Angiotensin converting enzyme inhibitory peptide as well as screening method and application thereof

Abstract

The invention belongs to the field of bioactive peptides. The invention provides an angiotensin converting enzyme inhibitory peptide as well as a screening method and application thereof. The amino acid sequence of the inhibitory peptide is GKGLW and / or GDGLKW. According to the screening method of the ACE inhibitory peptide, the soybean protein is taken as a raw material, and the ACE inhibitory peptide is prepared and screened by adopting a targeted alkaline protease and chymotrypsin double-enzymolysis technology, so that rapid screening of the ACE inhibitory peptide is realized, and an experimental basis is provided for the structure-function relationship of the ACE inhibitory peptide. The inhibitory peptide provided by the invention has strong antihypertensive activity, high safety and no toxic or side effect. Digestive tract stability determination shows that the inhibitory peptide has good gastrointestinal tract stability in gastrointestinal tract digestion, and can still keep good activity after entering blood.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and specifically relates to an angiotensin converting enzyme inhibitory peptide and a screening method and application thereof. Background Art

[0002] Bioactive peptides are organic substances composed of a specific arrangement of several to a dozen amino acids, connected by covalent bonds of amide bonds or peptide bonds. The study of peptides in diseases began in 1922, when insulin extracted from animal pancreas was first used in the medical field, which completely changed the treatment of type 1 diabetes. Subsequently, peptides were widely used in the treatment of various diseases, such as cardiovascular disease, antibacterial, immunomodulation, cancer, etc. Therefore, peptides play an important role in human physiology and pathology as therapeutic lead compounds, and have the potential to replace chemical drugs.

[0003] Hypertension, as one of the major diseases affecting human health, has brought major challenges to the global medical system. Among them, angiotensin converting enzyme (ACE) is a key mediator in the pathogenesis of hypertension. In the renin angiotensin system (RAS), ACE catalyzes the conversion of Ang I to Ang II, which has a strong vasoconstriction effect, resulting in aldosterone secretion and increased blood pressure. This suggests that inhibiting ACE activity is an effective strategy to prevent high blood pressure. At present, a number of ACE inhibitory drugs (such as lisinopril, captopril, enalapril, etc.) have been developed for the treatment of hypertension, but taking chemical drugs is often accompanied by many side effects, such as cough, headache, loss of taste, etc. In contrast, food-borne ACE inhibitory peptides (ACEIPs) are safe, mild, and easy to absorb, and are gradually being promoted as an alternative treatment for hypertensive diseases.

[0004] Soybeans are considered to be a good source for the development of ACE inhibitory peptides due to their rich protein content and their economic and environmentally friendly properties. For example, the soy protein-derived peptide LSW obtained by digestion with thermolysin has an anti-inflammatory effect on vascular smooth muscle cells; the active peptide VHVV obtained by enzymatic hydrolysis of soybean protease with flavor protease has been verified to have an improvement effect on hypertensive renal injury using the SHRs model.

[0005] The two-step enzymatic hydrolysis method has significant advantages in improving enzymatic hydrolysis efficiency, enhancing peptide stability and shortening enzymatic hydrolysis time. For example, a combination of neutral protease and alkaline protease was used to prepare tuna ACE inhibitory peptides. The results showed that the two-step enzymatic hydrolysis was superior to the single enzymatic hydrolysis in terms of hydrolysis degree, amino acid content and ACE inhibition ability.

[0006] Although there have been many studies on soybean ACE inhibitory peptides, the two-step enzymatic hydrolysis method has not been widely used in the preparation of soybean ACEIPs. Summary of the invention

[0007] In order to solve the above problems, the present invention provides an angiotensin converting enzyme inhibitory peptide and a screening method and application thereof. The screening method of the present invention uses soybean protein as a raw material, adopts a targeted alkaline protease and chymotrypsin double enzymatic hydrolysis technology to prepare and screen, realizes the rapid screening of ACE inhibitory peptides, and provides an experimental basis for their structure-activity relationship. The angiotensin converting enzyme inhibitory peptides GKGLW, GDGLKW, IC 50 The values ​​were 34.96μM and 33.98μM, respectively, showing strong hypotensive activity, high safety, and no toxic side effects. The digestive tract stability assay showed that GKGLW and GDGLKW had good gastrointestinal stability during gastrointestinal digestion and could still maintain good activity after entering the blood.

