Phage lyase and application thereof in preparation of antibacterial agent

Abstract

The invention discloses bacteriophage lyase and application thereof in preparation of an antibacterial agent. The bacteriophage lyase has a remarkable splitting effect on gram-negative bacteria, so that the bacteriophage lyase can be used as a broad-spectrum antibacterial protein. For example, the antibacterial effect on gram-negative bacteria such as escherichia coli, pseudomonas aeruginosa and klebsiella pneumoniae 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] The invention relates to a bacteriophage lytic enzyme and application thereof in the preparation of an antibacterial agent, belonging to the field of bacteriophage lytic enzymes. Background Art

[0002] With the widespread use of antibiotics, bacterial resistance is becoming an increasingly serious problem. Gram-negative bacteria (such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) in particular, due to their complex cell wall structures and multidrug resistance mechanisms, have become a major challenge in the treatment of clinical infections. Traditional antibiotics not only easily induce drug resistance but can also cause side effects such as intestinal flora imbalance and allergic reactions. Therefore, the development of new antimicrobial agents to replace or supplement antibiotics has become a research hotspot in the global biopharmaceutical field.

[0003] Phage lytic enzymes, a class of bacterial cell wall hydrolases encoded by bacteriophages, are considered potential alternatives to antibiotics due to their high efficiency, strong specificity, and resistance to drug resistance. Their mechanism of action is to hydrolyze the peptidoglycan layer, destroying the bacterial cell wall, leading to bacterial lysis and death. However, the application of existing phage lytic enzymes still faces many limitations: First, most lytic enzymes are effective against Gram-positive bacteria, but have weak antibacterial activity against Gram-negative bacteria because their outer membrane structure hinders contact between the lytic enzyme and the cell wall; second, some lytic enzymes are insufficiently stable in complex environments (such as high temperature and extreme pH), limiting their industrial application; and finally, the high production cost and immature large-scale preparation process of lytic enzymes also restrict their commercial promotion.

[0004] In recent years, research on lytic enzymes targeting Gram-negative bacteria has made some progress, for example, by combining the use of metal chelators (such as EDTA) to disrupt the outer membrane to enhance the permeability of lytic enzymes. However, the existing technology still lacks lytic enzymes that have broad-spectrum antibacterial properties, high stability, and efficient production processes. In addition, most reported lytic enzymes have a narrow host spectrum and are only effective against specific strains, making it difficult to meet the needs of multi-scenario applications. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a bacteriophage lytic enzyme and its application in the preparation of antibacterial agents.

[0006] Technical solution: To solve the above technical problems, the present invention provides a phage lytic enzyme, the amino acid sequence of which is shown in SEQ ID NO.1: MQLTKPEYEAMVADVALSKASVGDSSFIEATKSLKDKDGVSLYE.

[0007] Among them, the phage lytic enzyme is Klebsiella pneumoniae phage lytic enzyme, which is a bacterial cell wall hydrolase.

[0008] The present invention also provides a gene encoding the phage lytic enzyme.

[0009]

[0010] The present invention also provides the use of the phage lytic enzyme or its active fragments and analogs in the preparation of antibacterial agents.

[0011] The active fragments and analogs have at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 1 or have alternative amino acid sequences with the same functional groups.

[0012] Preferably, the bacteriophage lytic enzyme or its binding domain is used in combination with a signal molecule.

[0013] Preferably, the phage lytic enzyme or its binding domain is fused with the signal molecule to form a fusion through gene fusion or chemical coupling.

[0014] The replacement amino acid sequence is obtained by conservatively replacing amino acids in the same amino acid group, wherein the amino acid group includes aliphatic amino acids, hydroxyl- or sulfur / selenium-containing amino acids, cyclic amino acids, aromatic amino acids or basic acidic amino acids and their amides.

[0015] Among them, the aliphatic amino acids include glycine, alanine, valine, leucine or isoleucine; the hydroxyl or sulfur / selenium-containing amino acids include serine, cysteine, threonine or methionine; the cyclic amino acids include proline; the aromatic amino acids include phenylalanine, tyrosine or tryptophan; the basic amino acids include histidine, lysine or arginine; the acidic amino acids and their amides include aspartic acid, glutamic acid, asparagine or glutamine.

