As one embodiment, the topical formulation includes creams and emulsions. As an example, the cream or emulsion can be prepared by emulsifying a lyase chimera or a variant thereof in a water-in-oil or oil-in-water matrix. Common emulsifiers include cetyl alcohol, stearyl alcohol, and polysorbate.

[0121] As one embodiment, the topical formulation includes a spray formulation. As an example, a suitable propellant (for aerosol spraying) or a solvent system that promotes evaporation (such as ethanol or isopropanol) can be used to incorporate the lyase chimera or a variant thereof into the sprayable formulation.

[0122] As one embodiment, the topical formulation includes a powder formulation. As an example, dry powder formulations may be used in certain situations, particularly for wound care or treatment of skin conditions. Lysase chimeras or variants thereof may be combined with excipients such as starch or cellulose to achieve the desired consistency and ease of use.

[0123] In one embodiment of this invention, a mouthwash or mouthwash formulation containing a phage lysin chimera or a variant thereof is provided. As an example, the lysin chimera or variant thereof can be formulated into a mouthwash or mouthwash formulation and dissolved or suspended in an aqueous medium containing excipients such as glycerol, sorbitol, or alcohol to improve stability and solubility. The formulation may include flavoring agents, preservatives, and buffers to improve palatability and shelf life.

[0124] In one embodiment of this invention, a nasal spray or gel formulation containing a phage lysin chimera or a variant thereof is provided. As an example, the lysin chimera or variant thereof can be delivered via the nose in the form of a spray or gel. A method for preparing a nasal spray formulation typically involves placing the lysin chimera or variant thereof in a buffered saline solution, and penetration enhancers or stabilizers may be added to ensure optimal absorption and activity. The gel formulation can be designed to adhere to the nasal mucosa to prolong the release time.

[0125] In this invention, as one embodiment, a solid dosage form containing a phage lysin chimera or a variant thereof is provided:

[0126] Lysozyme chimeras or variants thereof can be formulated into solid dosage forms such as, but not limited to, tablets, capsules, or oral granules. As an example, lysozyme chimeras or variants thereof can be combined with excipients such as fillers (e.g., lactose, microcrystalline cellulose), binders (e.g., hydroxypropyl cellulose), disintegrants (e.g., croscarmellose sodium), and stabilizers to maintain the integrity of the lysozyme during storage and ensure effective release after administration.

[0127] As one implementation method, solid dosage forms containing phage lysin chimeras or variants also include sustained-release formulations: such as enteric coatings or controlled-release matrix systems, which can be used to protect the lysin from degradation in the acidic environment of the stomach and release it into the intestines or other target sites. Such formulations can be achieved by using excipients such as cellulose acetate, phthalates, or other enteric polymers.

[0128] In one embodiment of the present invention, the present invention provides a formulation of an antibacterial agent or disinfectant containing a phage lysin chimera or a variant thereof.

[0129] As an example, lyase chimeras or variants thereof can be formulated in combination with other antimicrobial agents, disinfectants, or antiseptics to produce a synergistic effect. This may include the addition of drugs such as chlorhexidine, benzalkonium chloride, or iodine-based compounds. This combination is particularly suitable for wound care (including open surgical wounds), oral hygiene products, or topical applications that require both lyases and antimicrobial agents to address infection and promote healing.

[0130] In this invention, as one embodiment, the invention provides another special formulation containing a phage lysin chimera or a variant thereof.

[0131] In addition to the formulations described above, the lyase chimera or variants of the present invention can also be applied to other specific dosage forms, such as transdermal patches, sustained-release formulations, or implantable drug delivery systems. These formulations will utilize a variety of polymers and excipients designed to release the lyase chimera or variants of the present invention in a controlled manner over time.

[0132] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to treat a variety of microbial infections.

[0133] In this invention, as one embodiment, the bacterial infection refers to a respiratory tract infection, such as pneumonia, bronchitis, sinusitis, pharyngitis, tonsillitis, and tuberculosis. This lung infection includes, but is not limited to, patients with lower respiratory tract and / or upper respiratory tract infections.

[0134] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to treat, control, or prevent bacterial infection or colonization in patients with an increased risk of bacterial infection or chronic bacterial colonization of the lungs. Such patients include, but are not limited to, patients with cystic fibrosis, patients with chronic obstructive pulmonary disease (COPD), patients with primary viral infections such as influenza or COVID-19, patients in intensive care units, patients on ventilators, patients with compromised immune systems, patients taking immunosuppressive drugs, and other patients with impaired lung function.

[0135] In this invention, as one embodiment, the lyase chimera or its variant is used to treat community-acquired pneumonia or hospital-acquired pneumonia.

[0136] In this invention, as one embodiment, the infectious diseases treated by the lyase complex or its variants include urinary tract infections such as cystitis, pyelonephritis, prostatitis, and urethritis.

[0137] In this invention, as one embodiment, the disease treated by the lyase chimera or its variant is a gastrointestinal infection, such as gastroenteritis, enteritis, enterocolitis, or food poisoning.

[0138] In this invention, as one embodiment, the bacterial infection treated by the lyase chimera or its variant is a skin and soft tissue infection, such as cellulitis, impetigo, folliculitis, furunculosis, carbuncle, erysipelas, necrotizing fasciitis, muscle necrosis, and wound infection.

[0139] In this invention, as one embodiment, the lyase chimera or its variant is used to treat bloodstream infections such as sepsis, bacteremia, endocarditis, and septicemia.

[0140] In this invention, as one embodiment, the lyase chimera or a variant thereof treats an infection of bone (osteomyelitis) or joint.

[0141] In this invention, as one embodiment, the infection treated by the lyase chimera or its variant is a sexually transmitted infection.

[0142] In this invention, as one embodiment, the infection refers to meningitis, encephalitis, osteomyelitis, purulent arthritis, peritonitis, pericarditis, abscess, or other types of Gram-positive bacterial infections.

[0143] In this invention, as one embodiment, the infection refers to an infection associated with the indwelling device. Such devices further include catheters, such as urinary catheters, venous catheters, arterial catheters, spinal catheters, peritoneal catheters, dialysis catheters; intravenous (IV) catheters, such as peripherally inserted venous catheters, central venous catheters, peripherally inserted central venous catheters (PICC), midline catheters; surgical mesh; ventilators; pacemakers; implantable cardioverter defibrillators; central venous catheters: tunneled central venous catheters, implantable infusion sets; gastrostomy tubes, such as percutaneous endoscopic gastrostomy (PEG) tubes, radially inserted... RIG tubes; tracheostomy tubes, such as tracheostomy tubes with and without cuffs; nerve stimulators, such as spinal cord stimulators, deep brain stimulators, and vagus nerve stimulators; artificial heart valves; vascular access devices, such as arteriovenous (AV) fistulas and AV grafts; tympanostomy tubes; hemodialysis catheters; feeding tubes, such as nasogastric tubes, nasojejunal tubes, and jejunostomy tubes; drainage tubes, such as chest tubes, abdominal drainage tubes, biliary drainage tubes, and pancreatic drainage tubes.

[0144] In this invention, as one embodiment, the lyase chimera or its variants are used to treat, control or prevent bacterial infections or colonization in patients whose risk of bacterial infection is increased due to impaired immune systems, including but not limited to patients taking immunosuppressive drugs, HIV patients, and patients whose immune systems are weakened due to genetic abnormalities.

[0145] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to prevent infection in high-risk individuals among patients with increased risk of infection with Gram-positive pathogens. Some non-limiting examples include patients with severe burns, cystic fibrosis, chronic obstructive pulmonary disease (COPD), mechanical ventilation, immunocompromised states, hospitalization, mechanical ventilation, indwelling catheters, and surgical procedures.

[0146] In this invention, as one embodiment, the lyase chimera or a variant thereof is used as a combination therapy together with another antibacterial agent or preservative.

[0147] In this invention, as one embodiment, the lysin chimera or a variant thereof is administered together with a reagent that enhances bacterial membrane permeability.

[0148] In this invention, as one embodiment, the lyase chimera or a variant thereof is administered co-administered with an antibacterial agent effective against the target bacteria. This includes co-formulation with standard therapeutic antibiotics to prevent the development of resistance to these antibiotics.

[0149] In this invention, as one embodiment, the lyase chimera or a variant thereof is administered co-administered with an antimicrobial agent. When the antimicrobial agent is administered alone, the organism is classified as resistant to that antimicrobial agent; however, the combination of the antimicrobial agent and the lyase chimera or a variant thereof is considered effective. This includes co-formulation with standard therapeutic antibiotics to resensitize resistant bacteria to the standard therapeutic antibiotics.

[0150] In this invention, as one embodiment, the lyase chimera or a variant thereof is used in combination with an antibacterial disinfectant.

[0151] In this invention, as one embodiment, the lyase chimera or a variant thereof is used as an antibacterial disinfectant. The use of such disinfectants may include, but is not limited to, eliminating targeted bacteria on inanimate object surfaces, hospital walls and equipment, surgical instruments, sutures, and other equipment. Similarly, the disinfectant can be used on living objects, including but not limited to the skin and mucous membrane surfaces of humans and animals, including the anterior nasal cavity.

[0152] In this invention, as one embodiment, the lyase chimera or a variant thereof is formulated into a topical preparation for use on the skin or mucous membrane surface. Such topical preparations include, but are not limited to, gels, creams, ointments, powders, and sprays.

[0153] In this invention, as one embodiment, the lyase chimera or a variant thereof is embedded in a wound dressing material.

[0154] In this invention, as one embodiment, the lyase chimera or a variant thereof is coated onto an implantable device.