[0008] The object of the present invention is to provide an angiotensin converting enzyme inhibitory peptide, the amino acid sequence of the inhibitory peptide is GKGLW and / or GDGLKW.

[0009] The present invention also aims to provide a composition, which comprises the above inhibitory peptide and excipients acceptable in medicine, food or health care products.

[0010] The present invention also aims to provide a use of the above-mentioned angiotensin converting enzyme inhibitory peptide or composition in the preparation of angiotensin converting enzyme inhibitor drugs and / or auxiliary antihypertensive health products or foods.

[0011] The object of the present invention is to provide a method for screening angiotensin converting enzyme inhibitory peptides, comprising the following steps:

[0012] (1) using alkaline protease and chymotrypsin to hydrolyze soybean protein step by step to obtain a protein peptide solution;

[0013] (2) ultrafiltration treatment is performed on the above protein peptide solution to obtain components with different molecular weights, and peptide segments with a molecular weight of <3 kDa are screened out;

[0014] (3) enriching and purifying the peptide fragment obtained in step (2) by gel chromatography column, collecting the components and determining the ACE inhibitory activity of different components, and screening the component with the strongest ACE inhibitory ability;

[0015] (4) The components obtained in step (3) were screened by LC-MS / MS identification, and peptides with ALC>90% were screened out. PeptideRanker (with a scoring threshold of >0.5) and BIOPEP-UWM (with a scoring threshold of >0.5) were used to further screen out peptides with potential ACE inhibitory effects. Seq logo was used to analyze the amino acid distribution of the peptides, and peptides containing W and F residues at the C-terminus were screened out;

[0016] (5) Among the peptides containing W and F residues at the C-terminus obtained in step (4), peptides with good water solubility, non-toxicity and non-sensitizing properties are selected, and the in vitro ACE inhibitory activity of the peptides is measured to screen out the peptides GKGLW and GDGLKW with the strongest ACE inhibitory ability.

[0017] Furthermore, the peptide sequences obtained by the above screening were virtually verified and evaluated, and the MOE 2019 software was used for molecular docking to evaluate the stability of the binding conformation between the peptide and the ACE molecule.

[0018] Furthermore, molecular dynamics simulation was performed on the peptide sequences obtained by the above screening and the ACE receptor. According to the Rg value, RMSD, RMSF, binding free energy, and Gibbs free energy data, it was verified that the screened peptide sequences GKGLW and GDGLKW formed more stable complexes with ACE.

[0019] Different functional peptides have unique amino acid sequence characteristics due to their specific biological activities. However, there is currently no standard to select proteases that produce specific bioactive hydrolysates. Therefore, if you want to develop an effective and rapid method to produce new antihypertensive peptides, it is crucial to deeply understand the correlation between peptide sequence and ACE inhibitor biological activity.

[0020] The present invention systematically evaluated the ACE inhibitory effects of 168,000 tripeptides and tetrapeptides based on molecular dynamics simulation, and found that the two ends of the ACE inhibitory peptides prefer aromatic amino acids, especially when the C-terminus contains tryptophan, the inhibitory effect is the best. Therefore, if a specific enzyme is used to cut and modify the end of the polypeptide, so that the C-terminal amino acid residue is as W or F as possible, it will help improve the efficiency of preparing ACE inhibitory peptides.

