Application of aeromonas hydrophila bacteriophage lyase Lys56 in preparation of bacteriostatic agent

Abstract

The invention provides application of aeromonas hydrophila bacteriophage lyase Lys56 in preparation of a bacteriostatic agent, and belongs to the technical field of biology. The aeromonas hydrophila bacteriophage lyase Lys56 disclosed by the invention has an inhibiting effect on certain gram-negative bacteria and gram-positive bacteria, and has no splitting effect on certain probiotics. The Lys56 is not resistant to high temperature and has high acid-base tolerance, the inhibition effect of the Lys56 can be enhanced by low-concentration divalent metal ions, the inhibition effect of the Lys56 can be enhanced by combined use of 1-10 mM of ethylenediamine tetraacetic acid (EDTA) and the Lys56, and the Lys56 can effectively inhibit and remove biofilms of host bacteria. In an artificial pollution model of the fish blocks, the antibacterial effect of the Lys56 is in direct proportion to the concentration of the Lys56. Lys56 has a good sterilization effect and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of an Aeromonas hydrophila phage lytic enzyme Lys56 in preparation of a bacteriostatic agent. BACKGROUND

[0002] Aeromonas hydrophila belongs to the genus Aeromonas of the family Vibrionaceae, is a gram-negative short bacillus, is a thermophilic and motile Aeromonas, is widely distributed in nature, is a primary pathogenic bacterium of various aquatic animals, and is a conditional pathogenic bacterium of human. Research shows that the pathogenicity of Aeromonas hydrophila is closely related to related virulence factors, mainly including adhesion factors, secretory proteins, secretion systems, quorum sensing systems and iron binding systems. Bacterial septicemia caused by Aeromonas hydrophila has a wide range of epidemic, a long epidemic time, a high incidence and mortality, and causes a major threat to aquaculture. The virulence factors of Aeromonas hydrophila can destroy the skin, gill and intestinal tissue of fish, cause hemorrhage, ulcer and organ failure, cause blood lymph function loss or infection of other tissues and organs of shrimps and crabs, and thus cause pathological changes and death. The most typical symptom of human infection with Aeromonas hydrophila is acute gastroenteritis. The act gene producing enterotoxin plays a role by combining with cholesterol, aggregates lipids, interacts with mucosal cells of the gastrointestinal system and internalizes, produces an inflammatory response, causes villus degeneration and produces mucus, and thus causes diarrhea. These virulence factors can help Aeromonas hydrophila to invade and colonize in the host, and can also inhibit the immune response of the host. In addition, drug-resistant strains of Aeromonas hydrophila increase year by year, and drug-resistant infection events occur frequently, which makes the treatment of Aeromonas hydrophila more difficult, and it has become an urgent matter to develop a treatment method that can effectively resist infection of multi-drug resistant strains.

[0003] Phage lysin is a kind of hydrolytic enzyme encoded by the gene of double-stranded DNA phage. Lysin is expressed in the late stage after phage infection of host, and the main action site is the peptidoglycan structure of bacterial cell wall, which can lyse bacteria from the inside, so it is also called endolysin. The lysin structures of phage of gram-positive bacteria and gram-negative bacteria are different. The phage lysin of gram-positive bacteria usually presents a modular structure, which contains two domains, namely N-terminal catalytic domain (CD) and C-terminal cell wall binding domain (CBD), and the N-terminal and C-terminal are connected by a small peptide. After the specific binding of CBD to the ligand on the bacterial peptidoglycan, CD specifically cuts the chemical bond in the peptidoglycan. The phage lysin of gram-negative bacteria is mostly a single domain of CD, and the size is about 15-20 kDa. Since the outer membrane exists in the cell wall of gram-negative bacteria, the phage lysin of gram-negative bacteria usually needs to work together with holin.

