Lyase and application thereof in antibiosis

Abstract

The invention relates to a lyase and a coding gene thereof, and application of the lyase in lysing bacteria. The bacteriophage capable of specifically splitting the psychrophilic pseudomonas is obtained through separation and purification, lyase is cloned from a genome of the bacteriophage, and after the lyase is subjected to prokaryotic expression and purification, the obtained protein shows the characteristics of the psychrophilic pseudomonas. The lyase disclosed by the invention has an obvious antibacterial effect on pseudomonas psychrophila, so that the lyase has a potential application value in the aspect of controlling pollution of the pseudomonas psychrophila.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial, and more particularly to a lytic enzyme and its application in antibacterial. Background Art

[0002] Pseudomonas is a common Gram-negative (G-) bacterium that is widely distributed in soil, water, plants and animal activities. Pseudomonas is one of the most threatening pathogens that cause fish ulcerative syndrome and hemorrhagic sepsis, and it is prevalent in a wide area and can be seen all year round. At the same time, Pseudomonas can cause spoilage of foods such as milk, eggs, meat and fish under low temperature conditions. Studies have shown that psychrophilic Pseudomonas is an important spoilage bacterium in fish meat. Due to the excessive use and abuse of antibiotics in the breeding process, as well as the widespread use of preservatives and disinfectants in the food industry, bacterial resistance has occurred, posing a serious food safety hazard.

[0003] Bacteriophages are natural enemies of bacteria and are considered to be the oldest and most abundant organisms on Earth. Lysins (endolysins) are peptidoglycan hydrolases encoded by bacteriophages. In recent years, lysins have been expected to be used as natural microbial antibacterial agents to replace antibiotics due to their high specificity, high safety and environmental friendliness.

[0004] Therefore, there is a need to identify useful lytic enzymes for lysing Pseudomonas. Summary of the invention

[0005] To solve the above problems, the present invention provides a lyase, the amino acid sequence of which is shown in SEQ ID NO:2.

[0006] The present invention also provides the use of the above lytic enzyme in lysing bacteria.

[0007] In a specific embodiment, the bacterium is Pseudomonas.

[0008] In a specific embodiment, the bacterium is Pseudomonas psychrophilus.

[0009] The present invention also provides a lytic enzyme gene, the encoded amino acid sequence is shown in SEQ ID NO:2.

[0010] In a specific embodiment, the nucleic acid sequence is shown as SEQ ID NO:1.

[0011] The present invention obtains a bacteriophage that can specifically lyse psychrophilic Pseudomonas by separation and purification, clones a lytic enzyme from its genome, and after prokaryotic expression and purification of the enzyme, the obtained protein exhibits the property of being able to lyse psychrophilic Pseudomonas. The lytic enzyme of the present invention has an obvious antibacterial effect on psychrophilic Pseudomonas, and therefore has potential application value in controlling the contamination of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Transmission electron microscope (TEM) image of bacteriophage P-2FD.

[0013] Figure 2 This is the lysis curve of bacteriophage P-2FD against psychrophilic Pseudomonas.

[0014] Figure 3 The SDS-PAGE photos of the induced expression products of the lytic enzyme expression strain. The samples in each lane are as follows: 1: protein standard (10-150KDa); 2: negative control without the introduction of recombinant plasmid; 3: protein supernatant obtained by BL21 induction at 16°C; 4: protein supernatant obtained by BL21 induction at 37°C; 5: protein precipitate obtained by BL21 induction at 16°C; 6: protein precipitate obtained by BL21 induction at 37°C; 7: protein supernatant obtained by T7E induction at 16°C; 8: protein supernatant obtained by T7E induction at 37°C; 9: protein precipitate obtained by T7E induction at 16°C; 10: protein precipitate obtained by T7E induction at 37°C.

[0015] Figure 4 This is the SDS-PAGE picture of the purified lytic enzyme.

