Polypeptide P3 truncated peptide with antioxidant activity and application thereof

Abstract

The invention provides a polypeptide P3 truncated peptide with antioxidant activity and application thereof, and belongs to the technical field of biology. The polypeptide P3 truncated peptide with antioxidant activity comprises a polypeptide P3V10 and / or a polypeptide P3V11, the amino acid sequence of the polypeptide P3V10 is CCNRHF, and the amino acid sequence of the polypeptide P3V11 is ISCCNRH. The two polypeptides have an excellent function of removing oxyradicals, the thermal stability, pepsin stability and biological safety of the two polypeptides are superior to those of a multifunctional peptide P3, the freeze-thaw stability of the two polypeptides is not reduced compared with that of the polypeptide P3, and the two polypeptides have high application potential.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to two artificially designed and optimized truncated peptides P3 with enhanced antioxidant activity and their uses. Background Art

[0002] Bioactive peptides are small proteins formed by the connection of two or more amino acids through peptide bonds, and have one or more biological activities in organisms, including antioxidant, antibacterial, antiviral, antitumor, immune regulation, blood pressure regulation, etc.

[0003] Oxidative stress is a major cause of many chronic diseases, including cardiovascular diseases, Alzheimer's disease and cancer. The excessive accumulation of reactive oxygen species (ROS) can lead to oxidative damage of cellular biomolecules, such as DNA fragmentation, protein denaturation and membrane lipid peroxidation. Research shows that supplementing antioxidants may help maintain the balance between free radical damage and antioxidant defense, thus contributing to the prevention of some chronic diseases. Therefore, the development of antioxidants is necessary. Antioxidant peptides have become one of the most widely studied natural source antioxidants due to their high safety, easy accessibility, good antioxidant effect and other advantages, and are applied in many fields such as medicine, healthcare, food and cosmetics. Currently, antioxidant peptides from different biological sources have been reported. Yeast cells are rich in proteins, and their content accounts for 30-60% of the cell dry weight. Relevant studies have mined antioxidant peptides from yeast through an enzymatic hydrolysis strategy (Mirzaei M, Shavandi A, Mirdamadi S, et al. Bioactive peptides from yeast: A comparative review on production methods, bioactivity, structure-function relationship, and stability. Trends in Food Science & Technology, 2021, 118: 297-315).

[0004] Before the present invention, a multifunctional peptide P3 consisting of 12 amino acids with anti-cancer, antibacterial, and antioxidant activities was obtained through genomic mining (CN 202410173076.8, a multifunctional bioactive peptide P3 derived from Saccharomyces cerevisiae and its applications). Generally speaking, the shorter a polypeptide is, the higher its solubility and the easier it is to be absorbed by the body. Considering that fewer amino acid residues in antioxidant peptides are beneficial to higher antioxidant activity (Liu X, Hu Q, Shen Y, et al. Research progress on antioxidant peptides from fish by-products: Purification, identification, and structure–activity relationship. Metabolites, 2024, 14(10):561). In recent years, many successes have been achieved in designing antioxidant peptides using structure-activity relationships. Density functional theory (DFT) can be used to explore electronic and geometric molecular properties as well as intermolecular interactions for accurate and precise quantum chemical calculations (Mahmoudi et al., Density functional theory studies of the antioxidants—a review. Journal of Molecular Modeling, 2021, 27, 271). Using DFT, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies of antioxidant peptides can be determined. Antioxidant peptides with higher electron transfer ability are usually more active (Igbokwe C J, Feng Y, Louis H, et al. Novel antioxidant peptides identified from coix seed by molecular docking, quantum chemical calculations and in vitro study in HepG2 cells. Food Chemistry, 2024, 440, 138234). However, there has been no report on whether the current related research can be used for optimizing polypeptides encoded by brewing genes. In addition, there are currently no good means to predict the stability and safety of the obtained polypeptides. Summary of the Invention

[0005] The present invention provides a truncated peptide of polypeptide P3 with antioxidant activity. These two polypeptides have excellent functions of scavenging oxidative free radicals, and their thermal stability, pepsin stability, and biosafety are superior to those of multifunctional peptide P3. Moreover, their freeze-thaw stability does not decrease compared with polypeptide P3, showing high application potential.