[0004] Lysin has many applications in the field of food. PlyEc2 can remove 99.7% of Shiga toxin-producing Escherichia coli O157:H7 in a lettuce leaf model; LysCSA13 is applied to polystyrene, glass and stainless steel surfaces to remove 80%-90% of staphylococcal biofilm; Lysqdvp001 combined with epsilon-polylysine (epsilon-PL) reduces the number of Vibrio parahaemolyticus in oriental shrimp, oyster and drum models; LysC02 is fused with an amphiphilic antibacterial peptide, and the fused lysin shows strong bactericidal activity against Cronobacter sakazakii without the assistance of outer membrane permeabilizers. The advantages of lysin as an antibacterial agent mainly include: wide lytic range, wider lytic spectrum than phage; high efficiency of sterilization, short action time; high safety, no harmful substances produced, no bacterial resistance, no interference with normal intestinal flora, etc. These characteristics provide a new idea and direction for solving bacterial resistance and bacterial prevention and control.

[0005] In-depth study of phage lysin not only helps to reveal the interaction mechanism between phage and bacteria, but also lays a solid foundation for the development of new antibacterial strategies and products, and shows broad application prospects in many fields such as medicine, food industry and agriculture. SUMMARY

[0006] Therefore, the purpose of the present application is to provide an application of Aeromonas hydrophila phage lysin Lys56 in preparation of bacteriostatic agent.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The application provides an Aeromonas hydrophila bacteriophage lytic enzyme Lys56, a gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56, or a vector containing the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56, and application of a recombinant bacterium in preparation of a bacteriostatic agent.

[0009] Preferably, the amino acid sequence of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is shown in SEQ ID No. 1, and the nucleotide sequence of the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is shown in SEQ ID No. 2.

[0010] Preferably, the bacteriostatic spectrum of the bacteriostatic agent comprises gram-positive bacteria and gram-negative bacteria; the gram-positive bacteria comprise Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and the gram-negative bacteria comprise Vibrio parahaemolyticus, Escherichia coli and Shewanella putrefaciens.

[0011] Preferably, the preparation method of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 comprises the following steps:

[0012] 1) PCR amplification is performed on the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56, restriction endonucleases are used to perform double enzyme digestion on the gp56 gene and a pET28a(+) vector after PCR amplification, and a recombination plasmid pET28a(+)-Lys56 is obtained by connecting the enzyme-digested products with T4 DNA ligase;

[0013] 2) The recombination plasmid pET28a(+)-Lys56 is transformed into a competent cell, and the recombination bacterium verified as positive is cultured, induced to express, and collected to extract and purify to obtain the Aeromonas hydrophila bacteriophage lytic enzyme Lys56.

[0014] Preferably, the primers for PCR amplification in step 1) are Lys56-F and Lys56-R, the nucleotide sequence of the Lys56-F is shown in SEQ ID No. 3, and the nucleotide sequence of the Lys56-R is shown in SEQ ID No. 4.

[0015] Preferably, the restriction endonucleases in step 1) are EcoRI and XhoI.

[0016] Preferably, the competent cell in step 2) is Escherichia coli BL21 (DE3).

[0017] The application also provides application of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 in inhibition of Aeromonas hydrophila.

[0018] The application also provides an application of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 in inhibiting biofilm of host bacteria.

[0019] The application also provides an application of metal ions or EDTA in enhancing bacteriostatic activity of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The Aeromonas hydrophila bacteriophage lytic enzyme Lys56 of the application has inhibiting effect on some gram-negative bacteria and gram-positive bacteria, and has no lytic effect on some probiotics. Lys56 is not resistant to high temperature, has strong acid and alkali resistance, and the inhibiting effect can be increased by low-concentration divalent metal ions. The inhibiting effect can be enhanced by combination of 1-10 mM ethylenediaminetetraacetic acid (EDTA) and Lys56. Lys56 can effectively inhibit and remove biofilm of host bacteria. In a fish block artificial pollution model, the bacteriostatic effect of Lys56 is proportional to the concentration of Lys56. Lys56 has good bactericidal effect and good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is an agarose gel electrophoresis diagram of a PCR amplification product of the lytic enzyme gp56 gene;

[0023] Figure 2 is an agarose gel electrophoresis diagram of positive identification of a recombinant expression strain;

[0024] Figure 3 is an expression and purification diagram of the lytic enzyme Lys56 (wherein a is small-scale expression, and b is protein purification);

