Phage lyase or mutant thereof and application thereof

Abstract

The invention provides bacteriophage lyase or a mutant thereof. The bacteriophage lyase comprises any one of the following items: (1) the bacteriophage lyase has an amino acid sequence as shown in SEQ ID NO.1 or has at least 80% identity with the amino acid sequence as shown in SEQ ID NO.1; (2) mutation in the amino acid sequence shown in (1) and in a polypeptide structural domain, a cell wall binding structural domain and / or an amidase structural domain with cysteine and histidine-dependent aminopeptidase (CHAP) activity; the temperature stability of the bacteriophage lyase and the cracking activity of the bacteriophage lyase on staphylococcus aureus are improved, dependence of the bacteriophage lyase on refrigeration conditions is reduced, side effects caused by antibiotics can be effectively avoided, and the bacteriophage lyase can be used for preventing or treating diseases caused by the staphylococcus aureus and has good application prospects. The growth of the staphylococcus aureus in the environment, production facilities, food or animal feed is inhibited, and biological membranes generated by the staphylococcus aureus are prevented and treated.

Description

A bacteriophage lytic enzyme or its mutant and use thereof Technical Field

[0001] The present application relates to the fields of microbial technology and enzyme engineering, and in particular to a bacteriophage lytic enzyme or a mutant thereof and uses thereof. Background Art

[0002] Staphylococcus aureus, also known as golden staphylococcus, is a bacterium that is widely present in the environment and commonly colonizes human skin and nasal cavities. Under normal circumstances, S. aureus does not cause harm to health. However, when it enters the human body, it can cause infection, especially in those with immunosuppression or other medical conditions. In mild cases, it can cause skin infections, while in severe cases, it can cause systemic diseases such as pneumonia and systemic infections, posing a threat to human life. Through its own evolution or mechanisms such as horizontal gene transfer, S. aureus has developed resistance to antibiotics such as methicillin and vancomycin, making it one of the six most antibiotic-resistant bacteria in the world. This creates a significant clinical burden, increases the recurrence of infection, increases clinical costs, and even increases the mortality rate of infected patients. Therefore, it is imperative to develop a clinical treatment plan that can effectively combine antibiotics or alternative antibiotics.

[0003] Bacteriophages are viruses that parasitize bacteria, invading bacterial cells and exploiting their metabolic machinery to replicate. During replication, phages use lytic enzymes to destroy the host cell wall, releasing newly formed phages. Since the late 1910s, phages have been used to prevent and treat bacterial diseases in humans and animals. Lytic enzymes are the primary tools phages use to lyse host cells. They break down the glucans in the bacterial cell wall, rupturing the cell wall and releasing the contents and newly formed phages. Due to the unique structure of the bacterial cell wall, common antibiotics often have difficulty penetrating the cell wall and directly acting on the bacterial cell. Therefore, using phages or their lytic enzymes to disrupt the bacterial cell wall has become a new antibacterial strategy. Compared to antibiotics, phages and their lytic enzymes can overcome primary bacterial resistance barriers (such as biofilms), resulting in more effective bacterial killing. Due to their host specificity, phages or lytic enzymes can eliminate pathogens while protecting other microorganisms, thereby maintaining a balanced microbiome.

[0004] Colonization with Staphylococcus aureus is a significant factor in the pathogenesis of atopic dermatitis (AD). S. aureus infection is highly correlated with the itching and skin lesions of AD and plays a significant role in exacerbating the course of AD. Unmet clinical needs for AD include the search for safe, cost-effective, and long-term treatments that minimize potential risks for patients with mild to moderate AD and those under 12 years of age. Common immunosuppressive drugs and systemic corticosteroids are limited by both short-term and long-term side effects in patients with moderate to severe AD. Dupilumab and JAK inhibitors are highly effective and approved for use in patients aged 12 years and older with moderate to severe AD. However, their use in patients with mild AD and those under 12 years of age is not yet approved. The FDA requires a black box warning for JAK inhibitors, warning of the potential risk of serious cardiac events, cancer, and thrombosis. Therefore, patients with mild to moderate atopic dermatitis and those under 12 years of age urgently need a long-term, cost-effective, and safe treatment that can prevent and control disease progression and relapses and combat S. aureus.

[0005] Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present application is to provide a phage lytic enzyme or a mutant thereof and uses thereof.

[0007] To this end, this application provides the following technical solutions:

[0008] A bacteriophage lytic enzyme or a mutant thereof, wherein the bacteriophage lytic enzyme comprises any one of the following:

[0009] (1) having an amino acid sequence as shown in SEQ ID NO.1, or having at least 80% identity with the amino acid sequence as shown in SEQ ID NO.1;

[0010] (2) The amino acid sequence shown in (1) has a mutation in the polypeptide domain having cysteine ​​and histidine-dependent aminopeptidase (CHAP) activity, the cell wall binding domain, and / or the amidase domain.

[0011] Optionally, the amino acid sequence shown in SEQ ID NO.1 undergoes at least one of the following mutations:

[0012] 1) Mutate the 150th amino acid of the phage lytic enzyme;

[0013] 2) mutation at amino acid 282 of the phage lytic enzyme;

[0014] 3) mutation at amino acid 419 of the phage lytic enzyme;

[0015] 4) simultaneous mutations at amino acids 246 and 301 of the phage lytic enzyme;

[0016] 5) deletion of amino acids 161 to 185 of the phage lytic enzyme;

[0017] 6) Simultaneous mutations at amino acids 246, 301, and 419 of the phage lytic enzyme;

[0018] 7) Based on 6), mutations were made at the 135th, 257th and 279th amino acids of the phage lytic enzyme.

[0019] Optionally, the amino acid sequence shown in SEQ ID NO.1 undergoes at least one of the following mutations:

[0020] 1) At the 150th amino acid of the phage lytic enzyme, the amino acid residue ASP was mutated to the amino acid residue PRO;

[0021] 2) at amino acid 282 of the phage lytic enzyme, the amino acid residue GLU was mutated to the amino acid residue ALA;

[0022] 3) at amino acid 419 of the phage lytic enzyme, the amino acid residue ASN was mutated to the amino acid residue VAL;

[0023] 4) Simultaneous mutations were made at amino acids 246 and 301 of the phage lytic enzyme, with amino acid 246 GLY and amino acid 301 VAL mutated to amino acid residues CYS;

[0024] 5) deletion of amino acids 161 to 185 of the phage lytic enzyme;

[0025] 6) Simultaneous mutations were made at amino acids 246, 301, and 419 of the phage lytic enzyme; amino acid 246 GLY and amino acid 301 VAL were mutated to amino acid residue CYS, and amino acid residue ASN was mutated to amino acid residue VAL;

[0026] 7) Based on 6), mutate the 135th, 257th, and 279th amino acids of the phage lytic enzyme gene; mutate the 135th amino acid GLN to PHE, the 257th amino acid ALA to CYS, and the 279th amino acid ALA to CYS;

[0027] 8) Based on 7), a PCNP tag KRKKRKKRK was added to the C-terminus of the protein.

[0028] Biological material, having any of the following:

[0029] (1) A nucleic acid molecule encoding the bacteriophage lytic enzyme or a mutant thereof according to any one of claims 1 to 3;

[0030] (2) Primers, expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines expressing the bacteriophage lytic enzyme or mutant thereof according to any one of claims 1 to 3;

[0031] (3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in (1);

[0032] (4) A recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in (2) or (3);

[0033] (5) A recombinant microorganism or transgenic cell line containing the recombinant vector described in (2) or (3) or (4).

[0034] The bacteriophage lytic enzyme or its mutant, and the biomaterial have any of the following uses:

[0035] a. Use in the inhibition of Staphylococcus aureus for non-disease treatment;

[0036] b. Use in the preparation of a product for broad-spectrum inhibition of Staphylococcus aureus;

[0037] c. Use in the preparation of products for preventing or treating diseases caused by Staphylococcus aureus;

[0038] d. Use in the preparation of products for preventing and treating biofilms produced by Staphylococcus aureus.

[0039] Optionally, in the use in preparing a product for inhibiting Staphylococcus aureus, the product includes cosmetics, antibacterial agents, disinfectants, and cleaning agents.

[0040] Optionally, in the use in preparing a product for preventing or treating a disease caused by Staphylococcus aureus, the product comprises a medical device kit and / or a drug; and / or

[0041] In the use of the invention in preparing a product for preventing, delaying and / or treating diseases caused by Staphylococcus aureus, the diseases include skin infection, respiratory tract infection, digestive system infection, urinary system infection, joint infection, blood infection, atopic dermatitis or diabetic complications;

[0042] Optionally, the skin infection includes skin abscess, eczema or folliculitis.

[0043] Optionally, in the use in preparing a product for preventing and treating biofilm produced by Staphylococcus aureus, the product comprises an antibacterial agent, a drug, a disinfectant or a cleaning agent.

[0044] A cosmetic, a skin care product, a daily chemical product, an antibacterial agent, a medical device or a medicine, comprising the phage lytic enzyme, the phage lytic enzyme mutant, the Staphylococcus aureus phage according to claim 3 or the biomaterial according to claim 4 as an active ingredient, with or without a carrier or excipient.

[0045] The cosmetics, toiletries, daily chemicals, antibacterial agents, medical devices or drugs, wherein the dosage forms of the cosmetics, toiletries, daily chemicals, antibacterial agents or drugs include microcapsule preparations, emulsions, ointments, solutions, powders, sprays, aerosols, capsules, solids or gels, and / or they can be attached to a solid surface;

[0046] Optionally, the binding to the solid surface is by immobilization with an affinity ligand, by ionic / hydrophobic interactions, or covalent immobilization.