[0006] To achieve the above technical effects, the present invention provides a truncated peptide of polypeptide P3 with antioxidant activity, including polypeptide P3_V10 and polypeptide P3_V11. The amino acid sequence of polypeptide P3_V10 is CCNRHF, and the amino acid sequence of polypeptide P3_V11 is ISCCNRH.

[0007] Preferably, it is obtained by a truncation method based on key amino acid sites of the polypeptide to remove structural redundancy of polypeptide P3 under the guidance of quantum energy calculation.

[0008] Preferably, the truncation method is specifically as follows:

[0009] Three active amino acids with strong free radical scavenging ability in the amino acid sequence of polypeptide P3 are selected, and taking these three amino acids as anchor points, one or more parts between or outside these anchor points are truncated to obtain a series of truncated peptides of polypeptide P3;

[0010] Then the energies of these truncated peptides are calculated using Gaussian software, and the truncated peptides with E HOMO higher than that of polypeptide P3 and smaller energy gap than that of polypeptide P3 are selected, namely the truncated peptides of polypeptide P3 are obtained.

[0011] Preferably, the amino acid sequence of polypeptide P3 is ISCFSLICNRHF, and the three amino acids serving as anchor points include two Cys and one His.

[0012] Preferably, the IC50 values of Vc, P3, P3_V10, and P3_V11 for scavenging ABTS free radicals are 25.2, 29.72, 18.58, and 26.64 μM respectively. The truncated peptides P3_V10 and P3_V11 of polypeptide P3 have stronger ability to scavenge ABTS free radicals than polypeptide P3.

[0013] Preferably, after incubation at 70 °C for 100 min, the ABTS free radical scavenging activities of polypeptide P3_V10 and polypeptide P3_V11 do not change relative to the initial state, and the ABTS free radical scavenging activity of polypeptide P3 is only 66.46% of the initial value. The truncated peptides P3_V10 and P3_V11 of polypeptide P3 have better thermal stability than polypeptide P3.

[0014] Preferably, after 5 freeze-thaw cycles, the ABTS radical scavenging activity of each polypeptide solution did not significantly decrease compared to the initial state, and the truncated peptides P3_V10 and P3_V11 of polypeptide P3 and polypeptide P3 all had good freeze-thaw stability.

[0015] Preferably, when in artificial gastric juice for 1 h and 2 h, the relative ABTS radical scavenging activities of polypeptides P3_V10 and P3_V11 were both higher than that of polypeptide P3, and the stability of polypeptide P3 against pepsin was improved after truncation.

[0016] Preferably, under the condition of 2000 μM, polypeptide P3 caused 4.84% of red blood cells to hemolyze, while the proportions of hemolyzed red blood cells of P3_V10 and P3_V11 were 0.69% and 0.10% respectively, and the hemolysis rate of P3_V11 was lower than that of Vc (0.27%). The biological safety of polypeptide P3 was enhanced after truncation.

[0017] The present invention also provides an antioxidant, which takes the truncated peptide of polypeptide P3 described in any one of the above technical solutions as the main component.

[0018] Compared with the prior art, the technical advantages / beneficial effects of the solution of the present invention:

[0019] Based on energy calculation, the present invention truncated, optimized and screened the multifunctional peptide P3 from Saccharomyces cerevisiae, and obtained two new antioxidant peptides. When the concentration was less than 45 μM, the ability of polypeptide P3_V10 to scavenge ABTS radicals was better than that of the control vitamin C. The IC50 values of VC, P3, P3_V10, and P3_V11 for scavenging ABTS radicals were 25.2, 29.72, 18.58, and 26.64 μM respectively. The IC50 values of polypeptides P3_V10 and P3_V11 were both smaller than that of the starting polypeptide P3. In particular, the IC50 of the mutant peptide P3_V10 decreased by 37.48% compared to P3, showing good application potential. Moreover, polypeptides P3_V10 and P3_V11 have excellent functions of scavenging oxidative radicals, and their thermal stability, pepsin stability, and biological safety are all superior to those of the multifunctional peptide P3. And their freeze-thaw stability did not decrease compared to polypeptide P3, having high application potential and can be effectively applied to food preservation, the development of cosmetics and drugs. Description of the Drawings

