A molecular binding mode of bacteriocin plantaricin EvF and application of its modified form plantaricin EsF

Abstract

The present invention relates to a molecular binding mode of a dimeric bacteriocin plantaricin EvF and the application of its modified form, plantaricin EsF, belonging to the field of microbial technology. The bacteriocin plantaricin EvF is composed of subpeptide chains plantaricin Ev and plantaricin F with a molecular ratio of 1:1. Plantaricin Ev is bound to the N-terminal region of plantaricin F through three groups of hydrogen bonds to form plantaricin EvF with antibacterial activity. The present invention also provides a modified dimeric bacteriocin plantaricin EsF. It has been verified that the modified plantaricin EsF has a significant inhibitory effect on Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli, and has good thermal and pH stability. It is sensitive to proteases and easily degrades, and has application potential in the fields of food and agricultural product preservation.

Description

Technical Field

[0001] The invention relates to a molecular binding mode of bacteriocin plantaricin EvF and application of its modified form plantaricin EsF, belonging to the technical field of microorganisms. Background Art

[0002] In recent years, with the widespread use of antibiotics, the emergence and spread of drug-resistant strains has become a major challenge to global public health. The inhibitory effects of traditional antibiotics on many pathogens have significantly decreased, necessitating the development of new antimicrobial agents. Bacteriocins, a class of antimicrobial peptides produced by microorganisms, have become an important area of ​​research and development for new antimicrobial agents due to their low toxicity and low resistance to drug development. In particular, bacteriocins produced by lactic acid bacteria, such as nisin, have been designated GRAS (Generally Recognized As Safe) by the U.S. Food and Drug Administration and are widely used in the food industry as natural preservatives.

[0003] Due to their unique molecular structure and mechanism of action, dimeric bacteriocins exhibit great potential for antimicrobial activity. However, the molecular binding mode and specific mechanism of action of dimeric bacteriocins remain insufficiently studied, limiting their practical applications. Further research into the molecular binding mode of dimeric bacteriocins, revealing their antimicrobial mechanism, and optimizing their structure based on this research could enhance their antimicrobial activity. This optimization approach would not only help improve the performance of existing dimeric bacteriocins but also, through rational structural modification, develop new modified bacteriocins to meet a wider range of application needs, further enhancing their application prospects in areas such as food preservation and medical hygiene. Summary of the Invention

[0004] To deepen the understanding of the antibacterial activity of dimeric bacteriocins and address the problem of the proliferation of drug-resistant bacteria, the present invention proposes a molecular binding mode of bacteriocin plantaricin EvF and a modified bacteriocin plantaricin EsF, which have application value in in vitro antibacterial properties.

[0005] The first object of the present invention is to provide a molecular binding method for the existing dimeric bacteriocin plantaricin EvF, wherein the daughter peptide chain plantaricin Ev is bound to the N-terminal region of the daughter peptide chain plantaricin F by forming a hydrogen bond.

[0006] Furthermore, the amino acid sequence of the daughter peptide chain plantaricin Ev of the dimeric bacteriocin plantaricin EvF is SEQ ID NO.1, and the amino acid sequence of the daughter peptide chain plantaricin F is SEQ ID NO.2.

[0007] Among them, there are three groups of hydrogen bonds formed between the daughter peptide chain plantaricin Ev and the daughter peptide chain plantaricin F, specifically: formed by ARG3 on the daughter peptide chain plantaricin Ev sequence and ARG8 on the daughter peptide chain plantaricin F sequence; formed by VAL12 on the daughter peptide chain plantaricin Ev sequence and ARG8 on the daughter peptide chain plantaricin F sequence; formed by ARG13 on the daughter peptide chain plantaricin Ev sequence and ALA4, TYR5, ALA7 and ARG8 on the daughter peptide chain plantaricin F sequence.