[0155] In this invention, as one embodiment, the lyase chimera or variant thereof is used in combination, or one or more lyase chimeras or variants thereof described in this invention are used together with additional lyase chimeras or variants thereof from different sources, or any of the above combinations may be used except for another antimicrobial agent or preservative.

[0156] In this invention, as one embodiment, the lyase chimera or its variants are encapsulated or modified in a manner that improves their pharmacokinetic properties.

[0157] In this invention, as one embodiment, the lyase chimera or a variant thereof is used as a food preservative to prevent or eliminate contamination by target bacteria.

[0158] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to combat bacterial biofilms, such as in host tissues, bones, mucosal tissues, lungs, or in connection with indwelling devices or catheters.

[0159] In this invention, as one embodiment, the lyase chimera or a variant thereof is used to alter the composition of bacterial communities in or on humans and / or animals and / or inanimate objects, such as objects that can be used as recolonization reservoirs. An example of this alteration of the bacterial community is the treatment of dysbiosis within a patient's microbiome, where an excess of target organisms causes or puts the patient at risk, and treatment with the lyase chimera or a variant thereof of the present invention can eliminate or reduce the abundance of target organisms, thereby resolving or reducing adverse symptoms or risks. Attached Figure Description

[0160] Figure 1: The amino acid sequences of ClyO and ClyF were compared with the chimeric polypeptide sequences ClyX-VER1 to ClyX-VER9 (including intermediate constructs) of the present invention using Clustal Omega and adjusted to clearly show the differences in the linker regions.

[0161] Figure 2: 2A: SDS-PAGE electrophoresis analysis of purified ClyO, ClyF and ClyX-VER1 to ClyX-VER7 lyase chimeric peptides; 2B: SDS-PAGE electrophoresis comparison of soluble and insoluble components of ClyX-VER8 and ClyX-VER9 induced expressed proteins;

[0162] Figure 3: Data on the lysis kinetics of ClyX-VER1 to ClyX-VER7 lysin chimeric peptides against Staphylococcus aureus using the OD reduction method;

[0163] Figure 4: Determination of the antibacterial activity of the ClyO, ClyF, ClyX-VER1 to ClyX-VER7, and ClyX-VER1.2.1 and ClyX-VER4.1.1 lyase chimeric peptides of the present invention using the minimum inhibitory concentration method;

[0164] Figure 5: Evaluation of the residual activity of the ClyX-VER1 to ClyX-VER7 lysin chimeric peptides of the present invention after incubation at 37°C for 2 months using the streak plate method;

[0165] Figure 6: Evaluation of the residual activity of the ClyX-VER1 to ClyX-VER7 lysin chimeric peptides of the present invention after incubation at 37°C for 2 months using the OD reduction method;

[0166] Figure 7: AlphaFold structure prediction based on the primary amino acid sequence of the lyase chimeric polypeptide;

[0167] Figure 8: 8A: Comparison of thermostability of ClyX-VER1 to ClyX-VER9, ClyX-VER1.2.1, ClyX-VER4.1.1, ClyF, and ClyO lyases; Figure 8B: Thermostability data of some modified ClyO variants, of which ClyO+C-ter-KK, ClyO+C-ter-KKK, ClyO+C-ter-RR, and ClyO+C-ter-RR maintained full activity, while ClyO+C-ter-K and ClyO+C-ter-R partially maintained activity; Figure 8C: Comparison of thermostability data of some modified ClyX variants;

[0168] Figure 9: SDS-PAGE electrophoresis analysis of purified ClyO+C-ter-K, ClyO+C-ter-KK, ClyO+C-ter-KKK, ClyO+C-ter-R, ClyO+C-ter-RR and ClyO+C-ter-RRR lyases.

[0169] Figure 10: A: SDS-PAGE electrophoresis analysis of purified ClyX-VER1.1, ClyX-VER1.2, ClyX-VER2.1, ClyX-VER3.1, ClyX-VER3.2, ClyX-VER4.1, ClyX-VER4.2, ClyX-VER4.3 and ClyX-VER6.1 lyases; B: SDS-PAGE electrophoresis analysis of purified ClyX-VER6.2 lyase; C: SDS-PAGE electrophoresis analysis of purified ClyX-VER1.2.1 and ClyX-VER4.1.1 lyases.

[0170] Figure 11: Determination of the antibacterial activity of the ClyX-VER1.1, ClyX-VER1.2, ClyX-VER2.1, ClyX-VER3.1, ClyX-VER3.2, ClyX-VER4.1, ClyX-VER4.2, ClyX-VER4.3, ClyX-VER6.1 and ClyX-VER6.2 lyase chimeric peptides of the present invention using the minimum inhibitory concentration method;

[0171] Figure 12: SDS-PAGE electrophoresis analysis of the lyase chimeric peptide;

[0172] Figure 13: SDS-PAGE electrophoresis analysis of the lyase chimeric peptide;

[0173] Figure 14: Determination of the antibacterial activity of the lysin chimeric polypeptide of the present invention (using the minimum inhibitory concentration method);

[0174] Figure 15: Determination of the antibacterial activity of the lysin chimeric polypeptide of the present invention (using the minimum inhibitory concentration method);

[0175] Figure 16: Activity retention rate assessment of the lysin chimeric polypeptides ClyF, ClyX-VER1, ClyX-VER1.1, ClyX-VER1.2, ClyX-VER1.2.1, ClyX-VER2, ClyX-VER2.1, ClyX-VER3, ClyX-VER3.1, ClyX-VER3.2, ClyX-VER4, ClyX-VER4.1, ClyX-VER4.1.1, ClyX-VER4.2, ClyX-VER4.3, ClyX-VER6, ClyX-VER6.1, ClyX-VER6.2, ClyX-VER7 and ClyO of the present invention: —After accelerated degradation at 25°C for 2 months under container sealing system specially designed for inducing stress, their activity was determined by absorbance (OD) reduction method;

[0176] Figure 17: Activity retention rate assessment of the lysin chimeric polypeptides ClyX-VER3.2, ClyX-VER3.2+C-ter-K, ClyX-VER3.2+C-ter-KK, ClyX-VER3.2+C-ter-KKK, ClyX-VER3.2+C-ter-R, ClyX-VER3.2+C-ter-RR, ClyX-VER3.2+C-ter-RRR, ClyX-VER1+C-ter-KK, ClyX-VER3+C-ter-KK, ClyX-VER4+C-ter-KK, ClyX-VER6+C-ter-KK, and ClyX-VER5+C-ter-KK of the present invention—their activity was determined by the absorbance (OD) reduction method after accelerated degradation at 45°C for one month. Detailed Implementation

[0177] The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the invention in any way.

[0178] Example 1: Bacterial strains and growth conditions

[0179] Escherichia coli DH5α strain was used for routine transformation. Escherichia coli BL21(DE3) was used for protein expression. Escherichia coli strains were routinely cultured on Luria Bertani (LB) agar or LB medium supplemented with appropriate antibiotics as an alternative. Protein expression was induced in LB medium by the addition of 1 mM IPTG (isopropyl β-D-1-thiogalactoside) in the presence of appropriate antibiotics. Alternatively, ZYM-5052 was used according to the published use of Studier et al. [studier, FW (2005). “Protein expression by auto-induction in high-density shaking culture” Protein expression purification 41(1): 207-234]. Staphylococcus aureus strain ATCC25923 was cultured on trypsin-soybean agar (TSA) plates or trypsin-soybean broth (TSB) medium. The media were purchased from Becton, Dickinson.

[0180] Example 2: Synthetic Design

[0181] This embodiment designed lyase chimeric peptides (ClyX-VER1-ClyX-VER9, SEQ ID NO:5-37, Table 1) containing a catalytic domain (SEQ ID NO:3), a binding domain (SEQ ID NO:4), and different types of linker regions. These novel lyase chimeric peptides were compared with two known lyase peptides in the art: ClyO (SEQ ID NO:1) and ClyF (SEQ ID NO:2). Notably, the ClyF linker also has two additional non-natural amino acids compared to the original domain, which are derived from the restriction sites used to connect the two domains.

[0182] To visually demonstrate the different lyase chimeric peptides used in this study, the amino acid sequences of ClyO, ClyF, and the constructed novel peptide sequences ClyX-VER1 to ClyX-VER9 (including intermediate constructs) were compared with Clustal Omega and adjusted to clearly show the differences in the linker regions, as shown in Figure 1.

[0183] Table 1:

[0184] Example 3 Construction of expression plasmid

[0185] Plasmids consisting of the pET28b(+) (MilliporeSigma, Novagen) vector and each nucleotide sequence of this invention were constructed using standard molecular cloning procedures, including standard techniques for DNA digestion, ligation, transformation, and screening, adhering to recommended reaction conditions and quality control, and following the established protocols outlined in *Molecular Cloning: A Laboratory Manual* [Green MR, Sambrook, J. (2012). *Molecular Cloning: A Laboratory Manual*. Vol. 3. 4th Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.]. Plasmid purification was performed using the EZNA Plasmid Mini Kit I (Omega Bio-tek). Restriction endonucleases used for DNA manipulation were obtained from ThermoFisher Sciences. PCR amplification was performed using PrimeSTAR high-fidelity DNA polymerase (TaKaRa). PCR purification was performed using the EZNA Cyclic Purity Kit (Omega Bio-tek). DNA gel extraction was performed using the EZNA Gel Extraction Kit (Omega Bio-tek). The plasmids were transformed into *E. coli* using a heat shock procedure, following the protocols described in the 4th edition of *Molecular Cloning: A Laboratory Manual*. Gene synthesis and sequencing were performed at Wuhan Sangon Biotech Co., Ltd. The plasmid was initially constructed in *Escherichia coli* DH5α strain and its sequence was verified by sequencing both DNA strands. Subsequently, a plasmid was produced for the host strain and transformed into *E. coli* strain BL21(DE3) to induce protein expression and protein purification.

