Cottonseed protein source ACE inhibitory peptide, screening and modification method and application thereof

Abstract

This invention belongs to the field of food biotechnology and bioactive peptides, and specifically relates to a cottonseed protein-derived ACE-inhibiting peptide, its screening, modification methods, and applications. The screening method of this application includes four core steps: virtual enzymatic library construction, multi-stage computer filtering, molecular docking screening, and in vitro activity verification, obtaining 194 high-confidence candidate peptides. Four cottonseed protein-derived peptides (SYF, DIF, IAF, and RFY) with in vitro ACE-inhibiting activity were verified, all exhibiting mixed ACE-inhibiting activity, with DIF showing the strongest inhibitory activity. Molecular dynamics simulations confirmed the structural stability of the ACE-DIF complex. A modified peptide FIF was obtained through a dipeptide module substitution strategy, with an IC50 value of [missing information]. 50 The activity was significantly reduced, approximately 3.7 times that of the parent peptide DIF, and the inhibition type changed from mixed to competitive. This invention provides a new approach for the development of natural antihypertensive functional peptides and the high-value utilization of plant protein resources.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology and bioactive peptides, and specifically relates to a cottonseed protein-derived ACE inhibitory peptide. Background Technology

[0002] cotton( Gossypium Cotton (spp.) is one of the world's most important industrial economic crops, with China being a major producer. In 2024, the cotton planting area reached 2.8383 million hectares, and the cotton wadding yield was 6.214 million tons, continuously generating a large amount of processing by-products. Cottonseed meal, produced after oil extraction from cottonseed, accounts for approximately 45% of the total raw materials, with a crude protein content as high as 30-50% (dry weight basis). However, due to the presence of anti-nutritional factors such as gossypol, cottonseed protein is currently mainly used for animal feed and has not yet achieved high-value utilization. The main storage proteins in cottonseed protein account for approximately 80% of the total protein content, and its amino acid composition is balanced, rich in hydrophobic amino acids, providing a good raw material basis for the development of bioactive peptides.

[0003] Hypertension affects more than 1 billion people worldwide and is a leading risk factor for cardiovascular disease, stroke, and kidney failure. The renin-angiotensin system (RAS) plays a central role in blood pressure regulation, with angiotensin-converting enzyme (ACE; EC3.4.15.1) catalyzing the conversion of inactive decapeptide angiotensin I into the potent vasoconstrictor angiotensin II, while simultaneously degrading the angiotensin-bradykinin. While clinically synthesized ACE inhibitors (such as captopril and enalapril) are effective, some patients experience adverse reactions such as dry cough, angioedema, and hyperkalemia, prompting researchers to seek naturally occurring ACE inhibitory peptides from food sources as a complementary antihypertensive strategy.

[0004] Dietary ACE inhibitory peptides (ACEIPs) have been studied from various protein sources such as dairy products, soybeans, rapeseed, and fish. However, research on ACEIPs derived from cottonseed protein remains scarce, and relevant structure-activity relationships and rational modification strategies have not yet been established. The traditional "enzymatic hydrolysis-screening-purification-identification" research paradigm is inefficient and costly, and often only obtains the main fragments from the enzymatic hydrolysis products, making it difficult to systematically explore the complete sequence space. For example, patent 201711000146.6 discloses a cottonseed protein polypeptide with high ACE inhibitory activity. Using dephenolized cottonseed meal as raw material, the protein is extracted by alkali dissolution and acid precipitation, and ACE inhibitory peptides are prepared by papain hydrolysis. Then, exogenous leucine is introduced for modification through the Plastein reaction, which significantly improves the ACE inhibitory activity of the cottonseed peptide. However, this technology still mainly relies on conventional enzymatic hydrolysis and reverse synthesis modification, without combining molecular design and rational modification methods, resulting in insufficient precise control of the peptide structure-activity relationship. Computer-aided virtual proteomics methods can significantly narrow down the candidate pool before experimental verification by virtually enumerating theoretical peptide libraries through enzymatic digestion, combined with multi-level computer filtering and molecular docking. These methods have the advantages of high throughput and low cost, but their application in the development of functional peptides from cottonseed protein is still in its early stages.