[0008] The second object of the present invention is to provide a modified dimeric bacteriocin plantaricin EsF with enhanced antibacterial activity based on the understanding of the binding mode between the sub-peptide chains of the dimeric bacteriocin plantaricin EvF, wherein the sub-peptide chain plantaricin Es is obtained by modifying the amino acid sequence shown in SEQ ID NO.1, and the sub-peptide chain plantaricin F remains unchanged.

[0009] Furthermore, the amino acid sequence of the daughter peptide chain plantaricin Es of the modified dimeric bacteriocin plantaricin EsF is SEQ ID NO. 3, and the modified dimeric bacteriocin plantaricin EsF exerts antibacterial activity as a whole.

[0010] The third object of the present invention is to provide the use of the modified dimeric bacteriocin plantaricin EsF in the preparation of antibacterial infection therapeutic drugs.

[0011] Furthermore, the anti-bacterial infection therapeutic drug is used to inhibit one or more of Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli.

[0012] Beneficial effects of the present invention:

[0013] The molecular binding mode of the dimeric bacteriocin plantaricin EvF provided by the present invention explains a source of antibacterial activity of the dimeric bacteriocin, and this understanding of the binding mode helps to further transform the antibacterial activity of the dimeric bacteriocin. Based on this, the present invention also provides a modified dimeric bacteriocin plantaricin EsF. In vitro antibacterial experiments show that the modified bacteriocin has a significant inhibitory effect on representative strains of Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli, with a minimum inhibitory concentration (MIC) of 2-8 μmol / L. It also has high thermal stability and pH stability, is sensitive to common proteases, and is easily degraded. Therefore, it is expected to become a substitute for antibiotics and chemical preservatives, and has significant application value in the fields of food and agricultural product preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The predicted results of potential docking site regions for the two sub-peptide chains of bacteriocin plantaricin EvF, where the orange regions indicate possible binding positions.

[0015] Figure 2 This is the molecular docking simulation structure between the two sub-peptide chains of bacteriocin plantaricin EvF. The yellow dotted line represents the formed hydrogen bond, and the marked amino acid residues are the amino acid residues involved in the formation of hydrogen bonds.

[0016] Figure 3 This is the time-kill curve of the modified bacteriocin plantaricin EsF against the indicator bacteria, where the square marks the control group, the circle marks the group treated with 2 times the MIC concentration, and the triangle marks the group treated with 1 times the MIC concentration.

[0017] Figure 4 Box plot of the antibacterial activity of the modified bacteriocin plantaricin EsF after different heat treatments, where black squares represent the mean and asterisks represent significant differences (P<0.05).

[0018] Figure 5 The box plot of the antibacterial activity of the modified bacteriocin plantaricin EsF after being treated with different pH values, where the black squares represent the mean. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0021] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0022] Example 1: Bacteriocin plantaricin EvF, plantaricin EsF sequences and sample preparation

[0023] Combined with the whole genome sequence (CP019348.1) of Lactobacillus plantarum KLDS1.0391 and the comparison analysis of bacteriocin-related gene clusters, the structural coding sequences of the daughter peptide chains plantaricin Ev and plantaricinF of the dimeric bacteriocin plantaricin EvF were confirmed and further translated into amino acid sequences. The amino acid sequence of the daughter peptide chain plantaricin Ev is shown in SEQ ID NO.1, specifically: FNRGGYNFGKSVRH, and the amino acid sequence of the daughter peptide chain plantaricin F is shown in SEQ ID NO.2, specifically: VFHAYSARGVRNNYKSAVGPADWVISAVRGFIHG.

[0024] By analyzing the amino acid sequence of the bacteriocin plantaricin EvF, the modified bacteriocin was designed using methods such as amino acid substitution and antibacterial activity evaluation. Four amino acid positions in the daughter peptide chain plantaricin Ev were selected for substitution. The resulting modified bacteriocin was named the daughter peptide chain plantaricin Es, and its amino acid sequence is shown in SEQ ID NO. 3: FNRKGYKFGKLKRH. The daughter peptide chain plantaricin F was not modified. Because the daughter peptide chains plantaricin Es and plantaricin F require mutual binding to exert antibacterial activity, they were treated as a whole and named the modified dimer bacteriocin plantaricin EsF.