[0021] The present invention has established a new targeted dual enzymatic hydrolysis technology. With soybean protein as raw material, the different enzymatic hydrolysis preferences of 5 kinds of proteases and the differences in the amino acid composition of the enzymatic hydrolysis products are analyzed, the optimal enzymatic hydrolysis combination of chymotrypsin and alkaline protease is determined, and the soybean protein hydrolysis product is prepared by targeting alkaline protease and chymotrypsin, and its ACE inhibition rate is 75.98%; Subsequently, multiple new C-terminal tryptophan-containing ACE inhibitory peptides are screened and identified by computer through ultrafiltration separation, gel chromatography separation and purification, LC-MS / MS identification and computer screening, and the peptide sequence with the best activity is screened and identified; Further, the ACE inhibition mechanism is explored from the perspective of binding structure through molecular simulation. The peptide mainly interacts with the key residues in the ACE active pocket through hydrogen bonds, proving that hydrophobicity and amino acids with cyclic structures are conducive to the ACE inhibitory activity of the peptide. Finally, the gastrointestinal digestion stability of these peptides is evaluated, providing useful information such as enzyme selection and mode of action for the development and application of soybean antihypertensive peptides.

[0022] The present invention has the following beneficial effects:

[0023] (1) The present invention discloses for the first time two angiotensin converting enzyme inhibitory peptides and a screening method thereof. The obtained two angiotensin converting enzyme inhibitory peptides have structures of GKGLW and GDGLKW, respectively, and the corresponding IC 50 The values ​​were 34.96 μM and 33.98 μM, respectively, showing good ACE inhibition ability, indicating that they are strong inhibitors of ACE;

[0024] (2) The present invention uses soybean protein as raw material, adopts alkaline protease and chymotrypsin two-step enzymatic hydrolysis method, and screens the enzymatic hydrolysis products to obtain ACEIPs. The targeted dual-enzyme hydrolysis technology of the present invention realizes the efficient preparation of soybean high-activity ACE inhibitory peptides, and provides an experimental basis for the rapid screening and structure-activity relationship of ACE inhibitory peptides;

[0025] (3) The angiotensin converting enzyme inhibitory peptide of the present invention has strong antihypertensive activity, high safety, no toxic side effects, small molecular weight and is easily absorbed by the human body, and has a wide source, thus having important market value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 amino acid composition of peptides prepared for different proteases;

[0027] Figure 2 The inhibitory effects of different single enzyme hydrolysis and tandem enzymatic hydrolysis with alkaline protease on ACE;

[0028] Figure 3 ACE inhibition capacity and IC of different ultrafiltration fractions 50 value;

[0029] Figure 4 Schematic diagram of partial purification using Sephadex G-25 gel;

[0030] Figure 5 The activity distribution, number of peptides and peak area of ​​G3 component are shown;

[0031] Figure 6 is the amino acid distribution diagram of 4-9 peptide;

[0032] Figure 7 This is the docking result diagram of potential bioactive peptides and ACE receptors;

[0033] Figure 8 It is a two-dimensional diagram of molecular docking of GKGLW binding to ACE molecule;

[0034] Fig. 9 It is a two-dimensional diagram of molecular docking of GDGLKW binding to ACE molecule;

[0035] Fig.10It is a two-dimensional diagram of molecular docking of EWEGF and ACE molecules;

[0036] Fig.11 Molecular dynamics analysis of the interaction of GKGLW, EWEGF and GDGLKW with ACE receptors;

[0037] Fig.12 ACE-GKGLW Gibbs free energy diagram, ACE-GDGLKW Gibbs free energy diagram (2D, 3D);

[0038] Fig.13 The flowchart of the in vitro digestion method for food is used as a reference, and the comparison chart of the digestive tract stability test data of GKGLW and GDGLKW. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1 Preparation of protease hydrolysate

[0041] Defatted soybeans (80 mesh, soybeans were from soybeans produced in Northeast China provided by Qingmei Group, and the variety was Heihe No. 43) were mixed with deionized water (1g / 15ml) and the pH was adjusted to 9.0 with 1.0M NaOH. The resulting mixture was stirred at 45°C for 1h with a magnetic stirrer, and then centrifuged 3 times at 4500×g for 10min using a centrifuge. The resulting supernatant was adjusted to pH 4.5 with 1.0M HCl, and then centrifuged at 4500×g at 25°C for 10min using a 5804R centrifuge (epppendorf, Germany). The resulting precipitate was redispersed in deionized water and adjusted to pH 7.0. The protein solution was freeze-dried and stored at -20°C.