[0005] The dipeptide-module substitution strategy is a rational design strategy that enhances the activity of a target peptide by replacing a low-contribution dipeptide module with a high-contribution module. This strategy has been shown to effectively enhance the activity of marine-derived ACE repressor peptides, but it has not yet been reported in the modification of ACE repressor peptides derived from plant proteins. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a cottonseed protein-derived ACE inhibitory peptide, its screening and modification methods, and its applications.

[0007] The technical solution of this invention is implemented as follows:

[0008] The cottonseed protein source ACE inhibitory peptide screening method provided by this invention includes four core steps: virtual enzymatic digestion library construction, multi-level computer filtering, molecular docking screening, and in vitro activity verification; the modular modification method includes three steps: dipeptide module contribution evaluation, alternative module system screening, and modified peptide activity verification.

[0009] Based on this, the present invention provides a cottonseed protein-derived ACE-inhibiting peptide, wherein the cottonseed protein-derived ACE-inhibiting peptide is selected from any one of peptide DIF, peptide SYF, peptide IAF, and peptide RFY, wherein the half-maximal inhibitory concentration (WMC) of peptide DIF is 0.28-0.38 mg / mL, the WMC of peptide SYF is 0.49-0.57 mg / mL, the WMC of peptide IAF is 0.41-0.65 mg / mL, and the WMC of peptide RFY is 0.59-0.63 mg / mL, all of which exhibit mixed-type inhibition of ACE.

[0010] Secondly, the present invention also provides a method for screening the above-mentioned cottonseed protein-derived ACE inhibitory peptide, the steps of which are as follows:

[0011] (1) The amino acid sequences of four major cottonseed storage proteins (Legumin A, Legumin B, Vicilin C72, and Vicilin GC72-A) were obtained from the UniProt database. Virtual enzymatic digestion was performed using the online tool BIOPEP-UWM. Fifteen commercial proteases and their single / multi-enzyme combination schemes (a total of 52 enzymatic digestion schemes) were selected to obtain 3,300 non-redundant cottonseed polypeptides.

[0012] (2) Bioactivity of peptides was predicted using the PeptideRanker program (retaining sequences with a score ≥0.5), toxicity was assessed using ToxinPred (retaining non-toxic peptides), ADMET properties were evaluated using ADMETSAR / ADMETlab 3.0 (excluding P-gp substrates and CYP450 inhibitors), and water solubility was predicted using Innovagen (retaining soluble peptides). After stepwise deduplication, 194 high-confidence candidate bioactive peptides were finally obtained.

[0013] (3) Using the ACE crystal structure (PDB ID: 1O8A) as the acceptor, molecular docking was performed using AutoDock software, with the docking binding free energy (|ΔG| ≥ 7 kcal·mol) as the target. -1 Candidate ACE inhibitory peptides were screened using multiple criteria, including the number of hydrogen bonds (≥4) and the participation of key active site residues.

[0014] (4) Candidate peptides (purity ≥95%) were synthesized using solid-phase chemical synthesis. The ACE inhibitory activity was verified in vitro using a colorimetric method based on FAPGG substrate, and the IC50 of the active peptide was determined. 50 The values ​​were determined, and the type of inhibition kinetics was identified by plotting using Lineweaver-Burk.

[0015] Preferably, the cottonseed storage proteins in step (1) above include Vicilin GC72-A, Vicilin C72, Legumin A and Legumin B; wherein Vicilin GC72-A has accession number P09799 in the UniProt database, Vicilin C72 has accession number P09801 in the UniProt database, Legumin A has accession number P09802 in the UniProt database, and Legumin B has accession number P09800 in the UniProt database.

