Aquatic antibacterial peptide with xanthine oxidase inhibiting function and application thereof

Abstract

The application discloses an aquatic antibacterial peptide with xanthine oxidase inhibiting function and application thereof, and belongs to the technical field of bioactive peptides. The amino acid sequence of the antibacterial peptide is Arg (Ph)-Trp-Leu-Ala-Arg-Ile-Arg-Val-Ile-Arg-(D-Val)-Ala-Arg-NH2. The antibacterial peptide provided by the application has broad-spectrum and strong antibacterial activity. Meanwhile, in the process of constructing the antibacterial peptide for efficiently inhibiting aquatic pathogenic bacteria, it is first found that the peptide has good xanthine oxidase inhibiting activity. The antibacterial peptide has good stability, and the antibacterial activity retention rate is maintained at more than 85% within 240 h of treatment in the range of-20 DEG C to 37 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an aquatic antimicrobial peptide that also has xanthine oxidase inhibitory function and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Currently, the mainstream intensive aquaculture model, especially the infectious diseases caused by bacterial pathogens, has caused huge economic losses to the industry. Common pathogens include Vibrio harveyi (belonging to the Vibrio genus), Aeromonas salmonicidae (belonging to the Aeromonas genus), Pseudomonas fluorescens, Aeromonas vesiculosus, and Aeromonas hydrophila, etc. Escherichia coli and Staphylococcus aureus can also have adverse effects. These pathogens have a wide range of hosts and spread rapidly, often leading to large-scale mortality of farmed animals.

[0004] Currently, the control of bacterial diseases in aquatic organisms mainly relies on the use of antibiotics. However, long-term and irregular use of antibiotics has led to the emergence of multidrug-resistant strains. Furthermore, drug residues are becoming increasingly serious, posing a potential threat to the ecological environment and food safety. Therefore, the development of novel, highly effective, safe, and non-resistant antimicrobial agents has become an urgent need in the aquaculture industry. Antimicrobial peptides (AMPs), as key effector molecules of the innate immune system, are considered one of the most promising candidates to replace traditional antibiotics due to their broad-spectrum antimicrobial activity, rapid action, and low resistance-inducing properties. However, currently reported antimicrobial peptides have a narrow antimicrobial spectrum, and most research focuses on the control of plant diseases, lacking stable, highly effective, and broad-spectrum antimicrobial peptides for aquatic pathogens.

[0005] In recent years, there have been research reports on antimicrobial peptides targeting aquatic pathogens, with some short peptides showing certain inhibitory effects on aquatic pathogens such as Aeromonas hydrophila and Aeromonas vesiculosus. However, the currently reported aquatic antimicrobial peptides still have the following shortcomings: their antibacterial activity needs to be further improved, and their functions are relatively singular, mainly focusing on antibacterial effects, lacking aquatic antimicrobial peptides with other physiological regulatory functions.

[0006] To better develop an antimicrobial peptide capable of inhibiting various aquatic pathogens, molecular docking verification combined with big data screening was used to evaluate the activity of the modified peptide. Unexpectedly, this revealed that the modified peptide possesses excellent xanthine oxidase inhibitory activity. Xanthine oxidase is a key enzyme regulating uric acid production. Currently, no aquatic antimicrobial peptides with xanthine oxidase inhibitory function have been reported in the literature. This unexpected discovery of dual function not only endows the antimicrobial peptide with the potential to regulate purine metabolism in farmed animals while controlling aquatic diseases, but also unexpectedly expands its application to the development of functional feed additives, demonstrating extremely high research value and broad application prospects.

[0007] In conclusion, developing novel antimicrobial peptides that possess both the dual functions of inhibiting aquatic pathogens and assisting in the regulation of uric acid metabolism can not only provide a green and efficient antibiotic alternative for the prevention and control of aquatic diseases, but also has the potential to expand their application in the field of functional feed additives. This has significant research value and broad market prospects. Summary of the Invention

[0008] To address the shortcomings of the prior art, this invention provides an aquatic antimicrobial peptide with xanthine oxidase inhibition function and its application.

[0009] The technical solution adopted in this invention is as follows:

[0010] In a first aspect, the present invention provides an aquatic antimicrobial peptide that also inhibits xanthine oxidase, wherein the amino acid sequence of the antimicrobial peptide is as follows:

[0011] Arg(Ph)-Trp-Leu-Ala-Arg-Ile-Arg-Val-Ile-Arg-(D-Val)-Ala-Arg-NH2.

