Phage lyase and use thereof

Abstract

A phage lyase with an amino acid sequence set forth in SEQ IN NO.2. The lyase can inhibit the growth of Staphylococcus, Escherichia, Klebsiella, and Salmonella bacteria, and can be used as an antibacterial substance in milk pollution control, belonging to the field of bioengineering. The phage lyase can be used for preparing an enzyme formulation alone or in a compounded manner to specifically inactivate bacteria such as Staphylococcus aureus, thereby providing an enzyme formulation source which is safe and free of toxic and side effects for controlling Staphylococcus aureus pollution in milk at present.

Description

Phage lytic enzyme and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a Staphylococcus aureus phage lytic enzyme and application thereof. BACKGROUND

[0002] Staphylococcus aureus is a pathogenic microorganism that poses a serious threat to human and animal health. In recent years, due to the widespread use of antibiotics, the drug resistance of Staphylococcus aureus has been increasing, especially the methicillin-resistant Staphylococcus aureus (MRSA). MRSA is not only resistant to methicillin, but also has varying degrees of resistance to some commonly used antibiotics (tetracyclines, cephalosporins), which poses a great challenge to anti-infection treatment and brings a great threat to health. At present, vancomycin can be used to treat infections caused by MRSA. However, studies have reported that with the increase of vancomycin dosage, the therapeutic effect of vancomycin on MRSA is weakened. Therefore, while reasonably using antibiotics, it is urgent to develop antibacterial drugs with different mechanisms of action from traditional antibiotics.

[0003] Phage lytic enzyme can efficiently lyse the cell wall of Staphylococcus aureus and has good bactericidal effect and does not produce drug resistance. Studies have shown that phage can specifically prevent and treat diseases caused by some bacteria by invading and lysing some types of bacteria, and has no adverse effects on the environment and organisms.

[0004] Phage lysins are a new type of antibacterial molecules encoded by phage genomes, and their bactericidal mechanism is to hydrolyze the peptidoglycan of the cell wall to make the bacteria lysed and die. Lysins of gram-positive bacteria usually include an enzyme active domain (EAD) at the N-terminus and a cell wall binding domain (CBD) at the C-terminus. Lysins usually target specific parts of the bacterial cell wall using CBD, and then EAD exerts a bactericidal effect by breaking the bonds of bacterial peptidoglycan, thereby making the bacterial cells lysed and die. Lysins are also considered as a potential antibacterial molecule according to this property. Since the action site of lysin is a conserved site in the structure of the bacterial cell wall, host bacteria are less likely to produce drug-resistant mutations, have high safety, and can effectively reduce the risk of bacterial drug resistance spreading (Nazir, A.; Xu, X.; Liu, Y.; Chen, Y. Phage Endolysins: Advances in the World of Food Safety. Cells 2023, 12, 2169. https: / / doi.org / 10.3390 / cells12172169), in addition, its strong specificity allows it to kill pathogenic bacteria without interfering with normal flora, and it directly acts on the cell wall of gram-positive bacteria from the outside, which is more efficient than phages, and has the potential to become a new type of antibacterial preparation.

[0005] SUMMARY

[0006] The present application provides a phage lysin and a preparation thereof, which has a wide lytic spectrum and can lyse not only Staphylococcus aureus, but also Klebsiella pneumoniae, Escherichia coli, Salmonella and Sakazii enterobacter. The lysin can be used alone or in combination with other substances to provide a safe and non-toxic phage lysin preparation for purifying the environment and ensuring food safety.

[0007] The above object is achieved by the following technical solutions:

[0008] [According to Rule 91 correction 28.08.2025] First, the present application provides a phage lysin with an amino acid sequence as shown in SEQ ID NO. 2, which is self-named by the applicant as lysin JS01.

[0009] [According to Rule 91 correction 28.08.2025] Second, the present application provides a coding gene of the above lysin JS01, and its nucleotide sequence is shown in SEQ ID NO. 1.

[0010] [According to the rules 91 correction 28.08.2025] Third, the application provides a biological material containing the above-mentioned lyase JS01 encoding gene sequence (SEQ ID NO. 1); the biological material includes but is not limited to vectors, engineering bacteria, transformants, transgenic cell lines; such as recombinant DNA, expression cassette, transposon, plasmid vector, bacteriophage vector, viral vector, etc.