[0016] Preferably, the 15 proteases selected for virtual enzymatic hydrolysis in step (1) above are one or more combinations of chymotrypsin (A), trypsin, proteinase K, pancreatic elastase, thermolysin, chymotrypsin C, closetripain, papain, ficin, leukocyte elastase, thrombin, stem bromelain, subtilisin, V-8 protease (Glutamyl endopeptidase), and pepsin; the combined enzymatic hydrolysis scheme covers a total of 52 schemes including single enzyme, double enzyme, and triple enzyme, excluding schemes with non-commercial enzymes, redundant cleavage modes, no fragment production, and low coverage (single enzyme ≤ 10 cleavage sites or multi-enzyme combination ≤ 10 non-redundant sites).

[0017] Preferably, the specific parameters for molecular docking in step (3) above are as follows: using the ACE crystal structure (PDB ID: 1O8A) as the acceptor, non-essential heteroatoms are removed, polar hydrogen is added, partial charge is assigned, and the PDBQT format is exported in AutoDock Tools; the three-dimensional conformation of the peptide is generated by OpenBabel 3.1.0 and converted into a PDBQT ligand after geometric optimization; the center coordinates of the docking box are x = 41.18, y = 34.44, z = 44.38, the size is 80×80×80 Å, and the spacing is 0.375 Å; the reliability of the docking results is verified by using lisinopril and 15 ACE inhibitory peptides reported in the literature as references; finally, the binding free energy |ΔG|≥7 kcal·mol -1 Candidate molecules with ≥4 hydrogen bonds and involving key hotspot residues (Gln281, Lys511, His353, Glu384, Tyr520, Tyr523) were selected based on the criteria.

[0018] Preferably, the method for determining ACE inhibitory activity in step (4) above is as follows: using FAPPGG (N-[3-(2-furanacryl)]-Phe-Gly-Gly) as the chromogen substrate, in 80 mM HEPES buffer (pH 8.3) at 37°C, in a 100 μL microplate system (final substrate concentration 3.0 mM, ACE 0.1 U / mL, peptide concentration 0.2-1.0 mg / mL, the inhibition rate is calculated by monitoring the absorbance change at 340 nm; ACE inhibition kinetic analysis is performed at substrate concentrations of 2.5, 3.0, 3.5, 4.0, 4.5 mM and inhibitor concentrations of 0, 0.4, 0.8 mg / mL, the inhibition type is determined by fitting the Michaelis-Menten equation by nonlinear regression and plotting a Lineweaver-Burk double reciprocal plot.

[0019] Thirdly, this invention also applies for protection of a method for modifying the above-mentioned cottonseed protein source ACE inhibitory peptide, the steps of which are:

[0020] 1) Dipeptide module contribution assessment: Dipeptide modules in the ACE repressor peptide sequence were molecularly docked with ACE, and high contribution modules were determined based on binding free energy;

[0021] 2) Screening of alternative modules: 19 XF dipeptides were molecularly docked with ACE, and high-contribution alternative modules were screened based on their binding free energy being lower than that of high-contribution modules to obtain candidate modified peptides;

[0022] 3) Validation of modified peptide activity: The ACE inhibitory activity of candidate modified peptides was chemically synthesized and validated in vitro to screen out the optimal modified peptide.

[0023] The detailed steps are as follows:

[0024] ① Evaluate the contributions of the dipeptide modules DI and IF in the DIF sequence: Perform molecular docking of DI and IF with ACE, respectively, and calculate the contribution based on the binding free energy (DI approximately 7.81 kcal·mol⁻¹). -1 IF is approximately 8.28 kcal·mol -1 The IF module is identified as the high-contribution module, and the DI module is identified as the low-contribution module.