[0186] Nucleotide sequences encoding ClyO (SEQ ID NO:38), ClyF (SEQ ID NO:39), and novel lyase chimeric peptides ClyX-VER1 to ClyX-VER9 (SEQ ID NO:42-74) were created in pET28b, allowing the sequences to be expressed in their native form without any fusion markers. The nucleotide sequences of the lyase chimeric peptides are shown in Table 2. In each case, a stop codon was introduced immediately following the presented sequence, but it is not shown in the sequences in Table 2.

[0187] Table 2

[0188] The amino acids of the relevant linker variants described in this invention are shown in Table 3:

[0189] Table 3:

[0190] Both ClyO and ClyF linker regions bind different portions of two linker regions derived from the parental lysin, thus each linker is associated with either the original catalytic domain or the binding domain of ClyO. In some embodiments, different portions of each of these parental linker regions are selected to form the final linker region. In some embodiments, the linker region is derived solely from the linker regions of the catalytic or binding domains.

[0191] Table 4 lists the different variants of adaptor peptides associated with the catalytic domain, including the amino acid sequences shown in P, PA, PAY and any one of SEQ ID NO:113 to SEQ ID NO:121. Table 5 lists the different variants of adaptor peptides associated with the binding domain, including the amino acid sequences shown in G, AG, LAG and any one of SEQ ID NO:125 to SEQ ID NO:137.

[0192] Therefore, a linker polypeptide selected from P, PA, PAY, or SEQ ID NO:113-121 is typically combined with a linker region selected from G, AG, LAG, or SEQ ID NO:125-137 to produce the final linker polypeptide for the lysin chimeric polypeptide of the present invention. In some cases, an additional linker amino acid is also included between the catalytic domain-associated linker region and the binding domain-associated linker region. Such added amino acids include, for example, glycine (G) and glycine-glycine (GG).

[0193] Table 4:

[0194] Table 5:

[0195] Example 4 Protein Purification

[0196] E. coli BL21(DE3) containing the expression plasmid with the polynucleotide sequence of this invention was streaked on LB agar plates containing 50 mg / L kanamycin and grown at 37°C for 16-20 hours to obtain single colonies. For each lyase chimeric polypeptide, colonies were inoculated into 10 mL of LB medium containing 50 mg / L kanamycin in a 125 mL flask and incubated at 37°C and 200 RPM for 16-20 hours. The overnight culture was diluted 1:200 into four 2 L flasks, each containing 250 mL of LB medium containing 50 mg / L kanamycin. The flasks were incubated at 37°C and 200 RPM for 3 hours, IPTG was added to the flasks to a final concentration of 1 mM, and the flasks were then incubated at 20°C and 300 RPM for 16-20 hours. Alternatively, under similar induction conditions, bacterial cultures were grown in ZYM-5052 medium + 50 mg / L kanamycin, but without IPTG. The cultures were harvested, resuspended in 20 mM HEPES pH 7.4, and homogenized. Cell debris was removed by centrifugation at 12000 RPM for 1 hour, and the cells were filtered through a 0.22 μm filter. Proteins were purified from the crude extract using an SCG chromatograph and a combination of multi-mode, hydrophobic interaction, and anion exchange columns, eluting the proteins in the first two columns using a salt gradient and then eluting the proteins in the last column using a straight-through elution. The purified lyase chimeric peptide was analyzed by SDS-PAGE as shown in Figure 2A. The peptides of the invention were purified according to the method of the invention. The purified lyase sample was added to a 10% SDS gel and run at 150 V for 1 hour. The gel was stained with Coomassie Brilliant Blue and the stain was removed for photographing. The proteins added to the gel from left to right are as follows: protein molecular weight marker, ClyO, ClyX-VER1, ClyF, ClyO-VER2, ClyO-VER3, ClyO-VER4, ClyO-VER5, ClyO-VER6, ClyO-VER7, protein molecular weight marker. All lyase chimeric peptides yielded bands of the expected size, indicating a purity >95% (Figure 2A). Protein concentrations were calculated using the extinction coefficients of individual proteins based on the absorbance at A280, and these concentration values ​​were used in all further experiments. As shown in Figure 2B, *E. coli* cells containing ClyX-VER8 and ClyX-VER9 expression plasmids were induced to express the proteins, lysed by sonication, and soluble and insoluble proteins were separated by centrifugation. Each fraction was loaded onto an SDS-PAGE, stained with Coomassie Brilliant Blue, and destained for photographing. The positions of the ClyX variant bands are marked with arrows. ClyX-VER8 and ClyX-VER9 have low solubility, resulting in a low content of soluble pure protein.These solubility issues were visible on SDS-PAGE gels, comparing the soluble and insoluble protein components of the cell lysates induced by these variants (Figure 2B). Nevertheless, a small amount of lyase chimeric peptides were purified, but the concentration of pure protein was low and not visible on SDS-PAGE.

[0197] Example 5: Evaluation of the cleavage activity of novel lysin chimeric peptides using the OD reduction method.

[0198] The preparation of Staphylococcus aureus for the OD reduction experiment was as follows: Staphylococcus aureus strain CMCC26003 was streaked onto trypsin-soy agar (TSA) plates and incubated at 37°C for 16-20 hours. Single, well-isolated colonies were transferred to 5 mL of trypsin-soy broth (TSB) in 50 mL test tubes and incubated overnight at 37°C and 200 RPM. The culture was diluted 1:100 in trypsin-soy broth (TSB) to a volume suitable for the number of assays, while maintaining a liquid-to-air ratio of at least 1:5 in the flask. The culture was incubated at 37°C and 200 RPM until an OD600 of 0.7 was achieved. The culture was then washed in PBS, resuspended in PBS with 15% glycerol, aliquoted, and frozen at -80°C. Before each use, the cells were thawed, centrifuged, resuspended in PBS until a final OD600 of 1.0 was achieved, and the cells were placed in a reservoir suitable for use with multichannel pipettes.

[0199] The experimental lysin chimeric peptide was diluted in PBS to a concentration of 0.6 μg / mL, with three replicates per concentration. 100 μL of each diluted lysin chimeric peptide was added to each clear 96-well polystyrene plate, with three replicates per group. As a control, wells containing only the assay buffer (without the lysin chimeric peptide) were also included. Then, 100 μL of Staphylococcus aureus prepared as described above was rapidly added to each well using a multichannel pipette, and the plate was immediately placed in a Multiskan GO microplate reader (Thermo Scientific). The microplate reader was pre-programmed to read the OD600 values ​​of all wells per minute, with 5 seconds of agitation between each reading, for a total of 15 minutes. The microplate reader temperature was set to 37°C. Lysis of Staphylococcus aureus by the lysin chimeric peptide was characterized by a decrease in the optical density at OD600.

[0200] This method was used to evaluate the relative lytic activities of ClyO, ClyF, and ClyX-VER1–ClyX-VER7. Staphylococcus aureus CMCC26003 cells were washed and mixed with different lysin chimeric peptides to a final concentration of 0.6 μg / mL. Each sample was placed in three replicate wells in a 96-well plate and immediately incubated at 37°C using a microplate reader. OD600 was measured every minute for 15 minutes, with a 5-second oscillation between measurements. The mean OD measurement and standard deviation were calculated from three replicates at each time point (see Figure 3). The average time for bacterial OD600 to decrease to 50% of its maximum reduction was also calculated (see Table 3B); shorter times indicated faster enzyme activity.

[0201] Table 3B

[0202] A significant relationship was observed between linker length and the cleavage kinetics of different lyase chimeric peptides. ClyO (the longest linker, OD reduction time of 2.87 min) exhibited the fastest cleavage rate, followed by ClyX-VER7 (one amino acid shorter, OD reduction time of 3.48 min). ClyX-VER6 (OD reduction time of 4.52 min), ClyX-VER5 (OD reduction time of 4.64 min), and ClyX-VER4 (OD reduction time of 4.64 min) showed slower cleavage kinetics. ClyX-VER3 (OD reduction time of 5.5 min), C1yX-VER2 (OD reduction time of 5.87 min), and ClyX-VER1 (OD reduction time of 6.29 min) showed the slowest cleavage kinetics. Finally, ClyF (OD reduction time of 6.91 min) showed the slowest cleavage kinetics. As expected, the control wells without the lyase chimeric peptide did not show any decrease in optical density during the assay.

[0203] Therefore, among the different lengths of linkers evaluated, longer linkers (ClyO) clearly translated to faster lysis kinetics, while progressively shorter linkers implied progressively slower lysis kinetics. Despite the slower lysis kinetics, it is important to note that at the end of the 15-minute reaction, the wells containing all the lyase chimeras reached approximately the same final OD. 600 The value indicates that the target cells were completely lysed.

[0204] Example 6 uses the minimum inhibitory concentration (MIC) assay to evaluate the antibacterial activity of the lyase chimeric peptide.