[0012] In this context, Arg(Ph) indicates that a phenylalanine residue is attached to the guanidinium group of the arginine side chain; D-Val indicates D-valine; and -NH2 indicates C-terminal amidation modification.

[0013] The amino acid sequence of the antimicrobial peptide described in this invention is shown in SEQ ID NO:1.

[0014] The antimicrobial peptide of this invention has the following structural features:

[0015] (1) It consists of 13 amino acid residues, of which the arginine at position 1 is modified with phenylalanine and the valine at position 11 is D-valine;

[0016] (2) The C-terminus is modified by amidation, which enhances the stability of the peptide in vivo;

[0017] (3) It has an overall α-helical conformation and typical amphiphilic structural features, which can interact with bacterial cell membranes to exert antibacterial effects.

[0018] The antimicrobial peptide described in this invention exhibits highly efficient activity against aquatic pathogens. Experiments show that this antimicrobial peptide is effective against Vibrio harveyi (…). Vibrio harveyi The minimum inhibitory concentration (MIC) of ) is as low as 2 μg / mL, which is effective against Aeromonas hydrophila ( Aeromonas hydrophila ) and Aeromonas versicolor ( Aeromonas veronii The MICs for all were 4 μg / mL, indicating that they were effective against Aeromonas salmonicida (Aeromonas salmonicida). Aeromonas salmonicida The MIC for ) is 8 μg / mL, which is effective against Pseudomonas fluorescens ( ). Pseudomonas fluorescens The MIC of the drug was 16 μg / mL, indicating broad-spectrum and potent antibacterial activity.

[0019] The antimicrobial peptide described in this invention also has xanthine oxidase inhibitory function. Experiments show that the half-maximal inhibitory concentration (IC50) of this antimicrobial peptide against xanthine oxidase is [value missing]. 50 At 1.8 mM, it can effectively inhibit xanthine oxidase, a key enzyme in uric acid production, and has the potential to regulate purine metabolism.

[0020] The antimicrobial peptides described in this invention exhibit good temperature stability. After treatment at temperatures ranging from -20°C to 37°C for 240 hours, their antimicrobial activity retention rate remains above 85%. Even after high-temperature treatment at 121°C for 1 hour, approximately 50% of the antimicrobial activity remains, facilitating storage and transportation.

[0021] In a second aspect, the present invention provides the application of the antimicrobial peptide described in the first aspect in the preparation of products that inhibit aquatic pathogens.

[0022] Preferably, the product is an aquatic feed additive or a veterinary drug for aquaculture.

[0023] The antimicrobial peptides described in this invention have strong inhibitory activity against major pathogens in aquatic organisms and can be used as antibiotic alternatives for the prevention and control of diseases in aquaculture.

[0024] Thirdly, the present invention provides the use of the antimicrobial peptide described in the first aspect in the preparation of products that inhibit xanthine oxidase.

[0025] Preferably, the product is a functional feed additive or a veterinary drug product.

[0026] The antimicrobial peptides described in this invention have xanthine oxidase inhibitory activity and can be used to prepare related products that regulate purine metabolism and help lower uric acid.

[0027] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0028] 1. The antimicrobial peptides provided by this invention have a minimum inhibitory concentration (MIC) of 2 μg / mL against Vibrio harveyi, 4 μg / mL against Aeromonas hydrophila and Aeromonas vesiculosus, 8 μg / mL against Aeromonas salmonicida, and 16 μg / mL against Pseudomonas fluorescens. These aquatic antimicrobial peptides effectively cover the major pathogens in aquaculture, exhibiting broad-spectrum and potent antimicrobial properties.

[0029] 2. In the process of constructing an antimicrobial peptide that effectively inhibits aquatic pathogens, this invention unexpectedly discovered for the first time that this peptide also possesses good xanthine oxidase inhibitory activity (IC50). 50 =1.8 mM). Currently, there are no reports in the field of aquatic antimicrobial peptides that also have xanthine oxidase inhibitory functions. The discovery of this dual function expands the application prospects of aquatic antimicrobial peptides, giving them potential value in regulating purine metabolism in farmed animals while preventing and controlling aquatic diseases.