[0025] ② Screening for alternative modules: Molecular docking was performed on 19 XF dipeptides (AF, CF, DF, EF, FF, GF, HF, IF, KF, LF, MF, NF, PF, QF, RF, SF, TF, VF, WF) with ACE. High-contribution alternative modules were screened based on binding free energy lower than DI. FIF, WIF, and GIF were predicted as the best candidates.

[0026] ③ Verification of the modified peptide synthesis and in vitro verification of its activity: The ACE inhibitory activity of FIF, WIF, and GIF was determined using the FAPEG colorimetric method, confirming that FIF (IC50) 50 = 0.09 ± 0.01 mg / mL)(Phe-Ile-Phe) is the optimal modified peptide, with an activity approximately 3.7 times that of the parent peptide DIF;

[0027] ④ The optimal modified peptide FIF was subjected to inhibition kinetic analysis, molecular docking and molecular dynamics simulation to comprehensively characterize its ACE inhibition mechanism.

[0028] Preferably, the molecular dynamics simulation parameters in step ④ above are as follows: using GROMACS 2022.1 software, CHARMM36 force field, TIP3P water model, dodecahedral periodic box (minimum distance between solute and box wall is 1.0 nm), adding an appropriate amount of counterion to neutralize the system charge; minimizing the energy through the steepest descent (maximum force < 10 kJ·mol⁻¹). -1 ·nm -1 Afterwards, the system was balanced for 100 ps under both the NVT and NPT ensembles (300 K, 1 bar, V-rescale thermostat, Parrinello-Rahman pressure regulator); the production simulation was run for 100 ns (time step 2 fs, one frame of compressed coordinates saved every 1 ps); the analysis included RMSD, RMSF, Rg, SASA and the number / occupancy of hydrogen bonds.

[0029] Fourthly, this invention also seeks to protect the modular modified peptides obtained by the above-mentioned modification method, wherein the modular modified peptides are peptide FIF, peptide WIF, or peptide GIF, wherein the half-maximal inhibitory concentration (WMC) of peptide FIF is 0.08-0.10 mg / mL, the WMC of peptide WIF is 0.17-0.19 mg / mL, and the WMC of peptide GIF is 0.33-0.39 mg / mL, all of which exhibit competitive inhibition of ACE.

[0030] Fifthly, this invention also seeks to protect the use of the above-mentioned cottonseed protein source ACE inhibitory peptide or the above-mentioned modular modified polypeptide in the preparation of blood pressure-lowering functional foods, health foods or drugs.

[0031] Sixthly, this invention also seeks to protect the use of the above-mentioned cottonseed protein-derived ACE-inhibiting peptide or the above-mentioned modularly modified polypeptide in the preparation of ACE inhibitors.

[0032] The present invention has the following beneficial effects:

[0033] 1. This invention is the first to systematically study ACE inhibitory peptides derived from cottonseed protein based on computer-aided virtual screening, disclosing four cottonseed protein-derived peptides (SYF, DIF, IAF, RFY) with in vitro ACE inhibitory activity, filling a gap in this field; and establishing an efficient multi-level virtual screening process, accurately screening 3300 candidate peptides down to 194 high-confidence candidates, with screening efficiency far exceeding that of traditional random enzymatic digestion-activity screening methods, reducing workload by more than 90%; through a combination of molecular docking, molecular dynamics simulation, and inhibition kinetic analysis, the binding mechanism of cottonseed-derived ACE inhibitory peptides was systematically revealed, and key interacting residues (Gln281, His353, Ala354, Glu384, Lys511, Tyr520, Tyr523) were identified, laying the foundation for subsequent rational design.

[0034] 2. This invention is the first to apply a dipeptide module replacement strategy to the modification of ACE inhibitory peptides derived from plant proteins. By replacing the low-contribution module DI in DIF with the high-contribution module FI, the modified peptide FIF is obtained. 50 The concentration was reduced from 0.33 mg / mL to 0.09 mg / mL (activity increased by about 3.7 times), and the inhibition type was successfully transformed from mixed to competitive, demonstrating that the module substitution strategy can reshape the inhibition mechanism while improving efficacy. The overall workflow of this invention is scalable and applicable to the discovery and optimization of functional peptides from other plant protein sources, providing a general technical route for the high-value utilization of agricultural by-products. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 A diagram illustrating the construction process of a cottonseed protein bioactive peptide library.