[0047] A method for preventing, delaying and / or treating diseases caused by Staphylococcus aureus, comprising administering the bacteriophage lytic enzyme or a mutant thereof or the biological material to a subject in need thereof.

[0048] Optionally, the disease includes skin infection, respiratory tract infection, digestive system infection, urinary system infection, joint infection, blood infection, atopic dermatitis or diabetic infection.

[0049] Optionally, the skin infection includes skin abscess, eczema or folliculitis.

[0050] Optionally, when the disease is atopic dermatitis, the concentration of the phage lytic enzyme is 10 ug / ml to 120 ug / ml, including but not limited to 10 ug / ml, 20 ug / ml, 30 ug / ml, 40 ug / ml, 50 ug / ml, 60 ug / ml, 70 ug / ml, 80 ug / ml, 90 ug / ml, 100 ug / ml, 110 ug / ml, and 120 ug / ml. Further, the concentration of the phage lytic enzyme is 30 ug / ml.

[0051] Furthermore, the bacteriophage lytic enzyme is administered topically.

[0052] Furthermore, the dosage is based on the fingertip unit principle and applied to the skin lesions.

[0053] Furthermore, the bacteriophage lytic enzyme is administered twice or more per day.

[0054] The technical solution of this application has the following advantages:

[0055] 1. The phage lytic enzyme or its mutant provided in the present application has improved temperature stability and lytic activity against Staphylococcus aureus, reducing its dependence on refrigeration conditions.

[0056] This phage and its lytic enzyme and lytic enzyme mutants, as alternative antibiotics, can effectively avoid the side effects caused by antibiotics; they can not only be used to prevent or treat diseases caused by Staphylococcus aureus, such as skin infections, respiratory tract infections, digestive system infections, urinary system infections, joint infections, blood infections, atopic dermatitis or diabetic complications, but can also be used to inhibit the growth of Staphylococcus aureus in the environment, production facilities, food or animal feed, and prevent and control biofilms produced by Staphylococcus aureus.

[0057] 2. The phage lytic enzyme gel combination provided in this application can efficiently lyse Staphylococcus aureus in patients with atopic dermatitis, safely and effectively improving skin lesions, itching and quality of life, and is expected to become an innovative method for the long-term control and treatment of skin diseases such as atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0059] FIG1 is a band of Lys-SA001 amplified by PCR in Example 1 of the present application;

[0060] FIG2 is a color diagram showing the change in vibrational entropy of Endolysin_D150P before and after mutation in Example 1 of the present application;

[0061] FIG3 is the PCR identification result of the pET28a-6His-pp-Endolysin_D150P plasmid in Example 1 of the present application;

[0062] FIG4 is the molecular sieve purification result and QC result of Endolysin_WT in Example 1 of the present application;

[0063] FIG5 is the protein purification result of Endolysin_D150P in Example 1 of the present application;

[0064] FIG6 is a color chart showing the change in vibrational entropy of the Endolysin_E282A mutant before and after mutation in Example 1 of the present application;

[0065] FIG7 is the PCR identification result of the pET28a-6His-pp-Endolysin_E282A plasmid in Example 1 of the present application;

[0066] FIG8 is the purification result of Endolysin_E282A protein in Example 1 of the present application;

[0067] FIG9 is a color diagram showing the change in vibration entropy of the Endolysin_N419V mutant before and after mutation in Example 1 of the present application;

[0068] Figure 10 is the PCR identification result of the pET28a-6His-pp-Endolysin_N419V plasmid in Example 1 of the present application;

[0069] Figure 11 is the purification result of Endolysin_N419V protein in Example 1 of the present application;

[0070] Figure 12 is the Wild-type position of the two residues G246 and V301 in the predicted three-dimensional structure of the Aim2 domain in Example 1 of the present application;

[0071] Figure 13 is the PCR identification results of the pET28a-6His-pp-Endolysin_G246C_V301C plasmid in Example 1 of the present application;

[0072] Figure 14 is the purification result of Endolysin_G246C_V301C protein in Example 1 of the present application;

[0073] Figure 15 is the sequence alignment of Endolysin and homologous proteins in Example 1 of the present application, where the red circled region is the I161-K185 truncated sequence;

[0074] Figure 16 is a diagram of the three-dimensional predicted structure of the I161-K185 truncated flexible sequence (blue) in Example 1 of the present application, circled in red;

[0075] Figure 17 is the PCR identification results of the pET28a-6his-pp-Endolysine_del_V163-K185 plasmid in Example 1 of the present application;

[0076] Figure 18 is the purification result of Endolysine_del_V163-K185 in Example 1 of the present application

[0077] Figure 19 is the PCR identification result of the pET28a-6his-pp-Endolysine_G246C-V301C_N419V plasmid in Example 1 of the present application;

[0078] Figure 20 is the purification result of Endolysine_G246C-V301C_N419V in Example 1 of the present application;

[0079] Figure 21 shows the purification results of Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP tag in Example 1 of the present application;

[0080] FIG22 is a diagram showing the effect of Endolysine Lys-SA001 and its mutants in inhibiting S. aureus-USA300 in Example 2 of the present application;

[0081] FIG23 is a diagram showing the effect of Endolysine Lys-SA001 and its mutants in inhibiting S. aureus-USA300 in Example 2 of the present application;

[0082] FIG24 is a diagram showing the effect of Endolysine Lys-SA001 and its mutants in inhibiting S. aureus-MS3 in Example 2 of the present application;

[0083] FIG25 is a diagram showing the effect of Endolysine Lys-SA001 and its mutants in inhibiting S. aureus-MS3 in Example 2 of the present application;

[0084] FIG26 shows the changes in antibacterial activity of non-mutated Endolysine Lys-SA001 at different concentrations over time in Example 2 of the present application;

[0085] FIG27 is the antibacterial test results of Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gel in Example 4 of the present application;

[0086] FIG28 is the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine_09, Q1, and Q2 lyase gels in Example 4 of the present application;

[0087] FIG29 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-USA 300 by Endolysine_09, Q1, and Q2 lyase gels in Example 4 of the present application;

[0088] FIG30 is the experimental results of the inhibition of Staphylococcus epidermidis S.epidermidis-RP62a by Endolysine_09, Q1, and Q2 lyase gels in Example 4 of the present application;

[0089] FIG31 shows the lytic activity results of Endolysine Lys-SA001 and its mutant lytic enzymes against Lactobacillus casei Zhang-1 in Example 5 of the present application;

[0090] FIG32 shows the lytic activity results of Endolysine Lys-SA001 and its mutant lytic enzymes against Lactobacillus casei Zhang-2 in Example 5 of the present application;

[0091] FIG33 shows the lytic activity of Endolysine Lys-SA001 and its mutant lytic enzymes against Lactococcus lactis BL19-1 in Example 5 of the present application;

[0092] FIG34 shows the lytic activity results of Endolysine Lys-SA001 and its mutant lytic enzymes against Lactococcus lactis BL19-2 in Example 5 of the present application;

[0093] FIG35 shows the lytic activity results of Endolysine Lys-SA001 and its mutant lytic enzymes against Lactobacillus plantarum P8-1 in Example 5 of the present application;

[0094] FIG36 shows the lytic activity results of Endolysine Lys-SA001 and its mutant lytic enzymes against Lactobacillus plantarum P8-2 in Example 5 of the present application;

[0095] FIG37 shows the lytic activity of Endolysine Lys-SA001 and its mutants against Salmonella in Example 5 of the present application;

[0096] FIG38 shows the lytic activity of Endolysine Lys-SA001 and its mutants against Escherichia coli in Example 5 of the present application;

[0097] FIG39 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-USA 300 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gels after the first week of storage in Example 6 of the present application;

[0098] FIG40 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-USA 300 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gel after the second week of storage in Example 6 of the present application;

[0099] FIG41 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-USA 300 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gel after the third week of storage in Example 6 of the present application;

[0100] FIG42 shows the experimental results of Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gels in Example 6 of the present application for the fourth week of inhibition of Staphylococcus aureus S. aureus-USA 300;

[0101] FIG43 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gels after the first week of storage in Example 6 of the present application;

[0102] FIG44 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gel after the second week of storage in Example 6 of the present application;

[0103] FIG45 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gel after the third week of storage in Example 6 of the present application;

[0104] FIG46 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gel after four weeks of storage in Example 6 of the present application;

[0105] FIG47 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine_09, Q1, and Q2 lyase gels after the first week of storage in Example 6 of the present application;

[0106] FIG48 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-MS3 by Endolysine_09, Q1, and Q2 lyase gels after the second week of storage in Example 6 of the present application;

[0107] FIG49 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-USA300 by Endolysine_09, Q1, and Q2 lyase gels after the first week of storage in Example 6 of the present application;

[0108] FIG50 shows the experimental results of the inhibition of Staphylococcus aureus S. aureus-USA300 by Endolysine_09, Q1, and Q2 lyase gels after the second week of storage in Example 6 of the present application;

[0109] FIG51 shows the experimental results of the inhibition of Staphylococcus epidermidis-RP62a by Endolysine_09, Q1, and Q2 lyase gels after the first week of storage in Example 6 of the present application;

[0110] FIG52 shows the experimental results of the inhibition of Staphylococcus epidermidis-RP62a by Endolysine-09, Q1, and Q2 lyase gels after the second week of storage in Example 6 of the present application;

[0111] FIG53 shows the experimental results of the inhibition of Staphylococcus epidermidis-BD40 by Endolysine_09, Q1, and Q2 lyase gels after the first week of storage in Example 6 of the present application;

[0112] FIG54 shows the experimental results of the inhibition of Staphylococcus epidermidis-BD40 by Endolysine_09, Q1, and Q2 lyase gels after the second week of storage in Example 6 of the present application;

[0113] Figure 55 shows the thermal stability results of Endolysine Lys-SA001 and its mutants in Example 7 of the present application;

[0114] FIG56 shows the lytic activity of Endolysine Lys-SA001 and its mutants against S. aureus-MS3 after heat treatment in Example 7 of the present application;

[0115] FIG57 shows the facial effects of the product on patients in the clinical trial of Example 8 of the present application; the left picture is the 0th day before use, and the right picture is the 3rd day of use;

[0116] FIG58 shows the effect of use on one side of a patient's foot in the clinical trial of Example 8 of the present application; the left picture is the 0th day before use, and the right picture is the 3rd day of use;

[0117] Figure 59 shows the effect of use on the other side of the patient's foot in the clinical trial of Example 8 of the present application; the left picture is the 0th day before use, and the right picture is the 3rd day of use. DETAILED DESCRIPTION

[0118] The following examples are provided to further better understand the present application, but are not limited to the best implementation mode described herein, and do not limit the content and protection scope of the present application. Any product identical or similar to the present application obtained by anyone under the inspiration of the present application or by combining the features of the present application with other prior arts shall fall within the scope of protection of the present application.

