Marine microorganism-derived antibacterial peptide, antibacterial composition and application

Abstract

The invention belongs to the field of polypeptides, and particularly relates to an antibacterial peptide derived from marine microorganisms, an antibacterial composition and application. Based on a deep learning algorithm, five potential antibacterial peptides are mined from a global marine microbiome database, and the five antibacterial peptides are expressed in yarrowia lipolytica. Experimental tests show that the five antibacterial peptides have differentiated antibacterial effects on different bacteria, and the antibacterial peptide shown in SEQ ID NO: 3 can have an obvious inhibition effect on escherichia coli, pseudomonas aeruginosa and staphylococcus aureus, has low hemolysis and has a positive application prospect. The antibacterial peptide disclosed by the invention has the potential of further developing novel antibacterial drugs or antibacterial additives.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides, and particularly relates to antibacterial peptides from marine microorganisms, antibacterial compositions and applications. BACKGROUND

[0002] The global spread of drug-resistant pathogens has attracted widespread attention from the medical and scientific communities. The extensive use of various antibiotics has led to the development of bacterial resistance to single antibiotics to multi-drug resistance and even pan-drug resistance. Antibacterial peptides mainly exert antibacterial effects by destroying bacterial cell membranes, and have broad-spectrum antibacterial activity and low drug resistance, making them potential substitutes for traditional antibiotics. At present, the antibacterial activity of natural antibacterial peptides is still limited, and higher activity and more targeted antibacterial peptides are needed to exert antibacterial effects in actual drug development.

[0003] The ocean, as the most biologically diverse ecosystem on Earth, has given birth to a large number of structurally unique and functionally diverse bioactive substances. Among them, antibacterial peptides from the ocean have become a research hotspot in the field of biological medicine in recent years due to their excellent antibacterial performance and unique mechanism of action. Compared with traditional antibiotics, antibacterial peptides from the ocean have broad-spectrum antibacterial activity, and can effectively inhibit not only bacteria such as gram-positive bacteria and gram-negative bacteria, but also fungi, viruses and some tumor cells. In addition, marine antibacterial peptides also have the characteristics of small molecular weight, good thermal stability, high water solubility and excellent biocompatibility, and show great application potential in the fields of clinical infection treatment, food preservation, and disease control in aquaculture. SUMMARY

[0004] Therefore, the purpose of the present application is to screen antibacterial peptides with high activity against specific pathogenic bacteria or broad-spectrum antibacterial activity. Five antibacterial peptides from marine microorganisms were screened in the present application, which were expressed in Yarrowia lipolytica and their antibacterial effects were verified.

[0005] The present application provides antibacterial peptides from marine microorganisms, the amino acid sequence of which is shown in any one of SEQ ID NO: 1-5.

[0006] Further, the amino acid sequence of the antibacterial peptide from marine microorganisms is shown in SEQ ID NO: 3.

[0007] The present application also provides the use of the antibacterial peptide from marine microorganisms in the preparation of an antibacterial composition, and the antibacterial composition is used for inhibiting gram-positive bacteria.

[0008] Further, the antibacterial composition is a broad-spectrum antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily washing product composition.

[0009] Further, the gram bacteria are gram positive bacteria or gram negative bacteria; preferably, the gram positive bacteria include, but are not limited to, one or both of Staphylococcus aureus and Bacillus subtilis, and the gram negative bacteria include, but are not limited to, one or more of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0010] The present application also provides an antibacterial composition comprising the marine microorganism-derived antibacterial peptide.

[0011] Further, the antibacterial composition is a broad-spectrum antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily chemical washing product, etc.

[0012] Further, the gram bacteria are gram positive bacteria or gram negative bacteria; preferably, the gram positive bacteria include, but are not limited to, one or both of Staphylococcus aureus and Bacillus subtilis, and the gram negative bacteria include, but are not limited to, one or more of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0013] Further, in the antibacterial composition, the effective concentration of the antibacterial peptide is ≥ 32 μg / mL.

[0014] Further, in the inhibition of specific bacteria, the effective concentration of the antibacterial peptide in the antibacterial composition is ≥ 8 μg / mL.