[0030] 3. The antimicrobial peptide of this invention retains over 85% of its antimicrobial activity after treatment at temperatures ranging from -20°C to 37°C for 240 hours, and still retains approximately 50% of its antimicrobial activity after treatment at 121°C for 1 hour. This linear peptide achieves excellent stability through the introduction of non-natural amino acids (Arg(Ph) and D-Val) and C-terminal amidation modification, facilitating storage and transportation. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 Schematic diagram of the secondary structure of antimicrobial peptide.

[0033] Figure 2 Figure 1: Results of inhibition zone experiments on different pathogens by antimicrobial peptides.

[0034] Figure 3 Temperature stability results of antimicrobial peptides.

[0035] Figure 4 : Schematic diagram of the docking of antimicrobial peptide and receptor molecule; A is a 3D interaction diagram: showing the binding conformation of antimicrobial peptide and xanthine oxidase in three-dimensional space; B is a 2D interaction diagram: showing specific hydrogen bonds, hydrophobic interactions and other interactions in the form of a two-dimensional planar diagram.

[0036] Figure 5 Standard curve of the linear relationship between antimicrobial peptide concentration and xanthine oxidase inhibition rate. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0039] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] Example 1: Virtual screening and structure prediction of antimicrobial peptides

[0042] 1.1 Virtual screening of potential antimicrobial peptides

[0043] A training dataset was constructed by integrating resources from multiple databases. The known antimicrobial peptide sequences were obtained from APD3 (Antimicrobial Peptide Database 3), CAMP (Collection of Antimicrobial Peptides), and Uniprot databases. The selection criteria were: clearly labeled antimicrobial activity (minimum inhibitory concentration MIC ≤ 64 μg / mL), sequence length of 10-50 aa (in line with the mainstream length range of natural antimicrobial peptides), and no obvious amino acid sequence redundancy (sequence similarity ≤ 80%). At the same time, globular protein fragments without antimicrobial activity were selected from the Swiss-Prot database as negative samples to ensure that the negative samples matched the positive samples in terms of sequence length and amino acid composition, thus avoiding dataset bias.

[0044] 1.2 Ligand and Receptor Processing

[0045] AlphaFold is a state-of-the-art protein structure prediction method from Google DeepMind. Its principle is based on deep learning technology and computer vision, predicting protein structure by simulating physical principles. Using AlphaFold2 to construct the 3D protein structure of an antimicrobial peptide, the results are as follows... Figure 1 As shown in the figure, the α-helix formed by the folding of the polypeptide chain is represented by a helical ribbon structure; the flexible connecting regions in the polypeptide chain are represented by thin chains / random coils. This illustration clearly reflects the folding pattern of the polypeptide backbone, the number, position, and spatial arrangement of secondary structural elements. This antimicrobial peptide is mainly composed of an α-helix structure. The helical structure is complete and continuous, with stable spatial conformation. The helical region is the key structural domain for the peptide to exert its antimicrobial activity. It can interact with the bacterial cell membrane through the amphiphilic helical structure, achieving membrane penetration and antibacterial effects. The flexible region ensures that the peptide has appropriate conformational adaptability when binding to the target, improving the efficiency of action.

[0046] The 3D structure of 1N5X was downloaded from the PDB database (https: / / www1.rcsb.org / ). The B chain in 1N5X was deleted using PyMOL software, along with any febuxostat (TEI) ligand molecules in a bound state. The processed molecule was saved in PDBQT format for later use. Autodock 1.5.7 was then used to perform dehydration and hydrogenation of the acceptor 1N5X, and Gasteiger charge calculations were performed. Finally, all atoms were classified as AD4 and saved in PDBQT format.

[0047] Example 2 Synthesis of antimicrobial peptides

[0048] The chemical reagents used in the experiment were purchased from Sinopharm Chemical Reagent Co., Ltd., and all were of analytical grade purity. The required antimicrobial peptide was synthesized by Shanghai Sangon Biotech Co., Ltd. using the Fmoc solid-phase synthesis method, with a purity greater than 95%. The sequence is Arg(Ph)-Trp-Leu-Ala-Arg-Ile-Arg-Val-Ile-Arg-(D-Val)-Ala-Arg-NH2. Here, Arg(Ph) indicates that a phenylalanine residue is attached to the guanidinium group of the arginine side chain; D-Val indicates D-valine; and the -NH2 at the C-terminus indicates amidation modification. This sequence is shown in SEQ ID NO:1.

[0049] Example 3: Determination of the antimicrobial peptide antibacterial spectrum and MIC

[0050] Source of strains used in the experiment: The strains mentioned in Table 1 were all provided by the Key Laboratory of Aquatic Product Bioprocessing of China Light Industry.