[0016] Wherein, the antibacterial agent is used to inhibit and / or kill Gram-negative bacteria.

[0017] Wherein, the Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

[0018] Wherein, the phage lytic enzyme is used in combination with EDTA.

[0019] Among them, the phage lytic enzyme has a strong inhibitory effect on Gram-negative bacteria, has broad-spectrum antibacterial properties, and has good thermal stability. It can effectively replace antibiotics and is widely used in the feed industry, medicine, food industry, animal husbandry, beer industry, aquaculture and other fields.

[0020] The products include medicines for external or internal use, feed or food additives, disinfectants, beauty products or medical devices.

[0021] Wherein, the concentration of the phage lytic enzyme is 0.1-1 mg / mL.

[0022] Wherein, the concentration of the EDTA is 0.1-10 mM.

[0023] Furthermore, the concentration of the EDTA is 0.5-10 mM.

[0024] Furthermore, the concentration of the EDTA is 1-10 mM.

[0025] Furthermore, the concentration of the EDTA is 5-10 mM.

[0026] Wherein, the applicable temperature of the antibacterial agent is 4-70°C.

[0027] Furthermore, the applicable temperature of the antibacterial agent is 4 to 50°C.

[0028] Wherein, the applicable pH of the antibacterial agent is 3-11.

[0029] Furthermore, the applicable pH of the antibacterial agent is 3-11.

[0030] The present invention also provides an antibacterial agent for inhibiting and / or killing Gram-negative bacteria, which contains the phage lytic enzyme.

[0031] The present invention also provides a method for preparing the phage lytic enzyme, comprising the following steps: centrifugation after culture, collecting the supernatant and separating and purifying the phage lytic enzyme using a Ni-TED column, then performing a dialysis replacement step, and vacuum concentrating and freeze-drying to obtain the phage lytic enzyme.

[0032] Preferably, the culture conditions are: 16-30°C, 180-220 rpm, 12-18 hours.

[0033] Specifically, the preparation method of the phage lytic enzyme is:

[0034] S1: obtain culture solution at 30°C and 180 rpm;

[0035] S2: The culture solution is centrifuged to collect the precipitate, the bacteria are broken and centrifuged, and the supernatant is taken, separated and purified by Ni-TED column, and then subjected to a dialysis replacement step, vacuum concentrated and freeze-dried to obtain the phage lytic enzyme.

[0036] During the induction expression of Klebsiella pneumoniae phage lytic enzyme S16a, the final concentration of IPTG is 0.2-1 mM and the temperature is 16-37°C.

[0037] The present invention provides a novel phage lytic enzyme derived from Klebsiella pneumoniae phage, which achieves efficient soluble production by optimizing gene expression and purification processes.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0039] (1) The present invention isolates a novel antimicrobial protein, which exhibits significant antibacterial activity against a variety of Gram-negative bacteria (including multidrug-resistant strains), and has a particularly good antibacterial effect against pathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and the like. It has broad-spectrum antimicrobial activity and good stability, overcoming the poor environmental adaptability of conventional lytic enzymes and providing a new, efficient and safe strategy for the prevention and control of bacterial infections. The antimicrobial protein can replace antibiotics and be applied to the prevention and treatment of bacterial diseases, as well as sterilization of medical devices and medical facilities. It can also be used as an additive in feed processing, food industry, animal husbandry, beer industry, aquaculture, cosmetics production, fruit preservation, and other fields.

[0040] (2) The antimicrobial protein disclosed in the present invention is a soluble protein, and the natural antimicrobial protein is produced from Escherichia coli, has the characteristics of rapid reproduction, strong vitality, safety and non-toxicity, etc., which facilitates the large-scale production of the natural antimicrobial protein and has huge economic value.