[0025] The bacteriocins plantaricin EvF and plantaricin EsF used in the following examples were synthesized by solid phase synthesis at Jier Biochemical (Shanghai) Co., Ltd., and the quality and purity of the synthesized samples were verified by electrospray ionization mass spectrometry (ESI-MS) and high performance liquid chromatography (HPLC).

[0026] Example 2: Three-dimensional structure and molecular binding mode of the dimeric bacteriocin plantaricin EvF subpeptide chain

[0027] The two-dimensional nuclear magnetic resonance (NMR) data of the daughter peptide chain plantaricin Ev (BMRB ID 52415) were obtained from the Biological Magnetic Resonance Database (BMRB). The three-dimensional structure of the daughter peptide chain plantaricin Ev was obtained by analyzing the NMR data and calculating its structure using the Collaborative Computational Nuclear Magnetic Resonance Project (CCPN) and Iterative Assignment of Fuzzy Constraints (ARIA) software. The three-dimensional structure of the daughter peptide chain plantaricin F (PDB ID 2RLW) was obtained directly from the PDB database.

[0028] The potential docking region of the dimeric bacteriocin plantaricin EvF was predicted using the binding site prediction tool DeepSite (URL: https: / / open.playmolecule.org / tools / deepsite). The prediction results are as follows: Figure 1 Then, based on the predicted docking region, the molecular docking software AutoDock Vina was used to calculate the docking conformation of the sub-peptide chains plantaricin Ev and plantaricin F. The best docking conformation result was as follows: Figure 2 shown.

[0029] Example 3: Minimum inhibitory concentration (MIC) test of the modified dimer bacteriocin plantaricin EsF

[0030] MICs were determined using the broth microdilution method. High-concentration stock solutions of bacteriocin samples were accurately prepared with sterile water and serially diluted twofold for testing. Indicator bacteria were grown to logarithmic phase in appropriate culture medium and conditions. A suspension of indicator bacteria equivalent to a 0.5 McFarland standard was prepared and washed three times with phosphate-buffered saline (PBS), pH 7.2. The suspension was then diluted 1:100 in Mueller-Hinton (MH) broth. In a 96-well plate, 50 μL of the bacterial suspension was mixed with an equal volume of the bacteriocin sample at varying concentrations to obtain the corresponding target treatment concentration. The last column of the plate was used for growth control, with plantaricin EvF and PBS buffer serving as positive and negative controls, respectively. The plates were incubated at the optimal temperature for each indicator bacterium for 24 hours. The absorbance was then measured at 600 nm using a microplate reader. The lowest sample concentration that inhibited bacterial growth was recorded as the MIC for the corresponding indicator bacterium.

[0031] The experimental results are shown in Table 1. Compared with the dimeric bacteriocin plantaricin EvF, the modified dimeric bacteriocin plantaricin EsF had lower MIC values ​​against all tested strains, indicating that it has stronger antibacterial activity against the tested strains. Specifically, the minimum inhibitory concentration of plantaricin EsF against Bacillus subtilis (ATCC6633) and Listeria monocytogenes (NICPBP54002) was 2 μmol / L, one-quarter that of the positive control group; and the minimum inhibitory concentration against Staphylococcus aureus (ATCC25923) and Escherichia coli (ATCC25922) was 8 μmol / L, one-half that of the positive control group.

