Multifunctional peptide as well as preparation method and application thereof

Abstract

The invention belongs to the field of protein engineering, and particularly relates to a multifunctional peptide and a preparation method and application thereof. According to the invention, on the basis of Raniseptin-3, the N-terminal structure of Raniseptin-3 is reserved, discontinuous deletion is carried out at the C terminal to form a 20-peptide core function framework, and further modification is carried out to obtain modified peptides 1-3, so that the following effects are achieved: the antibacterial spectrum is converted from broad-spectrum antibacterial spectrum to specific antifungal spectrum; the antioxidant activity is remarkably improved, the hydrophobic environment activity reaches the glutathione level, and the hydrophobic-hydrophilic dual-environment activity balance is better; the hemolytic activity is obviously reduced, and almost no hemolytic activity exists; the peptide length is reduced from 28 amino acid residues to 20, so that the production cost is reduced. Meanwhile, the modified peptide 1 also retains the heat resistance of the parent peptide Raniseptin-3; the modified peptide 2 has an enzymolysis-resistant effect. The antifungal and antioxidant capacities of the modified peptides are different, and the modified peptides can be accurately used for product development in the antifungal or antioxidant field.

Description

Technical Field

[0001] The present invention belongs to the field of protein engineering, and in particular relates to a multifunctional peptide and a preparation method and application thereof. Background Art

[0002] Fungi pose a serious threat to global health, agriculture, animal husbandry, aquaculture, food safety, and ecosystems. Compared to bacteria, the development of antifungal drugs is more challenging. This is primarily because fungi, like plants and animals, are eukaryotic organisms, resulting in a scarcity of specific targets and a limited range of available antifungal drugs. Currently reported antifungal drugs are primarily classified into polyenes, azoles, allylamines, pyrimidines, and echinocandins. These antifungal drugs are small-molecule chemicals and generally have toxic side effects. Antimicrobial peptides, on the other hand, have attracted considerable attention due to their low toxicity, minimal side effects, and resistance to drug resistance. However, many existing antifungal peptides have broad-spectrum activity, meaning they can fight both fungi and bacteria. In contrast, narrow-spectrum antifungal peptides, which are specifically effective against fungi, offer advantages in reducing host microbial imbalance, mitigating the spread of drug resistance, and managing ecological risks.

[0003] Antioxidants can effectively neutralize free radicals and mitigate oxidative stress damage to cells and molecules. They have broad application prospects in the health, food, cosmetics, and agricultural sectors. Currently, most antioxidants are small molecule chemicals that, while highly active, generally have potentially harmful side effects.

[0004] Given that microbial infection and oxidative stress often coexist and exacerbate each other, developing substances with both antimicrobial and antioxidant activity offers multiple advantages. On the one hand, such substances can directly inhibit pathogens and reduce pathogen invasion. On the other hand, by scavenging excess free radicals and alleviating oxidative damage to host tissues, they can effectively alleviate infection-induced inflammation and cell damage, thereby enhancing therapeutic efficacy and accelerating tissue repair. Furthermore, antioxidant activity can also, to a certain extent, protect the drug itself from oxidative degradation, thereby improving stability and bioavailability.

[0005] The APD3 database (Antimicrobial Peptide Database) contains 5414 peptides with clear activity (search deadline is May 20, 2025), of which only 33 are antibacterial and antioxidant dual-functional peptides, and of these, 23 antifungal peptides all have antibacterial activity. In addition, the antioxidant activity of peptides with antifungal activity reported so far is relatively low. Among them, the DPPH method antioxidant half-effective concentration (EC50) of the most active cyclotyro-isoleucine-leucine-color-suopeptide is 1. 50The value of the 500 μg / mL concentration was 820 μg / mL, while the rest were in the milligram range. Furthermore, natural antimicrobial peptides often easily cause hemolysis of red blood cells (a hemolysis rate > 5% is generally considered to have hemolysis risk), so the hemolysis rate is also a major factor limiting the application of antimicrobial peptides.

[0006] In summary, if new peptides with both specific antifungal and antioxidant activities and extremely low hemolysis rates can be developed, they will give rise to innovative products in multiple industrial fields such as medicine, agriculture, animal husbandry, aquatic products, food preservation, and cosmetics, and have broad application prospects. Summary of the Invention

[0007] The purpose of the present invention is to provide a multifunctional peptide and a preparation method and application thereof.

[0008] To achieve the above object, the technical solution adopted by the present invention is: a modified peptide 1, whose amino acid sequence is shown in SEQ ID NO: 2. The modified peptide 1 has non-diagnostic and therapeutic applications in antifungal and antioxidation.