[0041] (3) The antimicrobial protein disclosed in the present invention can be used in combination with EDTA to significantly enhance its antimicrobial effect, and the production process is simple and the cost is controllable, providing a technical basis for large-scale application in the fields of medical care, food, agriculture, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 To optimize the concentration of isopropyl-BD-thiogalactopyranoside (IPTG) for induction during lytic enzyme expression;

[0043] Figure 2 To optimize the induction temperature during the expression of lytic enzymes;

[0044] Figure 3 The figure is the SDS PAGE electrophoresis diagram of the lytic enzyme engineered bacteria after expression, fragmentation and purification; wherein: 1 is the marker, 2 is the total bacterial cells after fragmentation, 3 is the precipitate after fragmentation, and 4 is the supernatant after fragmentation;

[0045] Figure 4 For analysis of the optimal inhibitory concentration of lytic enzyme;

[0046] Figure 5 Analysis of optimal EDTA concentration for lytic enzyme;

[0047] Figure 6 For the lytic enzyme host spectrum;

[0048] Figure 7 To test the temperature tolerance of lyase;

[0049] Figure 8 This is a test for pH tolerance of lyase. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0051] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0052] Experimental Materials:

[0053] Enzymes and reagents: The enzymes used in the molecular biology operations in the examples were purchased from TakaRa, and the corresponding operation steps were carried out in full accordance with the relevant product instructions.

[0054] Klebsiella pneumoniae liquid and solid culture media were purchased from Solebao Biotechnology, and the corresponding preparation methods were carried out in full accordance with the relevant product instructions. Primer design and sequencing of the Klebsiella pneumoniae phage lytic enzyme and expression vector involved in the examples were completed by Sangon Biotechnology (Shanghai) Co., Ltd. All other raw materials and excipients whose sources are not indicated are commercially available products.

[0055] Example 1 Construction of Klebsiella pneumoniae phage lytic enzyme S16a expression vector

[0056] Using the DNA of Klebsiella pneumoniae phage pS16a (NCBI accession number PV239575) obtained by screening sludge collected from the sewage treatment plant in Xiqing District, Tianjin as a template, the primer pair S16a-F (5'-AAAACTGCAGatgcagctgaccaagccagagtatg-3') and S16a-R (5'-CCGCTCGAGgaccgaaagtgttcccactcgggag-3') were designed to amplify the lytic enzyme gene.

[0057] Table 1 PCR system

[0058]

[0059]

[0060] Table 2 PCR reaction procedure

[0061]

[0062] The amplified lytic enzyme gene and vector pET30a were digested with restriction endonucleases NdeI and XhoI, respectively, and then ligated with T4 ligase to successfully construct the recombinant plasmid pET30a-S16a. Subsequently, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to construct the recombinant Escherichia coli BL21 / pET30a-S16a.

[0063] Example 2 Optimization of expression and purification conditions of Klebsiella pneumoniae phage lytic enzyme S16a

[0064] 1. Optimization of IPTG concentration-induced expression conditions: Use a sterile pipette tip to randomly pick a single colony containing the recombinant plasmid, inoculate it into LB liquid culture medium containing 50μg / ml Kanamycin, and culture it in a 37°C shaker overnight; transfer it to 50ml LB liquid culture medium containing 50μg / ml Kanamycin at a 1% inoculation volume, and culture it in a 37°C, 220rpm constant temperature shaker until the logarithmic phase, add IPTG to a final concentration of 0.2mM, 0.4mM, 0.6mM, 0.8mM, and 1mM, respectively. Another bottle without IPTG is used as an uninduced control; culture it in a constant temperature shaker at 25°C and 180rpm overnight, collect the bacteria by centrifugation at 4°C, perform ultrasonic disruption (ultrasound conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution is transparent and clear; take the total protein for protein electrophoresis. The results are as follows Figure 1 As shown in the figure, the protein expression level increased significantly under the induction of IPTG. The expression level of S16a was slightly different under the induction of different IPTG concentrations. The optimal IPTG concentration in the following experimental expression process was 0.2 mM.

[0065] 2. Optimization of temperature-induced expression conditions: Use a sterile pipette tip to randomly pick a single colony containing the recombinant plasmid, inoculate it into LB liquid culture medium containing 50μg / ml Kanamycin, and culture it in a 37°C shaker overnight; transfer it to 50ml LB liquid culture medium containing 50μg / ml Kanamycin at a 1% inoculation volume, and culture it in a 37°C, 220rpm constant temperature shaker to the logarithmic phase, add the IPTG obtained in the above experiment to a final concentration of 0.2mM, and place it in a constant temperature shaker set to 16°C, 20°C, 25°C, 30°C, 37°C, and 180rpm for overnight culture. Collect the bacteria at 4°C by centrifugation; perform ultrasonic disruption (ultrasonic conditions: 46% power; 8min, ultrasonic 5s, interval 5s) until the bacterial solution is transparent and clear; take the total protein for protein electrophoresis. The results are as follows Figure 2 As shown in the figure, the protein expression levels were different under different temperature induction conditions. The protein expression level was the highest under the induction condition of 25℃, and the optimal temperature induction condition was 25℃.

