Production and application of an antimicrobial protein targeting Propionibacterium acnes

Abstract

This invention provides the production and application of an antimicrobial protein targeting Propionibacterium acnes. The invention provides the application of the antimicrobial protein in products for the prevention and treatment of acne or infections caused by Propionibacterium acnes or conditions related to Propionibacterium acnes. The antimicrobial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment thereof, or analogues thereof, which inhibit the growth of Propionibacterium acnes. The antimicrobial protein of this invention can specifically and effectively kill Propionibacterium acnes without affecting the host and other common skin microorganisms, providing a safe and effective acne solution suitable for long-term use without causing skin microecological imbalance or the risk of microbial resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the production and application of an antimicrobial protein targeting Propionibacterium acnes. Background Technology

[0002] Acne is a common skin condition, most severe in adolescents, causing significant psychological distress. Different degrees of acne present with different symptoms, leading to varying degrees of facial damage. Common manifestations include comedones, papules, pustules, and in severe cases, cysts, nodules, and scars, often accompanied by seborrhea.

[0003] Propionibacterium acnes is a common Gram-positive bacterium that frequently colonizes human skin and is an important component of the skin microbiome. The skin microbiome, together with the host, forms the human skin barrier, maintaining host health. Other important members of the skin microbiome include Staphylococcus epidermidis, Corynebacterium nigra, and Staphylococcus capitulata. Propionibacterium acnes has long been considered the microorganism most closely associated with acne, and substantial evidence suggests it plays a significant role in the pathogenesis of acne. Currently, the treatment of Propionibacterium acnes relies on traditional antibiotics. Clinically, for patients with mild to moderate acne, traditional antibiotics remain the first-line treatment. While this reduces the number of Propionibacterium acnes to some extent, it also affects other skin commensal bacteria and disrupts the skin barrier. Furthermore, antibiotics inevitably have cytotoxic effects. The problem of microbial resistance caused by antibiotic overuse is equally serious. For patients with severe acne, isotretinoin is the most commonly used retinoid, but its potential risks to male reproductive health are highly controversial, and it also has implications for liver and kidney function and the potential risk of causing depression, significantly limiting its use. Oral hormonal medications are another option, which can alleviate acne to some extent, but they also carry risks such as allergic reactions and hormonal imbalances.

[0004] Existing technical solutions:

[0005] Regarding the treatment of acne, Eichenfield DZ et al. proposed a combination of topical treatments such as retinoids (e.g., retinoic acid, adapalene), benzoyl peroxide, azelaic acid, and topical medications. In a randomized trial involving 207 patients, treatment with 0.025% retinoic acid gel as a monotherapy resulted in a 63% reduction in acne lesion counts at 12 weeks compared to the control group. This demonstrates the effectiveness of topical medications, but the problem is that topical treatments often lead to relapses and can cause some degree of skin irritation and damage. For more severe acne symptoms, oral antibiotics (e.g., doxycycline or minocycline), steroid therapy (e.g., in combination with oral steroids or spironolactone), or isotretinoin are most effective, but this also implies greater potential risks.

[0006] CN102482655A and CN114908077A both disclose phage lyases that can inhibit *Propionibacterium acnes*, but neither provides case studies or data demonstrating that these phage lyases specifically target and clear *Propionibacterium acnes*. Since the lyases disclosed in CN114908077A originate from phages of multiple different bacterial species, they are unlikely to be specifically targeted at *Propionibacterium acnes*. According to publicly available literature (PMID: 37239874), some phage lyases derived from the *Propionibacterium acnes* phage genome have not shown targeting specific *Propionibacterium acnes*. Furthermore, the disclosed lyases are all soluble proteins from the supernatant after heterologous expression in *E. coli*, and no phage lyases with activity and targeting specific *Propionibacterium acnes* obtained by inclusion body refolding have been disclosed.

[0007] In summary, the medical and skincare industries currently have a significant need for developing novel antibacterial agents that inhibit Propionibacterium acnes, especially those that can specifically inhibit the bacterium without affecting the normal skin flora. At present, there is still a lack of phage lysins with sufficient data and case studies to support their ability to target and eliminate Propionibacterium acnes. Summary of the Invention

[0008] The purpose of this invention is to provide an antibacterial agent that targets Propionibacterium acnes. This antibacterial agent can target and eliminate Propionibacterium acnes and inhibit its growth without disrupting the human skin microecology, thereby alleviating skin discomfort symptoms such as acne-related inflammatory reactions caused by Propionibacterium acnes, regulating the skin microecology, and improving skin condition.

[0009] This invention seeks potential antimicrobial proteins from the genome of bacteriophages that can target and inhibit various Propionibacterium acnes bacteria. Through heterologous expression, a bioactive antimicrobial protein can be obtained that can target and eliminate Propionibacterium acnes and inhibit its growth without disrupting the skin's microecology, thereby alleviating acne-related inflammatory reactions and other skin symptoms caused by Propionibacterium acnes, regulating the skin microecology, and improving skin condition.

[0010] The first aspect of the present invention provides the use of an antimicrobial protein in the preparation of products for the prevention, treatment of acne or inhibition of the growth of Propionibacterium acnes, wherein the antimicrobial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment thereof or analogue thereof, and the active fragment or analogue thereof can inhibit the growth of Propionibacterium acnes.

[0011] In this invention, the active fragment or analogue has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 6.

[0012] As one embodiment of the present invention, the active fragment or analogue has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.6 or is an alternative amino acid sequence having the same functional group.

[0013] In some embodiments, the alternative amino acid sequence is obtained by conservative substitution of amino acids from the same amino acid set, which includes:

[0014] Aliphatic: glycine, alanine, valine, leucine, or isoleucine;

[0015] Containing hydroxyl groups or sulfur / selenium: serine, cysteine, threonine, or methionine;

[0016] Cyclic: proline;

[0017] Aromatic compounds: phenylalanine, tyrosine, or tryptophan;

[0018] Basic: Histidine, lysine, or arginine;

[0019] Acidic substances and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

[0020] In one embodiment of the present invention, the nucleic acid encoding the antimicrobial protein comprises a sequence such as SEQ ID NO.1, or a synonymous codon sequence thereof.

[0021] As one embodiment of the present invention, the product includes a drug, a disinfectant, or a cosmetic product that inhibits the growth of Propionibacterium acnes.

[0022] As one embodiment of the present invention, the product is a cosmetic product that inhibits the growth of Propionibacterium acnes.

[0023] As one embodiment of the present invention, the cosmetic product for inhibiting the growth of Propionibacterium acnes also includes ionic additives and preservatives.

[0024] In this invention, the ionic additives include, but are not limited to, dipotassium glycyrrhizate, disodium EDTA, and sodium hyaluronate.

[0025] In this invention, the preservatives include, but are not limited to, benzoic acid or its salts.

[0026] As one embodiment of the present invention, the conditions associated with Propionibacterium acnes include prostatitis leading to cancer, SAPHO (synovitis, acne, impetigo, hypertrophy, osteitis) syndrome, sarcoidosis, or sciatica.