[0119] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0120] The Staphylococcus aureus phage in this application has a deposit number of CGMCC No. 45450, and the depositor is the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 2, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is March 23, 2023.

[0121] The sequences of the primers used in the following examples are shown in the following table:

[0122] Table 1. Primers

[0123] Example 1 Bacteriophage BCSAP-005 Lyase and 9 Mutants

[0124] 1. Identification of bacteriophage BCSAP-005 lytic enzyme

[0125] The protein sequences of 223 ORFs of bacteriophage BCSAP-005 were aligned using the online tool BLASTp and the Conserved Domain Database (CDD). Bacteriophage BCSAP-005 contains 167 known functional proteins, while the remaining ORFs are hypothetical. Furthermore, genome sequencing results revealed the amino acid sequence of the lysin enzyme (Phage lysin), which is associated with lytic ability, as shown in SEQ ID No. 1, and its encoding gene as shown in SEQ ID No. 2 (hereafter referred to as Lys-SA001).

[0126] 2. Amplification of the gene encoding the bacteriophage BCSAP-005 lytic enzyme

[0127] Based on the data of the lytic enzyme (Lys-SA001) of the BCSAP-005 phage, primers for the lytic enzyme Lys-SA001 were designed using Primer Premier 5.0 software. SacI and XhoI restriction sites were added to both ends of the primers, respectively. The nucleotide sequences of Lys-SA001-F and Lys-SA001-R are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0128] Using bacteriophage BCSAP-005 DNA as a template, a 25 μL PCR reaction system was set up: 1 μL of phage DNA; 1 μL of each upstream and downstream primer, 10 pmol; 0.5 μL of DNA polymerase, 0.04 U / μL; 0.5 μL of dNTP mix, 200 μmol / L of each base; 12.5 μL of 2× Mix buffer; and 8.5 μL of ddH2O. Reaction conditions included a 5-min initial denaturation at 94°C, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 61.4°C for 1 min, and extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min. PCR amplification products were examined by 1% agarose gel electrophoresis, yielding a target band corresponding to the expected size of 1488 bp (Lys-SA001) (see Figure 1). The PCR-amplified target fragment was recovered using the SanPrep column-based DNA gel extraction kit.

[0129] 3. Transformation, Construction, and Expression of Bacteriophage BCSAP-005 Lyase and Nine Mutants

[0130] 3.1. Endolysin_D150P mutant

[0131] (1) Endolysin_D150P mutant is a wild-type lysin Lys-SA001 mutant in which the aspartic acid (Asp, D) at position 150 is mutated to proline (Pro, P). The changes in protein folding free energy (ΔΔG or DDG) before and after the mutation were simulated and calculated using Rosetta and Fold X (based on the three-dimensional structure prediction of Lys-SA001 AlphaFold2) and five algorithms: mCSM, SDM, DUET, ENCoM, and DynaMut. The following are the results:

[0132] ΔΔG ENCoM:0.051kcal / mol(Destabilizing);

[0133] ΔΔG mCSM:2.038kcal / mol(Stabilizing);

[0134] ΔΔG SDM:-0.940kcal / mol (Destabilizing);

[0135] ΔΔG DUET:2.009kcal / mol(Stabilizing);

[0136] ΔΔG DynaMut: 2.031kcal / mol (Stabilizing);

[0137] The coloring of the vibrational entropy changes of the Endolysin_D150P mutant before and after mutation is shown in Figure 2. Blue represents an increase in structural rigidity, and red represents an increase in structural flexibility.

[0138] (2) Construction of wild-type lytic enzyme Lys-SA001 (denoted as Endolysin_WT) expression plasmid pET28a-6his-pp-Endolysine:

[0139] 1) Vector Enzyme Digestion: The expression vector pET28a plasmid (purchased from Takara) was subjected to a double enzyme digestion reaction using the following digestion system: 10 μL of pET28a plasmid (100 μg / mL); 1 μL of NcoI enzyme; 1 μL of Xho1 enzyme; 2 μL of 10×M buffer; and 6 μL of ddH2O. Incubate at 37°C in a water bath for 4 h. The digestion system was verified by 1% agarose gel electrophoresis and recovered from the gel to obtain a vector with sticky ends.

[0140] 2) Ligation of the linearized vector and target gene fragment: Based on the concentration of the target gene synthesized (SEQ ID NO. 2) fragment (Goldwiz) and the concentration of the pET28a plasmid double-enzyme digestion product, determine the 10 μL ligation system: 7 μL of gel-recovered product (target gene); 1 μL of template plasmid double-enzyme digestion product; 1 μL of 10× Ligation Buffer; 1 mL of T4 DNA ligase. Ligation is carried out at 50°C for 10 min. 3) Plasmid transformation: Add 10 μL of ligation product to 100 μL of E. coli DH5α competent cells, mix gently, and incubate on ice for 30 min. Heat shock the cells in a 42°C water bath for 90 s, then quickly place on ice for 2-3 min. Add 890 μL of LB medium and incubate at 37°C for 90 min. Centrifuge at 12,000 × g for 5 min, discard 800 μL of supernatant, and resuspend the cells in LB medium. Spread 100 μL of the bacterial suspension onto a LB agar plate containing kanamycin (Kana). After complete absorption of the liquid, invert and incubate at 37°C overnight. 4) Plasmid Extraction and Identification: Randomly select a single colony from the Kana plate and inoculate it into LB liquid medium containing Kana to obtain a bacterial suspension. Identify the positive bacterial suspension by PCR. Sequencing is performed to identify plasmids from the PCR-positive bacterial suspension. Sequencing results are aligned with the target gene sequence using the Alignment tool in the Geneious software package. Results indicate that the gene inserted into the recombinant plasmid is consistent with the target gene sequence.

[0141] (3) Construction of the expression plasmid pET28a-6his-pp-Endolysine_D150P of Endolysin_D150P mutant:

[0142] Using the pET28a-6his-pp-Endolysine plasmid as a template plasmid, circular PCR was performed using designed and synthesized primers 5 and 6 to introduce mutations. PCR amplification conditions were as follows:

[0143] Table 2. Amplification conditions

[0144] A small amount of PCR amplification product (2-5 μL) was run on a 1% agarose gel to verify product molecular weight. The remaining PCR product was then digested by adding 0.5-1 μL of Dpn1 enzyme (NEB, R0176L) and incubated at 37°C for 1 hour to enzymatically digest the template plasmid. The resulting linear DNA was then recovered using a PCR product recovery kit (FastPure Gel DNA Extraction Mini Kit, DC301-01).

[0145] The recovered linear DNA was mixed with an equal volume of Vazyme ClonExpress Ultra One Step Cloning Kit (C115-01 / 2xmix), incubated at 50°C for 15 minutes, cooled at 4°C for 5 minutes, and then transformed into homemade BL21Gold (DE3) or Fast T1 competent cells and plated onto LB agar plates containing kanamycin (Kana). Cultured at 37°C overnight. A single clone was picked and inoculated into LB liquid medium containing Kana to obtain a bacterial suspension. The plasmid was extracted and sent to Genewise for sequencing and identification to ensure that the gene sequence of the recombinant plasmid was consistent with the designed target gene sequence. The plasmid related information is as follows:

[0146] Table 3. Statistics of pET28a-6His-pp-Endolysin and pET28a-6His-pp-Endolysin_D150P expression plasmid information

[0147] The extracted target plasmid was used as a template and PCR identification was performed with primers 3 and 4. The PCR amplification conditions were as shown in Table 2 (the only difference was that the primers were replaced with primers 3 and 4). The amplified product was subjected to agarose gel electrophoresis. The identification results are shown in Figure 3. The size of the band obtained by PCR amplification identification was consistent with the size of the target sequence.

[0148] (4) Expression and purification of Endolysin_D150P or wild-type lysin Lys-SA001

[0149] Plasmid transformation:

[0150] 1) Prepare LB agar plates containing the corresponding antibiotics.

[0151] 2) LB culture medium (including agar): trypsin 10g / L, NaCl 10g / L, yeast extract 5g / L, agar 15g / L

[0152] 3) Take out the EP tube containing competent cells BL21(DE3) and thaw on ice.

[0153] 4) Add 1 μL of recombinant plasmid (pET28a-6His-pp-Endolysin_D150P or pET28a-6his-pp-Endolysine) to the competent cells and gently tap the tube wall to mix.