[0009] Correspondingly, the amino acid sequence of the modified peptide 2 is shown in SEQ ID NO: 3. The modified peptide 2 has non-diagnostic and therapeutic applications in antifungal and antioxidation.

[0010] Correspondingly, the amino acid sequence of the modified peptide 3 is shown in SEQ ID NO: 4. The modified peptide 3 has non-diagnostic and therapeutic applications in anti-oxidation.

[0011] Accordingly, the polypeptide Raniseptin-3 has non-diagnostic and therapeutic applications in anti-oxidation. The amino acid sequence of the polypeptide Raniseptin-3 is shown in SEQ ID NO: 1.

[0012] Correspondingly, foods, medicines (human drugs, veterinary drugs, etc.), health products, cosmetics, feed or fertilizers prepared using the modified peptide 1 and / or the modified peptide 2 and / or the modified peptide 3; or foods, medicines (human drugs, veterinary drugs, etc.), health products, cosmetics, feed or fertilizers comprising the modified peptide 1 and / or the modified peptide 2 and / or the modified peptide 3.

[0013] The present invention has the following beneficial effects: the present invention is the first to discover that the antimicrobial peptide Raniseptin-3 has antioxidant activity, so the peptide is an antimicrobial-antioxidant dual-functional peptide and can be used to develop antioxidant products.

[0014] This invention, based on raniseptin-3, retains its N-terminal structure and implements non-contiguous deletions at the C-terminus to form a 20-peptide core functional architecture. Further modifications were made to obtain modified peptides 1-3. Modified peptide 1 replaces all amino acids with D-amino acids to obtain modified peptide 2, and all lysine residues in modified peptide 1 are replaced with D-lysine to obtain modified peptide 3. This invention achieves the following benefits: Its antimicrobial spectrum is shifted from broad-spectrum antibacterial to specific antifungal; its antioxidant activity is significantly enhanced, reaching the level of glutathione in hydrophobic environments, and its balanced hydrophobic-hydrophilic activity is even better; its hemolytic activity is significantly reduced, becoming virtually non-hemolytic; and its peptide length is reduced from 28 amino acid residues to 20, which helps reduce production costs. Modified peptide 1 also retains the heat resistance of the parent peptide, raniseptin-3; modified peptide 2 is resistant to enzymatic degradation (including but not limited to trypsin). Each modified peptide exhibits distinct antifungal and antioxidant capabilities, allowing for targeted product development in the antifungal or antioxidant fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the secondary structure of Raniseptin-3, Raniseptin-3(1-22), Raniseptin-3(1-20) and RASEP20; Figure 2 This is a comparison chart of the DPPH scavenging effects of each polypeptide; Figure 3 This is a comparison chart of the ABTS scavenging effects of each peptide; Figure 4 This is a comparison chart of the difference in hemolysis rate of each polypeptide. DETAILED DESCRIPTION

[0016] The present invention provides a class of polypeptides with both antifungal and antioxidant effects. The polypeptides are modified from the parent peptide Raniseptin-3, whose amino acid sequence is shown in SEQ ID NO: 1 (Ala Trp LeuAsp Lys Leu Lys Ser Ile Gly Lys Val Val Gly Lys Val Ala Ile Gly Val Ala LysAsn Leu Leu Asn Pro Gln). Modified peptide 1, RASEP20, is obtained by targeted deletion of eight amino acids at positions 14, 16, and 23-28 of the parent peptide. This single-chain polypeptide has the amino acid sequence shown in SEQ ID NO: 2 (Ala Trp LeuAsp Lys Leu Lys Ser Ile Gly Lys Val Val Lys Ala Ile Gly Val Ala Lys). RASEP20 exhibits both antifungal and antioxidant effects, as well as extremely low hemolysis and excellent heat resistance.

[0017] The present invention further modified RASEP20 to obtain D-amino acid-derived peptides. Specifically, all amino acids in RASEP20 were replaced with D-amino acids to obtain modified peptide 2, designated RASEP20D, whose amino acid sequence is shown in SEQ ID NO: 3 (DAla DTrp DLeu DAsp DLys DLeu DLys DSer DIle DGlyDLys DVal DVal DLys DAla DIle DGly DVal DAla DLys). All lysine residues in RASEP20 were replaced with D-lysine residues to obtain modified peptide 3, designated RASEP20PD, whose amino acid sequence is shown in SEQ ID NO: 4 (Ala Trp Leu Asp DLys Leu DLys Ser Ile Gly DLys Val Val DLys Ala Ile Gly Val AlaDLys). After the all-D-amino acid modification, RASEP20D acquired resistance to protease degradation. RASEP20D also exhibits both antifungal and antioxidant effects, but its activity is slightly weaker and its heat resistance is reduced compared to RASEP20. RASEP20PD possesses only antioxidant activity and is an antioxidant peptide. Based on the differentiated functional properties of the modified peptides, they can be precisely targeted for product development in either the antifungal or antioxidant fields.