[0205] The minimum inhibitory concentration (MIC) was determined in cationic-regulated Mueller Hinton broth (CAMHB) medium according to CLSI manual M07-A10 (January 2015). In short, Staphylococcus aureus strain CMCC26003 was streaked onto TSA plates and incubated at 35°C for 16–20 hours to obtain isolated colonies. Colonies were scraped from the plates and resuspended in PBS, with a final OD600 of 0.1. The bacteria were further diluted to 1 × 10⁻⁶ in CAMHB. 6 CFU / mL, designated as the working inoculum. Add 100 μL CAMHB to all wells in rows 2-11 of the CAMHB 96-well plate, and add 200 μL CAMHB to all wells in row 12. Add 200 μL of each lyase chimeric peptide (diluted to 64 μg / ml in CAMHB) to column 1. Serially dilute 2-fold by transferring 100 μL from column 1 to column 2 and repeating this process up to column 10 (discard excess medium, leaving column 10 with 100 μL). Column 11 is a reagent-free control, and column 12 is a sterile control. Next, add 100 μL of working inoculum to each well in columns 1-11, with no bacteria added to column 12 – the sterile control. Incubate the plates at 35°C for 16–20 hours, and establish the MIC according to the principles established in CLSI manual M07-A10 (January 2015). Each lyase chimeric peptide assay was performed three or four times, and the results are shown in Figure 4.

[0206] The results showed that in all replicate assays, the MICs of ClyF, ClyO, ClyX-VER1, ClyX-VER1.2.1, ClyO-VER4.1.1, ClyX-VER6, and ClyX-VER7 were all 2 μg / mL, while the MICs of ClyX-VER2, ClyX-VER4, and ClyX-VER5 were 1 μg / mL or 2 μg / mL. The MIC of ClyX-VER3 was 1 μg / mL. Surprisingly, although the OD value decrease assay showed that lyase chimeric peptides with longer linker peptides exhibited faster cleavage kinetics, the MIC assays, which took 16-20 hours, did not continue this trend. Instead, the lyase peptides with medium linker peptide lengths (longer than ClyF but shorter than ClyO) – especially ClyX-VER2, VER3, VER4, and VER5 – showed superior antibacterial effects compared to ClyF and ClyO. Among them, VER3 had the lowest MIC value and no obvious tailing phenomenon, and the endpoint determination was clear and definite.

[0207] Example 7-1 Accelerated Degradation Assay of Lysing Enzyme Chimeric Peptides

[0208] To evaluate the ability of different lyase chimeric peptides to retain activity in accelerated degradation assays, each lyase peptide was diluted to 35 μg / mL in acetate buffer (test buffer) containing 0.05% calcium chloride and 0.9% sodium chloride. The different lyase chimeric peptides were aliquoted into 0.5 mL aliquots and incubated at 37°C. For each lyase chimeric peptide, its activity was assessed at two time points (before and after incubation). At each time point, two methods were used to assess its activity.

[0209] In the first test method, an overnight culture of Staphylococcus aureus strain CMCC26003 was prepared. The bacteria were evenly spread onto the surface of a fresh TSA plate using a sterile swab to form uniform colonies. Next, 10 μL of each lyase chimeric peptide to be tested was dropped onto a designated location on the plate and air-dried. The plates were incubated at 35°C for 16–20 hours, and the results were photographed (Figure 5). Samples tested before the accelerated degradation test at 37°C showed very good activity on the plates, as evidenced by the inhibition zones, indicating locations where Staphylococcus aureus cells were lysed and unable to form colonies. On the other hand, after two months of incubation at 37°C, the activity of many lyase chimeric peptides decreased significantly, as evidenced by the absence of inhibition zones in these lyase chimeric peptide samples. Specifically, ClyO (plate positions 1 and 44), ClyF (plate position 17), and ClyX-VER6 (plate position 6) exhibited weak and incomplete inhibition zones. ClyX-VER1 (plate position 2) exhibited a very weak inhibition zone. On the other hand, ClyX-VER7 (plate position 7) had a slightly stronger inhibition zone, while ClyX-VER2 (plate position 3), ClyX-VER3 (plate position 4), and ClyX-VER4 (plate position 5) had very strong inhibition zones, indicating that their stability was significantly better than that of ClyO and ClyF.

[0210] Example 7-2

[0211] A second assay for evaluating the activity of lyase chimeric peptides is the OD reduction assay as described in Example 5. At each time point, before incubation or after 2 months of incubation at 37°C, aliquots of each lyase chimeric peptide sample prepared as described above were serially diluted 2-fold in PBS in a 96-well plate, with a final volume of 100 μL per well. Each plate also contained serially diluted freshly thawed ClyO standard samples in the same manner, and negative control wells containing no lyase chimeric peptides. Then, 100 μL of Staphylococcus aureus cells prepared as described in Example 5 were added to each well, and the plate was placed in a microplate reader to obtain OD reduction curves as described in Example 5. Next, for each lyase chimeric peptide, its OD reduction curve was compared with the serial dilution curves of fresh ClyO standard to determine the catalytic activity value (expressed in μg / mL units equivalent to fresh ClyO standard). For each lyase chimeric peptide, the value obtained after 2 months of incubation at 37°C was divided by the value obtained at the starting point; the resulting value represents the percentage of remaining activity relative to the initial activity. These values ​​are plotted in a bar chart to show the differences between the lyase chimeric peptides (Figure 6) and the numerical values ​​(see Table 6B).

[0212] Table 6B

[0213] Compared to ClyO (residual activity 3.1%) and ClyF (residual activity 4.4%), ClyX-VER2 (residual activity 25%) and ClyX-VER3 (residual activity 25%) showed significantly improved stability, while ClyX-VER4 (residual activity 17.7%) also showed a slight improvement. On the other hand, the stability of other constructs tested did not significantly improve after incubation at 37°C for two months: ClyX-VER1 – residual activity 1.6%, ClyX-VER1.2.1 – residual activity 4.4%, ClyX-VER4.1.1 – residual activity 4.4%, ClyX-VER5 – residual activity 2.2%, ClyX-VER6 – residual activity 2.2%, and ClyX-VER7 – residual activity 3.1%. Given that ClyX-VER2, ClyX-VER3, and ClyX-VER4 are located in a contiguous sequence space, we predict that intermediate sequences within this sequence space will also possess improved stability properties, including but not limited to: ClyX-VER2.1 (SEQ ID NO:47), ClyX-VER3.1 (SEQ ID NO:49), and ClyX-VER3.2 (SEQ ID NO:50). Based on these data, we further predict that sequences adjacent to this identified sequence space will also possess improved stability properties, including but not limited to ClyX-VER1.1 (SEQ ID NO:43), ClyX-VER1.2 (SEQ ID NO:44), ClyX-VER4.1 (SEQ ID NO:52), ClyX-VER4.2 (SEQ ID NO:54), and ClyX-VER4.3 (SEQ ID NO:55). We further predict that using the correct formulation can further improve the preservation activity of the optimal ClyX variant.

[0214] Example 8: Structural prediction of various lysin chimeric peptides

[0215] The structures of the lysin chimeric peptides of the present invention with different linkers were analyzed using Alpha Fold, and the highest-ranking ones were selected for display (Figure 7).

[0216] The structures of different lyase chimeric peptides were visualized using PyMol. All lyase chimeric peptides are presented with the catalytic domain on the left, and all lyase chimeric peptides are presented in the same orientation (unless explicitly reversed). The binding domain is located on the right, and its specific conformation in space is determined by the conformation of the catalytic domain. As shown in the figure, the invented lyase chimeric peptides exhibit significant structural differences in domain conformation compared to known ClyO and ClyF lyase chimeric peptides in the art.

[0217] To better illustrate these differences, we labeled the RT loop of the SH3 binding domain involved in substrate recognition with cyan. In known ClyF lyase chimeric peptides, short linkers result in a rigid linear conformation that prevents the catalytic and binding domains from approaching each other closely. In this case, the RT loop of the binding domain faces outward, away from the catalytic domain, and is fully exposed to the solvent.

[0218] On the other hand, the ClyO lyase chimeric peptides known in the art have long linkers that allow for a large degree of freedom in the interaction between the two domains. The favorable interaction predicted by Alpha Fold is that the two domains interact with the RT ring closer to the catalytic domain, but are still exposed to the solvent (Figure 7, see ClyO horizontally rotated 180 degrees).

[0219] On the other hand, the novel lyase chimeric peptides disclosed herein exhibit different molecular conformations, some of which form more compact proteins. Furthermore, in some of these conformations, the RT loop faces the catalytic domain, and these conformations are significantly different from the ClyO and ClyF lyase chimeric peptides known in the art.

[0220] The lyase chimeric peptides ClyX-VER2 and ClyX-VER3 exhibit a unique structure in which the RT loop faces and is closely associated with the catalytic domain. The conformation of ClyX-VER4 is similar to that of ClyX-VER2 and VER3, but the RT loop faces laterally instead of directly towards the catalytic domain. The conformation of ClyX-VER2.1 is very similar to that of ClyX-VER2, while the conformations of ClyX-VER3.2 and ClyX-VER4.1 are similar to those of ClyX-VER3. ClyX-VER3.1 shows a slight twist in the linker, but the RT loop still faces directly towards the catalytic domain. With increasing linker length, the conformations of ClyX-VER4.2 and ClyX-VER4.3 remain largely similar to those of ClyX-VER3, with the RT loop facing the catalytic domain. ClyX-VER4.1.1 introduces two glycine amino acids into the linker, which has a kink, unlike ClyX-VER3, because the RT ring does not face the catalytic domain. The linker in ClyX-VER5 is shorter, and the RT ring is generally outward-facing. With the introduction of longer linkers in ClyX-VER5.0–ClyX-VER5.5, different conformations of the domain can be observed. In ClyX-VER6, ClyX-VER6.1, ClyX-VER6.2, and ClyX-VER7, the catalytic and binding domains are clearly separated, and in all these cases, the RT ring is exposed to the solvent and does not directly face the catalytic domain, similar to ClyO. In ClyX-VER8 and ClyX-VER9, the catalytic and binding domains are not clearly separated, which may be the reason for the low solubility of these lyase chimeric peptides. As the linker extends from ClyX-VER8.0 to ClyX-VER8.5, the separation of the catalytic and binding domains becomes increasingly clear.