[0025] Figure 4 is lytic activity of the lytic enzyme Lys56;

[0026] Figure 5 is bacteriostatic capacity of the lytic enzyme Lys56 on Aeromonas hydrophila ATCC7966;

[0027] Figure 6 is stability of the lytic enzyme Lys56 (wherein a is temperature stability, b is pH stability, c is metal ion stability, and d is influence of EDTA on the inhibiting effect);

[0028] Figure 7 is an effect of the lytic enzyme Lys56 on biofilm of Aeromonas hydrophila ATCC7966 (wherein a is inhibiting effect on biofilm, and b is removal capacity on biofilm formed at different times);

[0029] Figure 8is a scanning electron micrograph of the Lys56 enzyme acting on Aeromonas hydrophila ATCC 7966;

[0030] Figure 9 is the change of total number of colonies of artificially contaminated Aeromonas hydrophila during the storage of fish blocks.

[0031] Biological preservation instructions

[0032] The Aeromonas hydrophila phage vB_AhM_7 provided by the application is preserved in the Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 66525-B1, a preservation time of June 17, 2025, and a preservation address of 5th Floor, Building 59, 100, Martyrs' Road, Guangzhou. DETAILED DESCRIPTION

[0033] The application provides an Aeromonas hydrophila phage Lys56, a gene gp56 encoding the Aeromonas hydrophila phage Lys56, or a vector containing the gene gp56 encoding the Aeromonas hydrophila phage Lys56, and a recombinant bacterium in preparation of a bacteriostatic agent.

[0034] In the present application, the amino acid sequence of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is shown as SEQ ID No. 1, specifically MKALAAIGEFITGHVTWLVLAVGLLAGAAMGWSATDMVWKTKYALAEKDWTDQKAAQDRANLS HAEQTVAWERRSAELVAAADSAYQKGIEDGKKSLDADVAAIHDGSKRVRDKFRCPAPAKRPDPVPT ASPSPSGGDEAPQGGLQPADAEFLVRLGHEADGIVRQLTACQDYITQQQRPVSP; the nucleotide sequence of the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is shown as SEQ ID No. 2, specifically ATGAAGGCGCTCGCTGCCATCGGTGAGTTCATCACCGGCCACGTGACATGGCTCGTTCTCGCCGTGGGCCTGCTGGCTGGCGCGGCGATGGGGTGGTCTGCCACCGATATGGTCTGGAAGACCAAGTATGCGCTCGCTGAGAAAGATTGGACTGACCAGAAGGCGGCCCAAGACCGGGCGAACCTCTCCCACGCGGAGCAAACCGTGGCGTGGGAGAGGCGCTCTGCAGAGCTGGTTGCTGCGGCCGACAGTGCTTATCAAAAGGGAATTGAAGATGGCAAAAAGAGCCTTGATGCTGATGTTGCTGCTATCCATGACGGCAGCAAGCGGGTGCGCGACAAGTTCCGCTGCCCTGCCCCAGCCAAACGACCCGACCCTGTGCCCACGGCCAGCCCCTCCCCCAGCGGCGGTGATGAAGCCCCGCAAGGCGGACTTCAGCCAGCGGATGCAGAGTTTCTTGTTCGACTCGGACACGAAGCCGACGGCATCGTCAGGCAGCTAACTGCCTGCCAGGACTACATCACCCAGCAGCAGAGGCCAGTCTCTCCGTGA.

[0035] In the present application, the preparation method of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 comprises the following steps:

[0036] 1) PCR amplification is performed on a gene gp56 encoding Aeromonas hydrophila bacteriophage lyase Lys56, restriction endonucleases are used to double digest the PCR amplified gp56 gene and a pET28a(+) vector, and the digested product is connected by T4 DNA ligase to obtain a recombinant plasmid pET28a(+)-Lys56;

[0037] 2) The recombinant plasmid pET28a(+)-Lys56 is transformed into a competent cell, the recombinant bacteria verified as positive are cultured, expression is induced, bacterial liquid is collected, and Aeromonas hydrophila bacteriophage lyase Lys56 is obtained by extraction and purification.