[0018] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The data obtained are the average values ​​obtained after at least three repetitions, and the data obtained in each repetition are valid data.

[0019] Example 1: Construction and structural comparison of modified peptides

[0020] 1. The broad-spectrum antimicrobial peptide Raniseptin-3 (Uniprot database entry name: RNSP3_BOARA) is a member of the Dermaseptin subfamily of the frogskin active peptide (FSAP) protein family. Its amino acid sequence is shown in SEQ ID NO: 1. This peptide was found in species of the Hylidae family in South America. Boana raniceps (Cope, 1862) is a skin secretion of a species of sucrose. Numerous antimicrobial peptides have been discovered from this species. The amino acid sequences of the raniseptins (raniseptin-1 to raniseptin-9, mature peptides of 28-29 amino acids) share a high degree of similarity. Raniseptin-1, raniseptin-3, and raniseptin-6 have been shown to possess broad-spectrum antibacterial activity and some hemolytic activity. Raniseptin-3 and raniseptin-6 have also been shown to inhibit the growth of the yeast-like fungus Candida albicans. Furthermore, naturally truncated peptides of this class have been detected in the skin. These peptides are cleaved at positions 14-15 between glycine and lysine, forming hemi-peptides. The N-terminal hemi-peptide consists of 14 amino acids, while the C-terminal hemi-peptide consists of 14-15 amino acids. However, the activity of truncated hemi-peptides is significantly reduced: for example, the antibacterial activity of raniseptin-1(1-14) against various bacteria was reduced by 2.5- to 17-fold; C-terminal hemi-peptides such as raniseptin-1(15-29) showed no detectable activity. Therefore, the structure and function of these peptides are highly correlated, and simple amino acid additions, deletions, or substitutions may not effectively modulate their performance, and may even lead to complete loss of core function.

[0021] CI-TASSER was used to predict the secondary structure of peptides. The unmodified parent peptide Raniseptin-3, Raniseptin-3(1-22) obtained by continuous truncation of 6 amino acid residues at the C-terminus, Raniseptin-3(1-20) obtained by continuous truncation of 8 amino acid residues at the C-terminus, and RASEP20 obtained by non-continuous deletion of 8 amino acid residues at the C-terminus were constructed. The secondary structure prediction results of each peptide are shown in Figure 2. Figure 1 shown.

[0022] from Figure 1 As can be seen, these peptides share similar N-terminal structures: a loop region (coil) formed by alanine amino acids at the N-terminus, with an α-helix in the middle. These peptides differ at the C-terminus. Both RASEP20 and Raniseptin-3 have a loop region at their C-termini, and the proportion of amino acids in the loop region to the total amino acid count is similar: 15.0% for RASEP20 and 16.7% for Raniseptin-3. In contrast, the C-termini of Raniseptin-3(1-20) and Raniseptin-3(1-22), which have undergone simple C-terminal truncations, are both composed of a β-strand and a loop region. The proportion of amino acids in these two C-terminal non-helical structures is higher: 30.0% for Raniseptin-3(1-20) and 31.8% for Raniseptin-3(1-22). Therefore, RASEP20, whose secondary structure most closely resembles that of the parent peptide, was selected as the core structure for this study.

[0023] 2. Based on their amino acid composition, we commissioned a peptide synthesis company to chemically synthesize RASEP20, RASEP20D, RASEP20PD, and Raniseptin-3 for subsequent experiments. The synthesized peptides were all greater than 95% pure, desalted, and stored at -20°C. Immediately prior to use, the peptide stock solution (25 mg / mL) was prepared in deionized water and diluted to the desired concentration using the buffer or culture medium specified for the experiment.

[0024] Example 2: Demonstration of activity and effect of polypeptide

[0025] 1. Antibacterial activity detection Minimum inhibitory concentrations (MICs) against bacteria were determined using the Clinical Laboratory Standards Institute (CLSI) broth microdilution antibacterial assay (M07-A8). Test samples included RASEP20 and its derivative peptides (RASEP20D and RASEP20PD), with raniseptin-3 (the parent peptide), ampicillin sodium, and tetracycline hydrochloride as controls. The assay culture medium was calcium-regulated MH broth. The detection was carried out by the serial dilution method, i.e., the final concentrations of the peptide to be tested were diluted with calcium-regulated MH broth medium to 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL, respectively; the final concentrations of ampicillin sodium and tetracycline hydrochloride were diluted to 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively.