[0221] Example 9: The C-terminus of the lysin chimeric polypeptide has one or more additional positively charged amino acids, which can improve its thermal stability.

[0222] The thermostability assessment method for the lyase chimeric peptides of this invention is as follows: Each purified peptide was diluted to 100 μg / mL in 50 mM acetate buffer and placed into thin-walled PCR tubes, 100 μL per tube. The tubes were heated to 55°C, 57.5°C, or 60°C for 30 minutes, or placed at 4°C during this period (control). Next, each peptide was diluted to 1.6 μg / mL in PBS, and 100 μL of this diluted lyase chimeric peptide was placed in a 96-well plate. Each sample was measured four times, and the OD reduction method described above was used for assessment. In short, 100 μL of washed and resuspended Staphylococcus aureus cells were added to each well, and the plate was immediately placed in a microplate reader at 37°C. OD600 was recorded once per minute for 15 minutes, with a 5-second shake between readings. The OD600 reading at the 15-minute time point was subtracted from the control OD600 reading (without lyase), and the resulting value was called the "OD reduction delta". The final result is the average of the four replicates.

[0223] When comparing the lyase chimeric peptides with the linker variants—ClyX-VER1, ClyX-VER2, ClyX-VER3, ClyX-VER4, ClyX-VER6, ClyX-VER7, ClyX-VER8, ClyX-VER5, ClyX-VER9, ClyX-VER1.2.1, ClyX-VER4.1.1, ClyF, and ClyO (Figure 8A), it is clear that ClyX-VER8 and ClyX-VER9 exhibit lower stability compared to the other lyase chimeric peptides, as incubation at 55°C for 30 minutes leads to their inactivation, while the other lyase chimeric peptides retain activity after incubation. Other variations in linker length resulted in slight changes in residual activity after 30 minutes at 57.5°C, but none significantly improved activity compared to ClyO.

[0224] Surprisingly, the thermal stability of the polypeptides of the present invention is significantly improved when positively charged amino acids or positively charged oligopeptides are fused to the C-terminus. Various polypeptides of the present invention are modified with one or more positively charged amino acid residues added to their C-terminus.

[0225] Positively charged amino acids or polypeptides are selected from K, KK, KKK, R, RR, and RRR to obtain sequences SEQ ID NO:109~SEQ ID NO:112, SEQ ID NO:122~SEQ ID NO:124 and SEQ ID NO:138~SEQ ID NO:340 (Table 6).

[0226] When K, KK, KKK, R, RR, or RRR are fused to the C-terminus of ClyO (SEQ ID NO:1), resulting in ClyO+C-ter-K (SEQ ID NO:138), ClyO+C-ter-KK (SEQ ID NO:139), ClyO+C-ter-KKK (SEQ ID NO:140), ClyO+C-ter-R (SEQ ID NO:141), ClyO+C-ter-RR (SEQ ID NO:142), and ClyO+C-ter-RRR (SEQ ID NO:143), the new ClyO exhibits improved thermal stability in all cases. These modifications ensured that the ClyO variants retained full activity after incubation at 57.5°C for 30 minutes, and they retained full (ClyO+C-ter-KK, ClyO+C-ter-KKK, ClyO+C-ter-RR, ClyO+C-ter-RRR) or partial (ClyO+C-ter-K, ClyO+C-ter-R) activity (Figure 8B), while the original ClyO showed only weak activity after incubation under the same conditions. The SDS-PAGE electrophoresis results of these variants are shown in Figure 9.

[0227] Other peptides of the present invention also exhibit similar improvements in thermal stability. The activities of the modified peptides were compared with those of the unmodified peptides after incubation at 57.5°C for 30 minutes. The lyase chimeras or variant peptides evaluated included: ClyX-VER1 (SEQ ID NO:5), ClyX-VER1+C-ter-K (SEQ ID NO:150), ClyX-VER1+C-ter-KK (SEQ ID NO:151), ClyX-VER1+C-ter-RRR (SEQ ID NO:155), ClyX-VER3 (SEQ ID NO:11), ClyX-VER3+C-ter-K (SEQ ID NO:186), ClyX-VER3+C-ter-KK (SEQ ID NO:187), ClyX-VER3+C-ter-RRR (SEQ ID NO:191), ClyX-VER4 (SEQ ID NO:14), ClyX-VER4+C-ter-K (SEQ ID NO:204), and ClyX-VER4+C-ter-KK (SEQ ID NO:155). ID NO:205),ClyX-VER4+C-ter-RRR(SEQ ID NO:209),ClyX-VER5(SEQ ID NO:19),ClyX-VER5+C-ter-K(SEQ ID NO:234),ClyX-VER5+C-ter-KK(SEQ ID NO:235),ClyX-VER5+C-ter-RRR(SEQ ID NO:239),ClyX-VER6(SEQ ID NO:26),ClyX-VER6+C-ter-K(SEQ ID NO:276),ClyX-VER6+C-ter-KK(SEQ ID NO:277),ClyX-VER6+C-ter-RRR(SEQ ID NO:281),ClyX-VER1.2.1(SEQ ID NO:8),ClyX-VER1.2.1+C-ter-K(SEQ ID ClyX-VER1.2.1+C-ter-KK (SEQ ID NO:168), ClyX-VER1.2.1+C-ter-KK (SEQ ID NO:169), and ClyX-VER1.2.1+C-ter-RRR (SEQ ID NO:173). It was confirmed that the addition of K, KK, or RRR improved the thermal stability of ClyX-VER1, ClyX-VER3, ClyX-VER4, ClyX-VER5, ClyX-VER6, and ClyX-VER1.2.1. Furthermore, for many (but not all) modified peptides, activity after incubation at 60°C for 30 minutes also showed improved stability (Figure 8C).

[0228] Among the constructs tested in this invention, the ones showing the best improvement in stability after incubation at 60°C are ClyO+C-ter-KK (SEQ ID NO:139), ClyO+C-ter-KKK (SEQ ID NO:140), ClyO+C-ter-RR (SEQ ID NO:142), ClyO+C-ter-RRR (SEQ ID NO:143), ClyX-VER1+C-ter-KK (SEQ ID NO:151), ClyX-VER1.2.1+C-ter-KK (SEQ ID NO:169), ClyX-VER1.2.1+C-ter-RRR (SEQ ID NO:173), ClyX-VER3+C-ter-KK (SEQ ID NO:187), ClyX-VER3+C-ter-RRR (SEQ ID NO:191), and ClyX-VER4+C-ter-K (SEQ ID NO:187). ClyX-VER4+C-ter-KK (SEQ ID NO:204), ClyX-VER4+C-ter-RRR (SEQ ID NO:209), ClyX-VER5+C-ter-KK (SEQ ID NO:235), ClyX-VER6+C-ter-KK (SEQ ID NO:277), and ClyX-VER6+C-ter-RRR (SEQ ID NO:281). Based on the overall results, the optimal order for thermal stability is ClyX-VER3+C-ter-KK (SEQ ID NO:187).

[0229] We further predict that expanding the selection from K, KK, KKK, R, RR, RRR, KR, RK, KRR, KRK, KKR, RKK, RRK, and RKR will produce similar positive results. We further predict that adding a linker peptide between the C-terminus of the lyase chimera of the present invention and a positively charged amino acid or polypeptide can produce similar improvements in thermal stability.

[0230] We further predict that positively charged peptides containing more than three amino acids in length may lead to similar positive results. We further predict that, provided the added peptide is positively charged, using a peptide composed of one or more positively charged amino acids and one or more additional non-positively charged amino acids may lead to similar positive results. These predictions are based on the generalizability of our results and are derived directly from the scientific data provided.

[0231] Table 6

[0232] Example 10: Experimental characterization of variants of other lysin chimeras with intermediate-length linkers

[0233] After identifying the lyase variant with the most favorable properties, we constructed other variants with intermediate-length linkers to fully understand the effect of the linkers on lyase activity. To this end, we constructed the lyase variants ClyX-VER-1.1 (amino acid SEQ ID NO: 6, nucleotide SEQ ID NO: 43), ClyX-VER-1.2 (amino acid SEQ ID NO: 7, nucleotide SEQ ID NO: 44), ClyX-VER-2.1 (amino acid SEQ ID NO: 10, nucleotide SEQ ID NO: 47), ClyX-VER-3.1 (amino acid SEQ ID NO: 12, nucleotide SEQ ID NO: 49), ClyX-VER-3.2 (amino acid SEQ ID NO: 13, nucleotide SEQ ID NO: 50), ClyX-VER-4.1 (amino acid SEQ ID NO: 15, nucleotide SEQ ID NO: 52), ClyX-VER-4.2 (amino acid SEQ ID NO: 17, nucleotide SEQ ID NO: 54), ClyX-VER-4.3 (amino acid SEQ ID NO: 18, nucleotide SEQ ID NO: 55), and ClyX-VER-6.1 (amino acid SEQ ID NO: 27, nucleotide SEQ ID NO: 43). NO: 64), ClyX-VER-6.2 (amino acid SEQ ID NO: 28, nucleotide SEQ ID NO: 65). All of these ClyX variants were purified according to the method in Example 4, and the purity of all resulting proteins was assessed by 10% SDS-PAGE (Figure 10). For each purified protein, the minimum inhibitory concentration (MIC) was determined in cationic-adjusted Mueller Hinton broth (CAMHB) medium according to CLSI manual M07-A10 (January 2015) as described in Example 6. The results were as follows: the MIC of ClyX-VER-1.1 was 2 μg / mL, the MIC of ClyX-VER-1.2 was 2 μg / mL, the MIC of ClyX-VER-2.1 was 1 μg / mL, the MIC of ClyX-VER-3.1 was 2 μg / mL, the MIC of ClyX-VER-3.2 was 1 μg / mL, the MIC of ClyX-VER-4.1 was 1 μg / mL, the MIC of ClyX-VER-4.2 was 2 μg / mL, the MIC of ClyX-VER-4.3 was between 1 and 2 μg / mL, the MIC of ClyX-VER-6.1 was 1 μg / mL, and the MIC of ClyX-VER-6.2 was 1 μg / mL (Figure 11). Therefore, the MIC values ​​of the several construct variants tested here are superior to those of ClyO and ClyF, both of which have a MIC of 2 μg / mL.