[0154] 5) Place on ice for 30 minutes.

[0155] 6) Place the EP tube from step 5) in a 42°C circulating water bath for 90 seconds.

[0156] 7) Place the EP tube from step 6) on ice for 2 minutes.

[0157] 8) Add 1 mL of LB medium to the EP tube prepared in step 7).

[0158] 9) Place the EP tube in step 8) in a 37°C shaker (225 rpm) for 1 hour.

[0159] 10) Take an appropriate amount and evenly spread it on an LB plate containing 50 μg / mL kanamycin (Kan).

[0160] 11) Incubate the cells from step 10) at 37°C overnight.

[0161] Expression Test

[0162] 1) Three recombinant colonies were selected from each transformation plate and transferred to 5 mL of LB medium (containing 50 μg / mL Kan). The culture was incubated at 37°C, 250 rpm, and the OD600 reached 0.6-1.0. One tube was left without isopropyl-β-D-thiogalactopyranoside (IPTG) as a control. The other tubes were induced with 0.1 mM IPTG at a final concentration and incubated at 18°C ​​for 16 h.

[0163] 2) Transfer 2 mL of overnight culture to an EP tube and centrifuge at 12,000 rpm at 4°C for 1 min. Discard the supernatant and resuspend the pellet in 400 μL of lysis buffer. Sonicate the pellet in an ice bath (400 W, 1 s operation, 6 s rest, for a total of 5 cycles).

[0164] 3) Set aside 50 μL of the ultrasonically disrupted sample as the whole-bacteria sample, and centrifuge the remaining sample at 12,000 rpm, 4°C, for 5 min. Transfer the supernatant to another new EP tube, and resuspend the pellet in 350 μL of lysis buffer.

[0165] 4) Take out 50 μL of sample and place it in a new EP tube. Add an equal volume of 2× protein loading buffer. Heat in boiling water for 5 minutes and analyze by SDS-PAGE.

[0166] 5) Results: Bacteriophage BCSAP-005 lytic enzyme (Endolysin_WT) and Endolysin_D150P were expressed and were highly soluble.

[0167] Expand training

[0168] 1) Disperse a single clone into 100 μL of LB medium containing 1.5 mL of EP, spread evenly onto two 9 cm diameter Kan plates, and culture overnight in a 37°C biochemical incubator.

[0169] 2) Resuspend the cells on the Kan plate in 10 mL of LB medium and inoculate evenly into three LB culture bottles (5 L shake flask, 2 L LB medium); inoculate a total of three 2 L LB bottles from two plates; add Kan at a final concentration of 50 μg / mL, incubate at 37°C, 220 rpm to an OD600 of approximately 0.6-0.8, add IPTG to a final concentration of 0.1 mM, and induce at 18°C ​​for 16 h.

[0170] 3) Collecting bacteria: Centrifuge at 4000 rpm and 4°C for 15 min. Collect approximately 30 g of bacterial pellet and freeze at -20°C.

[0171] Protein purification

[0172] Nickel column purification:

[0173] 1) Purification column: Ni-NTA prepacked column (5mL / cv)

[0174] 2) Buffer:

[0175] Lysis Buffer: 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10% glycerol;

[0176] Ni-Wash Buffer: 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM / 50 mM imidazole, 10% glycerol;

[0177] Ni-Elution Buffer: 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 100 mM / 300 mM / 500 mM imidazole, 10% glycerol;

[0178] 3) Sample: Resuspend the cells in lysis buffer at a ratio of 1:20 (1 g of cells / 20 mL of buffer), sonicate twice in an ice bath, centrifuge at 12,000 rpm at 4°C for 90 min, and collect the supernatant.

[0179] 4) Procedure: Connect a Ni-NTA prepacked column to a 0.8 μm pore filter and equilibrate the column with lysis buffer for 2-3 column volumes. Load the sample at a flow rate of 2-3 mL / min. Rinse with Ni-wash buffer for 3-4 column volumes, then elute the target protein with Ni-elution buffer for 2-3 column volumes. Collect each fraction, take a 20 μL sample, add 4 μL of 5× loading buffer, boil at 95°C for 2 minutes, and analyze by SDS-PAGE. Collect the target fractions based on the results of the protein gel, add ppase at a 1:40 (w:w) ratio, and digest overnight at 4°C.

[0180] After enzyme digestion, reverse hanging nickel column

[0181] 1) Purification column: Ni-NTA prepacked column (5mL / cv)

[0182] 2) Buffer:

[0183] Ni-Wash Buffer: 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 30 mM imidazole, 10% glycerol;

[0184] Ni-Elution Buffer: 50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 300 mM imidazole, 10% glycerol;

[0185] 3) Sample: Dilute the imidazole concentration of the protein sample after enzyme digestion to 30 mM.

[0186] 4) Procedure: Connect a Ni-NTA prepacked column to a 0.8 µm pore filter. Equilibrate the column with Ni-wash buffer for 2-3 column volumes. Load the sample at a flow rate of 2-3 mL / min. Rinse with Ni-wash buffer for 1 column volume. Finally, elute any uncleaved proteins with Ni-elution buffer for 2-3 column volumes. Collect each fraction, aspirate 20 µL, add 4 µL of 5× loading buffer, boil at 95°C for 2 minutes, and analyze by SDS-PAGE. Verify protein cleavage and flow-through on the gel.

[0187] Cationic SP column purification

[0188] 1) Purification column: HiTrap SP HP (5mL / cv)

[0189] 2) Buffer:

[0190] SP-Buffer A: 50 mM Na2HPO4, pH 7.4, 5% glycerol;

[0191] SP-Buffer B: 50 mM Na2HPO4, pH 7.4, 1 M NaCl, 5% glycerol;

[0192] 3) Sample: The flow-through fraction after Ni column purification was diluted with SP-Buffer A to a final NaCl concentration of 100 mM.

[0193] 4) Procedure: Connect a 0.8 μm pore filter to the SP column and equilibrate the column for 5 column volumes with 50 mM Na₂HPO₄ pH 7.4, 1 M NaCl (or 100 mM NaCl for low-salt solutions), and 5% glucerol. Load the sample at a flow rate of 2-3 mL / min. Gradient elution is performed on the SP column using SP-Buffer B. Collect the fractions. After elution, sample 20 μL of each fraction and add 4 μL of 5× loading buffer. Boil at 95°C for 2 min. Analyze by SDS-PAGE. Collect the target fraction based on the protein gel results.

[0194] Molecular sieve purification

[0195] 1) Filler: Focudex 200PG;

[0196] 2) Column specifications: Generik FPLC 15x400mm;

[0197] 3) SEC-Buffer: 50 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20 (polysorbate 20), pH 7.4;

[0198] 4) Sample: Collect fractions using an SP column and concentrate to 1-2 mL.

[0199] 5) Procedure: Equilibrate the Focudex 200PG molecular sieve column with SEC-Buffer at a flow rate of 1 mL / min for one column volume. Once the salt concentration stabilizes, load the entire protein sample using a 2 mL sample loop. Continue rinsing the column with SEC-Buffer. Collect fractions in 1.4 mL increments according to UV values. After collection, remove a 2 μL sample and add 4 μL of 5× loading buffer. Boil at 95°C for 2 min. Analyze by SDS-PAGE. Collect the target fractions based on the protein gel results.

[0200] Concentration and QC identification

[0201] The target fractions of Endolysine_WT purified by molecular sieve P200 15 320 were collected, combined and concentrated, and detected by SDS-PAGE. Taking Endolysine_WT as an example, the purity was >90% (Figure 4); Implen N80 detection showed A280 = 38.46, A260 / 280 = 0.501, volume = 400 μL, quick-frozen in liquid nitrogen, and stored in a refrigerator at -80°C.

[0202] Table 4. Purification results of Endolysine_WT

[0203] Protein concentration = A280*MW / absorption coefficient = mg / mL, MW (MW stands for Molecular Weight).

[0204] The target fraction of Endolysin_D150P, which was digested and mounted on a nickel column, was collected and dialyzed against buffer (1XPBS 0.1% Tween 20, pH 7.4). The target fractions were combined and concentrated, and the purity was >90% after SDS-PAGE analysis (Figure 5). The Implen N80 analysis showed A280 = 2.19, A260 / 280 = 0.617. The volume was 1.5 mL, quick-frozen in liquid nitrogen, and stored in a refrigerator at -80°C.

[0205] Table 5 Endolysin_D150P purification results

[0206] Protein concentration = A280*MW / absorption coefficient = mg / mL.

[0207] 3.2 Endolysin_E282A mutant

[0208] (1) Endolysin_E282A mutant is a wild-type lysin Lys-SA001 in which the glutamic acid (Glu, E) at position 282 is mutated to alanine (Ala, A). The simulation calculation is as follows:

[0209] ΔΔG ENCoM:-0.369kcal / mol(Destabilizing);

[0210] ΔΔG mCSM:-1.564kcal / mol (Destabilizing);

[0211] ΔΔG SDM:0.260kcal / mol (Stabilizing);

[0212] ΔΔG DUET:-1.309kcal / mol(Destabilizing);

[0213] ΔΔG DynaMut:-0.588kcal / mol(Destabilizing);

[0214] The coloring of the vibration entropy changes before and after the Endolysin_E282A mutation is shown in Figure 6, where blue represents increased rigidity of the structure and red represents increased flexibility of the structure.

[0215] (2) Construction of the Endolysin_E282A mutant expression plasmid pET28a-6his-pp-Endolysine_E282A plasmid: The construction method is the same as the construction method of the pET28a-6his-pp-Endolysine_D150P plasmid in 3.1, except that primers 5 and 6 in the PCR system are replaced by primers 7 and 8, respectively. The constructed plasmid information is as follows:

[0216] Table 7. Statistics of pET28a-6His-pp-Endolysin_E282A expression plasmid information.