[0020] Figure 1 It is the molecular structure diagram of the polypeptide provided by the embodiment of the present invention. On the left is polypeptide P3_V10, and on the right is polypeptide P3_V11;

[0021] Figure 2 It is the identification of the chemically synthesized polypeptide P3_V10 by high performance liquid chromatography provided by the embodiment of the present invention;

[0022] Figure 3 Identification of chemically synthesized polypeptide P3_V10 by mass spectrometry provided by the embodiments of the present invention;

[0023] Figure 4 Identification of chemically synthesized polypeptide P3_V11 by high performance liquid chromatography provided by the embodiments of the present invention;

[0024] Figure 5 Identification of chemically synthesized polypeptide P3_V11 by mass spectrometry provided by the embodiments of the present invention;

[0025] Figure 6 ABTS radical scavenging rates of Vc, polypeptide P3, P3_V10, and P3_V11 at different concentrations provided by the embodiments of the present invention;

[0026] Figure 7 High temperature stabilities of Vc, polypeptide P3, P3_V10, and P3_V11 provided by the embodiments of the present invention;

[0027] Figure 8 Freeze-thaw stabilities of Vc, polypeptide P3, P3_V10, and P3_V11 provided by the embodiments of the present invention;

[0028] Figure 9 Pepsin stabilities of Vc, polypeptide P3, P3_V10, and P3_V11 provided by the embodiments of the present invention;

[0029] Figure 10 Visual characterization of hemolysis of Vc (a, e), P3 (b, f), P3_V10 (c, g), and P3_V11 provided by the embodiments of the present invention. Detailed implementation manners

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Example 1 Truncation and energy calculation of polypeptide

[0032] Although the polypeptide P3 has antioxidant activity, it has a relatively large number of amino acids, 12 in total. Compared with other antioxidant short peptides, its solubility decreases and the production cost increases. In order to improve the solubility of the polypeptide and reduce the production cost, it is necessary to truncate the polypeptide P3 while retaining or even enhancing its antioxidant activity. Generally, when streamlining the sequence of a relatively long antioxidant peptide, single amino acid truncation, double amino acid combination truncation, and multi - amino acid combination truncation need to be carried out step by step, which is time - consuming and laborious. Different from the prior art, the present invention proposes a truncation method for structural redundancy removal of relatively long antioxidant peptides based on key amino acid sites of the polypeptide under the guidance of quantum energy calculation, which can simplify the process of streamlining the sequence of relatively long antioxidant peptides. The specific operation is as follows.

[0033] Among the 20 common amino acids, Tyr, Cys, Trp, Met, and His have relatively strong free radical scavenging abilities (Wu RB, Huang JF, Huan R, et al. New insights into the structure - activity relationships of antioxidative peptide PMRGGGGYHY. Food chemistry, 2021, 337:, 127678). Generally speaking, the higher the E HOMO of a compound and the smaller its energy gap, the easier it is for the compound to donate electrons and be oxidized. The sequence of polypeptide P3 is ISCFSLICNRHF, which contains 3 active amino acids with relatively strong free radical scavenging abilities, including two Cys and one His. Taking these three amino acids as anchor points, one or more parts between or outside these anchor points were truncated to obtain a series of truncated peptides of polypeptide P3. Then, the Gaussian software (parameter settings: b3lyp, 6 - 31g(d)) was used to calculate the energies of these truncated peptides. The E HOMO is higher than that of P3, and the truncated peptides with an energy gap smaller than that of P3 may have better antioxidant activity than P3. Among the truncated peptide library of P3, the E Figure 1 of polypeptide P3_V10 ( Figure 1 left) and P3_V11 ( HOMO right) is higher than that of P3, and the energy gap is smaller than that of P3, indicating that they may have better antioxidant activity than P3. The sequence of polypeptide P3_V10 is CCNRHF, which is obtained by truncating the IS fragment at the front of the first Cys and the FSLI fragment between the two Cys from polypeptide P3 (ISCFSLICNRHF); the sequence of polypeptide P3_V11 is ISCCNRH, which is obtained by truncating the FSLI fragment between the two Cys from polypeptide P3. The E HOMO of polypeptide P3 is - 6.127eV, and the E LOMOis -0.687 eV, and the energy gap is 5.440 eV; polypeptide P3_V10 contains 6 amino acids, and the sequence is CCNRHF, E HOMO is -5.490 eV, E LOMO is -0.786 eV, and the energy gap is 4.704 eV; polypeptide P3_V11 is a heptapeptide, and the sequence is ISCCNRH, E HOMO is -5.503 eV, E LOMO is -1.131 eV, and the energy gap is 4.372 eV. The E of polypeptides P3_V10 and P3_V11 HOMO are both higher than that of P3, and the energy gaps are both smaller than that of P3, indicating that they may have good antioxidant activity.