[0011] Preferably, the above-mentioned vector is a recombinant expression vector containing pPIC9K-TLL.

[0012] Or preferably, the Pichia pastoris transformant containing the above-mentioned lyase JS01 encoding gene sequence; the Pichia pastoris transformant is constructed by linearizing the recombinant expression vector containing the lyase JS01 encoding gene sequence and transforming it into Pichia pastoris GS115.

[0013] [According to the rules 91 correction 28.08.2025] Fourth, the application provides the use of phage lyase JS01 with an amino acid sequence as shown in SEQ ID NO. 2 in inhibiting the growth of bacteria in the environment and food; wherein the bacteria include gram-positive bacteria and gram-negative bacteria; further, the above-mentioned bacteria include but are not limited to bacteria of Staphylococcus, Escherichia, Klebsiella, Salmonella. For example, Staphylococcus aureus, Escherichia coli, Klebsiella Pneumoniae, Salmonella. Such as the use of inhibiting the growth of Staphylococcus aureus in milk (including whole milk, skim milk).

[0014] [According to the rules 91 correction 28.08.2025] Fifth, the application provides the use of phage lyase JS01 with an amino acid sequence as shown in SEQ ID NO. 2 in the preparation of drugs for preventing and treating bacterial infections. The above-mentioned bacteria include but are not limited to bacteria of Staphylococcus, Escherichia, Klebsiella, Salmonella. For example, Staphylococcus aureus, Escherichia coli, Klebsiella Pneumoniae, Salmonella.

[0015] [Rule 91 correction 28.08.2025] Sixth, the present application provides a pharmaceutical composition containing a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO. 2; preferably, the pharmaceutical composition can also include a pharmaceutically acceptable carrier, or excipient. The pharmaceutical composition can be used to prevent bacterial infection, including but not limited to Staphylococcus, Escherichia, Klebsiella, Salmonella bacteria. For example, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella.

[0016] [Rule 91 correction 28.08.2025] Seventh, the present application provides a bactericide containing a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO. 2, which can be used to kill bacteria in the environment, or in food, or on the surface of the skin; the above-mentioned bacteria include but are not limited to Staphylococcus, Escherichia, Klebsiella, Salmonella bacteria. For example, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella.

[0017] [Rule 91 correction 28.08.2025] Eighth, the present application provides a food additive containing a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO. 2.

[0018] [Rule 91 correction 28.08.2025] Ninth, the present application provides a preparation method of the above-mentioned bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO. 2, which comprises the following steps: 1) linearizing a recombinant expression vector containing a gene sequence with a nucleotide sequence as shown in SEQ ID NO. 1, and transforming it into Pichia pastoris GS115 to construct a Pichia pastoris transformant; 2) activating the recombinant yeast strain (Pichia pastoris transformant) with BMGY medium, then inoculating it into BMMY liquid medium for fermentation and cell growth; 3) using methanol to induce the strain to express protein, i.e. to obtain the above-mentioned bacteriophage lytic enzyme.

[0019] The phage lytic enzyme obtained by the application has a broad-spectrum antibacterial effect, and still has a good bacteriostatic effect in a milk matrix. In the embodiments of the application, the enzyme can not only lyse Staphylococcus aureus, but also can lyse Klebsiella pneumoniae, Escherichia coli, Salmonella and Enterobacter sakazim. The lytic enzyme obtained in the embodiments of the application has been sequenced, and the lytic enzyme can be synthesized by using conventional techniques, and is used alone or in combination with other substances, thereby providing a safe and non-toxic bacterial infection prevention and treatment drug for purifying the environment and ensuring food safety, or a food (such as milk) additive. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the result of agarose gel electrophoresis detection of the product of PCR amplification of JS01 gene and extraction of empty plasmid pPIC9K in Example 1; wherein, M: DNA Marker; 1: TLL gene amplification product; 2: empty plasmid pPIC9K.

[0021] Figure 2 is the result of extraction and double enzyme digestion verification of recombinant plasmid pPIC9K-TLL in Escherichia coli DH5a;

[0022] Wherein, M: DNA Marker; 1 and 2: recombinant plasmid pPIC9K-TLL.