[0015] Compared with the prior art, the present application has at least the following beneficial effects:

[0016] The marine microorganism-derived antibacterial peptide screened by the present application is a newly discovered antibacterial peptide, which has good antibacterial effect; the antibacterial peptide with the sequence shown in SEQ ID NO: 3 has significant antibacterial effect on gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, and the antibacterial peptide has low hemolytic activity. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The plasmid map of the antibacterial peptide expression plasmid YLEp-leu-SP-AMP.

[0018] Figure 2 The hemolysis rate results of antibacterial peptides with different concentrations. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application will be further described in detail below in combination with the drawings and specific embodiments. The examples of the embodiments are shown in the drawings. It should be understood that the specific examples described in the following embodiments of the present application are only exemplary illustrations of the specific embodiments of the present application, and are intended to explain the present application, but do not constitute a limitation on the present application.

[0020] The endpoints of the ranges and any values disclosed herein are not limited to the precise values or the exact range, and the ranges should be interpreted as including approximate ranges.

[0021] Example 1: Screening of antibacterial peptides

[0022] Based on the deep learning model, an antibacterial peptide prediction model was constructed. The model features include the solvent accessibility of the first amino acid residue of the antibacterial peptide sequence, the proportion of aliphatic amino acid residues, the proportion of aromatic amino acid residues, the proportion of non-polar amino acid residues, the proportion of polar amino acid residues, the proportion of charged amino acid residues, the proportion of basic amino acid residues, the proportion of acidic amino acid residues, the relative volume occupied by aliphatic side chains in the peptide chain, the stability of the protein, the sum of the solubility of all amino acid residues, the hydrophobicity of the antibacterial peptide, and a total of 19 features. The model training set data includes 3306 antibacterial peptides and 135900 non-antibacterial peptides, and a random forest classifier in the scikit-learn machine learning library is used to construct a classification model. Through multiple rounds of validation set data prediction, the model constructed by the application has an accuracy of 0.91, a sensitivity of 0.92, a specificity of 0.91, and a precision of 0.91.

[0023] Based on the global marine microbiome database, smORFinder is used to identify smORF, and the amino acid sequence is obtained. Based on the constructed antibacterial peptide prediction model, antibacterial peptides are identified, and the top 5 candidate antibacterial peptides with the highest scores are screened. The amino acid sequences of the antibacterial peptides are shown in Table 1:

[0024] Table 1: Antibacterial peptide sequences and physicochemical properties

[0025]

[0026] Example 2: Expression and purification of antibacterial peptides

[0027] The expression host is Yarrowia lipolytica. The sequence of the antibacterial peptide is codon-optimized, and the pTEF1 promoter-signal peptide-antibacterial peptide sequence is directly synthesized by a sequencing company. The synthesized sequence is integrated into the YLEp-leu-SP-AMP plasmid (plasmid map as shown in Figure 1 The plasmid allows the signal peptide-antibacterial peptide tandem gene to be expressed in Yarrowia lipolytica. The promoter is a pTEF1 promoter. Under the guidance of the endogenous XPR2 signal peptide (MKLATAFTILTAVLA) in Yarrowia lipolytica, the antibacterial peptide is secreted outside the cell. The antibacterial peptide secreted into the extracellular fermentation broth is separated and purified by ion exchange chromatography.

[0028] Example 3: Determination of antibacterial activity of antibacterial peptides

[0029] The micro-broth dilution method was used to determine the antibacterial peptide, and the specific steps were as follows:

[0030] (1) Bacterial culture: E. coli, P. aeruginosa, K. pneumoniae, S. aureus, and B. subtilis were recovered and activated overnight, then streaked onto LB solid medium plates and incubated at 37°C for 16 h. Single colonies were picked and inoculated into 3 mL of LB medium in a shaking tube, which was incubated at 37°C for 4 h. The culture was then diluted with LB medium to 10 6 CFU / ml.

