Phage lyase Lys162 and expression and application thereof

Abstract

The invention discloses a bacteriophage lyase Lys162 and an application of the bacteriophage lyase Lys162 in preparation of an antibacterial product. The bacteriophage lyase has a remarkable splitting effect on gram-negative bacteria and gram-positive bacteria, so that the bacteriophage lyase can be used as a broad-spectrum antibacterial protein. For example, the antibacterial effect on pseudomonas aeruginosa, klebsiella pneumoniae and streptococcus is remarkable. The antibacterial protein has relatively high temperature tolerance and pH tolerance, can replace antibiotics, and is applied to prevention and treatment of bacterial diseases as an external medicine or an internal medicine and sterilization of medical instruments and medical places; and the product can also be used as an additive to be applied to the fields of feed processing, food industry, animal husbandry, beer industry, aquaculture, cosmetic production, fruit preservation and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a phage lysin Lys162 and its expression and application. Background Technology

[0002] Antimicrobial resistance (AMR) has become a major threat to global public health. The World Health Organization (WHO) has listed AMR as one of the top ten health challenges facing humanity in the 21st century. In recent years, the prevalence of multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), and multidrug-resistant Acinetobacter baumannii (MDR), has significantly reduced the effectiveness of traditional antibiotics, even leading to a situation where "no drugs are available."

[0003] Bacteriophages and their derivatives (such as lysins and endolysins) have become an important research direction in the post-antibiotic era due to their unique bactericidal mechanisms and low resistance induction potential. However, environmental bacteriophages may carry antibiotic resistance genes (ARGs), and their horizontal gene transfer (HGT) between bacteria may exacerbate the spread of resistance. Furthermore, the application of phage therapy still faces challenges such as strong host specificity, difficulties in large-scale production, and an imperfect regulatory framework.

[0004] In contrast, endolysins achieve rapid bactericidal action by specifically hydrolyzing the peptidoglycan layer, and due to the highly conserved target sites, bacteria are unlikely to develop resistance through a single mutation. However, some studies have shown that the outer membrane of Gram-negative bacteria limits the direct application of endolysins. The outer membrane can effectively prevent exogenously added lysins from reaching and acting on the peptidoglycan layer inside, resulting in a significant reduction in their antibacterial effect against Gram-⁻ bacteria. Therefore, despite the significant advantages shown by endolysins in laboratory studies, their clinical application still faces many challenges: (1) insufficient penetration efficiency against Gram-negative bacteria; (2) stability issues in complex physiological environments (such as blood and tissues); and (3) cost and quality control in large-scale production. Summary of the Invention

[0005] The present invention aims to overcome the above-mentioned defects and provide a lyase Lys162 encoded by the genome of Escherichia coli phage pEC-M2929.1AR.1. The expression vector of Lys162 was constructed by gene cloning to obtain the recombinant protein Lys162, and its physicochemical properties and antibacterial activity against a group of representative Gram-negative and Gram-positive bacteria were systematically evaluated.

[0006] The present invention provides a phage lysin Lys162, characterized in that:

[0007] The amino acid sequence of the lysin Lys162 is shown in SEQ ID NO.1;

[0008] SEQ ID NO.1:

[0009] Met-Ala-Lys-Val-Val-Asp-Val-Phe-Asp-Met-Leu-Arg-Phe-Asp-Glu-Gly-Leu-Lys-Leu-Thr-Val-Tyr-Pro-Asp-Thr-Glu-Gly-Tyr-Trp-Thr-Val-Gly-Ile-Gly-His-Leu-Leu-Thr-Lys-Leu-Lys-A sp-Lys-Ala-Glu-Ala-Ile-Arg-Ile-Leu-Asp-Asn-Leu-Val-Gly-Arg-Lys-Thr-Asn-Gly-Val-Ile- Thr-Glu-Ala-Glu-Ala-Arg-Arg-Ile-Phe-Glu-Ser-Asp-Val-Lys-Lys-Ala-Ile-Gln-Gln-Ile-His -Ser-Ser-Thr-Ile-Leu-Ser-Pro-Ile-Tyr-Asp-Lys-Val-Ser-Pro-Asn-Arg-Lys-Met-Ala-Ile-Il e-Asn-Met-Val-Phe-Gln-Met-Gly-Leu-Lys-Gly-Ala-Glu-Ser-Phe-Lys-Asn-Ser-Leu-Thr-Leu-V al-Ser-Asn-Ser-Tyr-Tyr-Thr-Gln-Ala-Ser-Ile-Asn-Leu-Arg-Lys-Ser-Lys-Trp-Tyr-Arg-Gln- Thr-Pro-Asn-Arg-Ala-Glu-Arg-Val-Ile-Gln-Val-Leu-Lys-Thr-Gly-Thr-Leu-Asp-Ala-Tyr-Asn