[0023] Figure 3 is the result of 1% agarose electrophoresis identification of pPIC9K-TLL linearized by Sac I;

[0024] Wherein, M: DNA Marker; 1 and 2: linearized recombinant plasmid pPIC9K-TLL.

[0025] Figure 4 is the result of specific PCR verification of recombinant yeast genome in Example 3;

[0026] Wherein, M: DNA Marker; 1-12: PCR amplification results of Pichia pastoris GS115 recombinants containing lytic enzyme coding sequences.

[0027] Figure 5 is the result of Tricine-SDS-PAGE electrophoresis detection of the fermentation supernatant of the recombinant yeast strain in Example 4 after 96h of induction fermentation; wherein, M: protein molecular weight marker; 1-5: electrophoresis bands of the fermentation supernatant induced for 0h, 24h, 48h, 72h and 96h, respectively.

[0028] Figure 6 is a schematic diagram of the change of total protein concentration and wet weight of the recombinant yeast strain in the example.

[0029] Figure 7 is the result of analysis of the bacteriostatic ability of the lytic enzyme JS01 in milk in the example;

[0030] A: bactericidal effect of lytic enzyme JS01 in skim milk at 4°C; B: bactericidal effect of lytic enzyme JS01 in skim milk at 25°C; C: bactericidal effect of lytic enzyme S01 in whole milk at 4°C; D: bactericidal effect of lytic enzyme JS01 in whole milk at 25°C. DETAILED DESCRIPTION

[0031] For the convenience of understanding the technical solutions of the present application, the present application will be further described below in combination with specific examples. The reagents and apparatuses involved in the following examples are commercially available unless otherwise specified. The strains, reagents and culture media involved in the examples are as follows:

[0032] 1. 2% LB agar solid medium (1 L): weigh 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast powder and 12 g of agar in 1 L of distilled water, heat to boiling until completely dissolved, 121°C high pressure sterilization for 20 min, cool for standby.

[0033] 0.6% LB agar solid medium (1 L): weigh 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast powder and 6 g of agar in 1 L of distilled water, heat to boiling until completely dissolved, 121°C high pressure sterilization for 20 min, cool for standby.

[0034] LB liquid medium (1 L): weigh 10 g of tryptone, 10 g of sodium chloride, 5 g of yeast powder in 1 L of distilled water, heat to boiling until completely dissolved, 121°C high pressure sterilization for 20 min, cool for standby.

[0035] YPD liquid medium (1 L): 50 g of YPD solid (purchased from Beijing Coolab Technology Co., Ltd.) is dissolved in 1 L of distilled water, the pH value is adjusted to 6.5, 121°C high pressure sterilization for 20 min, cool for standby.

[0036] 10×YNB (1 L): 134 g of YNB solid (purchased from Beijing Coolab Technology Co., Ltd.) is dissolved in 1 L of distilled water, sterilized by filtering with a 0.22 μm filter.

[0037] 500×Biotin (50 mL): 10 mg of Biotin solid is dissolved in 50 mL of distilled water, sterilized by filtering with a 0.22 μm filter, stored at 4°C.

[0038] BMGY liquid medium (1 L): add 10 g of yeast extract (UK OXOID Company) and 20 g of protein peptone to 879 mL of distilled water, 121°C for 20 minutes, after cooling, add 100 mL of 10×YNB, 1 mL of 500×Biotin, 20 mL of 50% glycerol in a clean bench.

[0039] BMMY liquid medium (1L): add yeast extract 10g, peptone 20g to 894mL distilled water, 121℃ for 20min, after cooling, add 100mL 10×YNB, 1mL 500×Biotin, 5mL methanol in the clean bench.

[0040] The gene amplification and transformation identification method involved in the following examples is PCR and DNA sequencing method.

[0041] The protein detection method involved in the following examples is Tricine-SDS-PAGE, which is a conventional method in the art, and the specific steps are disclosed in the literature "H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa. Anal Biochem. 1987 Nov 1;166(2):368-79. doi:10.1016 / 0003-2697(87)90587-2. PMID: 2449095." H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa. Anal Biochem. 1987 Nov 1;166(2):368-79. doi:10.1016 / 0003-2697(87)90587-2. PMID: 2449095."