[0066] 3. Expression and purification of Klebsiella pneumoniae phage lytic enzyme S16a: Recombinant Escherichia coli BL21 / pET30a-S16a was inoculated into LB liquid medium containing 50 μg / ml kanamycin and cultured at 37°C. A 1% inoculum was transferred to 50 ml of liquid LB containing 50 μg / ml kanamycin and cultured in a constant temperature shaker at 37°C and 220 rpm until the exponential growth phase. IPTG was added to a final concentration of 0.2 mM and cultured overnight in a constant temperature shaker at 25°C and 180 rpm. The cells were collected by centrifugation at 4°C and ultrasonicated (ultrasonication conditions: 46% power; 8 min, 5 s, 5 s intervals) until the cell solution was clear. After fragmentation, the expressed protein was purified using nickel-nitrotriacetic acid chromatography (Ni-NTA columns; Qiagen, Dusseldorf, Germany) with 2 ml of 20 mM imidazole and 2 ml of 30 mM imidazole to elute impurities, and 4 ml of 5 mM imidazole to elute impurities. The target protein was eluted with 100mM buffer. The protein was then dialyzed into PBS at pH 7. The results are shown in Figure 2. Figure 3 As shown, by constructing the expression strain BL21 / pET30a-S16a for the antimicrobial protein S16a, the lytic enzyme S16a was soluble expressed and purified in Escherichia coli, and its lytic activity was determined. The amino acid sequence of the antimicrobial protein S16a is shown in SEQ ID NO.1.

[0067] Example 3 Exploration of the Optimal Inhibitory Concentration of Klebsiella Pneumoniae Phage Lytic Enzyme S16a

[0068] Pick one monoclonal host bacterium and culture it in liquid LB medium to the logarithmic phase, collect the bacteria, treat it with 100mM, pH=7 EDTA at 37℃ for 10 minutes, and then resuspend the bacteria with PBS. Set up 4 groups: one group with antimicrobial protein S16a and EDTA, one group with only antimicrobial protein S16a, one group with only EDTA, and one group as a blank control. Among them, the final concentrations of antimicrobial protein S16a were set to 0.1, 0.5 and 1 mg / ml respectively, and the final concentration of EDTA in the group with EDTA was 10mM; each group was placed in a 37℃ constant temperature incubator for 6 hours, and the plates were smeared and counted. There were 3 parallel experiments for all of the above. The results are as follows Figure 4 As shown, when 0.5 mg / ml of the antimicrobial protein S16a was used alone, the survival rate of Klebsiella pneumoniae S16a in the logarithmic phase was reduced to 81.27%. When used in combination with EDTA, the ATCC survival rate was reduced to 5.28%. When used in combination with EDTA, the antimicrobial activity was further enhanced.

[0069] Example 4 Exploration of the Optimal EDTA Concentration for Klebsiella Pneumoniae Phage Lytic Enzyme S16a

[0070] Pick a monoclonal host bacteria and grow it in liquid LB medium until the logarithmic phase. Collect the bacteria, treat with 100mM, pH=7 EDTA at 37℃ for 10min, and resuspend the bacteria in PBS. Using the optimal protein concentration (0.5mg / ml) obtained in Example 3, set the final EDTA concentration to 0, 0.1, 0.5, 1, 5 and 10mM respectively; each group was placed in a 37℃ constant temperature incubator for 6h, and the plates were plated and counted every 2 hours. The above experiments were repeated 3 times. The results are as follows Figure 5 As shown in the figure, when the EDTA concentration was 10 mM, the concentration of S16 bacteria increased from 2.6×10 9 cfu / mL decreased to 4.1×10 8 cfu / mL, and the optimal EDTA concentration for the protein's antibacterial effect was 10 mM.