[0010] The DNA sequence of the lysin Lys162 is shown in SEQ ID NO.2:

[0011] SEQ ID NO.2:ATGGCTAAAGTAGTTGATGTGTTTGATATGTTACGTTTTGATGAAGGACTAAAGCTAACTGTATATCCTGATACCGAAGGATATTGGACAGTTGGTATTGGACACCTTCTGACAAAACTCA AAGATAAAGCAGAAGCTATACGCATTCTTGATAACTTAGTAGGTAGAAAAACCAATGGAGTTATTACCGAAGCGGAAGCTAGAAGAATCTTTGAGAGTGACGTAAAGAAAGCGATACAACAAATCCA TTCAAGTACCATAACTATCTCCTATTTACGATAAAGTAAGTCCTAATCGTAAAATGGCTATTATTAATATGGTATTTCAAATGGGCTTGAAAGGTGCAGAATCTTTCAAAAATAGCTTGACTTTAGTGAGTAATTCATATTATACTCAAGCCTCTATAAATTTACGTAAAAGTAAATGGTATCGCCAAACGCCTAATCGCGCAGAGCGTGTAATTCAGGTGCTTAAAACTGGAACATTAGACGCTTATAACTAA

[0012] The present invention also provides an expression of the phage lysin Lys162, characterized in that it includes primers as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] Furthermore, the present invention also provides the expression of the phage lysin Lys162, characterized in that it includes the promoters shown in SEQ ID NO.5 and SEQ ID NO.6.

[0014] Furthermore, this invention also suggests the application of this phage lysin Lys162 in the preparation of antibacterial products.

[0015] In addition, the present invention provides a formulation characterized in that it comprises the phage lysin Lys162 as claimed in claim 1 and an outer membrane permeation agent, wherein the outer membrane permeation agent is selected from any one of diaminetetraacetic acid, citric acid and ε-polylysine.

[0016] The aforementioned antibacterial products may be antibacterial drugs, which may also include pharmaceutically acceptable excipients.

[0017] The aforementioned antibacterial products are also disinfection / sterilization products, which further include at least one of the raw materials used to prepare the disinfection / sterilization products, such as stabilizers, pH adjusters, corrosion inhibitors, and solvents.

[0018] The aforementioned antibacterial products can also be used as additives in industries such as feed processing, food industry, animal husbandry, beer industry, aquaculture, cosmetics production, and fruit preservation. Attached Figure Description

[0019] Figure 1 Electrophoresis images of LyS162 PCR products and protein purification;

[0020] Wherein, (A)M is the DL2000 DNA Marker, and 1-2 are ORF162 PCR products;

[0021] (B) M is DL2000 DNA Marker, and 1-2 are PCR products of 162-BL21-28a colonies;

[0022] (C) M represents the standard relative molecular mass of the protein (100kDa). 1 is whole bacteria without pET28a / BL21 empty vector and not induced; 2 is whole bacteria induced with pET28a / BL21 empty vector; 3 is whole bacteria without pET28a-LyS162 / BL21 and not induced; 4 is whole bacteria induced with pET28a-LyS162 / BL21; 5 is bacterial supernatant after IPTG induction with pET28a-LyS162 / BL21; 6 is purified Lys162.

[0023] Figure 2 Results of LyS162 cleavage activity and stability;

[0024] Among them, (A) cleavage activity, (B) concentration, (C) temperature, and (D) pH.

[0025] Figure 3 Fragmentation spectrum of LyS162. Specific Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Experimental materials:

[0028] Bacteriophage pEC.M2929.1AR.1 is a laboratory-isolated product. Escherichia phage pEC-M2929- 1AR.1, complete genome - Nucleotide - NCBI );

[0029] Escherichia coli DH5α, Escherichia coli BL21(DE3), and plasmid extraction kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; restriction endonuclease EcoRI, restriction endonuclease XhoI, and gel recovery kits were purchased from Beijing TransGen Biotech Co., Ltd.; and sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparation kits were purchased from White Shark Biotechnology Co., Ltd.