[0027] As one embodiment of the present invention, the infections caused by Propionibacterium acnes include invasive infections, postoperative infections, and / or device-related infections.

[0028] A second aspect of the present invention provides the use of an antimicrobial protein or its binding domain in the preparation of a tool for diagnosing or detecting Propionibacterium acnes, wherein the antimicrobial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment thereof, or an analogue thereof.

[0029] As one embodiment of the present invention, the active fragment or analogue has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.6 or is an alternative amino acid sequence having the same functional group.

[0030] As one embodiment of the present invention, the active fragments and analogs can inhibit the growth of Propionibacterium acnes.

[0031] In some embodiments of the present invention, the alternative amino acid sequence is obtained by conservative substitution of amino acids from the same amino acid set, wherein the amino acid set includes:

[0032] Aliphatic: glycine, alanine, valine, leucine, or isoleucine;

[0033] Containing hydroxyl groups or sulfur / selenium: serine, cysteine, threonine, or methionine;

[0034] Cyclic: proline;

[0035] Aromatic compounds: phenylalanine, tyrosine, or tryptophan;

[0036] Basic: Histidine, lysine, or arginine;

[0037] Acidic substances and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

[0038] In one embodiment of the present invention, the antimicrobial protein or its binding domain is used in conjunction with a signaling molecule.

[0039] In one embodiment of the present invention, the antimicrobial protein or its binding domain is fused with a signaling molecule through gene fusion or chemical coupling to form a fusion product.

[0040] As one embodiment of the present invention, the fusion is used for direct detection of Propionibacterium acnes on a microscope slide by fluorescence or other means, for labeling Propionibacterium acnes by immunohistochemistry, as a detection reagent in ELISA assays, as a detection reagent in Western blot, for connection with magnetic beads in MACS or other pull-down assays, or as a detection reagent in assays where antibodies are used as detection reagents.

[0041] In this invention, the signaling molecules include, but are not limited to, proteins or chemical fluorescent dyes, protein tags, enzymes, avidin, streptavidin, ovalbumin, biotin, tags sensitive to click chemical labeling, peptides, or other molecules that can induce the recruitment of secondary proteins or molecules that generate signals.

[0042] In this invention, the fluorescent dyes include, but are not limited to, GFP, RFP, mCherry, FITC, TRITC, Alexafluor 488, Cy3, or Cy5.

[0043] In this invention, the protein tags include, but are not limited to, Flag-tags, myc-tags, halo-tags, his-tags, or any tags that can bind to antibodies or other high-affinity molecules to generate a signal.

[0044] In this invention, the enzymes include, but are not limited to, firefly luciferase, β-lactamase, alkaline phosphatase, horseradish peroxidase, or enzymes that cause reactions such as light, color changes, substrate deposition, or any other reactions that can be detected in experiments.

[0045] A third aspect of the present invention provides a method for inhibiting the growth of Propionibacterium acnes, the method comprising adding an effective amount of an antimicrobial protein to a system in need, wherein the antimicrobial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment thereof, or an analogue thereof, and the active fragment or analogue thereof can inhibit the growth of Propionibacterium acnes.

[0046] As one embodiment of the present invention, the active fragment or analogue has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.6 or is an alternative amino acid sequence having the same functional group.

[0047] In some embodiments of the present invention, the alternative amino acid sequence is obtained by conservative substitution of amino acids from the same amino acid group, wherein the amino acid group includes:

[0048] Aliphatic: glycine, alanine, valine, leucine, or isoleucine;

[0049] Containing hydroxyl groups or sulfur / selenium: serine, cysteine, threonine, or methionine;

[0050] Cyclic: proline;

[0051] Aromatic compounds: phenylalanine, tyrosine, or tryptophan;

[0052] Basic: Histidine, lysine, or arginine;

[0053] Acidic substances and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

[0054] In one embodiment of the present invention, the nucleic acid encoding the antimicrobial protein comprises the sequence shown in SEQ ID NO.1, or a synonymous codon sequence thereof.

[0055] A fourth aspect of the present invention provides a method for preventing or treating acne or infections caused by Propionibacterium acnes or conditions related to Propionibacterium acnes, the method comprising administering an antimicrobial protein to a subject with a corresponding need, wherein the antimicrobial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment thereof, or an analogue thereof. The active fragment or analogue inhibits the growth of Propionibacterium acnes.

[0056] As one embodiment of the present invention, the active fragment or analogue has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.6 or is an alternative amino acid sequence having the same functional group.

[0057] In some embodiments of the present invention, the alternative amino acid sequence is obtained by conservative substitution of amino acids from the same amino acid group, wherein the amino acid group includes:

[0058] Aliphatic: glycine, alanine, valine, leucine, or isoleucine;

[0059] Containing hydroxyl groups or sulfur / selenium: serine, cysteine, threonine, or methionine;

[0060] Cyclic: proline;

[0061] Aromatic compounds: phenylalanine, tyrosine, or tryptophan;

[0062] Basic: Histidine, lysine, or arginine;

[0063] Acidic substances and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

[0064] In one embodiment of the present invention, the nucleic acid encoding the antimicrobial protein comprises the sequence shown in SEQ ID NO.1, or a synonymous codon sequence thereof.

[0065] In this invention, the drug further comprises a pharmaceutically acceptable carrier and / or excipient, which refers to substances that do not cause significant irritation to the organism and do not affect the bioactivity and properties of the administered antimicrobial protein.

[0066] In this invention, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants. The diluents include, but are not limited to: lactose, sodium chloride, glucose, urea, starch, and water. The binders include, but are not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate, alginate, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. The surfactants include, but are not limited to: polyethylene glycol sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The humectants include, but are not limited to: glycerol and starch. The adsorbents include, but are not limited to: starch, lactose, bentonite, silica gel, kaolin, and soap clay. The lubricants include, but are not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc. The fillers include, but are not limited to: mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, sodium bicarbonate, etc. The disintegrants include, but are not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0067] In this invention, the subjects include humans or other mammals, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.

[0068] The cosmetic products for inhibiting the growth of Propionibacterium acnes described in this invention also contain any active ingredients with cosmetic activity. Examples of such active ingredients include emollients, moisturizers, free radical inhibitors, anti-inflammatory agents, vitamins, bleaching agents, anti-acne agents, keratolytic agents, slimming agents, skin pigments, and sunscreens, such as linoleic acid, retinol, retinoic acid, alkyl ascorbate, polyunsaturated fatty acids, nicotinic acid esters, tocopheryl nicotinic acid esters, unsaponifiable rice, soybean, or tallow, ceramides, hydroxy acids such as glycolic acid, selenium derivatives, antioxidants, beta-carotene, gamma-orizanol, and stearylglycerol.