[0217] The extracted target plasmid was used as a template and PCR identification was performed using primers 3 and 4. As shown in FIG7 , the size of the band obtained by PCR amplification and identification was consistent with the size of the target sequence.

[0218] (3) Expression and purification of Endolysin_E282A mutant

[0219] The method is the same as "(4) Expression and purification of Endolysin_D150P or wild-type lyase Lys-SA001" in 3.1, except that the recombinant plasmid was replaced with pET28a-6His-pp-Endolysin_E282A, and cationic SP column purification and molecular sieve purification were omitted. Expression test results showed that Endolysin_E282A was expressed and had good solubility. After enzyme digestion, the protein obtained by reverse hanging on nickel column was concentrated and QC: the target fractions after dialysis replacement buffer (1×PBS, 0.1% Tween 20, pH 7.4) were collected, combined and concentrated, and SDS-PAGE detection showed a purity of >90% (Figure 8); Implen N80 detection showed A280 = 13.85, A260 / 280 = 0.511, volume = 1.5mL, quick-frozen in liquid nitrogen, and stored in a refrigerator at -80℃.

[0220] Table 8. Endolysin_E282A purification results

[0221] Protein concentration = A280*MW / absorption coefficient = mg / mL.

[0222] 3.3 Endolysin_N419V mutant

[0223] (1) Endolysin_N419V mutant is a wild-type lysin Lys-SA001 in which the asparagine (Asn, N) at position 419 is mutated to valine (Val, V). The simulation calculation is as follows:

[0224] ΔΔG ENCoM:0.278kcal / mol(Destabilizing);

[0225] ΔΔG mCSM:-0.510kcal / mol (Destabilizing);

[0226] ΔΔG SDM:2.360kcal / mol (Stabilizing);

[0227] ΔΔG DUET:0.383kcal / mol(Stabilizing);

[0228] ΔΔDynaMut G:1.582kcal / mol (Stabilizing);

[0229] The coloring of the vibrational entropy changes before and after the Endolysin_N419V mutation is shown in Figure 9. Blue represents an increase in structural rigidity, and red represents an increase in structural flexibility.

[0230] (2) Construction of the Endolysin_N419V mutant expression plasmid pET28a-6his-pp-Endolysin_N419V plasmid: The construction method is the same as the construction method of the pET28a-6his-pp-Endolysine_D150P plasmid in 3.1, except that primers 5 and 6 in the PCR system are replaced by primers 9 and 10, respectively. The constructed plasmid information is as follows:

[0231] Table 9. Statistics of pET28a-6His-pp-Endolysin_N419V expression plasmid information.

[0232] The extracted target plasmid was used as a template and PCR identification was performed using primers 3 and 4. The identification results are shown in FIG10 . The size of the band obtained by PCR amplification identification was consistent with the size of the target sequence.

[0233] (3) Expression and purification of Endolysin_N419V mutant

[0234] The method is the same as "(4) Expression and purification of Endolysin_D150P or wild-type lyase Lys-SA001" in 3.1, except that the recombinant plasmid was replaced with pET28a-6His-pp-Endolysin_N419V and molecular sieve purification was omitted. Expression test results showed that Endolysin_N419V was expressed and had good solubility. The protein purified by cationic SP column was concentrated and QC: the target fractions after dialysis and replacement buffer (1XPBS, 0.1% Tween 20, pH 7.4) were collected, combined and concentrated, and tested by SDS-PAGE. The purity was >90% (Figure 11); Implen N80 test showed A280 = 23.11, A260 / 280 = 0.487, volume = 1.2 mL, quick-frozen in liquid nitrogen, and stored in a refrigerator at -80℃.

[0235] Table 10. Endolysine_N419V purification results

[0236] Protein concentration = A280*MW / absorption coefficient = mg / mL.

[0237] 3.5 Endolysin_G246C_V301C double-site mutant

[0238] (1) Based on the three-dimensional structure predicted by Alpha Fold 2, Rosetta and Fold X were used to screen disulfide bond mutations on a large scale, and the changes in structural free energy before and after the mutation were calculated. It was found that the simultaneous mutation of G246 and V301 in the catalytic center of the Aim_2 domain to cysteine ​​can form a disulfide bond between the β-helix and α-helix of the Aim_2 domain (Figure 12), increase the strength of the interaction force between the subdomains, and ultimately significantly reduce the free energy of protein folding (Table 2). Therefore, we chose to mutate G246 and V301 to cysteine ​​(Cysteine, C) to obtain the Endolysin_G246C_V301C double-site mutant: the glycine (Gly, G) at position 246 of the wild-type lysin Lys-SA001 was mutated to cysteine ​​(Cys, C), and the valine (Val, V) at position 301 was mutated to cysteine ​​(Cys, C). Rosetta and Fold X were used to screen the statistical table of protein folding free energy changes before and after the disulfide bond mutation.

[0239] Table 11. Statistics of protein folding free energy changes before and after disulfide bond mutation screening by Rosetta and Fold X.

[0240] (2) Construction of the expression plasmid pET28a-6his-pp-Endolysine_G246C_V301C for the Endolysin_G246C_V301C double-site mutant: The construction method was the same as that for the pET28a-6his-pp-Endolysine_D150P plasmid in 3.1, except that primers 5 and 6 in the PCR system were replaced with primers 11 and 12, respectively. The recovered linear DNA was used as a template, and the above PCR amplification experiment was repeated using primers 11 and 13. The kit (FastPure Gel DNA Extraction Mini Kit, DC301-01) was used to recover the linear DNA obtained in the new round of amplification. The linear DNA recovered from the second round of PCR was sequenced and identified. The constructed plasmid information is as follows:

[0241] Table 12. Statistics of pET28a-6His-pp-Endolysin_G246C_V301C expression plasmid information.

[0242] The extracted target plasmid was used as a template and PCR identification was performed using primers 3 and 4. The identification results are shown in FIG13 . The size of the band obtained by PCR amplification identification was consistent with the size of the target sequence.

[0243] (3) Expression and purification of the pET28a-6His-pp-Endolysin_G246C_V301C mutant

[0244] The method is the same as "(4) Expression and purification of Endolysin_D150P or wild-type lyase Lys-SA001" in 3.1, except that the recombinant plasmid was replaced with pET28a-6His-pp-Endolysin_G246C_V301C, and molecular sieve purification was omitted. Expression test results showed that Endolysin_G246C_V301C was expressed and had good solubility. The protein purified by cationic SP column was concentrated and QC: the target fractions after dialysis and replacement buffer (1XPBS, 0.1% Tween 20, pH 7.4) were collected, combined and concentrated, and SDS-PAGE detection showed a purity of >90% (Figure 14); Implen N80 detection showed A280 = 7.139, A260 / 280 = 0.479, volume = 1.5mL, quick-frozen in liquid nitrogen, and stored in a refrigerator at -80℃.

[0245] Table 13. Endolysin_G246C_V301C purification results

[0246] 3.6 Endolysin_del_V163-K185 mutant

[0247] If the N-terminus, C-terminus, and loop region of an enzyme contain irregular coil structures or potentially flexible regions, their excessive length can negatively impact protein folding and stability. Flexible sites, often found on the surface of enzyme structures, are potential targets for modifying enzyme thermal stability.

[0248] Reference XZ.700, after further deleting a 44-residue flexible region from SA.100, further improved the enzyme's activity and thermal stability. Homology alignment and structure prediction results (Figures 15 and 16) revealed that the flexible sequence deleted in XZ.700 corresponds to the I161-K185 region of the endolysin protein. The predicted three-dimensional structure also revealed this region to be flexible, so this sequence was truncated. The resulting Endolysin_del_V163-K185 mutant represents a deletion mutation from valine (Val, V) at position 163 to lysine (Lys, K) at position 185 of the wild-type endolysin, Lys-SA001.

[0249] (2) Construction of the Endolysin_del_V163-K185 mutant expression plasmid pET28a-6his-pp-Endolysine_del_V163-K185 plasmid: The construction method is the same as the construction method of the pET28a-6his-pp-Endolysine_D150P plasmid in 3.1, except that primers 5 and 6 in the PCR system are replaced by primers 16 and 17, respectively. The constructed plasmid information is as follows:

[0250] Table 14. pET28a-6his-pp-Endolysine_del_V163-K185 expression plasmid information statistics

[0251] The extracted target plasmid was used as a template and PCR identification was performed using primers 3 and 4. The identification results are shown in FIG17 . The size of the band obtained by PCR amplification identification was consistent with the size of the target sequence.

[0252] (3) Expression and purification of the Endolysine_del_V163-K185 mutant

[0253] The method was the same as "(4) Expression and purification of Endolysin_D150P or wild-type lyase Lys-SA001" in 3.1, except that the recombinant plasmid was replaced with pET28a-6his-pp-Endolysine_del_V163-K185, and molecular sieve purification was omitted. Expression test results showed that Endolysine_del_V163-K185 was expressed and had good solubility. The proteins purified by the cationic SP column were combined and concentrated, and the purity was >90% as determined by SDS-PAGE ( FIG18 ). The 08_Endolysin Implen N80 assay showed A280 = 3.836, A260 / 280 = 0.497, and the volume was 1.2 mL. The 08_Endolysin-SP-FT; Implen N80 assay showed A280 = 3.158, A260 / 280 = 0.644, and the volume was 1.5 mL. The samples were quickly frozen in liquid nitrogen and stored at −80°C.