[0034] Artificial synthesis of polypeptides in Example 2

[0035] According to the known amino acid sequences of polypeptides P3_V10 and P3_V11, these two peptides were artificially synthesized using the conventional solid-phase chemical synthesis method. Subsequently, high-performance liquid chromatography (results as shown in Figure 2 、 4 ) and mass spectrometry (results as shown in Figure 3 、 5 ) were used to identify the synthesized polypeptides. The results showed that the purities of the chemically synthesized polypeptides P3_V10 and P3_V11 were both greater than 95%, and the synthesized sequences were CCNRHF and ISCCNRH respectively, and the polypeptide sequences were accurate.

[0036] Example 3 Ability of polypeptides to scavenge ABTS· + Research

[0037] 179 μL of 140 mM potassium persulfate aqueous solution was added to 10 mL of 7 mM ABTS aqueous solution, and incubated for 14 hours at room temperature in the dark to prepare the ABTS mother liquor. Take 300 μL of the ABTS mother liquor and dilute it to 10 mL with distilled water to obtain the ABTS working solution. The absorbance of this working solution at 734 nm should be about 0.8. If the absorbance deviates too much, the dilution factor needs to be adjusted appropriately. During the detection, 170 μL of the ABTS working solution was added to the microplate, and then 30 μL of the polypeptide solution was added, mixed well, and left to stand at room temperature for 10 min, and then the absorbance A at 734 nm was measured i , using deionized water as the blank control, and recording its absorbance as A0.

[0038] ABTS radical scavenging rate = (A0 - A i ) / A0 × 100%

[0039] The results are as shown in Figure 6As shown, the ability of the three polypeptides to scavenge ABTS free radicals is P3_V10 > P3_V11 > P3. And when the concentration is between 7.5 - 37.5 μM, the ability of polypeptide P3_V10 to scavenge ABTS free radicals is stronger than that of Vc. The IC50 values of Vc, P3, P3_V10, and P3_V11 for scavenging ABTS free radicals are 25.2, 29.72, 18.58, and 26.64 μM respectively. The IC50 values of polypeptides P3_V10 and P3_V11 are smaller than that of P3, and the IC50 of P3_V10 is decreased by 37.48% compared to P3, indicating that truncating the multifunctional peptide P3 based on energy calculation obtained new polypeptides with stronger antioxidant ability.

[0040] Example 4 Study on the Thermal Stability of Polypeptides

[0041] Prepare 0.2 mM vitamin C and polypeptide solutions with ddH2O, incubate them at 70 °C, and measure the ABTS free radical scavenging ability of each solution at 0, 20, 40, 60, 80, and 100 min. When measuring the ABTS free radical scavenging ability, add 170 μL of ABTS working solution to the microplate, then add 30 μL of polypeptide solution, mix well, let it stand at room temperature for 10 min, and then measure its absorbance at 734 nm. Record the absorbance of the compound at 0 min as A1, and the absorbance after high-temperature incubation for a period of time as A i , using deionized water as a blank control, record its absorbance as A0, then the relative ABTS free radical scavenging activity of this compound compared to before incubation is:

[0042] Relative ABTS free radical scavenging activity = (A0 - A i ) / (A0 - A1) × 100%

[0043] The thermal stability of the polypeptides is as Figure 7 shown. Polypeptides P3_V10 and P3_V11 have good thermal stability. After incubation at 70 °C for 100 min, their ABTS free radical scavenging ability does not decrease; the thermal stability of vitamin C is also good, and its ABTS free radical scavenging ability decreases slightly with the increase of incubation time, and the relative scavenging ability at 100 min is 92.54%; the thermal stability of polypeptide P3 is less than that of vitamin C, polypeptide P3_V10, and P3_V11. With the increase of incubation time, its ABTS free radical scavenging ability gradually weakens, and the relative scavenging ability at 100 min is 66.46%. It shows that the thermal stability of polypeptide P3 is enhanced by truncating it.

[0044] Example 5 Study on the Freeze-Thaw Stability of Polypeptides

[0045] Freeze 0.2 mM of vitamin C and the polypeptide solution at -20 °C for 1 h, thaw at room temperature for 1 h, and evaluate its activity. This process is repeated 5 times. The calculation method of relative ABTS radical scavenging activity is as in Example 4.

[0046] The results are as Figure 8 shown. After 5 freeze-thaw cycles, the ABTS radical scavenging activity of each polypeptide solution relative to the initial state did not decrease significantly, indicating that the truncated polypeptides P3_V10 and P3_V11, like the original polypeptide P3, have good freeze-thaw stability.

[0047] Example 6 Study on the Pepsin Stability of Polypeptides

[0048] According to the Chinese Pharmacopoeia, take 16.4 mL of dilute hydrochloric acid (equivalent to 3.84 mL of hydrochloric acid), add about 800 mL of water and 10 g of pepsin, shake well, and then dilute with water to 1000 mL to obtain artificial gastric juice. Prepare 0.2 mM of vitamin C and polypeptide solutions with artificial gastric juice, and then incubate each solution at 37 °C. Detect the relative ABTS radical scavenging activity of each compound at 1 h and 2 h respectively to explore its pepsin stability.

[0049] The results are as Figure 9 shown. At 1 h and 2 h, the relative ABTS radical scavenging activities of polypeptides P3_V10 and P3_V11 are higher than that of polypeptide P3, indicating that the pepsin stability is improved after truncation of P3. The relative ABTS radical scavenging activities of polypeptides P3, P3_V10, and P3_V11 at 2 h are 75.02%, 85.83%, and 82.26% respectively.

[0050] Example 7 Study on the Hemolytic Activity of Polypeptides

[0051] Take 10 mL of anticoagulated whole blood in a 50 mL centrifuge tube, centrifuge at 1000 g for 5 min, and aspirate the upper plasma; add 20 mL of D-PBS and mix gently, centrifuge at 1000 g for 3 min and take out the test tube; discard the supernatant and repeat the operation 3 times, and increase the last centrifugation time to 5 min; after centrifugation of red blood cells, prepare a 5% RBC suspension according to the volume ratio of packed red blood cells to D-PBS of 1:19.

[0052] Prepare seven sets of 1.5 mL centrifuge tubes, with twelve tubes in each set. Distribute the red blood cell suspension into each tube and centrifuge again at 1000 g for 5 minutes. After centrifugation, remove the supernatant. Add vitamin C at five concentrations (100, 200, 500, 1000, 2000 μM) and the polypeptide samples to the test tubes as the experimental group. Add D-PBS to one group as the negative control, and add a D-PBS solution containing 2% Triton X-100 to another group as the positive control. After incubating in the incubator for 1 hour, centrifuge at 1000 g for 5 min, then transfer the supernatant to a 96-well plate, with 100 μL in each well. Measure the absorbance difference between the experimental group and the control group at 570 nm in triplicate and calculate the hemolysis rate. Denote the absorbance of the sample as A i , the absorbance of the positive control is A1, and the absorbance of the negative control is A0. The calculation formula for the hemolysis rate of the sample is as follows:

[0053] Sample hemolysis rate = (A i -A0) / (A1-A0)×100%

[0054] Combined Figure 10 and calculated, it can be seen that within the concentration range of 100 - 2000 μM, no obvious hemolysis occurred for Vc, P3_V10, and P3_V11. Polypeptide P3 can cause slight hemolysis of red blood cells under high concentration conditions, and as the concentration increases, the proportion of red blood cells undergoing hemolysis also gradually increases; under the condition of 2000 μM, P3 can cause 4.84% of red blood cells to undergo hemolysis, while the proportions of red blood cells undergoing hemolysis for P3_V10 and P3_V11 are 0.69% and 0.10% respectively. In particular, the hemolysis rate of P3_V11 is lower than that of Vc (0.27%), indicating that the biological safety has been enhanced after truncating the polypeptide P3.

Claims

1. A polypeptide P3 truncated peptide having antioxidant activity, characterized in that: It comprises polypeptide P3_V10 and polypeptide P3_V11, wherein the amino acid sequence of polypeptide P3_V10 is CCNRHF, and the amino acid sequence of polypeptide P3_V11 is ISCCNRH.

2. The polypeptide P3 truncated peptide according to claim 1, characterized in that The method was obtained by truncating the peptide P3 based on key amino acid sites of the peptide, guided by quantum energy calculation.

3. The polypeptide P3 truncated peptide according to claim 2, characterized in that: The truncation method is specifically as follows: Three active amino acids with strong free radical scavenging ability were selected from the amino acid sequence of polypeptide P3, and the three amino acids were used as anchor points, and one or more parts between or outside these anchor points were cut off to obtain a series of polypeptide P3 truncated peptides; Then, the energy of these truncated peptides was calculated using Gaussian software. HOMO A truncated peptide having a higher energy gap than polypeptide P3 and a smaller energy gap than polypeptide P3 is obtained, that is, a truncated peptide of polypeptide P3.

4. The polypeptide P3 truncated peptide according to claim 3, characterized in that The amino acid sequence of polypeptide P3 is ISCFSLICNRHF, and the three amino acids serving as anchors include two Cys and one His.

5. The polypeptide P3 truncated peptide according to any one of claims 1 to 4, characterized in that: The IC50 values ​​of Vc, P3, P3_V10 and P3_V11 for scavenging ABTS free radicals were 25.2, 29.72, 18.58 and 26.64 μM, respectively. The truncated peptides P3_V10 and P3_V11 of polypeptide P3 had stronger ability to scavenge ABTS free radicals than polypeptide P3.

6. The polypeptide P3 truncated peptide according to any one of claims 1 to 4, characterized in that: After incubation at 70℃ for 100min, the ABTS free radical scavenging activity of peptides P3_V10 and P3_V11 did not change compared with the initial state, and the ABTS free radical scavenging activity of peptide P3 was only 66.46% of the initial state. The truncated peptides P3_V10 and P3_V11 had better thermal stability than peptide P3.

7. The polypeptide P3 truncated peptide according to any one of claims 1 to 4, characterized in that: After five freeze-thaw cycles, the ABTS free radical scavenging activity of each polypeptide solution did not decrease significantly compared with the initial state, and the truncated peptides P3_V10, P3_V11 and polypeptide P3 all had good freeze-thaw stability.

8. The polypeptide P3 truncated peptide according to any one of claims 1 to 4, characterized in that: When exposed to artificial gastric juice for 1h and 2h, the relative ABTS free radical scavenging activities of peptides P3_V10 and P3_V11 were higher than that of peptide P3. The stability of peptide P3 in pepsin was improved after truncation.

9. The polypeptide P3 truncated peptide according to any one of claims 1 to 4, characterized in that: At 2000 μM, peptide P3 can cause hemolysis of 4.84% of erythrocytes, while the proportions of hemolyzed erythrocytes of P3_V10 and P3_V11 are 0.69% and 0.10%, respectively. The hemolysis rate of P3_V11 is lower than that of Vc (0.27%). The biosafety of peptide P3 is enhanced after truncation.

10. An antioxidant, characterized in that The main component is the polypeptide P3 truncated peptide described in any one of claims 1 to 4.

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