[0069] In this invention, the antimicrobial protein can be applied directly or formulated into various dosage forms using known pharmaceutical preparation methods. For example, as needed, the drug can be administered orally as sugar-coated tablets, capsules, elixirs, and microcapsules; or formulated into a sterile solution or suspension with water or any other pharmaceutically acceptable liquid for non-oral administration as an injection. For example, the compound can be mixed with a pharmaceutically acceptable carrier or medium, including but not limited to sterile water, physiological saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, mediators, preservatives, binders, etc., in the unit dosage form required for generally acceptable pharmaceutical administration. Appropriate dosages within a specified range can be obtained based on the content of the active ingredient in these formulations.

[0070] The dosage of the antimicrobial protein described in this invention is not limited, as long as the desired therapeutic or preventive effect can be achieved. It can be determined based on the subject's symptoms, gender, age, etc. The dosage of the antimicrobial protein described in this invention can be determined in detail using, for example, its therapeutic or preventive effect on a disease as an indicator.

[0071] In this invention, the treatment and / or prevention refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, conditions, illnesses, or diseases (e.g., acne). Desired therapeutic effects include, but are not limited to: preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, and improving or alleviating the disease state.

[0072] A fifth aspect of this invention provides a method for producing the aforementioned antimicrobial protein, wherein the antimicrobial protein comprises the amino acid sequence shown in SEQ ID NO. 6, or an active fragment thereof, or an analogue thereof. The method includes heterologous expression in *E. coli*, followed by denaturation and renaturation of inclusion bodies obtained by lysis with urea, and purification by affinity chromatography. In some embodiments, the lysis method includes ultrasonic disruption, high-pressure homogenization, and chemical disruption. In some embodiments, the affinity chromatography is performed using a metal ion column.

[0073] As one embodiment of the present invention, the antimicrobial protein comprises the amino acid sequence shown in SEQ ID NO.6, or its active fragments or analogs.

[0074] This invention, through simulation, experimentation, and verification, demonstrates that its antimicrobial protein effectively kills and inhibits the growth of *Propionibacterium acnes*, exhibits good targeting, and does not affect other common microorganisms on the skin surface. The effects of different environments on the efficacy of the antimicrobial protein and the minimum inhibitory concentration (MIC) of the antimicrobial protein against *Propionibacterium acnes* were also tested. Compared with other lysins and antibiotics, the antimicrobial protein of this invention possesses high efficiency, high specificity, and no risk of drug resistance.

[0075] (1) High efficiency: Compared with other lysins, the antimicrobial protein involved in this invention has a higher antimicrobial rate and a lower minimum inhibitory concentration (MIC).

[0076] (2) Immediate effect: Compared with antibacterial substances such as antibiotics and organic acids, the antibacterial protein involved in this invention can take effect in a short time (usually within a few minutes), as confirmed by the results of SEM electron microscopy.

[0077] (3) Universality: The antimicrobial protein involved in this invention is universally effective against Propionibacterium acnes and is effective against Propionibacterium acnes strains from different sources (including drug-resistant strains).

[0078] (4) No risk of drug resistance: The overuse of antibiotics has led to a serious problem of antibiotic resistance in Propionibacterium acnes. Based on the unique mechanism of action of phage lysins, they are unlikely to cause drug resistance or similar problems.

[0079] (5) Targeting: Compared with other lysins and antibiotics, the antimicrobial protein involved in this invention has high specificity against specific bacterial species or strains, which means that it can precisely kill Propionibacterium acnes without affecting other symbiotic flora on the skin.

[0080] (6) Safety: Due to the specificity of the antimicrobial proteins involved in this invention, they are generally harmless to mammalian cells, with higher safety and fewer side effects.

[0081] (7) pH compatibility: The antibacterial protein involved in this invention can tolerate different levels of acidity and alkalinity and can function in environments with pH = 5-7. Since human skin and sweat are neutral / slightly acidic, they are within the range where the antibacterial protein can function stably.

[0082] (8) Thermal stability: The antimicrobial protein involved in this invention can be treated at different temperatures. After being treated at 40-60℃ for a certain period of time, it can still function effectively, demonstrating good thermal stability. This greatly facilitates the application of antimicrobial proteins in the processing of skin care products and significantly increases the possibilities for the production and application of antimicrobial proteins.

[0083] (9) No need for long-term use: Compared with antibiotic treatment that requires long-term use, the antimicrobial protein involved in this invention may only require short-term application to achieve the therapeutic effect.

[0084] Compared to traditional antibiotic treatments, applying antimicrobial proteins to the skin surface allows for targeted eradication of Propionibacterium acnes without disrupting the skin's microbiome and affecting other common skin microorganisms. This is the greatest advantage and characteristic of antimicrobial proteins in skincare applications. This invention demonstrates the effects of antimicrobial proteins on other common skin microorganisms and proves the targeting ability of the antimicrobial protein of this invention. This provides strong evidence for using antimicrobial proteins to target and eliminate Propionibacterium acnes on the skin surface without disrupting the skin's microbiome. Attached Figure Description

[0085] Figure 1 This is the result of comparing LysCA001 with other phage lysin sequences.

[0086] Figure 2 This is a plasmid map of the LysCA001 lysin expression vector.

[0087] Figure 3 This is the result shown in Example 2, which indicates that the SDS-PAGE analysis of the lysin LysCA001 protein can be heterologously expressed in BL21(DE3).

[0088] Figure 4 This is the result shown in Example 3, which demonstrates that SDS-PAGE analysis of the lysin LysCA001 protein can purify inclusion bodies via affinity chromatography.

[0089] Figure 5 This is the result of Example 4 demonstrating the antibacterial activity of the phage lysin LysCA001 against Propionibacterium acnes.

[0090] Figure 6 This is the result shown in Example 5, which demonstrates that the lysin LysCA001 has lysing activity against Propionibacterium acnes in a buffer system.

[0091] Figure 7 This is a scanning electron microscope (SEM) image of the lysis process of Propionibacterium acnes by the lysin LysCA001.

[0092] Figure 8 This is a graph showing the targeting of the lyase LysCA001 to Propionibacterium acnes.

[0093] Figure 9 This is a statistical graph showing the tolerance of the lysin LysCA001 to different pH values.

[0094] Figure 10This is a statistical chart showing the tolerance of the lysin LysCA001 to different temperatures.

[0095] Figure 11 This is a statistical chart showing the tolerance of the lyase LysCA001 to different ionic additives and preservatives.

