Multifunctional peptide and its preparation method and application

Abstract

This invention belongs to the field of protein engineering, specifically relating to multifunctional peptides, their preparation methods, and applications. Based on Raniseptin-3, this invention retains its N-terminal structure and performs discontinuous deletion at the C-terminus to form a 20-peptide core functional architecture. Further modifications yielded modified peptides 1-3, achieving the following effects: the antibacterial spectrum shifted from broad-spectrum antibacterial to specific antifungal; antioxidant activity was significantly enhanced, with hydrophobic environment activity reaching glutathione levels, and a better balance between hydrophobic and hydrophilic dual-environment activity; hemolytic activity was significantly reduced, almost non-existent; and the peptide length was reduced from 28 amino acid residues to 20, facilitating lower production costs. Simultaneously, modified peptide 1 retains the heat resistance of the parent peptide Raniseptin-3; modified peptide 2 exhibits resistance to enzymatic degradation. Each modified peptide possesses different antifungal and antioxidant capabilities, allowing for precise application in product development in the antifungal or antioxidant fields.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering, specifically relating to multifunctional peptides, their preparation methods, and applications. Background Technology

[0002] Fungi pose a serious threat to global health, agriculture, aquaculture, food security, and ecosystems. Compared to bacteria, the development of antifungal drugs is more challenging, primarily because fungi, like plants and animals, are eukaryotes, resulting in a scarcity of specific targets and a limited range of available antifungal drugs. Currently reported antifungal drugs are mainly classified into polyenes, azoles, allylamines, pyrimidines, and echinocandins. These antifungal drugs are small-molecule chemicals and generally have toxic side effects. Antimicrobial peptides, however, have attracted considerable attention due to their low toxicity, fewer side effects, and lower likelihood of inducing drug resistance. However, most existing antifungal peptides possess broad-spectrum activity, meaning they can fight both fungi and bacteria; in contrast, narrow-spectrum antimicrobial peptides with specific antifungal activity are more advantageous in reducing host flora imbalance, minimizing the spread of drug resistance, and controlling ecological risks.

[0003] Antioxidants effectively neutralize free radicals, reduce oxidative stress damage to cells and molecules, and have broad application prospects in the health, food, cosmetic, and agricultural fields. Currently, most antioxidants are small-molecule chemicals; although highly active, they generally have potential harmful side effects.

[0004] Given that microbial infection and oxidative stress often coexist and exacerbate each other, developing substances with both antibacterial and antioxidant activities offers multiple advantages. On one hand, these substances can directly inhibit pathogens, reducing pathogen invasion; on the other hand, by scavenging excess free radicals and mitigating oxidative damage to host tissues, they effectively alleviate infection-induced inflammation and cell damage, thereby enhancing therapeutic efficacy and accelerating tissue repair. Furthermore, antioxidant function can, to some extent, protect the drug itself from oxidative degradation, thus improving stability and bioavailability.

[0005] The APD3 (Antimicrobial Peptide Database) contains 5414 peptides with definite activity (search as of May 20, 2025), of which only 33 are bifunctional antibacterial and antioxidant peptides. Of these, 23 peptides exhibiting antifungal activity also possess antibacterial activity. Furthermore, in existing reports, peptides with antifungal activity generally have low antioxidant activity. The highest-activity cyclic tyrosine-isoleucine-leucine-chromothreonide showed the highest antioxidant half-maximal concentration (EC50) using the DPPH method. 50The concentration of the active ingredient was 820 μg / mL, while the others were in the milligram range. Furthermore, natural antimicrobial peptides often cause hemolysis of red blood cells (a hemolysis rate >5% is generally considered a risk of hemolysis), therefore, the hemolysis rate is also an important factor limiting the application of antimicrobial peptides.

[0006] In conclusion, if new peptides with both specific antifungal and antioxidant activities and extremely low hemolysis rate can be developed, they will spur innovative products in multiple industries such as pharmaceuticals, agriculture, animal husbandry and aquaculture, food preservation, and cosmetics, and have broad application prospects. Summary of the Invention

[0007] The purpose of this invention is to provide multifunctional peptides, their preparation methods, and applications.