[0037] Figure 2 This is a diagram showing the docking results of potential ACE inhibitory peptides with ACE receptors.

[0038] Figure 3 The ACE inhibitory capacity of ACE inhibitory peptides, which are potential sources of cottonseed protein.

[0039] Figure 4 ACE inhibitory activity and IC50 of a novel cottonseed protein-derived ACE inhibitory peptide 50 value.

[0040] Figure 5This is a kinetic diagram of the inhibition of a novel cottonseed protein source ACE inhibitory peptide.

[0041] Figure 6 Molecular docking heatmaps for dipeptide modules and modularly modified peptides.

[0042] Figure 7 To improve the ACE inhibition capacity and IC50 of modularly modified peptides 50 value.

[0043] Figure 8 This is a diagram showing the inhibitory kinetics of modularly modified peptides.

[0044] Figure 9 This is a diagram showing the docking results of the ACE inhibitory peptide FIF with the ACE receptor. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0047] The 15 virtual enzymatic proteases used in this invention—chymotrypsin (A), trypsin, proteinase K, pancreatic elastase, thermolysin, chymotrypsin C, closetripain, papain, ficin, leukocyte elastase, thrombin, stem bromelain, subtilisin, V-8 protease (Glutamylendopeptidase), and pepsin—were all derived from the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / ).

[0048] Example 1: Construction of a bioactive peptide library by virtual enzymatic hydrolysis of cottonseed protein using computer technology

[0049] The major storage protein sequences of cottonseed were downloaded from the UniProt database, namely Vicilin GC72-A (P09799, 605 aa), Vicilin C72 (P09801, 588 aa), Legumin A (P09802, 509 aa), and Legumin B (P09800, 516 aa) (see Table 1). Virtual enzymatic digestion was performed using the BIOPEP-UWM platform.

[0050] Table 1 Raw Material Protein Information

[0051]

[0052] Table 2 Number of hydrolyzed peptides with different enzyme digestion combinations

[0053]

[0054]

[0055] During protease screening, commercially available proteases with abundant cleavage sites and good enzymatic hydrolysis coverage were selected, resulting in 15 single-enzyme schemes and various dual / triple-enzyme combinations, forming a total of 52 enzymatic hydrolysis strategies (Table 2). After deduplication, 3300 non-redundant peptide sequences were obtained. These 3300 non-redundant peptides were then subjected to PeptideRanker bioactivity prediction, ToxinPred toxicity prediction, AdmetSAR / ADMETlab property evaluation, and Innovagen water solubility prediction, ultimately yielding 194 candidate active peptides. The construction process of the cottonseed protein bioactive peptide library is shown in the figure below. Figure 1 .

[0056] Example 2: Molecular docking screening of candidate ACE inhibitory peptides

[0057] The ACE crystal structure (PDB ID: 1O8A) was used as the acceptor, and preprocessing was performed in AutoDock Tools: non-essential heteroatoms were removed, polar hydrogens were added, and partial charges were assigned, ultimately exporting the acceptor in PDBQT format. The peptide 3D conformation was generated using OpenBabel 3.1.0 and converted to a PDBQT ligand after geometry optimization. Molecular docking was performed using AutoDock software, with the docking box center coordinates at x = 41.18, y = 34.44, z = 44.38, dimensions of 80 × 80 × 80 Å, and a spacing of 0.375 Å to cover the ACE catalytic pocket. The docking conformations were ranked based on predicted binding affinity, and conformations with reasonable binding geometry were screened. Finally, candidate molecules were determined based on docking scores and conformational consistency related to ACE inhibition.