[0071] Example 5 Determination of host spectrum of Klebsiella pneumoniae phage lytic enzyme S16a

[0072] The present invention determines the host spectrum of different test strains by treating the antimicrobial protein S16a. In order to detect whether the antimicrobial protein has a wide host spectrum, three clinical Klebsiella pneumoniae strains (S21: SAMN43028728, S24: SAMN43028729, S34: SAMN43028732), one Escherichia coli BL21 (DE3), Staphylococcus aureus ATCC25923 and one clinical Pseudomonas aeruginosa PBW (NZCP081477.2) were selected. One monoclonal host bacterium was picked and incubated in liquid LB medium until the logarithmic phase, the cells were collected, treated with 100mM, pH=7 EDTA at 37°C for 10 minutes, and then resuspended in PBS; the most suitable protein concentration and EDTA concentration in Examples 3 and 4 were selected; each group was placed in a 37°C constant temperature incubator and cultured for 6 hours; the number of colonies on the plate was observed by spotting. The above experiments were repeated 3 times.

[0073] Table 3 Lysase host spectrum determination

[0074] Strain name Lyase ATCCa Clinical Klebsiella pneumoniae S21 + Clinical Klebsiella pneumoniae S24 + Clinical Klebsiella pneumoniae S34 + Clinical Pseudomonas aeruginosa P8W + Staphylococcus aureus ATCC25923 - Escherichia coli BL21(DE3) +

[0075] The results are shown in Table 3 and Figure 6 As shown, the protein has a significant antibacterial effect on Escherichia coli, Pseudomonas aeruginosa and drug-resistant Klebsiella pneumoniae (such as S21, S24 and S34), but has no antibacterial effect on Staphylococcus aureus SA.

[0076] Example 6 Temperature tolerance test of Klebsiella pneumoniae phage lytic enzyme S16a

[0077] Pick a monoclonal host bacteria and grow it in liquid LB medium until the logarithmic phase. Collect the bacteria, treat with 100mM, pH=7 EDTA at 37℃ for 10min, and then resuspend the bacteria in pH=7.4 PBS. Select the most suitable protein concentration in Example 3 and set different temperature gradients (4, 16, 25, 37, 50 and 70℃) for treatment. Select the most suitable EDTA concentration in Example 4. Each group is placed in a 37℃ constant temperature incubator for 6h and plated and counted. The above experiments are repeated 3 times. The results are as follows. Figure 7 As shown in the figure, in terms of temperature tolerance, the antimicrobial protein S16a remained stable after treatment at 4, 16, 25, 37 and 50°C. After exposure to 70°C for the same duration, the antimicrobial activity was reduced to approximately 50%. Therefore, the antimicrobial protein has good temperature tolerance.

[0078] Example 7 pH tolerance test of Klebsiella pneumoniae phage lytic enzyme S16a

[0079] Pick a monoclonal host bacteria and grow it in liquid LB medium until the logarithmic phase. Collect the bacteria, treat with EDTA for 10 minutes, and resuspend them in PBS. Select the most suitable protein concentration in Example 3 and set different pH gradients (3, 4, 5, 6, 7, 8, 9, 10, 11) for treatment. Select the most suitable EDTA concentration in Example 4. Each group is placed in a 37°C constant temperature incubator for 6 hours and the plate is plated and counted. The above experiments are repeated 3 times. The results are as follows. Figure 8 As shown in the figure, in terms of pH tolerance, the protein activity remains basically unchanged at pH 3-8, and the antibacterial activity decreases to about 80% at pH 9-11. Therefore, the antibacterial protein has good pH tolerance.

Claims

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

1.

2. A gene encoding the bacteriophage lytic enzyme according to claim 1.

3. The gene according to claim 2, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

2.

4. Use of the bacteriophage lytic enzyme according to claim 1 in the preparation of an antibacterial agent.

5. The application according to claim 4, characterized in that: The antimicrobial agent is used to inhibit and / or kill Gram-negative bacteria.

6. The application according to claim 4, characterized in that: The Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa.

7. The use according to claim 4, characterized in that The phage lytic enzyme is used in combination with EDTA.

8. The use according to claim 4, characterized in that: The concentration of the phage lytic enzyme is 0.1-1 mg / mL.

9. The application according to claim 7, characterized in that: The concentration of the EDTA is 0.1-10 mM.

10. An antibacterial agent for inhibiting and / or killing Gram-negative bacteria, characterized in that: It contains the bacteriophage lytic enzyme according to claim 1.

Images