[0008] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: modified peptide 1, the amino acid sequence of which is shown in SEQ ID NO: 2. The modified peptide 1 has non-diagnostic and therapeutic applications in antifungal and antioxidant activity.

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

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

[0011] Accordingly, the non-diagnostic and therapeutic applications of the peptide Raniseptin-3 in antioxidation, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0012] Accordingly, food, pharmaceuticals (human drugs, veterinary drugs, etc.), health products, cosmetics, feed or fertilizer prepared using the modified peptide 1 and / or the modified peptide 2 and / or the modified peptide 3; or; food, pharmaceuticals (human drugs, veterinary drugs, etc.), health products, cosmetics, feed or fertilizer including 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. Therefore, this peptide is an antimicrobial-antioxidant bifunctional peptide and can be used to develop antioxidant products.

[0014] This invention is based on Raniseptin-3. By retaining its N-terminal structure and performing discontinuous deletions at the C-terminus, a 20-peptide core functional architecture is formed. Further modifications yielded modified peptides 1-3. Specifically, all amino acids in modified peptide 1 were replaced with D-amino acids to obtain modified peptide 2; and all lysines in modified peptide 1 were replaced with D-lysines to obtain modified peptide 3. This invention achieves the following effects: the antibacterial spectrum is transformed from broad-spectrum antibacterial to specific antifungal; antioxidant activity is significantly improved, with hydrophobic environment activity reaching glutathione levels, and a better balance between hydrophobic and hydrophilic dual-environment activity; hemolytic activity is significantly reduced, with almost no hemolytic activity; and the peptide length is reduced from 28 amino acid residues to 20, which helps reduce production costs. Simultaneously, modified peptide 1 retains the heat resistance of the parent peptide Raniseptin-3; and modified peptide 2 exhibits resistance to enzymatic degradation (including but not limited to trypsin). Each modified peptide has different antifungal and antioxidant capabilities, allowing for precise application in product development in the antifungal or antioxidant fields. Attached Figure Description

[0015] Figure 1 A schematic diagram of the secondary structure of Raniseptin-3, Raniseptin-3(1-22), Raniseptin-3(1-20) and RASEP20;

[0016] Figure 2 A comparison chart showing the DPPH scavenging effects of various peptides;

[0017] Figure 3 A comparison chart showing the ABTS clearance effects of each peptide;

[0018] Figure 4 This is a comparison chart showing the differences in hemolysis rates among various peptides. Detailed Implementation

[0019] This invention provides a class of peptides possessing both antifungal and antioxidant effects. The peptides 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). After directionally deleting eight amino acids at positions 14, 16, and 23–28 of the parent peptide, modified peptide 1, RASEP20, is obtained. This is a single-chain peptide with 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 antifungal and antioxidant activities, along with extremely low hemolytic activity and good heat resistance.

[0020] This invention further modifies RASEP20 to obtain D-amino acid-derived peptides. Specifically, all amino acids in RASEP20 are replaced with D-amino acids to obtain modified peptide 2, named RASEP20D, with the amino acid sequence 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 lysines in RASEP20 are replaced with D-lysines to obtain modified peptide 3, named RASEP20PD, with the amino acid sequence shown in SEQ ID NO: 4 (Ala TrpLeu Asp DLys Leu DLys Ser Ile Gly DLys Val Val DLys Ala Ile Gly Val AlaDLys). After complete D-amino acid modification, RASEP20D acquires resistance to protease digestion. In addition, RASEP20D still possesses both antifungal and antioxidant effects, but its activity is slightly weaker and its heat resistance is reduced compared to RASEP20. RASEP20PD only has antioxidant function and is an antioxidant peptide. Based on the differentiated functional characteristics of different modified peptides, they can be precisely used for product development in the fields of antifungal or antioxidant applications.