[0051] 3.1 Determination of the antimicrobial peptide antibacterial spectrum

[0052] The selected test strains were cultured to OD200. 600 The concentration was approximately 0.8. The test strain was inoculated into solid LB agar plates at 1% (v / v) and then plated. Wells were then punched in the plates, and 100 μL of an antimicrobial peptide solution prepared in PBS (50 μg / mL) was added to each well. The plates were incubated at 4℃ for 12 h upright, then at 37℃ for 7–9 h. The antibacterial effect was observed. The inhibition zone results against *Escherichia coli*, *Vibrio harveyi*, *Aeromonas hydrophila*, *Staphylococcus aureus*, *Aeromonas salmonidae*, and *Pseudomonas fluorescens* are as follows: Figure 2 As shown, this antimicrobial peptide can effectively inhibit the growth of Gram-negative and Gram-positive bacteria such as Escherichia coli, Vibrio harveyi, Aeromonas hydrophila, Staphylococcus aureus, Aeromonas salmonidae, and Pseudomonas fluorescens.

[0053] 3.2 Determination of Minimum Inhibitory Concentration (MIC)

[0054] The suspensions of the aquatic pathogens and other common pathogens to be tested were diluted to 1×10⁻⁶ using LB medium. 6 CFU / mL, 100 μL of bacterial suspension and 100 μL of antimicrobial peptide solution were added to each well of a 96-well plate to achieve a final concentration gradient of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 0 μg / mL in each well. After incubation at 37℃ for 24 h, OD was measured. 600 OD was not measured in the end. 600 The corresponding concentration of the antimicrobial peptide is the MIC of the antimicrobial peptide for that strain, and its MIC values ​​are shown in Table 1.

[0055] Table 1. Minimum inhibitory concentrations of the antimicrobial peptides provided in this invention against pathogenic bacteria.

[0056] Types of pathogens Minimum inhibitory concentration (μg / mL) E. coli 8 Vibrio harveyi 2 Aeromonas hydrophila 4 Aeromonas vilvae 4 Aeromonas salmonid 8 Fluorescent Pseudomonas 16 Staphylococcus aureus 32

[0057] The results showed that the antimicrobial peptide of the present invention had a MIC as low as 2 μg / mL against Vibrio harveyi, a major pathogen in aquatic organisms, and a MIC of 4 μg / mL against Aeromonas hydrophila and Aeromonas vesiculosus, demonstrating strong inhibitory activity.

[0058] Example 4 Temperature stability analysis of antimicrobial peptides

[0059] Using Vibrio harveyi as a representative strain, the antimicrobial activity retention rate of antimicrobial peptides was determined at different temperatures (-20 ℃, 4 ℃, 25 ℃, 37 ℃, 121 ℃) and different treatment times. The antimicrobial peptide sample without temperature treatment (stored at -20 ℃ and freshly prepared before use) showed 100% antimicrobial activity, thus systematically evaluating its temperature stability. The results showed... Figure 3As shown, after treatment at -20 ℃, 4 ℃, and 25 ℃ for 240 h, the antibacterial activity retention rate remained above 90% without significant decrease; after treatment at 37 ℃ for 72 h, the activity retention rate was still above 90%, and slightly decreased to about 85% after 120 h. This antimicrobial peptide preparation can withstand high-temperature treatment for a certain period of time; after high-temperature treatment at 121 ℃ for 1 h, about 50% of the antibacterial activity remained, and the antibacterial activity was basically lost after 2 h.

[0060] Example 5: Molecular docking and visualization analysis

[0061] The Autodock Vina algorithm in PyRx was used to perform molecular docking between the processed receptor 1N5X and the ligand peptide to simulate the interaction between the receptor and ligand. Based on the position of febuxostat (TEI) in the 1N5X bound state, the center coordinates of the docking box were set to (96, 54, 39), the box size was set to 15×15×15 Å, the number of dockings was set to 10, and other parameters were set to default values.

[0062] The peptide with the highest Rank score was selected for visualization analysis. Specifically, the following steps were taken: The PyRx integration docking software output a file in peptide-out format. This output file, along with the modified 1N5X file, was simultaneously opened in Pymol software and exported as a ligand-receptor bound molecule. This file was saved as peptide-1N5X in pdb format. The bound molecule file was then opened in Discovery Studio Client software. The small molecule ligand was located and defined as a Ligand. The Ligand interaction function was used to view the 3D interaction between the small molecule ligand and the large molecule receptor. The Show 2D diagram function was used to represent the 2D interaction diagram between peptides 1N5X.