[0032] Table 1 Minimum inhibitory concentration of modified dimeric bacteriocin plantaricin EsF against test strains

[0033]

[0034] Example 4: Bactericidal kinetics of the modified dimeric bacteriocin plantaricin EsF

[0035] Bacillus subtilis (ATCC6633), which is relatively sensitive to bacteriocins, was selected as an indicator bacterium, and the time-kill curve method was used to evaluate the bactericidal kinetics of the modified dimeric bacteriocin plantaricin EsF. The cultured bacterial suspension was washed and resuspended in PBS buffer to reach an appropriate concentration (OD 600 =0.5), and then treated with bacteriocin samples at 1 and 2 times the MIC concentration. PBS buffer was used as a negative control. The treated bacterial suspension was incubated at 37°C for 4 hours, and samples were taken every 15 minutes. The bacterial survival rate at each time point was determined by plate colony counting.

[0036] The experimental results are as follows Figure 3 As shown in the figure, the time-kill curve shows the antibacterial efficiency of the modified dimeric bacteriocin plantaricin EsF. The sample with a concentration of 2 times the MIC can reduce the number of indicator bacteria by about 3.0 lg / CFU within 60 minutes, and reduce it to below 2.0 lg / CFU after 150 minutes; the sample with a concentration of 1 times the MIC can reduce the number of indicator bacteria by nearly 2.0 lg / CFU within 120 minutes.

[0037] Example 5: Stability evaluation of the modified dimer bacteriocin plantaricin EsF

[0038] Bacillus subtilis (ATCC 6633), which is sensitive to bacteriocins, was used as an indicator bacteria. The enzyme sensitivity, thermal stability, and pH stability of the modified dimeric bacteriocin plantaricin EsF were evaluated using the agar diffusion method. To ensure easy observation of the zone of inhibition and consistent results, the bacteriocin sample concentration used for stability testing was 128 μmol / L. The diameter of the zone of inhibition was measured after each treatment. Enzyme sensitivity was tested using trypsin, pepsin, and proteinase K. After treatment at 37°C and 40 U / mL for 1 hour, the enzymes were inactivated by incubation in an 80°C water bath for 10 minutes. Thermal stability was tested by placing the sample in 10 mmol / L PBS buffer and treating it in a water bath at 25°C, 50°C, 75°C, and 100°C, and then in an autoclave at 121°C for 30 minutes. The pH stability test method is to use HCl and NaOH to adjust the pH of the bacteriocin sample in 10 mmol / L PBS buffer to between 2 and 12 with a gradient of 1. The sample is then placed in a 37°C water bath for 2 hours. The pH value of the mixture is corrected back to 6.0-6.5 before performing the antibacterial test to ensure accurate activity assessment.

[0039] The results of enzyme sensitivity experiments are shown in Table 2. No inhibition zone was detected after the bacteriocin plantaricin EsF was treated with trypsin, pepsin, and proteinase K, indicating that the bacteriocin plantaricin EsF is very sensitive to the treatment of the assay enzymes and can therefore be inactivated by in vitro enzyme treatment. Figure 4 、 Figure 5 As shown, the antimicrobial activity of plantaricin EsF remained stable when treated at temperatures between 25°C and 100°C for 30 minutes and at pH values ​​between 2 and 12 for 2 hours, with no significant differences observed (P>0.05). However, exposure to 121°C for 30 minutes resulted in a decrease in antimicrobial activity (P<0.05), with an inhibition zone diameter of 14.85±0.35 mm. Overall, the modified dimeric bacteriocin plantaricin EsF exhibited excellent stability and was resistant to protease degradation.

[0040]

[0041] Table 2 Enzyme sensitivity of the modified dimeric bacteriocin plantaricin EsF

[0042] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A modified dimeric bacteriocin plantaricin EsF, characterized in that: The modified dimeric bacteriocin plantaricin EsF is composed of a daughter peptide chain plantaricin Es and a daughter peptide chain plantaricin F; the amino acid sequence of the daughter peptide chain plantaricin Es is shown in SEQ ID NO. 3; the amino acid sequence of the daughter peptide chain plantaricin F is shown in SEQ ID NO.

2.

2. A use of the modified dimeric bacteriocin plantaricin EsF according to claim 1 in the preparation of an antibacterial preparation, characterized in that: The bacteria are one or more of Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli.

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