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0022] Example 1: Construction and structural comparison of modified peptides

[0023] 1. Raniseptin-3, a broad-spectrum antimicrobial peptide (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 discovered in species of the Hylidae family in South America. Boana raniceps The skin secretions of *Cope* (1862). Several antimicrobial peptides have been discovered in this species. Among them, the amino acid sequences of the Raniseptins (Raniseptin-1 to Raniseptin-9, with mature peptides of 28–29 amino acids) members show high 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 detected to have a tendency to inhibit the growth of yeast-like fungi, *Candida albicans*. Furthermore, these peptides have been detected in naturally occurring truncated peptide forms in the skin, formed by cleavage at the 14–15 glycine-lysine residues, resulting in hemipeptides. The N-terminal hemipeptide has 14 amino acids, and the C-terminal hemipeptide has 14–15 amino acids. However, the activity of truncated hemipeptides is significantly reduced: for example, the antibacterial activity of Raniseptin-1 (1-14) against various bacteria is reduced by 2.5 to 17 times; C-terminal hemipeptides such as Raniseptin-1 (15-29) showed no detectable activity. Therefore, the structure and function of these peptides are highly correlated, and simple addition, subtraction, or substitution of amino acids may not effectively regulate their performance, or may even lead to the complete loss of their core function.

[0024] The secondary structures of peptides were predicted using CI-TASSER. The following peptides were constructed: the unmodified parent peptide Raniseptin-3; Raniseptin-3(1-22) obtained by sequentially removing 6 amino acid residues from its C-terminus; Raniseptin-3(1-20) obtained by sequentially removing 8 amino acid residues from its C-terminus; and RASEP20 obtained by non-contiguously deleting 8 amino acid residues from its C-terminus. The predicted secondary structures of each peptide are shown below. Figure 1 As shown.

[0025] from Figure 1 It can be seen that these peptides have similar N-terminal structures, namely, a coil formed by alanine residues at the N-terminus, with an α-helix in the middle. The differences between these peptides lie at the C-terminus. Both RASEP20 and Raniseptin-3 have a coil at the C-terminus, and the proportion of amino acids in the coil region to the total amino acids is similar: 15.0% for RASEP20 and 16.7% for Raniseptin-3. However, Raniseptin-3(1-20) and Raniseptin-3(1-22), which have their C-termini simply truncated, have a β-strand and a coil at the C-terminus. 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 is closest to the parent peptide, was chosen as the core structure for this study.

[0026] 2. Based on the amino acid composition, RASEP20, RASEP20D, RASEP20PD, and Raniseptin-3 were chemically synthesized by a peptide synthesis company for subsequent experiments. The synthesized peptides all had a purity greater than 95%, were desalted, and stored at -20℃. Before use, a peptide stock solution (25 mg / mL) was prepared with deionized water, and then diluted to the required concentration using the appropriate experimental buffer or culture medium.

[0027] Example 2: Demonstration of the activity and effects of the peptide

[0028] 1. Antibacterial activity test

[0029] The minimum inhibitory concentration (MIC) against bacteria was determined using the broth microdilution antibacterial assay (M07-A8) from the Clinical Laboratory Standards Institute (CLSI). Test samples included RASEP20 and its derivative peptides (RASEP20D, RASEP20PD), with Raniseptin-3 (the parent peptide), ampicillin sodium, and tetracycline hydrochloride serving as controls. The test medium was calcium-modified MH broth. The assay was performed using a serial dilution method, in which the final concentrations of the test peptides 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 calcium-modified MH broth medium; and 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.

[0030] The results are shown in Table 1. “ND” indicates that no activity was detected at the highest concentrations tested (512 μg / mL for the modified peptide and 64 μg / mL for ampicillin sodium).