[0058] Table 3. Docking score results and physicochemical properties between peptide inhibitors and ACE.

[0059]

[0060] Thirteen peptides with good ACE inhibitory effects were selected based on the scoring results (see Table 3). Among them, DIF had the lowest docking score, was stable in binding to ACE, and had high potential ACE inhibitory activity. It mainly forms hydrogen bond interactions with Glu384, His383, His387, Tyr523, Tyr520, His513, and Lys511 in ACE, and forms π-π stacking interactions with His353 (see Table 3). Figure 2 ).

[0061] Example 3: Detection of the ACE-inhibiting activity of ACE-inhibiting peptides

[0062] Thirteen candidate peptides obtained in Example 2 were synthesized using solid-phase chemical synthesis, with a purity of not less than 95%. In vitro ACE inhibitory activity was determined using the FAPGG colorimetric method. The total reaction volume was 100 μL, including 50 μL of 3.0 mM FAPGG solution, 10 μL of 0.1 U / mL ACE solution, and 40 μL of peptide sample; the buffer was 80 mM HEPES (pH 8.3). The reaction was initiated after pre-incubation at 37°C for 10 min, and continuous monitoring at 340 nm for 30 min was performed. At an initial screening concentration of 0.5 mg / mL, SYF, DIF, IAF, and RFY exhibited high ACE inhibitory activity, with inhibition rates of approximately 59%, 57%, 55%, and 50%, respectively. Figure 3 Further determination of IC 50 The values ​​were: DIF 0.33 ± 0.05 mg / mL, SYF 0.53 ± 0.04 mg / mL, IAF 0.53 ± 0.12 mg / mL, and RFY 0.61 ± 0.02 mg / mL. Figure 4 Therefore, DIF is the preferred target peptide.

[0063] Example 4: Inhibition kinetics of ACE inhibitory peptides

[0064] Inhibition kinetics of four bioactive peptides (SYF, DIF, IAF, and RFY) were analyzed. Initial velocities were determined at substrate concentrations of FAPGG (2.5, 3.0, 3.5, 4.0, 4.5 mM) and inhibitor concentrations of 0, 0.4, and 0.8 mg / mL. Apparent velocities were obtained by fitting the Michaelis-Menten equation using nonlinear regression. K m and V maxThe values ​​were calculated, and a Lineweaver-Burk double reciprocal plot was plotted for visualization. The results showed that the lines in the Lineweaver-Burk plots of the four peptides all intersected in the second quadrant (rather than the x-axis or y-axis), indicating that the concentration of the inhibitor increased with increasing inhibitor concentration. K m and V max All showed changes, a characteristic manifestation of mixed inhibition. This indicates that these peptides not only compete with the substrate at the catalytic site but also affect catalytic conversion through assisted binding to the ACE functional region. The results of molecular docking, showing that the peptide occupies the catalytic cavity and interacts with key residues (Gln281, His353, Ala354, Glu384, Lys511, His513, Tyr520, Tyr523), are consistent with these findings. Figure 5 ).

[0065] Example 5: Modification of ACE inhibitory peptides using a dipeptide module substitution strategy

[0066] To further enhance the ACE-inhibiting activity of DIF, the DIF sequence was decomposed into two constituent dipeptide modules, DI and IF, and their molecular docking with ACE (PDB ID: 1O8A) was evaluated. The results showed that the absolute binding free energies of DI and IF were approximately 7.81 kcal·mol⁻¹. -1 and 8.28 kcal·mol -1 The contribution of IF to ACE binding is stronger than that of DI. The overall binding free energy of DIF is approximately 9.36 kcal·mol⁻¹. -1 The individual values ​​of more than two modules are consistent with the synergistic contribution of the intact peptide. Therefore, DI was identified as a low-contribution module (alternative target), and IF was identified as a high-contribution module (retained). Using IF as the high-contribution module, molecular docking was performed on 19 XF dipeptides (AF, CF, DF, EF, FF, GF, HF, IF, KF, LF, MF, NF, PF, QF, RF, SF, TF, VF, WF, i.e., 20 standard amino acids excluding Phe combined with Phe) with ACE, with a binding free energy lower than DI as the candidate criterion. Figure 6 The FIF (corresponding to the FI substitution module), WIF (WI substitution module), and GIF (GI substitution module) predicted the lowest binding free energy and were identified as the optimal substitution candidates.