[0030] Based on the results of whole-genome prediction and functional analysis, the ORF162 gene fragment of phage pEC.M2929.1AR.1 was initially identified as an expression lyase fragment. Lyase digestion primers and pET-28a vector T7 promoter primers were designed using SnapGene software and synthesized and sequenced by Guangzhou Ruiboxingke Biotechnology Co., Ltd.

[0031] The primer sequences are as follows:

[0032] M2929.1AR.1-F 5′- TAA GAATTC ATGGCTAAAGTAGTTGATGTGTTTG-3′,

[0033] M2929.1AR.1-R 5′- TAA CTCGAG GTTATAAGCGTCTAATGTTCCAGT-3′.

[0034] *The sequences underlined and bolded are the EcoRI and XhoI restriction sites, respectively.

[0035] 28a-T7-F 5′-TAATACGACTCACTATAGGG-3′,

[0036] 28a-T7-R 5′-GCTAGTTATTGCTCAGCGG-3′.

[0037] Example 1. Cloning, expression, and purification of recombinant protein Lys162

[0038] Using cDNA from bacteriophage pEC.M2929.1AR.1 as a template, the target gene fragment was amplified using corresponding upstream and downstream primers. The PCR reaction program was as follows: 94.0 ℃ pre-denaturation for 5 min; 94.0 ℃ denaturation for 30 s, 57.5 ℃ annealing for 30 s, 72.0 ℃ extension for 1 min, for a total of 30 cycles; 72.0 ℃ extension for 5 min. The target gene fragment was recovered using a gel extraction kit.

[0039] The pET-28a(+) vector fragment was digested with EcoR I and Xho I, and then recovered by gel electrophoresis. The target gene fragment and the pET-28a(+) vector fragment were ligated using T4 ligase at 25 °C. The ligation product was transformed into Escherichia coli DH5α competent cells and cultured overnight at 37 °C. Positive clones were verified using corresponding upstream and downstream primers. PCR verification of positive clones was followed by sequencing.

[0040] like Figure 1 As shown in Figure A, a specific band was amplified at 500 bp (target band was 498 bp) by 1.0% agarose gel electrophoresis. After transformation, positive single colonies were picked and colony PCR was performed using upstream and downstream primers for verification. The results are as follows. Figure 1 As shown in Figure B, the size is consistent with the theoretical size. E. coli BL21(DE3) carrying pET-28a-Lys162 was induced to express protein using IPTG, and the protein expression was assessed using SDS-PAGE after high-pressure lysis of the bacteria. Figure 1 The electrophoresis results shown in Figure C indicate that a specific band was detected at 20.46 kDa in both the bacterial lysate and the supernatant, consistent with the theoretical size.

[0041] The verified positive recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent bacteria and cultured overnight at 37°C. The clones that were verified as positive by PCR were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0042] The DNA sequence of lyase Lys162 is as follows: ATGGCTAAAGTAGTTGATGTGTTTGATATGTTACGTTTTGATGAAGGACTAAAGCTAACTGTATATCCTGATACCGAAGGATATTGGACAGTTGGTATTGGACACCTTCTGACAAAACTCAAAGATAAAGCAGAAGCTATACGCATTCTTGATAACTTAGTAGGTAGAAAAACCAATGGAGTTATTACCGAAGCGGAAGCTAGAAGAATCTTTGAGAGTGACGTAAAGAAAGCGATACAACAAATCCATTCAAGTACCATACTATCTCCTATTTACGATAAAGTAAGTCCTAATCGTAAAATGGCTATTATTAATATGGTATTTCAAATGGGCTTGAAAGGTGCAGAATCTTTCAAAAATAGCTTGACTTTAGTGAGTAATTCATATTATACTCAAGCCTCTATAAATTTACGTAAAAGTAAATGGTATCGCCAAACGCCTAATCGCGCAGAGCGTGTAATTCAGGTGCTTAAAACTGGAACATTAGACGCTTATAACTAA;

[0043] The amino acid sequence is as follows:

[0044] Met-Ala-Lys-Val-Val-Asp-Val-Phe-Asp-Met-Leu-Arg-Phe-Asp-Glu-Gly-Leu-Lys-Leu-Thr-Val-Tyr-P ro-Asp-Thr-Glu-Gly-Tyr-Trp-Thr-Val-Gly-Ile-Gly-His-Leu-Leu-Thr-Lys-Leu-Lys-Asp-Lys-Ala-Gl u-Ala-Ile-Arg-Ile-Leu-Asp-Asn-Leu-Val-Gly-Arg-Lys-Thr-Asn-Gly-Val-Ile-Thr-Glu-Ala-Glu-Ala -Arg-Arg-Ile-Phe-Glu-Ser-Asp-Val-Lys-Lys-Ala-Ile-Gln-Gln-Ile-His-Ser-Ser-Thr-Ile-Leu-Ser-P ro-Ile-Tyr-Asp-Lys-Val-Ser-Pro-Asn-Arg-Lys-Met-Ala-Ile-Ile-Asn-Met-Val-Phe-Gln-Met-Gly-Le u-Lys-Gly-Ala-Glu-Ser-Phe-Lys-Asn-Ser-Leu-Thr-Leu-Val-Ser-Asn-Ser-Tyr-Tyr-Thr-Gln-Ala-Ser -Ile-Asn-Leu-Arg-Lys-Ser-Lys-Trp-Tyr-Arg-Gln-Thr-Pro-Asn-Arg-Ala-Glu-Arg-Val-Ile-Gln-Val- Leu-Lys-Thr-Gly-Thr-Leu-Asp-Ala-Tyr-Asn (i.e., MAKVVDVFDMLRFDEGLKLTVYPDTEGYWTVGIGHLLTKLKDKAEAIR ILDNLVGRKTNGVITEAEARRIFESDVKKAIQQIHSSTILSPIYDKVSPNRKMAIINMVFQMGLKGAESFKNSLTLVSNSYYTQASINLRKSKWYRQTPNRAERVIQVLKTGTLDAYN)

[0045] Escherichia coli BL21(DE3) carrying the verified recombinant plasmid was inoculated into 100 mL of LB broth containing a final concentration of 50 μg / mL kanamycin and cultured until OD. 600The pH value was approximately 0.6–0.8. IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 16°C for 18 h. The induced bacterial culture was centrifuged to collect the cells, washed twice with sterile PBS, and the precipitate was resuspended in Lysis Buffer (50 mM Tris-HCl (pH=7.4) and 300 mM NaCl). The lysate was homogenized using a high-pressure homogenizer, and the sample to be tested consisted of bacterial lysate, precipitate, and supernatant carrying the recombinant plasmid of *Escherichia coli* BL21(DE3). The sample was subjected to SDS-PAGE electrophoresis with a protein loading buffer. Afterward, the gel was stained with Coomassie stain and destained. The concentration of purified Lys162 was simultaneously measured. Lys162 was ultimately stored in a buffer containing 50 mM Tris-HCl (pH=7.4) and 20 mM NaCl (this buffer was used for all subsequent measurements unless otherwise specified).

[0046] Example 2. Structural Property Analysis

[0047] Physicochemical properties of the lyase, including its theoretical molecular weight, theoretical isoelectric point, and the number of positively and negatively charged amino acids, were predicted using online tools (ExPASy (https: / / www.expasy.org / )). Domain analysis of the lyase was performed using an online tool (http: / / www.ebi.ac.uk / interpro / ), and transmembrane domain analysis was conducted using the TMHMM-2.0 online tool (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). These methods revealed that the lyase Lys162 consists of 166 amino acids, has a relative molecular mass of 18.9 kDa, and an isoelectric point (pI) of 9.67. Its amino acid composition includes 17 negatively charged residues (Asp + Glu) and 25 positively charged residues (Arg + Lys). The protein has an instability index of 21.26 (Instability Index < 40), indicating good stability.

[0048] Example 3. Performance Analysis

[0049] 1. Method for Assaying Lys162 Enzyme Activity

[0050] The test strain (M2929.1AR.1) in logarithmic growth phase was centrifuged at 8000 rpm for 5 min at 4℃, and the supernatant was discarded. The precipitate was washed twice with PBS buffer and resuspended in Tris-HCl (pH 7.4). A 200 μL reaction system (100 μL bacterial suspension + 50 μL buffer + 50 μL test sample) was used. 100 μL of resuspended bacterial suspension, 50 μL buffer, and 50 μL Lys162 were added to each well of a 96-well plate and incubated at 37℃ for 2 h. The OD value at 600 nm was then measured using a microplate reader. Three replicates were set up, with the group without lyase serving as a negative control.