[0026] The results are shown in Table 1. "ND" indicates no activity was detected up to the highest concentration tested (512 μg / mL for the modified peptide and 64 μg / mL for ampicillin sodium).

[0027] Table 1 MIC values ​​of antibacterial agents (μg / mL)

[0028] The results showed that RASEP20, RASEP20D, and RASEP20PD showed no antibacterial activity at concentrations as high as 512 μg / mL, indicating that they had no antibacterial activity. However, Raniseptin-3 showed broad-spectrum antibacterial activity, consistent with literature reports.

[0029] 2. Antifungal activity, high temperature resistance and proteolysis resistance testing The minimum inhibitory concentration (MIC) against the yeast fungus Candida albicans was determined using the broth microdilution method (M27-A2) developed by the Clinical and Laboratory Standards Institute (CLSI). RASEP20 and its derivative peptides (RASEP20D and RASEP20PD) were tested, with Raniseptin-3 and amphotericin B used as controls. The assay medium was Sabouraud dextrose broth. The detection was carried out by the doubling dilution method, that is, the final concentrations of the peptides to be tested were diluted to 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL using Sabouraud dextrose liquid medium; the final concentrations of amphotericin B were diluted to 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL.

[0030] Three methods were used to test the antifungal activity: (1) without additional peptide treatment, the peptides were directly tested by the M27-A2 method; (2) heat treatment: the peptide stock solution was placed in a 90°C water bath for 1 h and then tested by the M27-A2 method; (3) proteolysis treatment: the peptide stock solution was diluted with 2× PBS to a 1× PBS buffer system in equal volumes. Trypsin was dissolved in 1× PBS to prepare a 1% (w / v) solution, and then added to the peptide solution at a trypsin to peptide mass ratio of 1:21. After mixing, the peptide solution was digested at 37°C for 20 h. The digested peptide solution was then tested by the M27-A2 method.

[0031] The antifungal test results under each treatment are shown in Table 2. "ND" means that no activity was detected at a peptide concentration as high as 512 μg / mL.

[0032] Table 2 Antifungal MIC values ​​(μg / mL)

[0033] The results showed that RASEP20 maintained the antifungal activity of Raniseptin-3 (32 μg / mL), while RASEP20D was slightly weaker (64 μg / mL). No antifungal activity was detected even at a concentration of RASEP20PD as high as 512 μg / mL.

[0034] The antifungal activity of RASEP20 and Raniseptin-3 after heating at 90°C for 1 hour only decreased slightly (64 μg / mL), indicating that RASEP20 and Raniseptin-3 have good thermal stability. However, no antifungal activity was detected even at a concentration of RASEP20D as high as 512 μg / mL, indicating that RASEP20D is significantly less heat-resistant than RASEP20 and Raniseptin-3.

[0035] After trypsin digestion, no antifungal activity was detected in RASEP20 and Raniseptin-3 at concentrations as high as 512 μg / mL, indicating that RASEP20 and Raniseptin-3 were not resistant to proteolysis, while RASEP20D maintained its activity, indicating that RASEP20D was resistant to proteolysis.

[0036] 3. Antioxidant activity detection The antioxidant activity of the samples was determined using the total antioxidant capacity (T-AOC) evaluation system, using the DPPH free radical scavenging assay (kit: Sangon Biotechnology D799296-0100) and the ABTS free radical scavenging assay (kit: Sangon Biotechnology D799298-0100). Test samples included raniseptin-3, RASEP20, and its derivative peptides (RASEP20D and RASEP20PD), with reduced glutathione (GSH) and sodium ascorbate (SA) used as controls.

[0037] EC were compared using one-way ANOVA and Tukey's multiple comparison test. 50 The values ​​were used to test the statistical differences in antioxidant capacity between different peptides, with p < 0.05 indicating significant differences. Figure 2 、 3 As shown in Table 3. Figure 2 、 3 There were no significant differences between the groups with the same letters in the columns, and no identical letters indicated significant differences between the groups. The data in Table 3 are formatted as "mean ± standard deviation" (n = 3).

[0038] Table 3 Antioxidant EC 50 Value (μg / mL)

[0039] according to Figure 2 As can be seen from Table 3: EC of RASEP20, RASEP20D and RASEP20PD 50 The values ​​(μg / mL) of RASEP20 and RASEP20D were significantly better than those of the parent peptide Raniseptin-3; and there was no statistical difference between RASEP20 and RASEP20D and glutathione, indicating that their antioxidant activity reached the level of strong antioxidants.