[0067] Example 6: Determination of the inhibitory activity of the modified ACE inhibitory peptide

[0068] Shanghai Sangon Biotech was commissioned to synthesize three modified peptides, FIF, WIF, and GIF (purity ≥95%). The ACE inhibitory activity was determined using the FAPGG colorimetric method at a series of concentrations (0.05-1.0 mg / mL). Figure 7The results showed that FIF exhibited the strongest ACE inhibitory activity, with the inhibition rate increasing from approximately 28% at 0.05 mg / mL to nearly 100% at 0.2 mg / mL, and the IC50 value was [missing information]. 50 The value was 0.09±0.01 mg / mL, which is approximately 3.7 times the activity enhancement of the parent peptide DIF (0.33±0.05 mg / mL), and significantly superior to WIF (IC). 50 = 0.18±0.01mg / mL) and GIF (IC 50 = 0.36±0.03 mg / mL p < 0.05). WIF achieved approximately 70% inhibition at 0.4 mg / mL, while GIF achieved approximately 60% inhibition at 1.0 mg / mL. Figure 7 ).

[0069] Example 7: Inhibition Kinetics of Modified Peptide FIF

[0070] Lineweaver-Burk analysis was performed on FIF at concentrations of 0, 0.05, and 0.1 mg / mL. Figure 8 The three straight lines converge at the same y-intercept, indicating that V increases with increasing inhibitor concentration. max K remains unchanged m The increasing inhibition pattern is consistent with competitive inhibition, meaning that FIF directly competes with the substrate for the ACE catalytic site. Compared to the mixed inhibition of the parent peptide DIF, the shift in inhibition type suggests that the peptide-ACE interaction interface changes after alteration of the N-terminal residues, with a greater tendency to directly occupy the substrate binding pocket. Figure 8 Molecular docking of FIF and ACE ( Figure 9 The results show that FIF is deeply contained within the ACE catalytic pocket, forming a rich interaction network with multiple key residues, including His353, Ala354, Ser355, Glu384, His387, Glu411, Tyr520, Tyr523, Lys511, and catalytic Zn. 2+ Metal-acceptor interactions are formed. Interaction types include multiple conventional hydrogen bonds, hydrophobic contacts (Val380, Phe457, Phe527), and π-cation and π-σ interactions, exhibiting a broader interaction spectrum compared to the parent peptide DIF. FIF possesses unique additional contacts with the S1 subpocket (Ala354, Phe457) and the zinc-binding motif (Glu411), as well as direct Zn... 2+ Coordination may be the structural basis for its enhanced inhibitory activity. Figure 9 ).

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cottonseed protein-derived ACE-inhibiting peptide, characterized in that: The cottonseed protein-derived ACE-inhibiting peptides are selected from any one of peptides DIF, SYF, IAF, and RFY, wherein the half-maximal inhibitory concentration (WMC) of peptide DIF is 0.28-0.38 mg / mL, that of peptide SYF is 0.49-0.57 mg / mL, that of peptide IAF is 0.41-0.65 mg / mL, and that of peptide RFY is 0.59-0.63 mg / mL, all of which exhibit mixed-type inhibition of ACE.