[0234] Example 11: Purification and Characterization of Minimum Inhibitory Concentration (MIC) of a Chimeric Leptozyme with Positively Charged Amino Acids Added to its C-Terminus

[0235] This invention relates to an expression plasmid for expressing a lyase chimera with a positively charged amino acid appended at the C-terminus, constructed using the pET28b(+) vector (Merck Sigma, Novartis). The construction process was performed as described in Example 3. After transforming the plasmid into *E. coli* BL21(DE3), the expression and purification of the lyase chimera with a positively charged amino acid appended at the C-terminus were performed as described in Example 4. The purified polypeptide containing the positively charged residues was added to a 12% SDS gel and electrophoresed at 150V for 1 hour, followed by Coomassie Brilliant Blue staining as described in Example 4.

[0236] The proteins added to the first gel, from left to right, are as follows:

[0237] ClyX-VER1+C-ter-K(SEQ ID NO:150)

[0238] ClyX-VER3+C-ter-K(SEQ ID NO:186)

[0239] ClyX-VER4+C-ter-K(SEQ ID NO:204)

[0240] ClyX-VER6+C-ter-K(SEQ ID NO:276)

[0241] ClyX-VER5+C-ter-K(SEQ ID NO:234)

[0242] ClyX-VER1+C-ter-KK(SEQ ID NO:151)

[0243] ClyX-VER3+C-ter-KK(SEQ ID NO:187)

[0244] ClyX-VER4+C-ter-KK(SEQ ID NO:205)

[0245] ClyX-VER6+C-ter-KK(SEQ ID NO:277)

[0246] ClyX-VER5+C-ter-KK(SEQ ID NO:241)

[0247] ClyX-VER2.1+C-ter-K(SEQ ID NO:180)

[0248] ClyX-VER2.1+C-ter-KK(SEQ ID NO:181)

[0249] ClyX-VER2.1+C-ter-KKK(SEQ ID NO:182)

[0250] ClyX-VER3.2+C-ter-K(SEQ ID NO:198)

[0251] Protein marker, the gel is shown in Figure 12.

[0252] The second gel contains the following proteins:

[0253] ClyX-VER3.2+C-ter-KK(SEQ ID NO:199)

[0254] ClyX-VER3.2+C-ter-KKK(SEQ ID NO:200)

[0255] ClyX-VER1+C-ter-RRR(SEQ ID NO:155)

[0256] Protein Marker

[0257] ClyX-VER3+C-ter-RRR(SEQ ID NO:191)

[0258] ClyX-VER4+C-ter-RRR(SEQ ID NO:209)

[0259] ClyX-VER6+C-ter-RRR(SEQ ID NO:281)

[0260] ClyX-VER5+C-ter-RRR(SEQ ID NO:239)

[0261] ClyX-VER2.1+C-ter-R(SEQ ID NO:183)

[0262] ClyX-VER2.1+C-ter-RR(SEQ ID NO:184)

[0263] ClyX-VER2.1+C-ter-RRR(SEQ ID NO:185)

[0264] ClyX-VER3.2+C-ter-R(SEQ ID NO:201)

[0265] ClyX-VER3.2+C-ter-RR(SEQ ID NO:202)

[0266] ClyX-VER3.2+C-ter-RRR(SEQ ID NO:203)

[0267] The gel is shown in Figure 13. All proteins were well expressed and purified.

[0268] Example 12: Characterization of the minimum inhibitory concentration (MIC) of a lyase chimeric variant with a positively charged amino acid added to its C-terminus.

[0269] The activity of the C-terminus-added positively charged amino acid lyase chimera of the present invention was evaluated using the minimum inhibitory concentration (MIC) assay. This assay was performed in cationic-regulated Mueller Hinton broth (CAMHB) medium according to the requirements of the Clinical and Laboratory Standards Institute (CLSI) M07-A10 manual (January 2015 edition), with specific procedures described in Example 6.

[0270] The MICs of the lyase chimeric strains ClyX-VER1, ClyX-VER3, ClyX-VER4, ClyX-VER5, and ClyX-VER6 (each with a positively charged amino acid selected from K, KK, or RRR added to its C-terminus) of this invention were determined, and the results are shown in Figure 14. The MIC values ​​of each variant are as follows:

[0271] ClyX-VER1+C-ter-K: 4μg / mL

[0272] ClyX-VER3+C-ter-K: 2μg / mL

[0273] ClyX-VER4+C-ter-K: 2μg / mL

[0274] ClyX-VER5+C-ter-K: 8μg / mL

[0275] ClyX-VER6+C-ter-K: 4μg / mL

[0276] ClyX-VER1+C-ter-KK: 2μg / mL

[0277] ClyX-VER3+C-ter-KK: 2μg / mL

[0278] ClyX-VER4+C-ter-KK: 2-4μg / mL

[0279] ClyX-VER5+C-ter-KK: 2-4μg / mL

[0280] ClyX-VER6+C-ter-KK: 1μg / mL

[0281] ClyX-VER1+C-ter-RRR: 4μg / mL

[0282] ClyX-VER3+C-ter-RRR: 4μg / mL

[0283] ClyX-VER4+C-ter-RRR: 4μg / mL

[0284] ClyX-VER5+C-ter-RRR: 4μg / mL

[0285] ClyX-VER6+C-ter-RRR: 4μg / mL

[0286] Therefore, lyase chimeras with K or KK added to the C-terminus have excellent MIC values ​​(good antibacterial activity), while lyase chimeras with RRR added have relatively poor MIC values ​​(weak antibacterial activity) (Figure 14).

[0287] In addition, the MICs of the lyase chimeras of the present invention, ClyX-VER2.1 and ClyX-VER3.2 (each with a positively charged amino acid selected from K, KK, KKK, R, RR, or RRR added to its C-terminus), were determined, and the results are shown in Figure 15. The MIC values ​​of each variant are as follows:

[0288] ClyX-VER2.1+C-ter-K: 2μg / mL

[0289] ClyX-VER2.1+C-ter-KK: 2μg / mL

[0290] ClyX-VER2.1+C-ter-KKK: 1μg / mL

[0291] ClyX-VER3.2+C-ter-K: 2μg / mL

[0292] ClyX-VER3.2+C-ter-KK: 2μg / mL

[0293] ClyX-VER3.2+C-ter-KKK: 2μg / mL

[0294] ClyX-VER2.1+C-ter-R: 2μg / mL

[0295] ClyX-VER2.1+C-ter-RR: 1μg / mL

[0296] ClyX-VER2.1+C-ter-RRR: 2μg / mL

[0297] ClyX-VER3.2+C-ter-R: 4 μg / mL

[0298] ClyX-VER3.2+C-ter-RR: 2μg / mL

[0299] ClyX-VER3.2+C-ter-RRR: 2μg / mL

[0300] Therefore, the lyase chimeras ClyX-VER2.1 and ClyX-VER3.2 with K, KK, KKK, R or RR added to the C-terminus both have excellent MIC values ​​(good antibacterial activity); among them, the ClyX-VER2.1 chimera with R added to the C-terminus has an excellent MIC value, but the ClyX-VER3.2 chimera with R added to the C-terminus has a slightly worse MIC value (slightly weaker antibacterial activity) (Figure 15).

[0301] Example 13: Accelerated Degradation Test of Lysing Enzyme Chimeric Peptides Using a Stress-Induced Container Sealing System