[0042] The strains and plasmids involved in the following examples are shown in Table 1:

[0043] Table 1 Test strains and plasmids

[0044] The strains involved in the following examples (Table 1, Table 6) are conventional strains in the art, as disclosed in the literature "Genomic epidemiology and characterization of Staphylococcus aureus isolates from raw milk in Jiangsu, China: emerging broader host tropism strain clones ST59 and ST398, Hui Liu etl, frontiers, 2023", which are now preserved in the laboratory of the applicant.

[0045] T4 ligase and NEB buffer are purchased from NEB company.

[0046] The pPIC9K vector is a conventional Pichia pastoris expression vector, which is purchased from Yubao Biology in the examples.

[0047] Example 1 Obtaining of lyase JS01 gene fragment

[0048] Optimization and design of lyase JS01 gene expression sequence:

[0049] [According to the rules 91 correction 28.08.2025] The coding gene TLL of Escherichia coli phage vB_EcoM_HANI is optimized according to the yeast codon bias, and an EcoR I restriction site is added at the 5' end of the optimized gene sequence, and a Not I restriction site is added at the 3' end by specific primer PCR amplification method, that is, the construction of gene expression frame is completed, and the lyase TLL gene fragment (as shown in SEQ ID NO. 1) is obtained.

[0050] The Escherichia coli phage vB_EcoM_HANI used in this example was deposited with the China Center for Type Culture Collection (CCTCC) on December 13, 2022, located at Wuhan University, Wuhan, China, with a postal code of 430072, and the deposit number is CCTCC M 20221948.

[0051] Example 2 Construction of yeast recombinant expression vector

[0052] The specific primer containing the EcoR I and Not I restriction site sequence of the lyase TLL gene fragment obtained in Example 1 was amplified and double-digested to recover and purify the fragment. At the same time, the pPIC9K vector was double-digested with EcoR I and Not I, and the product was verified by Tricine-SDS-PAGE electrophoresis (Figure 1).

[0053] 1. The double enzyme digestion system is as follows:

[0054] Table 2 Double enzyme digestion reaction system

[0055] After 1.5% agarose gel electrophoresis detection, the target band was found at about 559bp and 10000bp (Figure 1), which was consistent with the expected fragment size.

[0056] After the above enzyme digestion system was added, it was placed in a 37°C water bath for 4h, and 1% agarose gel electrophoresis detection was performed. The electrophoresis conditions were 135V for 20min. The enzyme digestion product was recovered with a DNA product recovery kit (purchased from Genview Biotech) to obtain the lyase gene and the double-digested pPIC9K vector.

[0057] 2. After the lyase gene fragment and the pPIC9K plasmid were double-digested with EcoR I and Not I, the lyase gene was ligated with the double-digested pPIC9K vector using T4 DNA ligase. The ligation system is as follows:

[0058] Table 3 Purpose gene and vector reaction connection system

[0059] The connection system is overnight at 16°C.

[0060] The obtained recombinant vector is transformed into E. coli DH5a (purchased from Genview Biotech Co., Ltd.), and the transformation steps are as follows:

[0061] 1) Take 100 μL of ice-melted competent cells, add 10 uL of ligation product, mix gently, and stand on ice for 30 min.

[0062] 2) 42°C water bath heat shock for 45 s, quickly transfer to ice bath, stand for 2 min.

[0063] 3) Add 700 μL of sterile liquid LB medium without antibiotics to the centrifuge tube, mix well, and recover at 37°C, 200 rpm for 60 min to obtain the recovery solution;

[0064] 4) Take the recovery solution and evenly spread it on 1.2% LB solid medium containing (final concentration) 100 μg / mL ampicillin, and place the plate in a 37°C incubator overnight culture.

[0065] 3, E. coli DH5a positive transformant identification:

[0066] Pick a single colony that grows on the 1.2% LB solid medium containing (final concentration) 100 μg / mL ampicillin in step 4) above and inoculate it in 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and incubate it at 37°C, 180 rpm overnight. Take an appropriate amount of plasmid extraction enzyme for digestion (the enzyme digestion program is the same as Table 2), and the detection results are shown in Figure 2.

[0067] The results show that the target fragment is amplified by PCR using the synthesized upstream primer and downstream primer, and the agarose gel electrophoresis detection result shows that the target gene is about 559 bp, which meets the expected result. Pick the positive transformant to extract the recombinant plasmid for double enzyme digestion verification, and find the target band at about 559 bp and 10000 bp by 1.5% agarose gel electrophoresis detection, which is consistent with the expected fragment size, indicating that the recombinant expression vector pPIC9K-TLL is successfully constructed.