[0016] Figure 5 To determine the cleavage ability of the lytic enzyme. DETAILED DESCRIPTION

[0017] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0018] 1. Phage Isolation and Purification and Host Spectrum Identification

[0019] Rotten fish and shrimp from the farmers' market near Wuhan University of Technology in China were used as samples. Weigh 25g of sample, add appropriate amount of physiological saline and homogenize for 2min. Take the supernatant and centrifuge at 12,000rpm for 10min, and filter the supernatant with a 0.22μm membrane. When the growth of Pseudomonas reaches the logarithmic phase, dilute the filtrate with LB medium and incubate it with the bacterial suspension at a ratio of 2:1 (v / v) at 30℃ for 16h. After centrifugation at 12000rpm for 10min, collect the supernatant for double-layer plate culture, and isolate the phage after overnight culture at 30℃. The phage titer (PFU / mL) is the number of plaques × dilution factor.

[0020] The host range of phages was determined by the spot method. Culture the bacteria overnight, take 100 μL of the bacterial solution and mix it with 3.7 mL of semi-solid culture medium and pour it into a culture dish. Take 10 μL of phage and spot it on the plate. Incubate Pseudomonas overnight at 30°C, Vibrio parahaemolyticus, Salmonella and Escherichia coli at 37°C overnight, and observe whether plaques appear on the plate.

[0021] Using psychrophilic Pseudomonas as the host bacteria, a phage P-2FD was screened and obtained. In order to determine the host range of Pseudomonas phage, spot tests were performed on 5 types of bacteria, including 20 Pseudomonas strains, 7 Vibrio parahaemolyticus strains, 7 Salmonella strains, 1 Escherichia coli strain, and 1 Staphylococcus aureus strain. The results showed that among the 20 Pseudomonas strains, phage P-2FD had the ability to lyse 3 of them, and had no lyse ability on Vibrio parahaemolyticus, Salmonella, Escherichia coli, and Staphylococcus aureus, indicating that the phage has a high degree of specificity.

[0022] 2. Morphology of bacteriophage P-2FD

[0023] Centrifuge the phage suspension at 4°C, 12,000rpm for 30min, take the supernatant, and filter the membrane. Ultracentrifuge the filtrate at 20,000rpm for 1h, take the precipitate, and resuspend it in PBS buffer. Spot the phage suspension on the copper mesh, let it absorb naturally for 5-10min, use filter paper strips to absorb excess droplets and dry. Drop 20μL of 2% phosphotungstic acid solution on the copper mesh, and negatively stain for 3-5min. After drying, use a transmission electron microscope to observe the stained phage.

[0024] The results are as follows Figure 1 As shown, bacteriophage P-2FD consists of an icosahedral head with a diameter of 55.38±4.6nm and a short tail with a length of 10.45±1.8nm, so bacteriophage P-2FD belongs to the family Brachyurae of the order Caudovirales.

[0025] 3. Lysis curve of bacteriophage P-2FD

[0026] The concentration of Pseudomonas P-2 was adjusted to about 1.0 × 10 5 CFU / mL, 200 μL of phage were added to 96-well plates according to MOI=1000, 100, 10, 1, 0.1, 0.01, and 0.001, and 200 μL of host bacterial solution were added. A blank control was added with 400 μL LB medium, and a negative control was added with 200 μL 1.0×10 5 CFU / mL of host bacterial solution and 200 μL LB medium. OD was measured every 1 h using an automatic cell growth curvature meter. 600 The values ​​were measured for a total of 36 h.

[0027] The results are as follows Figure 2 As shown in the figure, the number of Pseudomonas in the negative control group continued to increase with the extension of time. At the same time, the phage showed a certain lytic activity when infecting Pseudomonas at different MOIs. Among them, in the MOI=1000 experimental group, the growth of Pseudomonas was almost completely inhibited until 9 hours after inoculation, indicating that the phage P-2FD has a strong inhibitory effect on its host bacteria P-2. Even at a lower MOI (0.001), the growth of the host bacteria continued to be inhibited within 4 hours.

[0028] 4. Phage Genome Extraction and Whole-genome Analysis

[0029] The ZnCl2 method was used, referring to Guo Yangyi jun and slightly modified. DNase I, RNase A, ZnCl2 and proteinase K solution were added to the phage in sequence, and then treated with equal volumes of phenol / chloroform / isoamyl alcohol (25:24:1) and chloroform after centrifugation. Then isopropanol was added to the mixture to precipitate DNA, and the extracted DNA was verified by gel electrophoresis after dissolving with TE buffer.