[0038] In the present application, PCR amplification is performed on a gene gp56 encoding Aeromonas hydrophila bacteriophage lyase Lys56, restriction endonucleases are used to double digest the PCR amplified gp56 gene and a pET28a(+) vector, and the digested product is connected by T4 DNA ligase to obtain a recombinant plasmid pET28a(+)-Lys56. The primers for PCR amplification are Lys56-F and Lys56-R, the nucleotide sequence of Lys56-F is shown in SEQ ID No. 3, specifically CGGAATTCTGGTCTGCCACCGATATGGTC, the nucleotide sequence of Lys56-R is shown in SEQ ID No. 4, specifically CCGCTCGAGTCACGGAGAGACTGGCCTCT; and the restriction endonucleases are EcoRI and XhoI.

[0039] In the present application, the recombinant plasmid pET28a(+)-Lys56 is transformed into a competent cell, the recombinant bacteria verified as positive are cultured, expression is induced, bacterial liquid is collected, and Aeromonas hydrophila bacteriophage lyase Lys56 is obtained by extraction and purification. The competent cell is Escherichia coli BL21 (DE3); the induction expression preferably adopts isopropyl-β-D-thiogalactopyranoside (IPTG), and the purification preferably adopts nickel column affinity chromatography.

[0040] In the present application, the bacteriostatic spectrum of the bacteriostatic agent includes gram-positive bacteria and gram-negative bacteria; the gram-positive bacteria include Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and the gram-negative bacteria include Vibrio parahaemolyticus, Escherichia coli and Shewanella putrefaciens.

[0041] The present application also provides application of Aeromonas hydrophila bacteriophage lyase Lys56 in inhibiting Aeromonas hydrophila.

[0042] The application also provides the use of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 in inhibiting the biofilm of host bacteria. In the application, the host bacteria is Aeromonas hydrophila.

[0043] The application also provides the use of metal ions or EDTA in enhancing the bacteriostatic activity of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56. In the application, the metal ions include Mg 2+ and Ca 2+ , the concentration of the metal ions is preferably 0.1-1 mM, and the concentration of the EDTA is preferably 1-10 mM.

[0044] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0045] Example 1

[0046] Construction of the vector of the lytic enzyme Lys56

[0047] GAATTC TGGTCTGCCACCGATATGGTC-5' (SEQ ID No. 3, EcoRI), Lys56-R: 3'-CCG CTCGAG TCACGGAGAGACTGGCCTCT-5' (SEQ ID No. 4, Xhol), PCR amplification was performed and whether there was a band of interest was verified by 1.5% agarose gel electrophoresis, and the PCR product was purified. Experimental results: as shown in Figure 1

[0048] 2) Restriction enzyme digestion and ligation of the lytic enzyme gp56 gene: the gp56 gene and the pET28a(+) vector were double-digested using restriction endonucleases EcoRI and Xhol, and the digested products were ligated with T4 DNA ligase.

[0049] 3) Transformation of the recombinant vector: Escherichia coli DH5a was selected as the cloning strain, 10 μL of the ligation product was transferred into DH5a by heat shock transformation, and was plated on LB solid plates (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C for 16 h. Multiple single colonies were picked and inoculated in LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL), and after shaking culture at 37°C, the plasmid was extracted and sequenced. The recombinant plasmid with correct sequencing results was named pET28a(+)-Lys56 and was transferred into BL21(DE3), which was plated on LB solid plates (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C for 16 h. Multiple single colonies were picked from the plate and inoculated in LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL), and after shaking culture at 37°C, the method of bacterial liquid PCR was used for verification. Electrophoresis verification of a band at the predicted length position indicated that the expression strain pET28a(+)-Lys56-BL21(DE3) was successfully constructed. Experimental results: as shown in Figure 2

[0050] Example 2

[0051] Preparation of recombinant lytic enzyme

[0052] 1) Prokaryotic expression of lytic enzyme Lys56: pET28a(+)-Lys56-BL21(DE3) was inoculated in LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C at 180 r / min overnight. The next day, 10% of the bacterial solution was added to fresh LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C with shaking until the logarithmic phase (OD 595nm ​​=0.4-0.6), add IPTG solution (final concentration 1 mmol / L), and continue culturing for 4-6 h. Centrifuge the bacterial culture at 10000 r / min for 5 min at 4℃, remove the culture medium, resuspend the bacterial cells in PBS, and sonicate to disrupt the bacterial cells. Observe the protein band positions of E. coli transformed with empty vector and E. coli transformed with recombinant vector before and after induction by SDS-PAGE gel electrophoresis.