[0031] Table 1. MIC values ​​for antibacterial activity (μg / mL)

[0032]

[0033] The results showed that RASEP20, RASEP20D, and RASEP20PD showed no antibacterial activity even at concentrations up to 512 μg / mL, indicating no antibacterial activity. Raniseptin-3, however, exhibited broad-spectrum antibacterial activity, consistent with previous reports.

[0034] 2. Tests for antifungal activity, high-temperature resistance, and resistance to protein hydrolysis.

[0035] The minimum inhibitory concentration (MIC) against yeast-like fungi (Candida albicans) was determined using the broth microdilution method (M27-A2) of the Clinical Laboratory Standards Institute (CILSI). Test samples included RASEP20 and its derivative peptides (RASEP20D, RASEP20PD), with Raniseptin-3 and amphotericin B used as controls. The test medium was Sabouraud dextrose broth. The assay was performed using a serial dilution method, in which the final concentrations of each analyte were diluted with Sabouraud dextrose liquid 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; and 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.

[0036] Three methods were used to detect antifungal activity: (1) no additional treatment was performed on the peptides, and the M27-A2 method was used directly; (2) heat treatment: each peptide stock solution was heated in a water bath at 90℃ for 1 hour, and then the M27-A2 method was used for detection; (3) proteolytic digestion: the peptide stock solution was diluted with 2×PBS to a 1×PBS buffer system in an equal volume. 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 solution was digested at 37℃ for 20 hours, and the digested peptide solution was then detected by the M27-A2 method.

[0037] The antifungal test results for each treatment are shown in Table 2. “ND” indicates that no activity was detected even at a peptide concentration as high as 512 μg / mL.

[0038] Table 2. MIC values ​​for antifungal activity (μg / mL)

[0039]

[0040] The results showed that RASEP20 maintained the antifungal activity of Raniseptin-3 (32 μg / mL), while RASEP20D was slightly weaker (64 μg / mL), and no antifungal activity was detected even at a high concentration of RASEP20PD (512 μg / mL).

[0041] The antifungal activity of RASEP20 and Raniseptin-3 decreased only slightly (64 μg / mL) after heating at 90℃ for 1 hour, indicating that RASEP20 and Raniseptin-3 have good thermal stability. However, no antifungal activity was detected even at a concentration of 512 μg / mL of RASEP20D, indicating that the heat resistance of RASEP20D is significantly weaker than that of RASEP20 and Raniseptin-3.

[0042] Even at concentrations as high as 512 μg / mL, no antifungal activity was detected in RASEP20 and Raniseptin-3 after trypsin digestion, indicating that RASEP20 and Raniseptin-3 are not resistant to protease digestion. However, RASEP20D retained its activity, indicating that RASEP20D is resistant to protease digestion.

[0043] 3. Antioxidant activity detection

[0044] The total antioxidant capacity (T-AOC) evaluation system was used, and the antioxidant activity of the samples was determined by the DPPH free radical scavenging method (kit: Sangon Biotech D799296-0100) and the ABTS free radical scavenging method (kit: Sangon Biotech D799298-0100). The test samples included Raniseptin-3, RASEP20 and its derivative peptides (RASEP20D, RASEP20PD), with reduced glutathione (GSH) and sodium ascorbate (SA) used as controls.

[0045] EC was compared using one-way ANOVA and Tukey's multiple comparison test. 50 The values ​​were used to test the statistical differences in antioxidant capacity among different peptides; p < 0.05 indicated a significant difference. The results are as follows: Figure 2 , 3 As shown in Table 3. Figure 2 , 3 Groups with the same letter on the column showed no significant difference, while the absence of the same letter indicated a significant difference between groups. The data in Table 3 are in the format "mean ± standard deviation" (n=3).

[0046] Table 3 Antioxidant EC 50 Value (μg / mL)

[0047]

[0048] according to Figure 2 As can be seen from Table 3, the EC values ​​of RASEP20, RASEP20D, and RASEP20PD are... 50 The values ​​(μg / mL) were significantly better than those of the parent peptide Raniseptin-3; and there was no statistically significant difference between RASEP20 and RASEP20D and glutathione, indicating that their antioxidant activity reached the level of strong antioxidants.