[0040] according to Figure 3 As can be seen from Table 3, the EC values ​​of RASEP20 and RASEP20PD 50 The values ​​were significantly better than those of the parent peptide Raniseptin-3.

[0041] ABTS EC of all peptide samples 50 The values ​​were significantly lower than the EC values ​​of DPPH method. 50 Given that the DPPH method and ABTS method simulate hydrophobic and hydrophilic environments, respectively, it indicates that the activity of these peptides is stronger in hydrophilic environments than in hydrophobic environments. 50 The EC values ​​of Raniseptin-3, RASEP20 and its derivative peptides (RASEP20D, RASEP20PD) were analyzed by the ratio of the mean (DPPH / ABTS). 50 The ratio (2.6±0.2~4.2±0.5) was significantly lower than that of glutathione (11.4±1.5). 50 The ratio shows that Raniseptin-3, RASEP20 and their derivative peptides have a better activity balance in hydrophobic-hydrophilic dual environments than glutathione. This property gives them an advantage in developing broad-spectrum antioxidant products suitable for complex or changing environments.

[0042] In addition, sodium ascorbate showed stronger free radical scavenging ability than all tested peptides (except glutathione's ABTS system) in both DPPH and ABTS systems, which is consistent with the rule that small molecule chemical antioxidants generally have higher direct activity in in vitro tests.

[0043] 4. Hemolytic activity detection Hemolytic activity was assayed spectrophotometrically. Test samples included raniseptin-3, RASEP20, and its derivative peptides (RASEP20D and RASEP20PD). 0.1% Triton × 100 was used as a positive control, and blank PBS was used as a negative control. Fresh blood from healthy adults (supplemented with the anticoagulant EDTA-K2) was diluted with PBS to an 8% (v / v) packed red blood cell concentration. The blood was then plated into a U-bottom 96-well plate. Equal volumes of serially diluted peptide solutions in PBS were then added. The final concentrations of the peptide samples were 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, and 32 μg / mL, respectively, with a final packed red blood cell concentration of 4% (v / v). After incubation in a 37°C incubator for 1 hour, the U-bottom 96-well plate was centrifuged at 1000×g for 5 minutes, and the supernatant was transferred to a new flat-bottom 96-well plate. The plate was placed in a microplate reader and the OD value of each well at 540 nm was read.

[0044] Hemolysis rate (%) = (ODt - ODnc) ÷ (ODpc - ODnc) × 100.

[0045] Wherein, ODt is the absorbance of the test well, ODnc is the absorbance of the PBS negative control well, and ODpc is the absorbance of the 0.1% Triton × 100 positive control well.

[0046] The results are as follows Figure 4 The results showed that the parent peptide, Raniseptin-3, had a hemolytic rate (%) of 5.7 ± 1.6 (mean ± SD, n = 3) at 256 μg / mL, indicating a risk of hemolysis. However, the hemolytic rate of RASEP20 and its derivative peptides (RASEP20D and RASEP20PD) was less than 1% at concentrations as high as 2048 μg / mL, indicating virtually no hemolytic potential.

[0047] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. Modified peptide 1, characterized in that: The amino acid sequence of the modified peptide 1 is shown in SEQ ID NO:

2.

2. Non-diagnostic and therapeutic applications of the modified peptide 1 according to claim 1 in antifungal treatment.

3. Non-diagnostic and therapeutic applications of the modified peptide 1 according to claim 1 in anti-oxidation.

4. Modified peptide 2, characterized in that: The amino acid sequence of the modified peptide 2 is shown in SEQ ID NO:

3.

5. Non-diagnostic and therapeutic use of the modified peptide 2 according to claim 4 in antifungal treatment.

6. Non-diagnostic and therapeutic applications of the modified peptide 2 according to claim 4 in anti-oxidation.

7. Modified peptide 3, characterized in that: The amino acid sequence of the modified peptide 3 is shown in SEQ ID NO:

4.

8. Non-diagnostic and therapeutic applications of the modified peptide 3 according to claim 7 in anti-oxidation.

9. Non-diagnostic and therapeutic applications of the polypeptide Raniseptin-3 for antioxidant purposes, characterized by: The amino acid sequence of the polypeptide Raniseptin-3 is shown in SEQ ID NO:

1.

10. Food, medicine, health product, cosmetic, feed or fertilizer prepared using the modified peptide 1 according to claim 1 and / or the modified peptide 2 according to claim 4 and / or the modified peptide 3 according to claim 7; or; food, medicine, health product, cosmetic, feed or fertilizer comprising the modified peptide 1 according to claim 1 and / or the modified peptide 2 according to claim 4 and / or the modified peptide 3 according to claim 7.

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