[0053] 2) Purification of the lysin Lys56: 10% of overnight cultured pET28a(+)-Lys56-BL21(DE3) was inoculated into 400 mL LB liquid medium (containing kanamycin at a final concentration of 50 μg / mL) and cultured at 37°C with shaking until the logarithmic growth phase (OD50). 595nm =0.4~0.6), add IPTG solution (final concentration 1mmol / L), and induce expression at 37℃ for 4~6h. Centrifuge the bacterial culture at 10000r / min for 25min at 4℃, add 4~5mL of Binding Buffer to resuspend the precipitate and sonicate. After sonication, centrifuge and collect the precipitate, add 4~5mL of Binding Buffer to dissolve the precipitate. Take 1mL of the dissolved precipitate and add it to a centrifuge tube equipped with a nickel column, mix well, and rotate overnight at 4℃ to ensure that the protein is fully bound to the nickel column. Remove the centrifuge tube, centrifuge at 800r / min for 5min at 4℃, discard the supernatant, add 2mL of Binding Buffer to wash once, centrifuge and discard the supernatant. Add 200 μL of solution buffer, rotate at 4°C for 1–2 hours, then centrifuge. The supernatant is the purified protein, namely the lysin Lys56 (amino acid sequence as shown in SEQ ID No. 1, specifically MKALAAIGEFITGHVTWLVLAVGLLAGAAMGWSATDMVWKTKYALAEKDWTDQKAAQDRANLS HAEQTVAWERRSAELVAAADSAYQKGIEDGKKSLDADVAAIHDGSKRVRDKFRCPAPAKRPDPVPT ASPSPSGGDEAPQGGLQPADAEFLVRLGHEADGIVRQLTACQDYITQQQRPVSP). The protein band positions and concentration were observed by SDS-PAGE gel electrophoresis. Experimental results: Figure 3 As shown.

[0054] Example 3

[0055] Determination of the cleavage activity of the lysin Lys56

[0056] Aeromonas hydrophila ATCC 7966 (purchased from American Type Culture Collection) was cultured to logarithmic phase (OD 595nm = 0.4-0.6), 20 μL of the bacterial solution was added to LB semi-solid medium, mixed well and then poured onto the lower layer of agar medium. After solidification, 50 μL of Lys56 was added dropwise, dried and then cultured at 30°C for 6-8 h, and the inhibition zone was observed.

[0057] The experimental results are shown in Table 1. Figure 4 As shown in Table 1, 50 μL of Lys56 can form a lysis zone on LB solid medium, and the diameter of the lysis zone can reach 21 mm.

[0058] Example 4

[0059] Determination of the bacteriostatic ability of the lytic enzyme Lys56

[0060] Aeromonas hydrophila ATCC 7966 was cultured to logarithmic phase (OD 595nm = 0.4-0.6), and 100 μL of the bacterial solution was added to a 96-well plate. The experimental group was added with 100 μL of Lys56, and the control group was added with 100 μL of LB liquid medium, and then cultured at 30°C for 12-14 h. The OD 595nm was measured three times. 100 μL of the co-culture was diluted 10 times with sterile PBS, 5 μL of the diluted solution was dropped on LB solid medium, and 100 μL of the diluted solution was plated and cultured at 37°C. The colonies were observed and counted.

[0061] The experimental results are shown in Table 2. Figure 5 As shown in Table 2, the OD 595nm of the Lys56-treated group was 0.319 after 12 h of treatment, while the OD 595nm value of the control group was close to 1, with an inhibition rate of 89.95%. The total number of colonies in the control group was 4.2 x 10 8 CFU / mL, while the total number of colonies in the Lys56-treated group was 4.4 x 10 6 CFU / mL, which decreased by two orders of magnitude.