[0096] Figure 12 This is a graph showing the results of the MTT cytotoxicity assay of the lyase LysCA001 on human immortalized keratinocytes. Detailed Implementation

[0097] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0098] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0099] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0100] Example 1: Identification of the lyase gene targeting Propionibacterium acnes and construction of its expression vector

[0101] Genomic sequences of phages targeting *Propionibacterium acnes* were collected, and lyase genes were identified using BLAST and Pfam sequences through sequence alignment and domain alignment. A total of 124 potential phage lyase gene sequences were discovered. Cluster analysis was performed on the identified lyases based on sequence similarity, and they were classified according to a set threshold (both sequence similarity and sequence coverage are greater than or equal to 90%). The identified phage-derived lyase genes were divided into five major categories, and one representative sequence from each category was selected for expression vector construction. One preferred representative sequence was named LysCA001, which has less than 80% amino acid sequence similarity to publicly available phage lyases. The representative sequences from the remaining categories were named LysCA002, LysCA003, LysCA004, and LysCA005, respectively. The maps of the selected phage lyases are shown below. Figure 1 As shown, LysCA001 has an extra segment in the sequence from amino acid 150 to amino acid 185 compared to other lyases, making it significantly different from other phage lyases. Among the 124 discovered phage lyase genes, only LysCA001 has a unique sequence and structure.

[0102] The identified lyase gene was codon-optimized in *E. coli*, and the corresponding gene was cloned into the pET28a(+) vector with a 6x-His tag added to the C-terminus. The gene is controlled by the lactose operon. The pET28a(+) plasmid was used as the expression vector for the identified lyase gene, and it was constructed using BamHI / XhoI restriction sites. Figure 2 This is a map of the plasmid expressing the lysin.

[0103] The following are the codon-optimized nucleic acid sequences of E. coli by the five major classes of lysins:

[0104] LysCA001 nucleic acid sequence:

[0105] ATGCGTTTATCCCGGCGGCACATCACAGTGCGGGTAGCAATTCTCCTGTGAATCGGGTTGTTATT

[0106] CATGCGACATGTCCTGACGTTGGCTTTCCGAGTGCTTCACGTAAAGGCCGCGCGGTTTCAACCGC

[0107] CAATTATTTCGCGTCACCTTCATCTGGCGGCAGCGCGCATTACGTCTGCGATGTGGGTGAAACCG

[0108] TGCAATGTTTATCTGAAGGTACAATTGGTTGGCATGCACCGCCCAACCCACACTCCTTGGGTATC

[0109] GAGATTTGCGCCGATGGAGGCAGTCACGCGAGCTTCCGCGTCCCAGGTCATGCATATACCCGCGA

[0110] GCAGTGGTTAGATCCACGTGTTTGGCCTGCAGTCGAGAAGGCCGCAATCCTGTGCCGTCGCTTAT

[0111] GTGATAAATATAATGTGCCAGGCCACGCTTATACTCGCGAACAGTGGCTTGATCCTCGTGTGTGG

[0112] CCGGCCGTGGAGAAGGCAGCAATCTTATGCCGCCGCTTATGTGATAAGTATAATGTTCCAAAGCG

[0113] TAAACTGAGCGCAGCCGATCTGAAAGCGGGTCGCCGGGGAGTGTGTGGACATGTCGATGTAACT

[0114] GATGCCTGGCATCAGAGCGATCATGATGACCCGGGCCCGTGGTTTCCGTGGGATAAATTTATGGC

[0115] TGTCGTCTGTGGAGGTAGTGGTGAGAGTGGGGAACTGACCGTGGCCGACGTGAAGGCGTTGCAT

[0116] GACCAGATTAAGCAGCTGTCAGCACAGCTTTCCGGCTCTGTTAACAAGCTTCATCATGATGTTGG

[0117] AGTTGTGCAAGTTCAGAACGGAGATCTGGGTAAACGGGTTGAAGCCCTGAGCTGGGTAAAGAAT

[0118] CCGGTTACAGGTAAATTGTGGCGTACTAAAGACGCACTTTGGTCTATTTGGTACTATGTGCTGGAATGTCGTTCCCGGATTGATCGTCTGGAATCCGCCGTCAATGGCCTGAAGAAG(SEQ ID NO.1)

[0119] LysCA002 nucleic acid sequence:

[0120] ATGCGCTATATCCCTGCGGCGCACCATAGCGCCGGCAGTAACAGTCCGGTGAATCGTGTGGTTAT

[0121] ACATGCGACATGTCCGGATGTCGGATTTCCCTCTGCCAGTCGTAAAGGTCGCGCAGTTTCAACAG

[0122] CGAATTATTTCGCCAGCCCATCGGCTGGAGGTTCTGCGCATTACGTCTGCGATGTTGGAGAAACT

[0123] GTTCAGTGCTTATCTGAATCTACCATTGGTTGGCATGCACCGCCTAACCCGCATAGCTTAGGTATT

[0124] GAAATCTGTGCCGACGGTGGCTCCCACGCCAGCTTTCGTGTTCCTGGACACGCCTATACCCGCGA

[0125] GCAGTGGTTGGATCCGCGTGTTTGGCCTGCAGTAGAGAAAGCGGCTGTGTTATGTCGGCAACTGT

[0126] GCGACAAATACAATGTCCCAAAACGCAAACTGTCAGTGAGCGATCTGAAAGCAGGCCGCCGTGG

[0127] CGTGTGTGGCCATACCGATGTGACGGATGCATGGCATCAAAGTGATCATGATGATCCGGGCCCAT

[0128] GGTTTCCATGGGACCGGTTTATGGCGGTTGTGAACGGTGGCTCCGGTGAGTCCGGTGAACTGAGC

[0129] ATGGCCGATGTGAAAGCGCTGCATGATCAGATTAAACAGCTGTCCGGACAGGTCGCACAGTCAG

[0130] TTAACAAGCTTCACCATGATGTTGGGGTCGTCCAAGTGCAGAATGGCAATTTAAAAAAGCAGGT

[0131] GGACTCTCTTAGTATGGTGAAGAACCCGGTTACAGGGAAGCTGTGGCGCACTCGTGATGCAATCT

[0132] GGTCAATTTGGTATTATGTTCTTGAATGTCGTTCACGGATTGGTAAATTGGAAGCCGAAGTTGCACGGCTGAAGAAG(SEQ ID NO.2)

[0133] LysCA003 nucleic acid sequence:

[0134] ATGGTGCGGTTTATCCCTGCCGCACATCATAGTGCGGGAAGTAATAGCCCGGCAAACCGTGTGGT

[0135] TATACATGCGACATGCCCGGATGTGGGTTTCCCATCTGCAAGTCGCAAAGGTCGCGCAGTTTCCA

[0136] CAGCCAATTACTTCGCATCTCCATCTGCCGGCGGTAGCGCCCACTATGTTTGTGATATTGGGGAA

[0137] ACCGTTCAGTGCCTTTCTGAAGCAACCATCGGCTGGCATGCCCCGCCGAACCCGCATAGCCTGGG

[0138] AATTGAAATTTGTGCAGACGGTGGATCCCACGCCAGCTTCCGGGTGCCTGGTCATGCGTATACTC

[0139] GCGAACAGTGGCTGGATCCACGGGTATGGCCCGCGGTGGAAAAGGCCGCAATTTTGTGTCGTCG

[0140] CTTATGCGATAAACATGGTGTCCCTAAACGTAAATTGTCAGCAGCTGATCTGAAAGCGGGTCGTC

[0141] GGGGTGTTTGCGGACATGCCGATGTCACTGACGCCTGGCATCAATCAGACCATGACGATCCGGG

[0142] ACCATGGTTTCCGTGGGATCGTTTTATGGCGGTGGTTTGTGGAGGCAGTAGTTCAGATTCAGGCG

[0143] AACTGACTGTCGCGGACGTTAAGGCTCTGCACGATCAGATCAAGCAGCTGAGCGCGCAGTTAAC

[0144] CGGCTCAGTAAACAAGCTGCACCATGATGTGGGCGTGGTCCAGGTGCAGAATGGTGATCTGGGT

[0145] AAACGCGTTGATGCTCTTAGCTGGGTGAAGAACCCTGTTACAGGCAAACTTTGGCGTACCAAAGA

[0146] TGCGTTATGGTCCGTCTGGTACTATGTTCTGGAATGTCGTTCTCGCATTGGCAAGTTAGAGGCCGAGCTTGCACAGTTGAAGAAGTGA(SEQ ID NO.3)

[0147] LysCA004 nucleic acid sequence:

[0148] ATGACCTATGTTGATGGCGTTTGGGGCTGGTGTGTTGTCCGCTATATCCCTGCCGCACATCATTCA

[0149] GCGGGCTCTAACAAGCCTGTGAATCGCGTTGTGATTCACGCAACCTGCCCGGATGTCGGTTTTCC

[0150] ATCAGCTAGCCGTAAAGGCCGCGCGGTGTCAACCGCGAATTATTTCGCATCCCCGTCATCAGGTG

[0151] GAAGCGCGCACTACGTTTGTGATATTGGTGAAACAGTCCAGTGCCTGTCTGAGAGTACTATTGGT

[0152] TGGCACGCACCACCGAATCCTCATTCCCTGGGCATAGAAATCTGCGCCGATGGCGGGTCACACGC

[0153] GTCGTTCCGTGTGCCGGGTCATGCGTATACACGTGAACAGTGGTTAGATCCTCGGGTATGGCCGG

[0154] CCGTCGAGCGTGCAGCCATTCTGTGTCGCCAGCTGTGTGATAAACATGGTGTTCCCAAGCGCAAA

[0155] CTGAGTGTGGCTGATCTGAAAGCTGGCAAACGTGGCGTTTGTGGTCATGTGGATGTTACTGATGC

[0156] CTGGCATCAAAGCGATCATGACGATCCAGGACCATGGTTTCCGTGGGACAAATTTATGGCGGTTG

[0157] TCAATGGACATGGTGGAGGTAGTAGCTCTGAAGAACTTTCTATGGCCGACGTGCAGGCGCTGCAT

[0158] AACCAGATCAAGCAGCTGTCCGCACAAGTCGCCCAGTCTGTGAACAAGCTGCATCATGACGTAG

[0159] GAGTTGTCCAGGTGCAGAATGGCGACCTTGGCAAGCGCGTCGATGCACTTAGCTGGGTGAAGAA

[0160] TCCGGTGACGGGTAAATTATGGCGTAGCAAGGATGCATTATGGAGTGTTTGGTATTACGTTCTTGAATGCCGGAGTCGTTTAGATCGGTTGGAAAGTGCCGTGAACGATTTGAAGAAGTGA(SEQ ID NO.4)

[0161] LysCA005 nucleic acid sequence:

[0162] ATGCGGTTCATACCTGCAGCTCATCATTCAGCGGGATCCAATAGCCCTGTAAACCGCGTTGTCAT

[0163] TCATGCTACATGCCCTGATGTTGGCTTTCCTAGCGCGTCACGCAAAGGTCGCGCCGTTAGTACCG

[0164] CAAACTACTTCGCCAGTCCGAGCTCTGGCGGTAGTGCGCATTATGTGTGTGATGTCGGTGAGACC

[0165] GTTCAGTGTTTGAGTGAAGGTACCATCGGCTGGCACGCGCCGCCAAATCCGCATTCTCTGGGAAT

[0166] CGAAATTTGCGCGGATGGAGGTAGCCACGCATCATTTCGGGTGCCAGGCCATGCATATACTAGA

[0167] GAACAGTGGCTGGATCCGCGTGTCTGGCCGGCAGTGGAGAAGGCCGCCATTTTATGTCGTCGTTT

[0168] ATGTGATAAATACAATACACGTGGGTCTTCTGGCCTGATTCTTGCCTGTGGTCTGCGTTGGCGGC

[0169] GCCTGCCGTCATGCGTGGACGTTTGCGTGACTAATATTATGTTTCGCAAGGGAAATTGTCGTCTTCCAATC(SEQ ID NO.5)

[0170] LysCA001 Amino acid sequence:

[0171] MRFIPAAHHSAGSNSPVNRVVIHATCPDVGFPSASRKGRAVSTANYFASPSSGGSAHYVCDVGETVQCLSEGTIGWHAPPNPHSLGIEICADGGSHASFRVPGHAYTREQWLDPRVWPAVEKAAILCRRLCDKYNVPGHAYTREQWLDPRVWPAVEKAAILCRRLCDKYNVPKRKLSAADLKAGRRGVCGHVDVTDAWHQSDHDDPGPWFPWDKFMAVVCGGSGESGELTVADVKALHDQIKQLSAQLSGSVNKLHHDVGVVQVQNGDLGKRVEALSWVKNPVTGKLWRTKDALWSIWYYVLECRSRIDRLESAVNGLKK(SEQ ID NO.6)

[0172] Amino acid sequence of LysCA002:

[0173] MRYIPAAHHSAGSNSPVNRVVIHATCPDVGFPSASRKGRAVSTANYFASPSAGGSAHYVCDVGETVQCLSESTIGWHAPPNPHSLGIEICADGGSHASFRVPGHAYTREQWLDPRVWPAVEKAAVLCRQLCDKYNVPKRKLSVSDLKAGRRGVCGHTDVTDAWHQSDHDDPGPWFPWDRFMAVVNGGSGESGELSMADVKALHDQIKQLSGQVAQSVNKLHHDVGVVQVQNGNLKKQVDSLSMVKNPVTGKLWRTRDAIWSIWYYVLECRSRIGKLEAEVARLKK(SEQ IDNO.7)

[0174] Amino acid sequence of LysCA003:

[0175] MVRFIPAAHHSAGSNSPANRVVIHATCPDVGFPSASRKGRAVSTANYFASPSAGGSAHYVCDIGETVQCLSEATIGWHAPPNPHSLGIEICADGGSHASFRVPGHAYTREQWLDPRVWPAVEKAAILCRRLCDKHGVPKRKLSAADLKAGRRGVCGHADVTDAWHQSDHDDPGPWFPWDRFMAVVCGGSSSDSGELTVADVKALHDQIKQLSAQLTGSVNKLHHDVGVVQVQNGDLGKRVDALSWVKNPVTGKLWRTKDALWSVWYYVLECRSRIGKLEAELAQLKK(SEQ IDNO.8)