[0068] Example 3 Construction of recombinant yeast strain containing lyase gene

[0069] 1. Linearization of recombinant vector pPIC9K-TLL

[0070] The recombinant expression vector pPIC9K-TLL is digested with restriction endonuclease Sac I (purchased from NEB), and the enzyme digestion system and reaction conditions are as follows:

[0071] Table 4 Restriction enzyme digestion conditions for linearizing recombinant vector pPIC9K-TLL

[0072] After the above enzyme digestion system was added, it was placed at 37°C for 4h of reaction, and 1% agarose gel electrophoresis was used for detection. The electrophoresis conditions were 135V for 30min. The electrophoresis results showed that the pPIC9K-TLL recombinant vector was completely linearized (Figure 3), and then the linearized plasmid was purified using the Omega DNA pure kit (Omega).

[0073] The results showed that there was a band in lanes 1 and 2, and the electrophoresis results showed that the pPIC9K-TLL recombinant vector was completely linearized.

[0074] 2. Preparation of Pichia pastoris GS115 competence

[0075] 1) A single colony of Pichia pastoris GS115 (preserved in the laboratory) was inoculated into 5mL YPD liquid medium, and cultured at 30°C and 200rpm for overnight.

[0076] 2) The inoculum was transferred into 100mL YPD liquid medium at a 1% inoculation amount (V / V), and placed in a 30°C and 200rpm shaker until the medium OD = 1.3-1.5. 24h

[0077] 3) Centrifuged at 4°C and 5000rpm for 5min, and the supernatant was discarded.

[0078] 4) The bacterial body was resuspended with 100mL ice-precooled sterile water.

[0079] 5) Centrifuged at 4°C and 5000rpm for 10min, and the supernatant was discarded.

[0080] 6) The bacterial body was resuspended with 50mL ice-precooled sterile water.

[0081] 7) Centrifuged at 4°C and 5000rpm for 10min, and the supernatant was discarded.

[0082] 8) The bacterial body was resuspended and washed with 20mL ice-precooled 1mol / L sorbitol.

[0083] 9) Centrifuged at 4°C and 5000rpm for 5min, and the supernatant was discarded.

[0084] 10) The bacterial body was resuspended with 1mL ice-precooled 1mol / L sorbitol, and the GS115 yeast competent cells were obtained; 100μL was aliquoted in 1.5mL EP tubes for ready-to-use electroporation.

[0085] 3. Electroporation of Pichia pastoris

[0086] 1) In 100 μL GS115 yeast competent cells, add 1 μg of linearized recombinant plasmid, mix well with pipette gun, place on ice for 15 minutes, quickly add 0.2 cm ice pre-cooled electric transformation cup, gently shake the liquid to the bottom of the cup, immediately ice bath.

[0087] 2) Set the electric transformation parameters, voltage 2000V, capacitance 25 μF, resistance 200Ω, general discharge time between 4-5 ms, immediately after electric transformation, add 1 mL of ice pre-cooled 1M sorbitol, mix well.

[0088] 3) Take 200 μL of the above bacteria liquid and spread on YNB plate (YNB solid medium), invert culture in 30°C constant temperature incubator for 2-3 days, until the colony diameter is about 1 mm, then pick single colony for next experiment.

[0089] 4, Screening and identification of positive transformants

[0090] Inoculate the recombinant GS115 bacteria in 5 mL BMGY liquid culture, 30°C 220 rpm shaking culture for 16-18 hours. Centrifuge at 5000 rpm for 5-10 min at room temperature, discard the supernatant. Extract the yeast genome with the fungal genome DNA rapid extraction kit (Kangke Biological), then perform PCR amplification with the above primers.

[0091] PCR system as follows:

[0092] Table 5 Screening and identification of positive clones of Pichia pastoris GS115 recombinant strain PCR system conditions

[0093] PCR conditions: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, for a total of 35 cycles; finally 72°C for 10 min. And use 1% agarose gel electrophoresis for identification (Figure 4).

[0094] The results show that there is a target band at 559 bp, indicating that the recombinant plasmid pPIC9K-TLL is successfully introduced into Pichia pastoris GS115. The corresponding colony is the positive colony.