[0302] For ClyF (amino acid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:39), ClyX-VER1 (amino acid sequence SEQ ID NO:42, nucleotide sequence SEQ ID NO:5), ClyX-VER1.1 (amino acid sequence SEQ ID NO:6, nucleotide sequence SEQ ID NO:43), ClyX-VER1.2 (amino acid sequence SEQ ID NO:7, nucleotide sequence SEQ ID NO:44), ClyX-VER1.2.1 (amino acid sequence SEQ ID NO:8, nucleotide sequence SEQ ID NO:45), ClyX-VER2 (amino acid sequence SEQ ID NO:9, nucleotide sequence SEQ ID NO:46), ClyX-VER2.1 (amino acid sequence SEQ ID NO:10, nucleotide sequence SEQ ID NO:47), ClyX-VER3 (amino acid sequence SEQ ID NO:11, nucleotide sequence SEQ ID NO:48), ClyX-VER3.1 (amino acid sequence SEQ ID NO:12, nucleotide sequence SEQ ID NO:49), ClyX-VER3.2 (amino acid sequence SEQ ID NO:39), ClyX-VER1 (amino acid sequence SEQ ID NO:42, nucleotide sequence SEQ ID NO:5), ClyX-VER1.1 (amino acid sequence SEQ ID NO:6, nucleotide sequence SEQ ID NO:43), ClyX-VER1.2 (amino acid sequence SEQ ID NO:7, nucleotide sequence SEQ ID NO:44), ClyX-VER1.2.1 (amino acid sequence SEQ ID NO:8, nucleotide sequence SEQ ID NO:45), ClyX-VER2 (amino acid sequence SEQ ID NO:9, nucleotide sequence SEQ ID NO:46), ClyX-VER2.1 (amino acid sequence SEQ ID NO:10, nucleotide sequence SEQ ID NO:47), ClyX-VER3 (amino acid sequence SEQ ID NO:11, nucleotide sequence SEQ ID NO:48), ClyX-VER3.1 (amino acid sequence SEQ ID NO:1 ClyX-VER4 (amino acid sequence SEQ ID NO:13, nucleotide sequence SEQ ID NO:50), ClyX-VER4 (amino acid sequence SEQ ID NO:14, nucleotide sequence SEQ ID NO:51), ClyX-VER4.1 (amino acid sequence SEQ ID NO:15, nucleotide sequence SEQ ID NO:52), ClyX-VER4.1.1 (amino acid sequence SEQ ID NO:16, nucleotide sequence SEQ ID NO:53), ClyX-VER4.2 (amino acid sequence SEQ ID NO:17, nucleotide sequence SEQ ID NO:54), ClyX-VER4.3 (amino acid sequence SEQ ID NO:18, nucleotide sequence SEQ ID NO:55), ClyX-VER6 (amino acid sequence SEQ ID NO:26, nucleotide sequence SEQ ID NO:63), ClyX-VER6.1 (amino acid sequence SEQ ID NO:27, nucleotide sequence SEQ ID NO:64), ClyX-VER6.2 (amino acid sequence SEQ ID NO:28, nucleotide sequence SEQ ID NO:65), ClyX-VER7 (amino acid sequence SEQ ID NO:13, nucleotide sequence SEQ ID NO:50), ClyX-VER4 (amino acid sequence SEQ ID NO:14, nucleotide sequence SEQ ID NO:51), ClyX-VER4.1 (amino acid sequence SEQ ID NO:15, nucleotide sequence SEQ ID NO:52), ClyX-VER4.1.1 (amino acid sequence SEQ ID NO:16, nucleotide sequence SEQ ID NO:53), ClyX-VER4.2 (amino acid sequence SEQ ID NO:28, nucleotide sequence SEQ ID NO:65), ClyX-VER7 (amino acid sequence SEQ ID NO:13, nucleotide sequence SEQ ID NO:50), ClyX-VER4 (amino acid sequence SEQ ID NO:14, nucleotide sequence SEQ ID NO:51), ClyX-VER4.1 (amino acid sequence SEQ ID NO:15, nucleotide sequence SEQ NO:29 (nucleotide sequence SEQ ID NO:66) and ClyO (amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:38) were subjected to accelerated degradation tests in a container-sealed system designed to induce protein stress, and their activity retention was determined.It should be noted that the experimental conditions were specially designed to induce stress through a mechanism different from temperature stress; therefore, the combination of these conditions and temperature stress can more comprehensively reflect the optimal stability of the lyase chimera in this invention.

[0303] In these accelerated degradation assays for container sealing systems, each lyase peptide was diluted to 35 μg / mL in acetate buffer (test buffer) containing 0.05% calcium chloride and 0.9% sodium chloride, and then aliquoted into stress-induced containers. The containers were incubated at 25°C for 2 months. The activity of each lyase chimeric peptide was assessed at two time points (before and after accelerated degradation incubation) using the quantitative absorbance (OD) reduction assay method described in Examples 7-2. At each time point (before incubation or after 2 months of incubation at 25°C), samples of each lyase chimeric peptide prepared as described in Examples 7-2 were aliquoted and serially diluted 2-fold with phosphate-buffered saline (PBS) in 96-well plates, with a final volume of 100 μL per well. Each well also contained freshly thawed ClyO standards serially diluted in the same manner, and negative control wells containing no lyase chimeric peptides. Subsequently, 100 μL of Staphylococcus aureus bacterial suspension prepared as described in Example 5 was added to each well, and the plate was placed in a microplate reader to obtain absorbance (OD) reduction curves as described in Example 5. Next, the absorbance (OD) reduction curves of each lyase chimeric peptide were compared with those of serially diluted fresh ClyO standards to determine their catalytic activity values ​​(expressed in μg / mL units equivalent to fresh ClyO standards). For each lyase chimeric peptide, the activity value obtained after 2 months of incubation under the above stress-induced conditions was divided by the activity value recorded at the initial time point; the result was the activity retention rate (expressed as a percentage of initial activity). This value is presented in bar chart form to show the differences between the various lyase chimeric peptides (Figure 16).

[0304] The experimental results show that, under the specific stress-induced conditions described above, the following chimeric peptides exhibited higher activity retention rates compared to ClyO and ClyF: ClyX-VER1.2, ClyX-VER2.1, ClyX-VER3, ClyX-VER3.1, ClyX-VER3.2, ClyX-VER4, ClyX-VER4.1, ClyX-VER4.1.1, ClyX-VER4.2, ClyX-VER4.3, ClyX-VER6, ClyX-VER6.1, ClyX-VER6.2, and ClyX-VER7.

[0305] Therefore, under the stress conditions of this container sealing system, the various peptides of the present invention outperform the parent molecules known in the art, indicating that they are more suitable for application scenarios with higher requirements for protein stability.

[0306] Example 14 Accelerated degradation test of C-terminally positively charged lyase chimeric peptide at 45°C for 1 month

[0307] The following lysin chimeric polypeptides of the present invention are: ClyX-VER3.2 (SEQ ID NO:13), ClyX-VER3.2+C-ter-K (SEQ ID NO:198), ClyX-VER3.2+C-ter-KK (SEQ ID NO:199), ClyX-VER3.2+C-ter-KKK (SEQ ID NO:200), ClyX-VER3.2+C-ter-R (SEQ ID NO:201), ClyX-VER3.2+C-ter-RR (SEQ ID NO:202), ClyX-VER3.2+C-ter-RRR (SEQ ID NO:203), ClyX-VER1+C-ter-KK (SEQ ID NO:151), ClyX-VER3+C-ter-KK (SEQ ID NO:187), ClyX-VER4+C-ter-KK (SEQ ID NO:187). ClyX-VER6+C-ter-KK (SEQ ID NO:205) and ClyX-VER5+C-ter-KK (SEQ ID NO:277) were subjected to an accelerated degradation test at 45℃ for one month, and their activity retention rate was determined.

[0308] To evaluate the activity retention of different lyase chimeric peptides in an accelerated degradation assay at 45°C for one month, each lyase peptide was diluted to 35 μg / mL in acetate buffer (test buffer) containing 0.05% calcium chloride and 0.9% sodium chloride, aliquoted into 0.5 mL centrifuge tubes, and incubated at 45°C. The activity of each lyase chimeric peptide was assessed at two time points (before and after incubation), using the quantitative absorbance (OD) reduction assay method described in Examples 7-2. At each time point (before incubation or after one month of incubation at 45°C), the lyase chimeric peptide samples prepared as described above were aliquoted and serially diluted 2-fold with phosphate-buffered saline (PBS) in 96-well plates, with a final volume of 100 μL per well. Each well also contained freshly thawed ClyO standards serially diluted in the same manner, and negative control wells containing no lyase chimeric peptides. Subsequently, 100 μL of Staphylococcus aureus bacterial suspension prepared as described in Example 5 was added to each well, and the plate was placed in a microplate reader to obtain absorbance (OD) reduction curves as described in Example 5. Next, the absorbance (OD) reduction curves of each lyase chimeric polypeptide were compared with those of a series of diluted fresh ClyO standards to determine their catalytic activity values ​​(expressed in μg / mL units equivalent to fresh ClyO standards).

[0309] For the lyase polypeptide ClyX-VER3.2 of the present invention, compared with ClyX-VER3.2 without added positive charge, the activity retention rate after incubation at 45°C for 1 month was increased by 50% after adding K, KK, or KKK to the C-terminus; in addition, the activity retention rate after incubation at 45°C for 1 month was increased by 100% after adding R to the C-terminus, and the activity retention rate after adding RR was increased by 50%, but the activity decreased after adding RRR. Other lyase polypeptides of the present invention (including ClyX-VER1+C-ter-KK, ClyX-VER3+C-ter-KK, ClyX-VER4+C-ter-KK, ClyX-VER6+C-ter-KK, and ClyX-VER5+C-ter-KK) all showed high levels of stability after incubation at 45°C for 1 month compared with the control molecule ClyX-VER3.2 without added positive charge at the C-terminus.

[0310] These results further confirm the conclusions shown in Example 9 (Figures 8B and 8C), indicating that the “improved stability of the construct with a positively charged C-terminus under short-term exposure to high temperatures (up to 60°C)” observed in Example 9 can be translated into the “improved long-term stability under high-temperature conditions” shown in this example. This improved stability at high temperatures is significant for a variety of commercial and medical applications. As a non-limiting example, such applications could include the transport and storage of consumer products or pharmaceuticals in many regions around the world—regions frequently exposed to the aforementioned high-temperature environments. Therefore, the peptides of the present invention offer significant advantages in such and similar products. We further speculate that the stability advantage conferred by the modified peptides (lysin peptides) of the present invention may also bring clinical benefits when used to treat humans or other mammals: the stability of the peptides of the present invention is improved in scenarios where body temperature (normal body temperature or high temperatures during fever) may adversely affect the stability of the lysin peptides (Figure 17).

Claims

1. A phage lysin chimera, characterized in that, The lyase chimera includes a catalytic domain, a binding domain, and a linker. The catalytic domain has the amino acid sequence shown in SEQ ID NO:3; The binding domain has the amino acid sequence shown in SEQ ID NO:4; The connector includes a connector of one or more catalytic domains or a connector of one or more binding domains or a combination thereof. The linker of the catalytic domain is selected from any linker in the amino acid sequence shown in SEQ ID NO:113 to SEQ ID NO:121; The linker of the binding domain may optionally include any linker selected from the amino acid sequences shown in SEQ ID NO:125 to SEQ ID NO:137; The connector optionally includes one, two or more glycine residues; Furthermore, the chimera does not include the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:

2.