[0176] Amino acid sequence of LysCA004:

[0177] MTYVDGVWGWCVVRYIPAAHHSAGSNKPVNRVVIHATCPDVGFPSASRKGRAVSTANYFASPSSGGSAHYVCDIGETVQCLSESTIGWHAPPNPHSLGIEICADGGSHASFRVPGHAYTREQWLDPRVWPAVERAAILCRQLCDKHGVPKRKLSVADLKAGKRGVCGHVDVTDAWHQSDHDDPGPWFPWDKFMAVVNGHGGGSSSEELSMADVQALHNQIKQLSAQVAQSVNKLHHDVGVVQVQNGDLGKRVDALSWVKNPVTGKLWRSKDALWSVWYYVLECRSRLDRLESAVNDLKK(SEQ ID NO.9)

[0178] Amino acid sequence of LysCA005:

[0179] MRFIPAAHHSAGSNSPVNRVVIHATCPDVGFPSASRKGRAVSTANYFASPSSGGSAHYVCDVGE TVQCLSEGTIGWHAPPNPHSLGIEICADGGSHASFRVPGHAYTREQWLDPRVWPAVEKAAILCRRLC DKYNTRGSSGLILACGLRWRRLPSCVDVCVTNIMFRKGNCRLPI(SEQ ID NO.10)

[0180] Heterologous expression of the lyase protein LysCA001 in Example 2

[0181] The constructed plasmid containing the LysCA001 lysin gene was transformed into BL21(DE3) competent cells, then evenly spread onto LB agar plates (containing 50 μg / mL kanamycin sulfate) and incubated overnight at 37°C. Single colonies were selected from the transformed plates and inoculated into 1 L of TB medium (containing 50 μg / mL kanamycin sulfate) and cultured until OD500 reached. 600 To induce LysCA001 protein expression, add IPTG to the culture medium in test tubes to a final concentration of 0.1-1 mM, and then incubate at 16-37℃, 100 rpm, for 18 h.

[0182] Centrifuge the induced culture medium at 12000 rpm for 5 min to remove the culture medium. Resuspend the strain in PBS to wash it. Finally, add SDS-PAGE loading buffer and heat the sample at 100℃ for 30 min. Centrifuge and collect the supernatant for electrophoresis. For the first 10 min of electrophoresis, maintain a constant voltage of 100-150 V. After the bromophenol blue indicator enters the separating gel, maintain a constant voltage of 200 V until the bromophenol blue band migrates to 1 cm from the bottom of the gel. Remove the gel and stain it with Coomassie Brilliant Blue staining solution. Then transfer it to destaining solution and destain until the background is clear.

[0183] Whole bacteria were added to PBS buffer and homogenized by sonication or high-pressure grinding. Simultaneously, the Ni-IDA affinity chromatography column was equilibrated with PBS buffer at least three times. The target protein was then eluted with equilibration buffers containing different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE analysis. SDS results are shown below. Figure 3 (In the figure, M is the SDS-PAGE protein marker, 0 is the control, 1 is the low temperature induction for 16-24h, 2 is the induction expression supernatant, and 3 is the induction expression inclusion body). Figure 3 This indicates that LysCA001 can be heterologously expressed in E. coli. After disruption and separation, it was shown that the lysin LysCA001 protein was expressed in large quantities in E. coli in the form of inclusion bodies.

[0184] Example 3: Denaturation, refolding, and purification of the lysin protein LysCA001

[0185] Inclusion bodies were washed with 200 ml of washing buffer and centrifuged at 12000 rpm for at least 15 min at 4°C, then the supernatant was discarded. The inclusion bodies were dissolved in approximately 2 L of denaturing buffer and stirred completely with a magnetic stirrer at 4°C (approximately 30 min). Simultaneously, the Ni-IDA column was equilibrated with denaturing buffer. The denaturing buffer was thoroughly mixed with the metal ion column using a four-dimensional mixer. After at least 1 h of thorough equilibration, the target protein was eluted with equilibration buffers of different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE analysis. After purification analysis by Ni-IDA affinity chromatography, Lane3-9 with high purity was collected and added to a treated dialysis bag. The protein was dialyzed into buffer at 4°C using a 5-10 KD dialysis bag for renaturation, and then renatured overnight at low temperature. After renaturation, the dialysis buffer was replaced, and LysCA001 protein was dialyzed again at low temperature for 6-8 h. The supernatant was collected, and 1 ml was taken for SDS electrophoresis identification. SDS results are shown below. Figure 4 (In the figure, M is the SDS-PAGE protein marker, 1 is the supernatant from whole-cell lysis and centrifugation, 2 is the supernatant incubated with Ni-IDA, and 3-9 are the elution fractions of imidazole). The protein concentration was determined using the Nanodrop and Bradford methods, showing that a high concentration of LysCA001 protein was finally obtained. Figure 4 This indicates that LysCA001 can be renatured via inclusion bodies and purified by affinity chromatography. A large amount of the lysin LysCA001 protein was isolated and purified from the precipitate.

[0186] After dialysis and refolding, the supernatant was filtered through a 0.22µm filter, aliquoted, and stored at -80°C. One aliquot of LysCA001 protein, frozen at -80°C, was placed in an ice-water mixture to thaw slowly. If no abnormalities were observed after thawing, it indicates that the LysCA001 protein freeze-thaw cycle was normal.

[0187] Example 4: Determination of the antibacterial activity of lysin protein against Propionibacterium acnes

[0188] Propionibacterium acnes was inoculated into anaerobic BHI liquid medium and cultured to the plateau phase. The bacterial culture was then diluted to 10⁵ CFU / ml and added to the BHI medium. LysCA001 and four other lysins (LysCA002, LysCA003, LysCA004, and LysCA005) were added to a final concentration of 100 μg / ml. The plates were then placed in 96-well plates and continuously monitored for absorbance changes using a microplate reader. The absorbance changes were calculated after 24 hours.

[0189] Under conditions of 100 μg / mL enzyme concentration, initial inoculum of 10⁵ CFU / mL, 5% H₂, 5% CO₂, 90% N₂ atmospheres, and 37°C, LysCA001 exhibited complete inhibitory activity against Propionibacterium acnes, indicating that LysCA001 possesses extremely strong antibacterial properties against Propionibacterium acnes. The results of its antibacterial activity are shown in [Figure 1]. Figure 5 Compared to other lyases, LysCA001 exhibits stronger antibacterial activity against Propionibacterium acnes.

[0190] Example 5: Determination of the lytic activity of LysCA001 against Propionibacterium acnes in a buffer system.

[0191] Propionibacterium acnes was inoculated into anaerobic BHI liquid medium and cultured to OD. 600 =0.45. Centrifuge the bacterial culture from the culture medium at 4000 rpm for 5 min, wash twice with buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and resuspend the cells in the same buffer after washing. Add the buffer containing the cells to a 96-well plate, add LysCA001 lyase protein to a final concentration of 100 μg / ml, and the final volume of each well is 200 μl. Organize the experimental data and calculate the lysis activity.