[0254] Table 15. Endolysine_del_V163-K185 purification results

[0255] 3.7 Endolysin_G246C-V301C_N419V mutant

[0256] Activity experiments in this application have shown that the G246C-V301C mutation increases the protein's thermal stability, while the N419V mutation increases its cleavage activity compared to the wild-type. Therefore, these two mutations were combined to create a triple mutant protein. The Endolysin_G246C-V301C_N419V mutant is a wild-type cleavage enzyme, in which the glycine (Gly, G) at position 246 of Lys-SA001 is mutated to cysteine ​​(Cys, C), the valine (Val, V) at position 301 is mutated to cysteine ​​(Cys, C), and the asparagine (Asn, N) at position 419 is mutated to valine (Val, V).

[0257] (2) Construction of the Endolysin_G246C-V301C_N419V mutant expression plasmid pET28a-6his-pp-Endolysine_G246C-V301C_N419V plasmid: The construction method is the same as the construction method of the pET28a-6his-pp-Endolysine_D150P plasmid in 3.1, except that primers 5 and 6 in the PCR system are replaced by primers 3 and 10, respectively, and the template plasmid is pET28a-6his-pp-Endolysine_G246C_V301C. The constructed plasmid information is as follows:

[0258] Table 16. pET28a-6his-pp-Endolysine_G246C_V301C_N419V expression plasmid information statistics

[0259] Using the extracted target plasmid as a template, PCR identification was performed using primers 3 and 4. The identification results are shown in Figure 19. The size of the band obtained by PCR amplification identification is consistent with the size of the target sequence.

[0260] (3) Expression and purification of the Endolysine_G246C_V301C_N419V mutant

[0261] The method was the same as "(4) Expression and purification of Endolysin_D150P or wild-type lyase Lys-SA001" in 3.1, except that the recombinant plasmid was replaced with pET28a-6His-pp-Endolysin_D150P and molecular sieve purification was omitted. Expression test results showed that Endolysine_G246C_V301C_N419V was expressed and had good solubility. The proteins obtained after purification on a cationic SP column were combined and concentrated, and SDS-PAGE analysis showed a purity of >90% (Figure 20); Endolysine_G246C_V301C_N419V Implen N80 analysis showed A280 = 64.82, A260 / 280 = 0.504, volume = 1.12 mL, quick-frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0262] Table 17, Endolysine_G246C_V301C_N419V purification results

[0263] Protein concentration = A280*MW / absorption coefficient = mg / mL.

[0264] 3.8 Endolysin_G246C-V301C_N419V_Q135F_A257C-A279C_PCNP tag mutant

[0265] Based on the Endolysin_G246C-V301C_N419V mutant, mutations at the A257C and A279C sites were added to further increase the disulfide bond formation within the Aim2 domain. At the same time, mutations at the Q135F site were added. Reference was made to other endolysins that adding a PCNP tag (KRKKRKKRK) to the C-terminus of the protein can increase protein stability. Therefore, Endolysin_G246C-V301C_N419V_Q135F_A257C-A279C_PCNP was constructed. Tag mutant: The glycine (Gly, G) at position 246 of the wild-type lyase Lys-SA001 is mutated to cysteine ​​(Cys, C), the valine (Val, V) at position 301 is mutated to cysteine ​​(Cys, C), the asparagine (Asn, N) at position 419 is mutated to valine (Val, V), the glutamine (Gln, Q) at position 135 is mutated to phenylalanine (Phe, F), the alanine (Ala, A) at position 257 is mutated to cysteine ​​(Cys, C), and the alanine (Ala, A) at position 279 is mutated to cysteine ​​(Cys, C). The PCNP tag KRKKRKKRK (K is lysine and R is arginine) is added to the C-terminus of the protein.

[0266] (2) Construction of the expression plasmid pET28a-6his-pp-Endolysin_G246C-V301C_N419V_Q135F_A257C-A279C_PCNP tag of the Endolysin_G246C-V301C_N419V_Q135F_A257C-A279C_PCNP tag mutant: The construction method is the same as that of (2) Construction of the expression plasmid pET28a-6his-pp-Endolysine of the wild-type lysin Lys-SA001 (denoted as Endolysin_WT) in 3.1, except that the target gene in "2) Connection of linearized vector and target gene fragment" is replaced by the Endolysin_G246C-V301C_N419V_Q135F_A257C-A279C_PCNP tag encoding DNA sequence, which is directly synthesized by Jinweizhi. The plasmid construction information is as follows:

[0267] Table 18. pET28a-6his-pp-Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP tag plasmid information

[0268] (3) The expression and purification method of the Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP tag mutant (Endolysine_11) was the same as that described in "(4) Expression and purification of Endolysin_D150P or wild-type lyase Lys-SA001" in 3.1, except that the recombinant plasmid was replaced with pET28a-6his-pp-Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP tag, and molecular sieve purification was omitted. Expression test results showed that Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP tag was expressed and had good solubility. The proteins obtained after purification by the cationic SP column were combined and concentrated, and the purity was >90% as determined by SDS-PAGE ( FIG. 21 ). 11_Endolysin SP Elution 1 (250 mM NaCl elution fraction) was detected by Implen N80 with A280 = 2.631, A260 / 280 = 0.584, and the volume was 0.5 mL. 11_Endolysin_SP Elution 2 (500 mM NaCl elution fraction) was detected by Implen N80 with A280 = 1.913, A260 / 280 = 0.64, and the volume was 1.0 mL. The samples were quickly frozen in liquid nitrogen and stored in a −80°C refrigerator.

[0269] Table 19 Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP tag purification results

[0270] Protein concentration = A280*MW / absorption coefficient = mg / mL.

[0271] Example 2 Antibacterial experiments of Endolysine Lys-SA001 and its mutants

[0272] The minimum inhibitory concentration of the lytic enzyme at different concentrations was determined. Staphylococcus aureus (purchased from Beijing Biobowei Biotechnology Co., Ltd.) and Staphylococcus epidermidis (isolated from Yangzhou University) stored at -80°C were removed. A small amount of bacterial liquid was dipped into a sterile 10μL inoculating loop in a biosafety cabinet. Three zones were drawn on TSA solid medium and incubated at 37°C for 12-18 hours.

[0273] Pick a single colony and place it in 2 mL of MH liquid medium. Measure the turbidity using a turbidimeter and adjust the turbidity of the culture solution to 0.5 using a McFarland tube. Use a sterile cotton swab to smear the culture solution and evenly spread it on TSA solid medium using the cross-spreading method. Dilute the lytic enzyme in the corresponding protein buffer (0.1 M phosphate buffer, pH 7.4, 10% glycosides, 200 mM NaCl, 5 mM imidazole) in half dilutions to 64, 32, 16, 8, 4, and 2 μg / mL. For each gradient, apply 10 μL of the lytic enzyme dilution solution to a plate. After the plate has dried, place it upside down in a 37°C incubator and incubate for 9 hours.

[0274] The results are shown in Figures 22 to 25. It can be seen that the unmutated Endolysine Lys-SA001, V301C, and N419V have the same lytic activity as XZ700. At an extremely low concentration of 2ug / ml, they still have lytic activity against Staphylococcus aureus S.aureus-MS3 or S.aureus-USA300. In the figure, Endolysine represents unmutated Endolysine Lys-SA001, X2700 represents Micreos' endolysin lyase, D150P represents Endolysin_D150P, E282A represents Endolysin_E282A, V301C represents Endolysin_G246C_V301C, K185 represents Endolysin_del_V163-K185, K185SP-FT represents 08_Endolysin-SP-FT, and N419V represents Endolysin_G246C-V301C_N419V.

[0275] Figure 26 Method: Determination of the antibacterial activity of Endolysine Lys-SA001 over time. A sterile 96-well microplate was placed on a clean bench. 50 μL of fresh TSB medium was added to the plate. 100 μL of Staphylococcus aureus suspension (10 8 CFU / mL) and diluted to 60, 30, 15, and 7.5 μg / mL, were added to a 96-well plate, mixed, and incubated in a 37°C incubator for 24 hours. The solution was observed for turbidity and the OD value was measured at 600 nm on a microplate reader to compare the antibacterial stability of the lyase at different time points. OD600 values ​​were read and recorded at one-hour intervals, and a 7-hour growth curve was plotted. This experiment was performed in triplicate.

[0276] FIG26 shows Endolysine Lys-SA001 without mutation. It can be seen that Endolysine Lys-SA001 lyase still has cleavage activity at an extremely low concentration of 7.5 μl / ml and for 5 hours.

[0277] Example 3 Lysine enzyme gel composition of Endolysine Lys-SA001 and mutants

[0278] This embodiment provides a lytic enzyme gel composition, comprising: lytic enzyme concentration of 30 μg / ml, 0.5 wt% sodium alginate, 3 wt% glycerol, 0.05 wt% calcium chloride, and the balance being water. The above-mentioned lytic enzyme is selected from Lys-SA001 in Example 1, XZ700 represents the endolysin lytic enzyme developed by Micreos, D150P represents Endolysin_D150P, E282A represents Endolysin_E282A, V301C represents Endolysin_G246C_V301C, 09 represents Endolysine_G246C_V301C_N419V, Q1 represents Endolysine_G246C-V301C_N419V_Q135F_A257C / A279C_PCNP tag protein Q column low salt elution fraction (11_Endolysin_SP Elution 1), Q2 represents Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP Tag protein Q column high salt elution fraction (11_Endolysin_SP Elution 2).

[0279] The preparation method of the above-mentioned lyase gel composition is to weigh the raw materials according to the formula:

[0280] 1. Add calcium chloride to water and stir until completely dissolved;

[0281] 2. Add water to the pre-sterilized and cleaned reactor according to the above ratio and keep it at 80-85℃ for 30 minutes. Then add sodium alginate and glycerin in sequence and homogenize for 3-10 minutes until the material is completely dissolved and no particles are left.