[0049] according to Figure 3 As can be seen from Table 3, the EC values ​​of RASEP20 and RASEP20PD are... 50 The values ​​were all significantly better than those of the parent peptide Raniseptin-3.

[0050] ABTS EC method for all peptide samples 50 The values ​​were all significantly lower than the EC values ​​obtained by the DPPH method. 50 The values, given that the DPPH and ABTS methods simulate hydrophobic and hydrophilic environments respectively, indicate that these peptides are more active in hydrophilic environments than in hydrophobic environments. Combined with EC... 50 Analysis was performed on the ratio of means (DPPH / ABTS): EC5 of Raniseptin-3, RASEP20 and its derivative peptides (RASEP20D, RASEP20PD). 50 The ratio (2.6±0.2~4.2±0.5) was significantly lower than that of glutathione (11.4±1.5). The EC50 was also significantly lower. 50 The ratio indicates that Raniseptin-3, RASEP20 and their derivative peptides have better activity balance in both hydrophobic and hydrophilic environments than glutathione. This characteristic makes them more advantageous in developing broad-spectrum antioxidant products suitable for complex or variable environments.

[0051] In addition, sodium ascorbate showed stronger free radical scavenging ability in both DPPH and ABTS systems than all tested peptides (except for glutathione in the ABTS system), which is consistent with the general rule that small molecule chemical antioxidants usually have higher direct activity in in vitro tests.

[0052] 4. Hemolytic activity detection

[0053] Hemolytic activity was detected 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 as a negative control. Fresh blood from healthy adults (with added anticoagulant EDTA-K2) was prepared into 8% (v / v) hematocrit with PBS, then added to U-bottom 96-well plates. Equal volumes of peptide solutions prepared with PBS were then added, resulting in final peptide concentrations of 2048 μg / mL, 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, and 32 μg / mL. The final hematocrit concentration was 4% (v / v). After incubating at 37°C for 1 hour, the U-shaped bottom 96-well plate was centrifuged at 1000×g for 5 minutes, and the supernatant was transferred into a new flat-bottom 96-well plate. The plate was then placed in a microplate reader to read the OD value of each well at 540 nm.

[0054] Hemolysis rate (%) = (ODt - ODnc) ÷ (ODpc - ODnc) × 100.

[0055] 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.

[0056] The results are as follows Figure 4 As shown in the figure. The results showed that the hemolysis rate (%) of the parent peptide Raniseptin-3 at 256 μg / mL was 5.7 ± 1.6 (mean ± standard deviation, n=3), indicating a risk of hemolysis. In contrast, the hemolysis rate of RASEP20 and its derivative peptides (RASEP20D, RASEP20PD) was less than 1% even at a concentration as high as 2048 μg / mL, indicating almost no hemolysis.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined 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. The non-diagnostic and therapeutic application of the modified peptide 1 according to claim 1 in antifungal treatment, characterized in that: The fungus in question is Candida albicans.

3. The non-diagnostic and therapeutic use of the modified peptide 1 according to claim 1 in antioxidation.

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

3.

5. The non-diagnostic and therapeutic application of the modified peptide 2 according to claim 4 in antifungal treatment, characterized in that: The fungus in question is Candida albicans.

6. The non-diagnostic and therapeutic use of the modified peptide 2 of claim 4 in antioxidation.

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

4.

8. The non-diagnostic and therapeutic use of the modified peptide 3 of claim 7 in antioxidation.

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

1.

10. Pharmaceuticals, health products, cosmetics, feeds, or fertilizers prepared using the modified peptide 1 of claim 1 and / or the modified peptide 2 of claim 4 and / or the modified peptide 3 of claim 7; or; pharmaceuticals, health products, cosmetics, feeds, or fertilizers comprising the modified peptide 1 of claim 1 and / or the modified peptide 2 of claim 4 and / or the modified peptide 3 of claim 7.

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