[0062] Example 5

[0063] Determination of the lysis spectrum of the lytic enzyme Lys56

[0064] The pathogenic Vibrio parahaemolyticus ATCC 17802, Pseudomonas fluorescens ATCC 13525, Shigella sonnei ATCC 29930 and Listeria monocytogenes ATCC 7644 were purchased from American Type Culture Collection;

[0065] Escherichia coli GDMCC1.366, Shewanella putrefaciens GDMCC1.4594, Bacillus cereus GDMCC1.2764, Staphylococcus aureus GDMCC1.2442 were purchased from Guangdong Microbial Culture Collection Center;

[0066] Lactobacillus plantarum GNL3 was described in the literature (Gu Nai, Wu Wenyu, Liu Shuilin, et al. Screening, identification and fermentation characteristics of salt-tolerant lactic acid bacteria in northeast traditional pickled vegetable [J]. China Brewing, 2024, 43(08): 31-37.);

[0067] Lactobacillus paracasei CICC 6244, Lactobacillus acidophilus CICC 6081, Lactobacillus plantarum CICC 6240 were purchased from China General Microbiological Culture Collection Center;

[0068] Gram-negative bacteria such as pathogenic Vibrio parahaemolyticus ATCC17802, Escherichia coli GDMCC1.366, Pseudomonas fluorescens ATCC13525, Shigella sonnei ATCC29930, Shewanella putrefaciens GDMCC1.4594 and Gram-positive bacteria such as Bacillus cereus GDMCC1.2764, Listeria monocytogenes ATCC7644, Staphylococcus aureus GDMCC1.2442 and probiotics such as Lactobacillus acidophilus CICC 6081, Lactobacillus plantarum GNL3, Lactobacillus plantarum CICC 6240, Lactobacillus paracasei CICC 6244 were selected and cultured to logarithmic phase (OD 595nm =0.4-0.6), 20μL of activated Gram-negative bacteria and Gram-positive bacteria were added to the semi-solid medium, mixed evenly, then poured onto the lower layer of agar medium, the probiotics were plated on MRS solid plate, after solidification and drying, 20μL of Lys56 was added, after absorption and drying, the culture was observed for 6-8h.

[0069] The experimental results are shown in Table 1. Lys56 has a wide lytic spectrum. Among Gram-negative bacteria, it has strong lytic effect on Vibrio parahaemolyticus ATCC17802, weak lytic effect on Escherichia coli GDMCC1.366 and Shewanella putrefaciens GDMCC1.4594, and cannot lyse Pseudomonas fluorescens ATCC13525 and Shigella sonnei ATCC29930. Among Gram-positive bacteria, it has weak lytic effect on Bacillus cereus GDMCC1.2764, Listeria monocytogenes ATCC7644 and Staphylococcus aureus GDMCC1.2442. It has no lytic effect on the four probiotics.

[0070] Table 1 Lytic spectrum of lytic enzyme Lys56

[0071]

[0072] Note: ++: Clear and transparent fracture zone; +: Slightly opaque fracture zone; -: No fracture zone.

[0073] Example 6

[0074] Determination of the stability of the lysin Lys56

[0075] 1) Effect of temperature on the activity of lysin Lys56: Aeromonas hydrophila ATCC7966 was cultured to the logarithmic growth phase (OD56). 595nm =0.4~0.6), Lys56 was placed at 4℃, 37℃, 45℃, 55℃, 65℃, and 75℃ for 10 min. 100 μL of bacterial culture and 100 μL of Lys56 treated at different temperatures were added to a 96-well plate and incubated at 30℃. OD was measured at 0h, 1h, 2h, 4h, and 12h. 595nm The experiment was repeated 3 times.

[0076] Experimental results: such as Figure 6 As shown in a, Lys56's lytic ability gradually decreased after incubation at high temperature for 10 minutes, and its antibacterial effect was significant at 4℃.