[0095] Example 4 Induced expression of recombinant yeast

[0096] 1) Use a inoculation ring to pick the positive single colony identified in Example 3, place it in a 250 mL conical flask containing 25 mL of BMGY liquid medium, and shake culture at 28-30°C and 220-250 rpm until the OD 600 = 2.

[0097] 2) Centrifuge at 5000g for 5 min at room temperature, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells with BMMY liquid medium to OD600 = 1.0 (about 100-200 mL).

[0098] 3) Place the bacterial solution obtained in step 2) in a 1 L flask, seal with double-layer gauze, and continue to shake culture at 28-30°C and 220 rpm on a shaker.

[0099] 4) Add analytical grade methanol to the culture medium every 24 h to a final concentration of 1.0%.

[0100] 5) Take bacterial solution samples at different time points, with a sample size of 1 mL, centrifuge at 8000 rpm for 2 min, collect the supernatant, and filter using a 0.22 μm filter for the purpose of analyzing the expression of the target protein, the biological activity of the lyase, and the optimal harvesting time of the bacterial solution. The sampling time points are: 24 h, 48 h, 72 h, and 96 h.

[0101] 6) Centrifuge at 8000 rpm for 10 min at the optimal harvesting time, take the supernatant, and filter using a 0.22 μm filter for preservation.

[0102] 7) Identify the expression of the recombinant protein at each time point using SDS-PAGE, Coomassie staining, and sample spotting experiments (Figure 5).

[0103] 8) Purify the recombinant fusion protein using a HIS-Trap HP affinity column (cytiva).

[0104] 9) Measure the protein concentration of the fermentation supernatant of the recombinant yeast strain using the BCA method (Thermo Fisher) (Figure 6).

[0105] [Corrected according to Rule 91 on 28.08.2025] The results show that the SDS-PAGE analysis results are shown in Figure 5. After methanol induction of the recombinant Pichia pastoris, there is an induced protein band at about 25 kD in the supernatant, which is consistent with the expected size. This indicates that the recombinant bacteria are successfully constructed, and the expressed lyase protein product is a soluble protein. The applicant self-named this protein as phage lyase JS01, whose amino acid sequence is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 1. The expression amount is as high as 61.175 μg / mL.

[0106] Example 5 Bactericidal effect of phage lyase JS01 in culture medium

[0107] The lysis effect of the phage lytic enzyme JS01 obtained in Example 4 on different bacteria was evaluated by using double-layer plate spotting method to explore the lysis spectrum of the phage lytic enzyme JS01. The spotting method is a conventional evaluation method in the art, and the specific evaluation steps in this example are disclosed in the literature "Lu Z, Breidt F Jr, Fleming H P, et al. Isolation and characterization of a Lactobacillus plantarum bacteriophage, phiJL-1, from a cucumber fermentation [J]. Int J Food Microbiol, 2003, 84(2): 225-235."

[0108] Table 6 Lysis spectrum of phage lytic enzyme JS01 Note: "+" represents that the lytic enzyme JS01 has lysis effect on bacteria; "-" represents that the lytic enzyme JS01 has no lysis effect on bacteria.

[0109] The results show that the lytic enzyme JS01 can effectively lyse Staphylococcus aureus, Salmonella, Enterobacter sakazim, Klebsiella pneumoniae and Escherichia coli, and has broad-spectrum lytic activity.

[0110] Example 6 Bacteriostatic application experiment of phage lytic enzyme JS01 in milk

[0111] 1. Staphylococcus aureus YZ17 was cultured to the logarithmic growth phase, and the bacterial concentration was measured by dilution plating method;

[0112] 2. 1 mL of bacterial solution was centrifuged at 8000 rpm for 1 min, 1 mL of PBS was used to wash away the LB liquid medium, the supernatant was discarded after centrifugation, and 1 mL of sterile milk was added;

[0113] 3. The bacteria were diluted to a final concentration of 10 5 CFU / mL with whole milk and skim milk, respectively;

[0114] 4. Set up experimental groups and control groups, each with three parallel samples to increase the reliability of the experimental results. The experimental groups were added with diluted bacterial solution (4x10 4 CFU / mL), phage lytic enzyme (100 μg / mL) and 50 μL PBS buffer, respectively, to observe the inhibitory effect of the lytic enzyme on bacterial growth. The control group was added with the same concentration of bacterial solution and PBS (the amount of PBS added was controlled to be the same volume as the experimental group) to evaluate the natural growth of bacteria in the absence of lytic enzyme. By incubating at different temperatures (4°C and 25°C), the bacteriostatic effect of the lytic enzyme in different storage environments was simulated.