[0063] The molecular docking results of the antimicrobial peptide and xanthine oxidase described in this invention are as follows: Figure 4As shown, this antimicrobial peptide can form a stable complex with xanthine oxidase through multiple non-covalent synergistic interactions, thereby significantly inhibiting its activity. Specifically, the antimicrobial peptide is embedded in the active pocket or allosteric regulatory region of xanthine oxidase in an extended conformation. Its main chain and side chain groups form multiple hydrogen bonds, salt bridges, and π-alkyl / alkyl interactions with key amino acid residues of the enzyme (such as GLU A:879, SER A:876, PHE A:649, etc.), while van der Waals forces further enhance the stability of the complex. This binding mode directly hinders the binding of the substrate xanthine to the enzyme's active site through steric hindrance, and also disrupts the integrity of its catalytic function by inducing conformational perturbation of the enzyme, ultimately achieving effective inhibition of xanthine oxidase activity. This provides a clear molecular mechanism basis for the application of this antimicrobial peptide in the prevention and treatment of hyperuricemia and related diseases.

[0064] Example 6: Determination of xanthine oxidase inhibition rate and IC50

[0065] Prepare 1.5 mM xanthine solution and 0.1 U / mL xanthine oxidase solution using PBS buffer. Set up sample group, blank control group and enzyme control group in 96-well microplate, with 3 duplicates in each group.

[0066] For the sample group, 50 μL of xanthine oxidase solution and 50 μL of antimicrobial peptide solutions of different concentrations were added to each well sequentially; for the enzyme control group, 50 μL of xanthine oxidase solution and 50 μL of PBS buffer were added to each well; and for the blank control group, 100 μL of PBS buffer was added to each well. The microplates were incubated at 37°C for 20 min. Then, 100 μL of 1.5 mM xanthine solution was quickly added to each well and mixed by pipetting. The absorbance was measured at 290 nm, and data were recorded every 10 s over 3 min. The xanthine oxidase inhibition rate was calculated as [1 − (OD sample − OD blank) / (OD enzyme control − OD blank)] × 100%, and the results are shown in Table 2.

[0067] Table 2. Inhibition rate of this peptide on xanthine at different concentrations.

[0068] Concentration / mM The peptide provided by this invention 2 55.67% 1 27.67% 0.5 14.32% 0.25 8.23% 0.125 3.14%

[0069] The peptide was dissolved in PBS to prepare five concentrations: 2 mM, 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM. The in vitro xanthine oxidase inhibitory activity of the synthesized peptide was determined according to the prescribed method, and the 50% inhibitory concentration (IC50) of the active peptide's xanthine oxidase activity was calculated. 50 A standard curve was plotted to show the linear relationship between concentration and inhibition rate (value), as shown in the figure. Figure 5 As shown: y = 0.0043 + 27.58x (R 2= 0.9992), the IC50 of this peptide 50 It is 1.8 mM.

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An aquatic antimicrobial peptide with xanthine oxidase inhibitory function, characterized in that, The amino acid sequence of the antimicrobial peptide is as follows: Arg(Ph)-Trp-Leu-Ala-Arg-Ile-Arg-Val-Ile-Arg-(D-Val)-Ala-Arg-NH2; Arg(Ph) indicates that a phenylalanine residue is attached to the guanidinium group of the arginine side chain; D-Val indicates D-valine; -NH2 indicates C-terminal amidation modification.

2. The application of the aquatic antimicrobial peptide with xanthine oxidase inhibition function as described in claim 1 in the preparation of products that inhibit aquatic pathogens, characterized in that, The aquatic pathogens were selected from Vibrio harveyi ( Vibrio harveyi Aeromonas hydrophila ( ) Aeromonas hydrophila Aeromonas versicolor ( Aeromonas veronii Aeromonas salmonidae ( ) Aeromonas salmonicida ), fluorescent pseudomonas ( Pseudomonas fluorescens At least one of the following; The product is an aquatic feed additive or a veterinary drug for aquaculture.

3. The application of the aquatic antimicrobial peptide with xanthine oxidase inhibition function as described in claim 1 in the preparation of feed additives or veterinary drugs for lowering uric acid.

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