[0077] 2) Effect of pH on the activity of lysin Lys56: The pH of PBS buffer was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, and 11 using 2 mol / L NaOH and 1 mol / L HCl. 2 mL of bacterial culture was centrifuged, the supernatant was discarded, and the cells were resuspended in PBS at different pH values. 100 μL of the resuspended bacterial culture and 100 μL of Lys56 were added to each well of a 96-well plate and incubated at 30 °C. OD values ​​were measured at 0 h, 1 h, 2 h, 4 h, and 12 h. 595nm The experiment was repeated 3 times.

[0078] Experimental results: such as Figure 6 As shown in b, Lys56 exhibits significant antibacterial effects at pH 6, pH 7, and pH 8, while overall Lys56 is relatively stable at pH 5–10.

[0079] 3) Effect of metal ions on the activity of lysin Lys56: MgCl2, CaCl2, and KCl were added to Lys56 at a volume ratio of 10:1 to achieve final concentrations of 0, 0.1, 1, and 10 mM, respectively. 100 μL of bacterial culture and 100 μL of Lys56 containing different concentrations of metal ions were added to 96-well plates and incubated at 30℃. OD values ​​were measured at 0 h and 12 h. 595nm And calculate ΔOD 595nm The experiment was repeated 3 times.

[0080] Experimental results: such as Figure 6 As shown in c, low concentrations (0.1–1 mM) of Mg 2+ and Ca2+ The bacteriostatic effect of Lys56 can be increased, K + The bacteriostatic effect of Lys56 can be reduced.

[0081] 4) Effect of EDTA on the activity of lysin Lys56: 1 mM, 5 mM, 10 mM, 20 mM, 25 mM EDTA were prepared respectively, 50 μL of bacterial solution, 50 μL of EDTA of different concentrations and 100 μL of Lys56 were added in a 96-well plate, and no EDTA was used as a control, and incubation was carried out at 30°C, and OD 595nm was measured at 0 h, 1 h, 2 h, 4 h and 12 h respectively, and the experiment was repeated 3 times.

[0082] Experimental results: as shown in d of Figure 6 , Lys56 can effectively lyse host bacteria when used in combination with 1-10 mM EDTA.

[0083] Example 7

[0084] Effect of lysin Lys56 on biofilm of host bacteria

[0085] 1) Inhibition of lysin Lys56 on biofilm of host bacteria: 10 μL of Aeromonas hydrophila ATCC7966 bacterial solution and 90 μL of LB liquid medium were added in a 96-well plate, 100 μL of Lys56 of different concentrations were added in the experimental group, and 100 μL of sterile PBS was added in the control group, and incubation was carried out at 30°C for 48 h, and then crystal violet staining was carried out, and OD 595nm was measured, and the experiment was repeated 3 times.

[0086] Experimental results: as shown in a of Figure 7 , the OD 595nm values of the Lys56 treatment groups of 800-50 μg / mL were significantly lower than those of the control group, and the inhibition rate was 80.71%-77.48%, and the OD 595nm value of the Lys56 treatment group of 25 μg / mL was significantly lower than that of the control group, and the inhibition rate was 70.18%, and the inhibition effect was slightly lower than that of the other five groups.

[0087] 2) Removal of lysin Lys56 on biofilm of host bacteria: 2 μL of Aeromonas hydrophila ATCC7966 bacterial solution and 198 μL of LB liquid medium were added in a 96-well plate, and incubation was carried out for 12 h, 24 h, 36 h and 48 h respectively. The planktonic bacteria were discarded and washed with PBS, 200 μL of Lys56 was added in the experimental group, and 200 μL of sterile PBS was added in the control group, and incubation was carried out at 30°C overnight. The next day, crystal violet staining was carried out, and OD 595nm was measured, and the experiment was repeated 3 times.

[0088] Experimental results: as shown in Figure 7As shown in b, 50 μg / mL Llys56 has a significant removal effect on biofilms formed at different time periods.

[0089] Example 8

[0090] Scanning electron microscopy was used to observe the cleavage effect of the lysin Lys56.