[0115] 5. Repeat the above test twice at 4°C and 25°C constant temperature standing culture, collect samples at 0, 4, 8, 12, 24, 48, 72, 96, 120h to calculate the bacterial concentration (dilution plating method)

[0116] The results are shown in Figure 7, the experiment shows that: in the 4°C environment, the bactericidal rate of phage lytic enzyme in whole milk and skim milk is 99.99%; at 25°C, the bactericidal effect on whole milk and skim milk is not obvious, and there is no significant difference between whole milk and skim milk. It can be seen that the lytic enzyme JS01 provided in the application can effectively kill Staphylococcus aureus in food, and because the phage lytic enzyme itself has safety, therefore, the lytic enzyme JS01 can be used as a food additive to kill bacteria (such as Staphylococcus aureus) in food (such as milk).

[0117] Example 7 Bactericidal test of phage lytic enzyme JS01 on Staphylococcus aureus turbidity

[0118] The broth microdilution method was used to determine the minimum inhibitory concentration of lytic enzyme JS01 on all bacteria in Table 6.

[0119] Prepare a sterile 96-well bacterial culture plate, use a micropipette to suck 50μL TSB liquid medium into the 2-12th rows of holes, add 8mg / mL lytic enzyme solution to the first row of holes, and use a pipette to suck 50μL drug solution from the first row to gradient dilute in turn. Then add 50μL of the diluted bacteria solution (final concentration 5×10 5 CFU / mL) from low concentration to high concentration in turn to rows 2-11, of which row 11 is the bacterial growth control and row 12 is the blank broth control. At this time, the drug concentrations in rows 1-10 are 4mg / mL, 2mg / mL, 1mg / mL, 0.5mg / mL, 0.25mg / mL, 0.125mg / mL, 62.5μg / mL, 31.25μg / mL, 15.625μg / mL, 7.8125μg / mL. After the operation, cover the 96-well plate cover, gently shake horizontally to make it uniform, and place it in a 37°C constant temperature incubator for overnight culture for 16h, and observe the MIC results the next day.

[0120] The results show that the MIC (minimum inhibitory concentration) of lytic enzyme JS01 on different bacteria is as follows in Table 7.

[0121] Table 7 MIC of phage lytic enzyme JS01 on different bacteria

[0122] The above experimental results can be seen that the lower concentration of lytic enzyme JS01 has a good inhibitory effect on a variety of bacteria, which can be prepared as a broad-spectrum bactericide for killing a variety of bacteria on the environment, utensils, and skin surface.

Claims

1. A bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO.

2.

2. [Rule 91 correction 28.08.2025] A gene encoding a bacteriophage lytic enzyme as claimed in claim 1, characterized in that, The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

3. The lytic enzyme of claim 1 for use in inhibiting the growth of bacteria in an environment or food; the bacteria including at least one of Staphylococcus, Escherichia, Klebsiella, Salmonella.

4. The lytic enzyme of claim 1 for use in the preparation of a drug for preventing and treating bacterial infection; the bacteria including at least one of Staphylococcus, Escherichia, Klebsiella, Salmonella.

5. A vector, transformant, engineered bacterium or transgenic cell line containing the coding gene of claim 2.

6. Use according to claim 3, characterized in that, The bacteria include Staphylococcus aureus.

7. Use according to claim 4, characterized in that, The bacteria include Staphylococcus aureus.

8. [Amended according to Rule 91 on 28.08.2025] A pharmaceutical composition characterized in that, The pharmaceutical composition contains a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO.

2.

9. [Amended according to Rule 91 on 28.08.2025] A bactericide, characterized in that, The bactericide contains a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO.

2.

10. [Amended according to Rule 91 on 28.08.2025] A food additive, characterized in that, The additive contains a bacteriophage lytic enzyme with an amino acid sequence as shown in SEQ ID NO. 2.

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