[0282] 3. During the homogenization process, gradually add the pre-dissolved solution in step 1 and stir evenly;

[0283] 4. Cool down to below 10℃, add corresponding lyase, stir evenly, and adjust to 30ug / ml;

[0284] 5. Test the physical and chemical indicators of the material (appearance: transparent colorless gel; pH: (5-7); viscosity mPa.s: (No. 4 rotor 60rpm) 2500-3500) and discharge the material only after it passes the test.

[0285] Example 4 Antibacterial Experiment of Endolysine Lys-SA001 and Mutant Lysase Gel Composition

[0286] The antibacterial test method of Endolysine Lys-SA001 and the mutant lyase gel in Example 3 is as follows:

[0287] The minimum inhibitory concentration of the lytic enzyme at different concentrations was determined. Staphylococcus aureus S. aureus-MS3 (Yangzhou University isolate) or S. aureus-USA300 (purchased from Beijing Biobowei Biotechnology Co., Ltd.) and Staphylococcus epidermidis S. epidermidis-RP62a (Yangzhou University isolate) stored at -80°C were removed. A small amount of bacterial liquid was dipped into a sterile 10μL inoculating loop in a biosafety cabinet, and three-zoned on TSA solid medium was incubated at 37°C for 12-18 hours.

[0288] Pick a single colony and place it in 2 mL of MH liquid medium. Measure the turbidity using a turbidimeter and adjust the turbidity of the bacterial solution to McFarland 0.5. Use a sterile cotton swab to smear the bacterial solution and evenly spread it on TSA solid medium using the cross-coating method. Dilute the lyase gel with the corresponding protein buffer (0.1 M phosphate buffer, pH 7.4, 10% diacylglycerol, 200 mM NaCl, 5 mM imidazole) and dilute it in half to achieve a final lyase concentration of 64, 32, and 16 μg / mL. For each gradient, apply 10 μL of the lyase dilution solution to the plate. After the plate has dried, place it upside down in a 37°C incubator and incubate for 9 hours. Among the test samples, 64, 32, and 16 μg / ml (preservative-free gel) were the lyase gels prepared according to Example 3; 64, 32, and 16 μg / ml (preservative gels) were the lyase gels prepared according to Example 3, to which a preservative was added. The preservative was added after step "5. During the homogenization process, gradually add the predissolved solution in step 1 and stir evenly." The preservative in the obtained gel was ethylparaben, with a final concentration of 0.2%.

[0289] In the antibacterial experiments using Endolysine, XZ700, V301C, DP150, E282A, and V301C lyase gels, preservative-free lyase gels were also used. Control groups included only preservative gel, preservative-free gel, and buffer (protein buffer). The results are shown in Figure 27 , indicating that Endolysine and V301C exhibited the best lysing activity.

[0290] The results of the antibacterial experiment of Endolysine_09, Q1, and Q2 lyase gels are shown in FIG30 . It can be seen that the 64 ug / ml lyase+gel composition has no antibacterial activity against Staphylococcus epidermidis, which is beneficial to the human body.

[0291] The results are shown in FIG28 , where it can be seen that the 09 lytic enzyme has good lytic activity against Staphylococcus aureus S. aureus-MS3 (still has lytic activity at a concentration of 32 ug / ml).

[0292] The results are shown in Figure 29. It can be seen that the 09 lyase has good lysis activity against Staphylococcus aureus S.aureus-USA300 (still has lysis activity at a concentration of 32ug / ml). In the figure, protein + blank gel (protein + gel with added preservatives), protein + no-additive gel (protein + gel with no added preservatives), protein refers to the lyase alone, blank gel + no-additive gel (gel with added preservatives + gel with no added preservatives) and gel without lyase and preservatives.

[0293] The results are shown in FIG30 . It can be seen that the 09, Q1, and Q2 lyase+gel compositions at 64 ug / ml have no lysing activity against Staphylococcus epidermidis S. epidermidis-RP62a and do not inhibit beneficial microorganisms.

[0294] Example 5 Experiments on the effects of Endolysine Lys-SA001 and its mutants on six probiotics and two pathogenic bacteria

[0295] Experimental Methods: The lysis activity of the lytic enzyme against six probiotics (all purchased from Beijing Ketuo Hengtong Biotechnology Co., Ltd.) and two pathogenic bacteria was tested. The corresponding strains were frozen at -80°C. A small amount of bacterial suspension was taken with a sterile 10μL inoculating loop in a biosafety cabinet. The suspension was then streaked onto TSA solid medium and incubated at 37°C for 12-18 hours.

[0296] Pick a single colony and place it in 2 mL of MH liquid medium. Measure the turbidity using a turbidimeter and adjust the turbidity of the culture solution to 0.5. Use a sterile cotton swab to smear the culture solution and evenly spread it onto TSA solid medium using the cross-spreading method. Dilute the lytic enzyme to 32 μg / mL using the corresponding protein buffer (0.1 M phosphate buffer, pH 7.4, 10% glycosides, 200 mM NaCl, 5 mM imidazole). Apply 10 μL of the sample to a plate. After the plate has dried, invert it in a 37°C incubator and incubate for 9 hours. 1-11 in Figures 31-38 represent XZ700, Endolysine, Endolysine_D150P, Endolysine_E282A, Endolysine_V301C, Endolysine_N419V, Endolysine_185 (Endolysine_del_V163-K185), Endolysine_185FT (Endolysine_del_V163-K185-SP-FT), 11-Endolysine_Q1 (Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP), respectively. Tag Protein Q Column Low Salt Elution Fraction), 11-Endolysine_Q2 (Endolysine_G246C / V301C_N419V_Q135F_A257C / A279C_PCNP Tag Protein Q Column High Salt Elution Fraction), 09-Endolysine. The black areas near the red values ​​in Figures 32-36 are the corresponding values ​​marked in black.

[0297] The results are shown in FIG31 . The conclusion is that Endolysine Lys-SA001 and its mutants have no lytic activity against Lactobacillus casei Zhang-1.

[0298] The results are shown in Figure 32. The conclusion is that Endolysine Lys-SA001 and its mutants have no lytic activity against Lactobacillus casei Zhang-2.

[0299] The results are shown in FIG33 . The conclusion is that Endolysine Lys-SA001 and its mutants have no lytic activity against Lactococcus lactis BL19-1.

[0300] The results are shown in FIG34 . Lactococcus lactis BL19-2. The conclusion is that Endolysine Lys-SA001 and its mutants have no lytic activity against Lactococcus lactis BL19-2.

[0301] The results are shown in Figure 35. The conclusion is that Endolysine Lys-SA001 and its mutants have no lytic activity against plant P8-1 bacteria.

[0302] The results are shown in Figure 36. The conclusion is that Endolysine Lys-SA001 and its mutants have no lytic activity against plant Lactobacillus P8-2.

[0303] The results are shown in FIG37 . For Salmonella (Yangzhou University isolate), it was concluded that Endolysine Lys-SA001 and its mutants had no lytic activity against Salmonella.

[0304] The results are shown in FIG38 . Escherichia coli (purchased from Shanghai Qincheng Biotechnology Co., Ltd.) were tested. The conclusion was that Endolysine Lys-SA001 and its mutants had no lytic activity against Escherichia coli.

[0305] Example 6 Temporal Stability of Endolysine Lys-SA001 and Mutants

[0306] To determine the stability of the lytic enzyme in a gel environment, remove Staphylococcus aureus and Staphylococcus epidermidis from -80°C freezer. In a biosafety cabinet, use a sterile 10μL inoculating loop to siphon off a small amount of bacterial liquid. Striking the culture medium with three zones is performed on TSA solid medium and incubated at 37°C for 12-18 hours.

[0307] Single colonies were picked and placed in 2 mL of MH liquid medium. Turbidity was measured using a turbidimeter and adjusted to a McFarland turbidity of 0.5. A sterile cotton swab was used to smear the bacterial solution and evenly spread onto TSA solid medium using the cross-spreading method. Lysase was diluted to 64 μg / mL using the corresponding protein buffer (0.1 M phosphate buffer, pH 7.4, 10% glycosides, 200 mM NaCl, 5 mM imidazole). 100 μL of lysase was mixed with 100 μL of either preservative-free gel or preservative gel, respectively, to achieve a final concentration of 32 μg / mL of lysase with either preservative-free gel or preservative gel. Controls containing lysase protein, preservative gel, or preservative gel at corresponding dilutions were also prepared. 10 μL of sample was spotted onto a plate. After the plate dried, it was inverted and incubated at 37°C for 9 hours. The preservative-free gel (abbreviated as additive-free gel or preservative-free gel) is prepared by removing the lyase from the lyase gel composition of Example 3. The preservative-free gel is prepared by removing the lyase from the lyase gel composition of Example 3, and then adding the preservative after the step "5. Gradually add the pre-dissolved solution from step 1 during homogenization and stir evenly." The preservative in the resulting gel is ethylparaben, with a final concentration of 0.2%.

[0308] The results are shown in Figures 39 to 46, and it was concluded that Endolysine Lys-SA001 and V301C had the best lytic activity against S. aureus-USA300 or S. aureus-MS3 compared to other mutants within four weeks (stored at 4°C) using a lytic enzyme + gel composition (64ug / ml).

[0309] The results are shown in Figures 47 to 50, showing that the 09 (09_Endolysine_G246C_V301C_N419V) lytic enzyme + gel composition (64ug / ml) had lytic activity against S. aureus-USA300 or S. aureus-MS3 within two weeks.