2. The lyase chimera according to claim 1, characterized in that, The lyase chimera includes a catalytic domain of the amino acid sequence shown in SEQ ID NO:3, a binding domain of the amino acid sequence shown in SEQ ID NO:4, and a linker selected from GG or any of the amino acid sequences shown in SEQ ID NO:77 to SEQ ID NO:

108.

3. The lyase chimera according to claim 1, characterized in that, The lyase chimera comprises the amino acid sequence shown in any one of SEQ ID NO:5 to SEQ ID NO:37; preferably, the lyase chimera is the amino acid sequence shown in any one of SEQ ID NO:5 to SEQ ID NO:18 or SEQ ID NO:26 to SEQ ID NO:29; more preferably, the lyase chimera is the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:14; more preferably, the lyase chimera is the amino acid sequence shown in SEQ ID NO:

11.

4. The lyase chimera according to claim 3, characterized in that, The lyase chimera comprises any nucleotide sequence shown in SEQ ID NO:42 to SEQ ID NO:74; preferably, the lyase chimera comprises any nucleotide sequence shown in SEQ ID NO:42 to SEQ ID NO:55 or SEQ ID NO:63 to SEQ ID NO:66; more preferably, the lyase chimera comprises any nucleotide sequence shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, or SEQ ID NO:51; and more preferably, the nucleotide sequence shown in SEQ ID NO:

48.

5. A variant of a lyase chimera, characterized in that, The variants of the lyase chimera include the C-terminus of the lyase chimera according to any one of claims 1 to 4 modified with a positively charged amino acid or with a polypeptide consisting of one or more positively charged amino acids, wherein the positively charged amino acid is selected from lysine (K), arginine (R), or histidine (H), or more preferably from lysine (K) and arginine (R); preferably, the added polypeptide is selected from K, KK, KKK, R, RR, RRR, KR, RK, KRR, KRK, KKR, RKK, RRK, or RKR; more preferably, the added polypeptide is selected from K, KK, KKK, R, RR, or RRR.

6. The lyase chimeric variant according to claim 5, characterized in that, The variants of the lyase chimera include the amino acid sequences shown in any one of SEQ ID NO:109–SEQ ID NO:112, SEQ ID NO:122–SEQ ID NO:124, or SEQ ID NO:138–SEQ ID NO:340; preferably, the variants of the lyase chimera include the amino acid sequences shown in any one of SEQ ID NO:138–SEQ ID NO:299; preferably, the variants of the lyase chimera include SEQ ID NO:138–143, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:198–SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205 ... The amino acid sequence is any one of SEQ ID NO:209, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:239, SEQ ID NO:276, SEQ ID NO:277, or SEQ ID NO:281; more preferably, the variant of the lyase chimera includes any one of SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:187, SEQ ID NO:191, SEQ ID NO:198 to SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:209, SEQ ID NO:235, SEQ ID NO:277, or SEQ ID NO:281; even more preferably, the variant of the lyase chimera is SEQ ID NO:187, SEQ ID NO:199, or SEQ ID NO:

281. NO:205; Most preferably, the variant of the lyase chimera has the amino acid sequence shown in SEQ ID NO:

187.

7. The lyase chimera or a variant thereof according to any one of claims 1 to 6, characterized in that, The lyase chimera or its variants further include an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more preferably 100% identity.

8. A nucleic acid molecule encoding a lyase chimera or a variant thereof as described in any one of claims 1 to 7, wherein the nucleic acid molecule is DNA or RNA.

9. An expression vector comprising the nucleic acid molecule of claim 8, characterized in that, The expression vector is a plasmid, bacteriophage, virus, or artificial chromosome; plasmids are preferred; pET matrix particles or pBAD matrix particles are even more preferred.

10. The expression vector according to claim 9, characterized in that, The expression vector further includes a promoter, preferably having constant expression or inducible expression; more preferably an arabinose-inducible promoter, a lactose-inducible promoter or an isopropyl β-D-1-thiogalactoside (IPTG)-inducible promoter; even more preferably an IPTG-inducible promoter.

11. A host cell comprising the expression vector of claim 10, characterized in that, The host cell is a microbial cell, preferably a bacterial cell, and most preferably an Escherichia coli.

12. A method for producing the lyase chimera or a variant thereof according to any one of claims 1-7, characterized in that, The method comprises culturing the host cell population of claim 11 under conditions expressing a lyase chimera or a variant thereof, and isolating therefrom.

13. A purification method comprising the lyase chimera or a variant thereof as described in any one of claims 1-7.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the lyase chimera or a variant thereof as described in any one of claims 1-7, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, characterized in that, The pharmaceutical composition is an injection, an inhalation preparation, a topical preparation, a surgical cleanser, a spray, a mouthwash, a nasal spray, a direct nasal application preparation, a solid preparation, or a dosage form coated on clips, patches, implantable devices, sutures, wound dressings, or other objects; preferably, the injection includes an injection solution or a lyophilized powder for injection, and the topical preparation includes a gel, ointment, spray, cream, or powder.

16. The use of any one of the lyase chimeras or variants thereof, the nucleic acid molecule of claim 8, the expression vector of claim 10, the host cell of claim 11, or the pharmaceutical composition of claim 14 or 15 in the preparation of a medicament for treating or preventing the colonization and / or infection of pathogens.

17. The application according to claim 18, characterized in that, The pathogen is a Gram-positive bacterial pathogen; preferably, the pathogen is selected from one or more of the genus Staphylococcus; more preferably, it is selected from one or more of the following: Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus-MRSA and vancomycin-resistant Staphylococcus aureus-VRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus equorum, Staphylococcus capitulata, Staphylococcus albus, Staphylococcus xylose, Staphylococcus aureus, Staphylococcus hemolyticus, or Staphylococcus chromogenicus; most preferably, it is Staphylococcus aureus (including MRSA and VRSA); the pathogen includes those with drug resistance, multidrug resistance (MDR), or extensively drug-resistant (XDR); the infection includes multimicrobial infection.

18. The application according to claim 17, characterized in that, The lyase chimera or its variants are formulated or co-administered with another antimicrobial agent, antifungal agent, preservative or disinfectant, including formulation with standard care antibiotics.

19. The use of any one of the lyase chimeras or variants thereof according to claims 1-7, the nucleic acid molecule according to claim 8, the expression vector according to claim 10, the host cell according to claim 11, or the pharmaceutical composition according to claim 14 or 15 in the preparation of medicaments for treating or preventing infectious diseases, respiratory infections, gastrointestinal infections, blood infections, urinary tract infections, reproductive tract infections, skin and soft tissue infections, nervous system and brain infections, structural system and bone infections, device-related infections, immunodeficiency infections, or diseases caused by bacterial infections; wherein, The urinary tract infection further includes cystitis, pyelonephritis, prostatitis, or urethritis; the gastrointestinal infection includes gastroenteritis, enteritis, enterocolitis, food poisoning, typhoid fever, cholera, or Helicobacter pylori infection; The diseases caused by the bacterial infection include pneumonia, bronchitis, sinusitis, pharyngitis, tonsillitis, tuberculosis, meningitis, encephalitis, osteomyelitis, suppurative arthritis, peritonitis, pericarditis, abscess, botulism, tetanus, diphtheria or pertussis, as well as skin or soft tissue infections, further including cellulitis, impetigo, folliculitis, furunculosis, carbuncle, erysipelas, necrotizing fasciitis, myonecrosis or wound infection; The diseases caused by the bloodstream infection include sepsis, bacteremia, endocarditis, or septicemia; The diseases caused by the reproductive tract infections include gonorrhea, chlamydia, syphilis, chancroid, or lymphogranuloma venereum; The indwelling devices include catheters, intravenous catheters, ventilators, pacemakers, implantable cardioverter defibrillators, central venous catheters, gastrostomy tubes, tracheostomy tubes, nerve stimulators, artificial heart valves, vascular access devices, tympanostomy tubes, hemodialysis catheters, feeding tubes, or drainage tubes.

20. The use of the lyase chimera or a variant thereof as described in any one of claims 1-7, the nucleic acid molecule as described in claim 8, the expression vector as described in claim 10, the host cell as described in claim 11, or the pharmaceutical composition as described in claim 14 or 15 as a food preservative, disinfectant, bactericide, medical device, or cosmetic.

21. The use of any one of the lyase chimeras or variants thereof as claimed in claims 1-7, the nucleic acid molecule as claimed in claim 8, the expression vector as claimed in claim 10, the host cell as claimed in claim 11, or the pharmaceutical composition as claimed in claim 14 or 15 in the treatment or prevention of bacterial biofilm formation.

22. The use of any lyase chimera or variant thereof of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 10, the host cell of claim 11, or the pharmaceutical composition of claim 14 or 15 for the treatment or prevention of the formation of bacterial persistent cells or metabolically dormant cells in the presence or absence of a biofilm.

23. The use of the lyase chimera or a variant thereof as described in any one of claims 1-7, the nucleic acid molecule as described in claim 8, the expression vector as described in claim 10, the host cell as described in claim 11, or the pharmaceutical composition as described in claim 14 or 15 to decolonize target bacteria.

24. The application of the lyase chimera or variant thereof of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 10, the host cell of claim 11, or the pharmaceutical composition of claim 14 or 15 to alter the microbiome of an individual by removing unwanted bacteria while retaining desired bacteria.

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