[0030] The Illumina TruSeq DNA Nano Library Prep Kit was used for library construction and sequencing, and the sequences were spliced ​​using MicrobeTrakr plus v.0.9.1.

[0031] The full genome length of bacteriophage P-2FD was measured to be 40435bp, with a GC content of 59%, and a total of 42 open reading frames (ORFs).

[0032] Its ORF2 was identified to encode a lytic enzyme, the nucleic acid sequence of which is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:2.

[0033] 5. Construction of lytic enzyme expression plasmid and its inducible expression

[0034] ORF2 was amplified by PCR, the vector pET28a was linearized by PCR amplification, and the PCR products of ORF2 and pET28a were mixed. HD cloning Kitn (TaKaRa, Dalian, China) used seamless ligation to connect the target band to the vector, and the ligation product was transformed into BL21 and T7E competent cells by heat shock method. Single colonies were picked in LB medium, cultured overnight and sent for sequencing, and the correct sequence was determined as a positive transformant.

[0035] Add the transformed competent cells to LB agar medium containing kanamycin (50 μg / mL) and spread evenly, and culture at 37°C overnight. Pick a single colony containing the recombinant plasmid and inoculate it into 5 mL LB medium containing kanamycin (50 μg / mL), and culture it at 37°C overnight. Take 200 μL and transfer it to 20 mL LB medium containing resistance, and culture it at 37°C until OD 600nm =0.6-0.8, add IPTG (final concentration 1mM) and culture overnight at 16°C or 37°C. Resuspend the cells with PBS, disrupt the cells with ultrasound, and take samples for SDS-PAGE to verify the expression. Equilibrate the nickel column to room temperature, balance it with washing solution, pass the collected supernatant sample through the column, rinse the nickel column with a buffer containing 20mM imidazole, elute the target protein with an eluent containing 250mM imidazole, dialyze to remove excess imidazole, and take samples for SDS-PAGE detection.

[0036] The results are as follows Figure 3 As shown: the protein was expressed in large quantities in both E. coli BL21 and T7E, and the induction effect at 37°C was significantly better than that at 16°C. The protein concentration in the supernatant was higher. Therefore, the protein in the supernatant of the E. coli BL21 expression system at 37°C was selected for separation and purification.

[0037] The protein in the supernatant was purified and then verified by SDS-PAGE gel electrophoresis. Figure 4 As shown, after elution with protein eluents of different imidazole concentrations, a single band at 15-20 KDa was finally obtained, which was the same size as the target protein.

[0038] 6. Antibacterial effect of lyase on bacteria

[0039] After overnight culture of the host bacteria, the cells were inoculated into 200 mL of LB medium at a ratio of 1%, cultured at 30°C and 200 rpm for 3 h, centrifuged at 6,000 rpm for 10 min, and washed twice with sterile deionized water. The pellet was resuspended in Tris-HCl buffer containing EDTA (50 mmol / L), allowed to stand for 5 min, centrifuged at 6,000 rpm for 10 min, and washed with Tris-HCl buffer to adjust the OD 600 The value is between 0.8 and 1.0. Mix the target protein with different concentrations and the bacterial solution. As a negative control, use Tris-HCl buffer instead of the target protein and measure the change in OD600 value.

[0040] The results are as follows Figure 5As shown, the ORF2 protein lysis ability was measured with Pseudomonas P-2 as the host bacteria, and it was found that the lysis effect was better with the increase of concentration. When the ORF2 protein concentration reached 20 μmol / L, the OD600 value was reduced by 0.787 compared with the control group, and the lysis rate was 78.17%. Therefore, this lysis protein was named LysP-2FD. The above results show that LysP-2FD has a high lysis ability.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A lytic enzyme, characterized in that The amino acid sequence is shown in SEQ ID NO:

2.

2. Use of the lytic enzyme according to claim 1 in lysing bacteria.

3. The use according to claim 2, characterized in that: The bacteria is Pseudomonas.

4. The use according to claim 3, characterized in that: The bacterium is psychrophilic Pseudomonas.

5. A lytic enzyme gene, characterized in that The encoded amino acid sequence is shown in SEQ ID NO:

2.

6. The lytic enzyme gene according to claim 5, characterized in that The nucleic acid sequence is shown in SEQ ID NO:1.

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