[0192] Significant changes in colony count were observed in the buffer solution containing LysCA001 lysin. The curve showed the greatest curvature around 30 minutes, gradually slowing down after 60 minutes. OD 600 It hardly changes anymore. This demonstrates that LysCA001 can effectively lyse Propionibacterium acnes in this buffer, proving the lytic performance of LysCA001 against Propionibacterium acnes.

[0193] Figure 6 This indicates that LysCA001 can effectively kill Propionibacterium acnes in the Tris-HCl buffer system.

[0194] Figure 6 The results showed that LysCA001 significantly reduced the number of Propionibacterium acnes colonies in the buffer solution within two hours.

[0195] The lysis rate is calculated as follows: [ΔOD600 of the test (with added lyase) - ΔOD600 control (buffer only)] / initial OD600.

[0196] Example 6: Determination of the minimum inhibitory concentration (MIC) of LysCA001

[0197] The expression and purification of LysCA001 followed the method described above. In short: E. coli BL21(DE3) containing each recombinant expression plasmid were cultured in LB medium for 2-3 hours until OD... 600 =0.6, then IPTG was added to a final concentration of 0.1 Mm, and the cells were cultured at 16 °C and 100 rpm for 18 h. Cells were collected by centrifugation, lysed by sonication, and purified by affinity chromatography. The antimicrobial protein obtained according to the above method was used to determine the minimum inhibitory concentration (MIC).

[0198] The minimum inhibitory concentration (MIC) was determined as follows: Propionibacterium acnes was streaked onto brain heart extract agar plates (1.5% agar BHI medium). The plates were anaerobically incubated at 37°C for 72 hours. Single colonies were then picked and transferred to liquid BHI medium and incubated for 48 hours until OD (digesterone incubation). 600 =0.6, diluted 10 times with BHI liquid medium, at which point each milliliter of liquid contains 10 7 Colony forming units (CFU / ml) were then inoculated into liquid BHI medium and diluted 20-fold to 5 x 10⁻⁶. 5 CFU / ml. Prepare serially diluted 2-fold doses of each lyase using sterile PBS buffer, adding no more than one-tenth of the total culture volume. Incubate the cultures anaerobically at 37°C for 72–96 hours. The MIC is the lowest concentration of lyase at which no growth of Propionibacterium acnes is observed.

[0199] Table 1 records the minimum inhibitory concentration (MIC) of LysCA001 against different Propionibacterium acnes strains. C. acnes strain 1 is the type strain, ATCC6919. C. acnes strains 2 and 3 were obtained from collected samples. C. acnes strain 4 is the standard strain, ATCC11827. Both LysCA001 and LysCA004 exhibited strong inhibitory effects against different C. acnes strains. The MIC test revealed that the MIC of LysCA001 was lower than that of LysCA004, indicating that LysCA001 has a stronger inhibitory effect against C. acnes strains than other phage lyases.

[0200] The MIC results of phage lysins against Propionibacterium acnes are shown in Table 1:

[0201] Table 1

[0202]

[0203] Example 7: SEM images of LysCA001 lysed Propionibacterium acnes in buffer solution.

[0204] Propionibacterium acnes was inoculated into BHI liquid medium and cultured to the plateau phase, followed by sequencing verification. After centrifugation at 4000 rpm for 10 min, the medium was discarded, and the bacteria were washed three times with 50 mM Tris-HCl buffer. The bacteria were then resuspended in a buffer solution equal to the medium volume. Phage lysin was added to a final concentration of 100 μg / ml, and the mixture was incubated at room temperature. For the control group, an equal volume of lysin preservation solution without phage lysin was added. Bacterial cells from both the experimental and control groups were collected at 15 min, 30 min, and 1 h and placed in 2 ml centrifuge tubes. After centrifugation to remove the buffer, the cells were fixed with 2% glutaraldehyde solution to maintain their morphology. After 6-8 h of fixation, the samples were thoroughly dried to avoid artifacts caused by water vapor during SEM observation. A conductive coating was applied to the sample surface to enhance conductivity and obtain clear images in SEM. The samples were placed on the SEM sample holder, and their position and focus were adjusted. The high-energy electron beam of the SEM was used to scan the sample surface to acquire images of its morphology.

[0205] like Figure 7 As shown, LysCA001 effectively lyses the cell wall of *Propionibacterium acnes* in a Tris-HCl buffer system, causing cell rupture and release of contents, resulting in the loss of the cell's intact morphology. Under the same environmental conditions, the lysis efficiency of LysCA001 against *Propionibacterium acnes* per unit time is significantly superior to that of lysozyme (derived from egg white). These results demonstrate the high efficiency of LysCA001 in the in vitro lysis of *Propionibacterium acnes*. This has significant implications for the application of LysCA001 in skincare products, biomedicine, and medical devices.

[0206] Example 8: Targeting determination of LysCA001

[0207] The antibacterial targeting of LysCA001 was determined in solid culture medium. The test strains were evenly spread on BHI plates, LysCA001 was added, and its location was recorded. Antibiotics were used as a control. The plates were incubated anaerobically at 37°C for 3 days, and colony growth was observed. No inhibition zones were observed near the antibiotic addition site, serving as the control group. A clear inhibition zone appeared near the antibiotic addition site, demonstrating that LysCA001 had no inhibitory effect on other common skin strains and protein-expressing Escherichia coli.

[0208] The antibacterial targeting of LysCA001 and other lysins was determined in liquid culture medium. Common skin microbial strains were inoculated into BHI liquid medium and cultured for 24 hours to the logarithmic growth phase. Then, 20 μL of bacterial culture containing 10^6 CFU / mL was transferred to BHI medium containing 25 μg / mL LysCA001, for a final volume of 200 μL. The plates were then placed in 96-well plates and the absorbance was continuously measured using a microplate reader. A control group without LysCA001 was also included. The absorbance changes were calculated after 24 hours. Figure 8 As shown, LysCA001 can significantly inhibit the growth of Propionibacterium acnes in vitro. However, LysCA001 did not exhibit significant inhibitory activity against other microorganisms. This indicates that LysCA001 has significant targeting properties, capable of targeting and eliminating Propionibacterium acnes on the skin surface, thus maximizing the protection of the skin's microecology from disruption. This is of great significance for the application of LysCA001 in skincare products and biomedicine.

[0209] Example 9: pH tolerance test of LysCA001

[0210] LysCA001 was placed in the same Tris-HCl buffer, and the pH was adjusted to 5, 6, 7, and 8 with concentrated hydrochloric acid and sodium hydroxide, respectively. Propionibacterium acnes was then inoculated into BHI medium and cultured to the logarithmic growth phase, followed by dilution to 10⁻⁶. 5 Add 100ug / ml of LysCA001 at different pH values ​​to a power of CFU / ml, place it in an ELISA reader to continuously measure its absorbance, and observe its growth status.