[0051] 2. Stability test of Lys162

[0052] The enzyme activity of Lys162 (concentration of 125 μg / mL was used for all non-specific values) under different conditions was evaluated by stability experiments.

[0053] (1) Temperature stability: 200 μL of Lys 162 lyase (500 μg / mL) was added to a 1.5 mL EP tube and incubated at 4, 20, 30, 37, 50, 60, and 70 °C for 30 min, respectively. Then, 50 μL of the incubated lyase and 50 μL of buffer were added to a 96-well plate (the final concentration of lyase in each well was 125 μg / mL), and 100 μL of bacterial resuspension was added to each well. The control group consisted of 100 μL of Tris-HCl (pH 7.4) buffer + 100 μL of bacterial resuspension, and the blank group consisted of 100 μL of Lys 162 lyase and 100 μL of Tris-HCl (pH 7.4) buffer. Three replicates were set up.

[0054] like Figure 2 As shown in Figure D, Lys162 exhibits good tolerance in temperature stability tests, with pyrolysis activity still detectable at 60℃ (pyrolysis rate of 36.42%). These results indicate that Lys162 maintains strong pyrolysis activity and demonstrates strong tolerance under high-temperature conditions.

[0055] (2) pH stability: 200 μL of Lys 162 lyase (500 μg / mL) was added to a 1.5 mL EP tube and incubated for 30 min in Tris-HCl at pH 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively. Then, 50 μL of the incubated lyase and 50 μL of buffer were added to a 96-well plate (final concentration of lyase in each well was 125 μg / mL), and 100 μL of bacterial resuspension was added to each well. The control group consisted of 100 μL Tris-HCl (pH 7.4) buffer + 100 μL bacterial resuspension, and the blank group consisted of 100 μL Lys 162 lyase and 100 μL Tris-HCl (pH 7.4) buffer. Three replicates were set up.

[0056] like Figure 2 As shown in Figure C, in the pH stability experiment, Lys162 exhibited cleavage activity in the pH range of 4.0-11.0, and showed the highest activity at pH 7.0. As can be seen from the figure, at pH 7.0, Lys162 (cleavage rate 41.97%) had a higher cleavage rate for the host bacterium M2929.1AR.1 than at pH 10.0 (41.57%). Its activity gradually decreased with increasing or decreasing pH, which may be due to the destruction of the structure of the lyase Lys162 under excessively low or high pH conditions, leading to protease denaturation.

[0057] 3. Effects of different final concentrations of enzyme solution on the activity of host bacteria

[0058] Different concentrations of Lys 162 lyase (200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL) were added to 1.5 mL EP tubes. 100 μL of bacterial resuspension was added to each well. 50 μL of lyase and 50 μL of buffer were added to each well of a 96-well plate (final concentrations of lyase in each well were 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL, respectively). The control group consisted of 100 μL Tris-HCl (pH 7.4) buffer + 100 μL bacterial resuspension, and the blank group consisted of 100 μL Lys 162 lyase and 100 μL Tris-HCl (pH 7.4) buffer. Three replicates were set up.

[0059] The activity of enzyme solutions at different final concentrations against host bacteria was measured, and it was found that, for example... Figure 2As shown in Figure B, the concentration gradient experiment showed that the cleavage activity of the lysin Lys162 increased with increasing enzyme concentration, with cleavage rates of 21.92% (final concentration 50 μg / mL), 32.41% (final concentration 100 μg / mL), 31.06% (final concentration 150 μg / mL), and 31.95% (final concentration 200 μg / mL). However, the cleavage rate tended to level off as the concentration increased, suggesting that further increases in enzyme concentration had limited effect on improving cleavage activity.

[0060] 4. Combined effect of Lys162 and EDTA

[0061] The test strain (M2929.1AR.1) in logarithmic growth phase was centrifuged at 8000 rpm for 5 min at 4℃, and the supernatant was discarded. The precipitate was washed twice with PBS buffer and resuspended in Tris-HCl (pH 7.4) buffer. Group 1 (Lys162+EDTA group): 50 μL (final concentration 125 μg / mL) Lys162 and 50 μL EDTA (final concentration 0.5 mM) were added to each well of a 96-well plate. Group 2 (EDTA control group): 50 μL Tris-HCl and 50 μL EDTA (0.5 mM) were added to each well. Group 3 (Lys162): 50 μL (final concentration 125 μg / mL) Lys162 and 50 μL Tris-HCl were added to each well. Group 4 (Tris-HCl control group): 100 μL Tris-HCl was added. 100 μL of conditionally pathogenic bacteria (M2929.1AR.1) grown to the logarithmic phase was added to each well in each group. OD values ​​were measured for all groups after incubation at 37°C for 2 hours. 600 Set three sets of repeated parallel values.