[0091] Silica wafers were added to 24-well plates. The experimental group received 1 mL of LB broth, 10 μL of *Aeromonas hydrophila* ATCC7966 culture, and 100 μL of Llys56. The control group received only 1 mL of LB broth and 10 μL of *Aeromonas hydrophila* ATCC7966 culture. Incubation was carried out overnight at 30°C. The wafers were gently rinsed with sterile PBS, and one drop of pre-chilled 2.5% glutaraldehyde fixative was placed on each wafer. Fixation was carried out overnight at 4°C. The wafers were then gently rinsed with sterile PBS and dehydrated with 50%, 70%, 90%, and 100% ethanol for 10 min each time. After dehydration, the wafers were dried and observed under a scanning electron microscope.

[0092] Experimental results: such as Figure 8 As shown, the control group (e.g.) Figure 8 (As shown in a and b) The host bacteria are rod-shaped, plump, and smooth without depressions; after Lys56 treatment (as shown in a and b), the host bacteria are rod-shaped, plump, and smooth without depressions; Figure 8 (As shown in c and d in the figure), the bacteria broke apart, the contents leaked out, the surface became sunken and wrinkled, and there was no complete bacterial morphology.

[0093] Example 9

[0094] Application of lyase Lys56 in the antibacterial activity of artificially contaminated Aeromonas hydrophila in fish fillets

[0095] Cut fresh grass carp fillets into 5±0.5g pieces and sterilize both sides with ultraviolet light for 15 minutes. Dilute the logarithmic-phase Aeromonas hydrophila ATCC7966 bacterial suspension to 10... 5 CFU / mL, fish pieces were evenly coated with the bacterial solution on both sides and then removed and placed in a sterile sealed bag. In the experimental group, 500 μL of Lys56 at different concentrations was evenly added to the surface of the fish meat, while in the control group, 500 μL of sterile PBS was added. All samples were stored at 4°C. Total bacterial count was measured every 3 days.

[0096] Experimental results: such as Figure 9 As shown, Lys56 can slow down the growth of bacterial colonies in fish meat samples, and the inhibition efficiency increases with increasing concentration.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of an Aeromonas hydrophila bacteriophage lytic enzyme Lys56, a gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56, or a vector containing the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56, or a recombinant bacterium in preparation of a bacteriostatic agent.

2. Use according to claim 1, characterized in that, The amino acid sequence of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is shown as SEQ ID No. 1, and the nucleotide sequence of the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is shown as SEQ ID No.

2.

3. Use according to claim 1, characterized in that, The bacteriostatic spectrum of the bacteriostatic agent includes gram-positive bacteria and gram-negative bacteria; the gram-positive bacteria include Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, and the gram-negative bacteria include Vibrio parahaemolyticus, Escherichia coli, and Shewanella putrefaciens.

4. Use according to claim 1, characterized in that, The preparation method of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 comprises the following steps: 1) PCR amplification is performed on the gene gp56 encoding the Aeromonas hydrophila bacteriophage lytic enzyme Lys56, restriction endonucleases are used to double-digest the gp56 gene after PCR amplification and a pET28a(+) vector, and a T4 DNA ligase is used to ligate the digested products to obtain a recombinant plasmid pET28a(+)-Lys56; 2) the recombinant plasmid pET28a(+)-Lys56 is transformed into a competent cell, the recombinant bacterium verified as positive is cultured, expression is induced, bacterial liquid is collected, and the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 is extracted and purified.

5. The use according to claim 1, characterized in that, The primers for the PCR amplification in step 1) are Lys56-F and Lys56-R, the nucleotide sequence of the Lys56-F is shown as SEQ ID No. 3, and the nucleotide sequence of the Lys56-R is shown as SEQ ID No.

4.

6. Use according to claim 1, characterized in that, The restriction endonucleases in step 1) are EcoRI and XhoI.

7. The use according to claim 1, characterized in that, The competent cell in step 2) is Escherichia coli BL21(DE3).

8. An application of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 in inhibition of Aeromonas hydrophila.

9. An application of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56 in inhibition of a biofilm of a host bacterium.

10. An application of metal ions or EDTA in enhancing bacteriostatic activity of the Aeromonas hydrophila bacteriophage lytic enzyme Lys56.

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