[0211] Figure 9 The effects of different pH values ​​on the efficacy of LysCA001 were recorded. LysCA001 exhibited good antibacterial properties under pH conditions ranging from 5 to 7. Considering that human skin has a slightly acidic environment, its ability to inhibit the growth of Propionibacterium acnes under acidic conditions is of great significance for subsequent applications and production.

[0212] Example 10 Temperature tolerance test of LysCA001

[0213] LysCA001 was heated in a water bath at temperatures of 40℃, 50℃, and 60℃ for 120 min. After heating, LysCA001 was removed. Propionibacterium acnes was inoculated into BHI medium and diluted to 10⁵ CFU / ml. 100 μg / ml of LysCA001 treated at different temperatures was added, and its absorbance was continuously measured using a microplate reader to observe its growth status.

[0214] After treatment at different temperatures, with the highest temperature being 60℃ for 120 minutes, LysCA001 still exhibited in vitro antibacterial activity, proving that LysCA001 can withstand high in vitro temperatures and has good in vitro thermal stability. This has important reference value for the application of LysCA001 in production and processing.

[0215] Figure 10 The effects of different temperature conditions on the efficacy of LysCA001 were recorded. LysCA001 was treated at different temperatures for 2 hours, demonstrating that LysCA001 stored at low temperatures had better bactericidal activity.

[0216] Example 11: Determination of LysCA001's tolerance to different ionic additives and preservatives

[0217] Propionibacterium acnes was inoculated into BHI liquid medium and cultured to the plateau phase. Sequencing verification was performed, and the bacterial culture was then diluted to 10⁵ CFU / ml and added to BHI medium. Different concentrations of LysCA001 (to a final concentration of 100 μg / ml) and different concentrations of ionic additives or preservatives were added. A control group was established with only ionic additives or preservatives added, without LysCA001. The absorbance changes were continuously measured in 96-well plates using a microplate reader. The absorbance changes were calculated after 48 hours.

[0218] like Figure 11 As shown, ionic additives such as dipotassium glycyrrhizate, disodium EDTA, and sodium hyaluronate, as well as the preservative sodium benzoate, did not affect the inhibitory effect of LysCA001 on Propionibacterium acnes, indicating that LysCA001 can coexist with common ionic agents and preservatives while maintaining its activity. Ionic additives such as dipotassium glycyrrhizate, disodium EDTA, and sodium hyaluronate are commonly used to adjust the pH and stability of skincare products and have water-locking and moisturizing effects, making them the most important additives in skincare products. Benzoic acid and its salts are commonly used broad-spectrum antimicrobial agents in skincare products, showing good effects against yeasts, molds, and some bacteria. These results are of significant reference value for the application of LysCA001 in skincare products.

[0219] Example 12: MTT cytotoxicity assay of LysCA001 on human immortalized keratinocytes

[0220] HaCat cells were cultured in T75 flasks until 80% confluence. Cells were then digested with 2 mL of 0.25% TE enzyme at 37°C for 4 min to resuspend the cells. The culture medium was removed at 900 rpm for 5 min, and the cells were collected. The cells were resuspended in 10% FBSDMEM medium to a concentration of 3 x 10⁻⁶ cells / mL. 5 / mL. Seed 100µl of cell resuspension in each well of a 96-well plate and fix for 18-24 hours. Dilute the lyase stock solution with DMEM basal medium to prepare a test medium containing 20% ​​lyase stock solution, and then perform 8 serial dilutions at 2-fold concentrations. Remove the original culture medium from each well of the 96-well plate to remove suspended cells, and wash with 1X PBS to remove residual FBS. Add 100µl of different concentrations of test medium to each well, with 3 replicates for each concentration. Incubate for 24 hours. Aspirate the culture medium to remove floating dead cells, and wash once with PBS. Add 100µl of cell lysis buffer to each well and incubate at 37°C for 30 minutes. Collect the lysis supernatant at 9000 rpm for 5 minutes and transfer it to a new well. Prepare MTT reagent, add it to the well, incubate for 30 minutes, and measure OD. 570 The result is as follows Figure 12 As shown, LysCA001 did not exhibit cytotoxicity to human immortalized keratinocytes even at a concentration 10 times the effective dose, indicating that LysCA001 has good safety for human cells.

[0221] Example 13: The LysCA001 binding domain binds to fluorescent proteins for the detection of Propionibacterium acnes.

[0222] Primers were designed to amplify these two genes based on the binding domain of LysCA001 and the gene sequence of GFP (green fluorescent protein). Using the designed primers, PCR amplification was performed on the LysCA001 binding domain and the GFP gene. The two fragments obtained from PCR amplification were recombined to generate a gene fusion fragment containing the LysCA001 binding domain and the fluorescent protein. The recombinant gene fragment was ligated using restriction enzymes to construct the complete fusion gene. The constructed fusion gene was inserted into an expression vector, and the fusion protein was expressed at low temperature in *E. coli* host cells containing the fusion gene using IPTG. The purified fusion protein was then purified using affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was used to test the specific binding ability of the phage lysin binding domain-fluorescent protein fusion protein to *Propionibacterium acnes*. The fusion protein labeled with the fluorescent protein was co-treated with *Propionibacterium acnes*, and the sample was then observed under a microscope to observe the intensity and location of the fluorescence signal to confirm the specific binding of the fusion protein to *Propionibacterium acnes*.

[0223] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of a targeted antimicrobial protein in the preparation of products for treating acne or targeting and inhibiting the growth of Propionibacterium acnes, wherein, The amino acid sequence of the antimicrobial protein is shown in SEQ ID NO.6; The antimicrobial protein was expressed in Escherichia coli in the form of inclusion bodies.

2. The application according to claim 1, wherein, The nucleic acid encoding the antimicrobial protein comprises the sequence shown in SEQ ID NO.1, or a synonymous codon sequence thereof.

3. The application according to claim 1, wherein, The products mentioned include pharmaceuticals, disinfectants, or cosmetic products that inhibit the growth of Propionibacterium acnes.

4. The application according to claim 3, wherein, The product described is a cosmetic product that inhibits the growth of Propionibacterium acnes.

5. The application according to claim 4, wherein, The cosmetic products that inhibit the growth of Propionibacterium acnes also contain ionic additives and preservatives.

6. The application according to claim 5, wherein, The ionic additives include one or more of dipotassium glycyrrhizate, disodium EDTA, and sodium hyaluronate.

7. The application according to claim 5, wherein, The preservatives mentioned above contain benzoic acid or its salts.

8. A method for inhibiting the growth of Propionibacterium acnes for non-therapeutic purposes, comprising adding an effective amount of an antimicrobial protein to a system in which it is desired, wherein, The amino acid sequence of the antimicrobial protein is shown in SEQ ID NO.6, and the antimicrobial protein is expressed in Escherichia coli in the form of inclusion bodies.

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