[0062] like Figure 2 As shown in Figure A, in the system where Lys162 was used in combination with EDTA (0.5 mM), the data showed that the lysis activity of the Lys162+EDTA combination group was significantly higher than that of the group without EDTA, and was 1.6 times higher than that of the group without EDTA.

[0063] Example 4. Determination of the fragmentation spectrum of Lys162

[0064] Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis were cultured to the logarithmic growth phase, respectively. 100 μL of each bacterial culture was added to 96-well plates, and 50 μL of Lys162 (500 μg / mL) was added to each well. A control without Lys162 lyase was used. After incubation for 2 hours, the OD600 value was measured, and the lysis profile of Lys162 was analyzed by observing changes in turbidity. Results are as follows: Figure 3 As shown in Table 1:

[0065] Table 1. List of bacterial strains to be tested

[0066]

[0067] The table above shows that Lys162 not only exhibits lytic activity against the original host *Escherichia coli* M2929.1AR.1, but also against other *Escherichia coli* strains such as M2951-2AR and N3229-3DT.1. Furthermore, Lys162 also shows lytic activity against *Klebsiella pneumoniae* Δ4M186-2, N3262-3AT, BM419-3, *Salmonella enterica* N1094-1At, *Streptococcus* N3161-2dt, *Enterococcus faecalis* N3680-1At, and *Pseudomonas aeruginosa* 3103-3aT and S3687-2Ab.

[0068] The function and effect of this embodiment:

[0069] This embodiment presents a broad-spectrum phage lysin Lys162 with strong lytic activity, providing a strategy for combating Gram-negative pathogens, bacterial resistance, and antibiotic scarcity. Experimental results from this embodiment show that Lys162 not only exhibits good antibacterial activity against *Escherichia coli*, but also demonstrates antibacterial activity against various pathogens, such as *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, and *Streptococcus*. In other words, this phage lysin exhibits significant lytic effects against both Gram-negative and Gram-positive bacteria, and therefore can be used as a broad-spectrum antibacterial protein. Furthermore, this antimicrobial protein has high temperature and pH tolerance, and can replace antibiotics as a topical or internal medicine for the prevention and treatment of bacterial diseases. When used as an antimicrobial drug, it can be combined with pharmaceutically acceptable excipients. It can also be used as a disinfectant / sterilization product for the sterilization of medical devices and medical facilities. When used as a disinfectant / sterilization product, it also contains commonly used auxiliary materials for sterilization and disinfection, such as stabilizers, pH adjusters, corrosion inhibitors, and solvents. It is also used as an additive in feed processing, food industry, animal husbandry, brewing industry, aquaculture, cosmetics production, and fruit preservation.

Claims

1. A phage lysin Lys162, characterized in that: The amino acid sequence of the lysin Lys162 is shown in SEQ ID NO.

1.

2. A phage lysin Lys162, characterized in that: The DNA sequence of the lysin Lys162 is shown in SEQ ID NO.

2.

3. The expression of a phage lysin Lys162, characterized in that, This includes primers as shown in SEQ ID NO. 3 and SEQ ID NO.

4.

4. Expression of a phage lysin Lys162, characterized in that, This includes promoters as shown in SEQ ID NO. 5 and SEQ ID NO.

6.

5. The use of the phage lysin Lys162 as described in claim 1 or 2 in the preparation of antibacterial products.

6. A formulation, characterized in that: Includes the phage lysin Lys162 as described in claim 1 or 2 and an outer membrane permeabilizer.

7. The application of the formulation as described in claim 6 in the preparation of antibacterial products.

8. An antibacterial product as described in claim 5 or 7, characterized in that: It is an antibacterial drug; The antimicrobial drug also includes pharmaceutically acceptable excipients.

9. An antibacterial product as described in claim 5 or 7, characterized in that: For disinfection / sterilization products; The disinfection / sterilization product also contains at least one of the following: stabilizer, pH adjuster, corrosion inhibitor, and solvent.

10. An antibacterial product as described in claim 5 or 7, characterized in that: As an additive; The additives are used in feed processing, food industry, animal husbandry, beer industry, aquaculture, cosmetics production, and fruit preservation.

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