2. The method for screening cottonseed protein-derived ACE-inhibiting peptides according to claim 1, characterized in that, The steps are as follows: (1) The amino acid sequence of cottonseed storage protein was virtually digested using the BIOPEP-UWM tool to obtain non-redundant cottonseed polypeptides; (2) Non-redundant cottonseed peptides were scored for bioactivity in the PeptideRanker program, and sequences with scores ≥0.5 were retained. Then, toxicity was predicted by ToxinPred, ADMET properties were evaluated by ADMETSAR / ADMETlab 3.0, and water solubility was predicted by Innovagen. High-confidence candidate bioactive peptides were obtained by deduplication step by step. (3) Using the ACE crystal structure as the acceptor, molecular docking was performed using AutoDock software to screen for candidate ACE inhibitory peptides; (4) Candidate ACE inhibitory peptides with a purity of ≥95% were synthesized by solid-phase chemical synthesis. The ACE inhibitory activity was verified in vitro by colorimetric method based on FAPPGG substrate, the half-inhibition concentration was determined, and the inhibition kinetics were determined by Lineweaver-Burk plotting. Finally, the target peptide with ACE inhibitory activity was obtained.

3. The screening method according to claim 2, characterized in that: In step (1), the cottonseed storage proteins include Vicilin GC72-A, Vicilin C72, Legumin A, and Legumin B; wherein Vicilin GC72-A has accession number P09799 in the UniProt database, Vicilin C72 has accession number P09801 in the UniProt database, Legumin A has accession number P09802 in the UniProt database, and Legumin B has accession number P09800 in the UniProt database.

4. The screening method according to claim 3, characterized in that: In step (1), the protease selected for virtual enzymatic hydrolysis is one or a combination of several of the following: chymotrypsin A, trypsin, proteinase K, pancreatic elastase, thermolysin, chymotrypsin C, clostripain, papain, ficin, leukocyte elastase, thrombin, stembromelain, subtilisin, V-8 protease, and pepsin.

5. The screening method according to claim 4, characterized in that: The criterion for molecular docking screening in step (3) is a docking binding free energy ≥ 7 kcal·mol⁻¹. -1 It has ≥4 hydrogen bonds and can specifically interact with key active site residues, which include Gln281, Lys511, His353, Glu384, Tyr520 and Tyr523.

6. The method for modifying the cottonseed protein-derived ACE-inhibiting peptide according to claim 1, characterized in that, The steps are as follows: 1) Dipeptide module contribution assessment: Dipeptide modules in the ACE repressor peptide sequence were molecularly docked with ACE, and high contribution modules were determined based on binding free energy; 2) Screening of alternative modules: 19 XF dipeptides were molecularly docked with ACE, and high-contribution alternative modules were screened based on their binding free energy being lower than that of high-contribution modules to obtain candidate modified peptides; 3) Validation of modified peptide activity: The ACE inhibitory activity of candidate modified peptides was chemically synthesized and validated in vitro to screen out the optimal modified peptide.

7. The modification method according to claim 6, characterized in that: In step 2), the 19 XF dipeptides are AF, CF, DF, EF, FF, GF, HF, IF, KF, LF, MF, NF, PF, QF, RF, SF, TF, VF, and WF.

8. The modular modified polypeptide obtained by the modification method according to claim 7, characterized in that: The modularly modified peptides are peptide FIF, peptide WIF, or peptide GIF, wherein the half-maximal inhibitory concentration (WMC) of peptide FIF is 0.08-0.10 mg / mL, the WMC of peptide WIF is 0.17-0.19 mg / mL, and the WMC of peptide GIF is 0.33-0.39 mg / mL, all of which exhibit competitive inhibition of ACE.

9. The use of the cottonseed protein-derived ACE-inhibiting peptide of claim 1 or the modularly modified polypeptide of claim 8 in the preparation of blood pressure-lowering functional foods, health foods or drugs.

10. The use of the cottonseed protein-derived ACE-inhibiting peptide of claim 1 or the modularly modified polypeptide of claim 8 in the preparation of ACE inhibitors.

Images