[0310] The results are shown in Figures 51 to 54, indicating that the lytic enzyme No. 09 (09_Endolysine_G246C_V301C_N419V) had slight lytic activity against S. epidermidis-RP62a or S. epidermidis-BD40.

[0311] Example 7 Thermal Stability of Endolysine Lys-SA001 and Mutants

[0312] The antibacterial activity of lytic enzymes was determined in the temperature range of 4-100℃. Eight different lytic enzymes with the same concentration (concentration 64μg / mL) were placed at 4, 20, 40, 60, 80, and 100℃ for 30 minutes. After cooling, a sterile 96-well microplate was placed on the clean bench.

[0313] 50 μL of fresh TSB medium was added, and then 100 μL of Staphylococcus aureus bacterial suspension (10 8 Equal volumes of 100 μg / mL (CFU / mL) and the treated lytic enzyme were added to a 96-well plate, mixed thoroughly, and incubated in a 37°C incubator for 24 hours. The solution was observed for turbidity, and the OD value was measured at 600 nm on a microplate reader to compare the antibacterial stability of the lytic enzyme at different temperatures. OD600 values ​​were read and recorded at one-hour intervals, and a 24-hour growth curve was plotted. This experiment was performed in triplicate. The results are shown in Figure 55, concluding that mutant V301C maintained strong lytic activity at 20°C for 9 hours, demonstrating excellent temperature stability.

[0314] Pick a single colony of Staphylococcus aureus and place it in 2 mL of MH liquid medium. Measure the turbidity using a turbidimeter and adjust the turbidity of the culture solution to a McFarland 0.5. Use a sterile cotton swab to smear the culture solution and evenly spread it on TSA solid medium using the cross-spreading method. Dilute the treated lytic enzyme in the corresponding protein buffer (0.1 M phosphate buffer, pH 7.4, 10% Glyeerol, 200 mM NaCl, 5 mM imidazole), diluting it in half to achieve a final lytic enzyme concentration of 32. Apply 10 μL of the lytic enzyme dilution to a plate. After the plate has dried, place it upside down in a 37°C incubator and incubate for 30 minutes.

[0315] The results are shown in Figure 56 (the figure corresponds to Staphylococcus aureus), which shows that the 09 (09_Endolysine_G246C_V301C_N419V) and 185 (Endolysine_del_V163-K185) lytic enzymes still have lytic activity against S. aureus-MS3 after treatment at 37°C for 30 minutes, and have good temperature stability.

[0316] Example 8 Clinical Trial

[0317] This application is for a single-center, open-label, investigator-initiated clinical trial conducted at Shenzhen Qianhai Shekou Free Trade Zone Hospital to evaluate the safety and efficacy of Endolysin Gel for the treatment of mild to moderate atopic dermatitis in adults. Ethics approval number: 2024KY-005-01K. Subject 04HSY experienced significant resolution of facial and foot erythema after three days of application of Endolysin Gel (lyase concentration: 30 μg / ml, corresponding to Endolysine-09, with the gel formulation as described in Example 3) (topically applied at least twice daily). The results are shown in Figures 57-59.

[0318] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A bacteriophage lytic enzyme or a mutant thereof, characterized in that: The bacteriophage lytic enzyme includes any of the following: (1) having an amino acid sequence as shown in SEQ ID NO.1, or having at least 80% identity with the amino acid sequence as shown in SEQ ID NO.1; (2) The amino acid sequence shown in (1) has a mutation in the polypeptide domain having cysteine-histidine-dependent aminopeptidase (CHAP) activity, the cell wall binding domain, and / or the amidase domain.

2. The bacteriophage lytic enzyme or a mutant thereof according to claim 1, characterized in that The amino acid sequence shown in SEQ ID NO.1 undergoes at least one of the following mutations: 1) Mutation at the 150th amino acid of the bacteriophage lytic enzyme; 2) mutation at the 282nd amino acid of the bacteriophage lytic enzyme; 3) mutation at the 419th amino acid of the bacteriophage lytic enzyme; 4) simultaneous mutations at amino acids 246 and 301 of the phage lytic enzyme; 5) deletion of amino acids 161 to 185 of the bacteriophage lytic enzyme; 6) Simultaneous mutations at amino acids 246, 301, and 419 of the phage lytic enzyme; 7) Based on 6), mutations are made at the 135th, 257th and 279th amino acids of the bacteriophage lytic enzyme.

3. The bacteriophage lytic enzyme or a mutant thereof according to claim 2, characterized in that: The amino acid sequence shown in SEQ ID NO.1 undergoes at least one of the following mutations: 1) At the 150th amino acid of the bacteriophage lytic enzyme, the amino acid residue ASP was mutated to the amino acid residue PRO; 2) at the 282nd amino acid of the bacteriophage lytic enzyme, the amino acid residue GLU was mutated to the amino acid residue ALA; 3) at the 419th amino acid of the bacteriophage lytic enzyme, the amino acid residue ASN was mutated to the amino acid residue VAL; 4) Simultaneous mutations were made at the 246th and 301st amino acids of the phage lytic enzyme, mutating the 246th amino acid GLY and the 301st amino acid VAL to the amino acid residue CYS; 5) deletion of amino acids 161 to 185 of the bacteriophage lytic enzyme; 6) Simultaneous mutations were made at the 246th, 301st, and 419th amino acids of the phage lytic enzyme; the 246th amino acid GLY and the 301st amino acid VAL were mutated to the amino acid residue CYS, and the amino acid residue ASN was mutated to the amino acid residue VAL; 7) Based on 6), mutate the 135th, 257th and 279th amino acids of the bacteriophage lytic enzyme gene; mutate the 135th amino acid GLN to PHE, the 257th amino acid ALA to CYS and the 279th amino acid ALA to CYS; 8) Based on 7), add PCNP tag KRKKRKKRK to the C-terminus of the protein.

4. Biomaterial, characterized in that (1) A nucleic acid molecule encoding the bacteriophage lytic enzyme or a mutant thereof according to any one of claims 1 to 3; (2) Primers, expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines expressing the bacteriophage lytic enzyme or mutant thereof according to any one of claims 1 to 3; (3) An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule described in (1); (4) A recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette described in (2) or (3); (5) A recombinant microorganism or transgenic cell line containing the recombinant vector described in (2) or (3) or (4).

5. The bacteriophage lytic enzyme or its mutant according to any one of claims 1 to 3 and the biomaterial according to claim 4 have any of the following uses: a. Use in the inhibition of Staphylococcus aureus for non-disease treatment; b. Use in the preparation of a product for broad-spectrum inhibition of Staphylococcus aureus; c. Use in the preparation of products for preventing, delaying and / or treating diseases caused by Staphylococcus aureus; d. Use in the preparation of products for preventing and treating biofilm produced by Staphylococcus aureus.

6. The use according to claim 5, characterized in that In the use in preparing products for inhibiting Staphylococcus aureus, the products include cosmetics, antibacterial agents, disinfectants, and cleaning agents.

7. The use according to claim 5, characterized in that In the use of preparing a product for preventing or treating a disease caused by Staphylococcus aureus, the product comprises a medical device kit and / or a drug; and / or In the use of preparing a product for preventing, delaying and / or treating a disease caused by Staphylococcus aureus, the disease includes skin infection, respiratory tract infection, digestive system infection, urinary system infection, joint infection, blood infection, atopic dermatitis or diabetic complications; Optionally, the skin infection comprises skin abscess, eczema or folliculitis.

8. The use according to claim 5, characterized in that In the use in preparing a product for preventing and treating biofilm produced by Staphylococcus aureus, the product includes an antibacterial agent, a medicine, a disinfectant or a cleaning agent.

9. A cosmetic, a cleaning product, a daily chemical product, an antibacterial agent, a medical device or a medicine, characterized in that: The composition comprises the bacteriophage lytic enzyme, the phage lytic enzyme mutant according to claim 1 or 2, the Staphylococcus aureus phage according to claim 3 or the biomaterial according to claim 4 as an active ingredient, with or without the addition of a carrier or excipient.

10. The cosmetic, toiletries, daily chemicals, antibacterial agent, medical device or medicine according to claim 9, characterized in that: The dosage forms of the cosmetics, toiletries, daily chemicals, antibacterial agents or drugs include microcapsule preparations, emulsions, ointments, solutions, powders, sprays, aerosols, capsules, solids or gels, and / or they can be bonded to a solid surface; Optionally, the binding to the solid surface is by immobilization with an affinity ligand, by ionic / hydrophobic interactions, or covalent immobilization.

11. A method for preventing, delaying and / or treating a disease caused by Staphylococcus aureus, comprising administering the bacteriophage lytic enzyme or a mutant thereof according to any one of claims 1 to 3 or the biological material according to claim 4 to a subject in need thereof.

12. The method according to claim 11, characterized in that The diseases include skin infection, respiratory tract infection, digestive system infection, urinary system infection, joint infection, blood infection, atopic dermatitis or diabetic complications.

13. The method according to claim 12, characterized in that The skin infections include skin abscesses, eczema or folliculitis.

14. The method according to claim 12, characterized in that When the disease is atopic dermatitis, the concentration of the bacteriophage lytic enzyme is 10 ug / ml to 120 ug / ml, and can be optionally 30 ug / ml.

15. The method according to claim 14, characterized in that The bacteriophage lytic enzyme is administered in a local manner.

16. The method according to claim 14, characterized in that The dosage is based on the fingertip unit principle and applied to the skin lesions.

17. The method according to claim 14, characterized in that The bacteriophage lytic enzyme is administered twice or more per day.

Images