[0031] (2) Antibacterial test: The test was performed using a sterilized 96-well culture plate, with three parallel controls. The antibacterial peptide was diluted to the highest concentration to be tested using LB medium, and different concentrations of the antibacterial peptide solution were obtained using a two-fold micro-dilution method. 50 μL of the solution was added to the micro-wells of the culture plate, followed by the addition of 50 μL of the prepared bacterial solution. The control without the antibacterial peptide was added with 50 μL of LB medium and 50 μL of the bacterial solution. The blank control was added with 100 μL of LB medium. The culture was incubated at 37°C in a constant-temperature incubator for 18 h. After incubation, the OD600 of each well was determined using a multifunctional microplate reader. The concentration that significantly inhibited microbial growth was the minimum inhibitory concentration of the antibacterial peptide.

[0032] The antibacterial results of the antibacterial peptides are shown in Table 2. The antibacterial peptides represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 had obvious antibacterial effects on E. coli, P. aeruginosa, and other gram-negative bacteria.

[0033] Table 2: Minimum inhibitory concentration of antibacterial peptides (μg / mL)

[0034]

[0035] Example 4: Hemolytic assay of antibacterial peptides

[0036] The hemolytic toxicity was evaluated using defibrillated sheep blood. 2 mL of blood was centrifuged at 800 x g for 10 min, and the supernatant was discarded. The red blood cell suspension was prepared by diluting the blood with PBS buffer to 2% and washing twice with PBS buffer. The antibacterial peptide dispersion was prepared at concentrations of 8, 16, 32, 64, and 128 μg / mL. In a 2 mL centrifuge tube, 200 μL of the red blood cell suspension and 200 μL of the antibacterial peptide dispersion were added. The negative control was 200 μL of the red blood cell suspension and 200 μL of PBS buffer, and the positive control was 200 μL of the red blood cell suspension and 200 μL of 2% Triton X-100 solution. The mixture was incubated at 37°C for 1 h. After incubation, the mixture was centrifuged at 800 x g for 10 min, and 200 μL of the supernatant was added to a 96-well plate. The absorbance value was measured at 540 nm using a microplate reader, and the hemolysis rate was calculated. Hemolysis rate (%) = (A sample -APBS ) / (A tritonX-100 -A PBS )×100%;A sample : absorbance of the mixture of antibacterial peptide and red blood cell PBS : absorbance of negative control tritonX-100 : absorbance of positive control

[0037] The results are shown in Table 3, Figure 2 The antibacterial peptide shown in SEQ ID NO: 3 has low hemolysis, and the hemolysis rate is less than 4% at the experimental concentration, and has no hemolysis effect.

[0038] Table 3: Hemolysis experiment results of antibacterial peptides

[0039]

[0040] In summary, the five antibacterial peptides have different bacteriostatic effects on different bacteria, and the antibacterial peptide shown in SEQ ID NO: 3 has strong inhibitory effect on E. coli, P. aeruginosa and S. aureus, and has low hemolysis rate, which is the most preferred antibacterial peptide.

[0041] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. An antibacterial peptide of marine microbial origin, characterized in that, The amino acid sequence of which is shown in any one of SEQ ID NOs: 1-5.

2. Use of the marine microorganism-derived antibacterial peptide according to claim 1 for the preparation of an antimicrobial composition, characterized in that, The bacteriostatic composition is used for inhibiting gram bacteria.

3. Use according to claim 2, wherein the compound is ###0002### The bacteriostatic composition is a broad-spectrum antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily washing product.

4. The use according to claim 2, wherein the compound is ###0002### The gram bacteria are gram-positive bacteria or gram-negative bacteria.

5. The use according to claim 4, wherein the compound is ###0002### The gram-positive bacteria are selected from one or both of Staphylococcus aureus and Bacillus subtilis; the gram-negative bacteria are selected from one or more of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

6. A bacteriostatic composition characterized in that, The bacteriostatic composition is a broad-spectrum antibacterial drug, a disinfectant, a preservative, a feed additive, or a daily washing product.

7. The bacteriostatic composition of claim 6, wherein The bacteriostatic composition is used for inhibiting gram bacteria.

8. The bacteriostatic composition of claim 6, wherein The gram bacteria are gram-positive bacteria or gram-negative bacteria.

9. The bacteriostatic composition of claim 8, wherein, The gram-positive bacteria are selected from one or both of Staphylococcus aureus and Bacillus subtilis; the gram-negative bacteria are selected from one or several of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

10. The bacteriostatic composition of claim 9, wherein ​

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