[0042] To prepare soy protein hydrolysate, SPI (soy protein isolate) was suspended in deionized water (5%, w / w), and five different proteases were used to enzymatically hydrolyze the soy protein hydrolysate. By repeated stirring, the enzyme amount was introduced into alkaline protease (pH 9.0, 50℃), flavor protease (pH 7.0, 50℃), papain (pH 7.0, 50℃), trypsin (pH 8.0, 37℃), and chymotrypsin (pH 7.0, 37℃) at a ratio of 2000U / g (E / S) for hydrolysis. The enzyme was inactivated by heating at 90℃ for 10min to terminate the reaction. The resulting hydrolysate was centrifuged at 8000×g for 15min. The supernatant was collected and stored at -20℃ until further use.

[0043] Example 2 Determination of amino acid composition of polypeptides after different enzyme treatments

[0044] The preparation of highly effective ACE inhibitory peptides is closely related to the specific cleavage site of the enzyme and the amino acid composition of the substrate protein. In order to study the cleavage preference of each protease, the amino acid composition and content of the peptide solution after treatment with different enzymes were determined.

[0045] Amino Acid Determination Method

[0046] Take 0.01g sample and dissolve it in 10mL HCl (6M), and hydrolyze it at 110℃ for 23h (filled with nitrogen). After the volume is fixed in a 50mL volumetric flask, dilute it 5 times with 0.02M HCl, and filter it (0.22μm filter membrane). Then put the solution into an amino acid automatic analyzer (L-8900) for analysis. The tryptophan content is determined by alkaline hydrolysis method.

[0047] The amino acid composition of peptides prepared by different proteases was determined as follows Figure 1 As shown, the results showed that the content of hydrophobic amino acids in the alkaline protease hydrolysate was 311.42 mg / g, which was higher than that of other hydrolysates (286.36-301.12 mg / g), especially the Leu content accounted for 20.94% of the total hydrophobic amino acids. According to previous studies, when the content of hydrophobic amino acids in the ACE-I peptide chain reaches 19.8%, the ACE inhibition effect can reach 65.09%. Therefore, alkaline protease may help to improve ACE inhibitory activity by specifically cleaving hydrophobic amino acids. The content of Trp tryptophan in the chymotrypsin hydrolysate is 138 mg / 100 g, which is higher than that of other hydrolysates (27-91 mg / 100 g), indicating that the cleavage site of chymotrypsin may be related to Trp. The steric hindrance brought by the indole group in tryptophan can effectively increase ACE activity and Zn 2+ The binding force.

[0048] Example 3: Step-by-step enzymatic hydrolysis of different proteases and alkaline protease

[0049] The alkaline protease with excellent performance and other proteases were used for step-by-step combined enzymolysis. In the two-step enzymolysis method, alkaline protease (E / S = 1000U / g) was first added, incubated at 50°C for 1h, and then the enzyme was inactivated by heating at 90°C for 10min, and then other proteases (E / S = 1000U / g) were added for further enzymolysis for 1h, and finally heated at 90°C for 10min to terminate the reaction. The obtained hydrolyzate was centrifuged at 8000×g for 15min, and the supernatant was collected and stored at -20°C. The above combined enzymolysis product and the single enzyme enzymolysis product prepared in Example 1 were tested for ACE inhibitory activity and compared.

[0050] Method for determination of ACE inhibitory activity

[0051] ACE and HHL were dissolved in 0.1M sodium borate buffer (pH 8.3) for determination. The synthetic peptide was dissolved in distilled water to 6 concentration levels. The peptide to be tested (20μL) was mixed evenly with 2mM HHL (20μL), preheated at 37°C for 5 minutes, and then 20μL ACE (0.1U) was added to start the reaction, and the reaction was kept at 37°C for 30min. Finally, 50μL 1M HCl was added to terminate the reaction, and the obtained reaction solution was subjected to HPLC analysis. The analysis conditions were: C18 liquid chromatography column (4.6mm×250mm×5μm), column temperature 35°C, mobile phase (acetonitrile / water=1:3 (v / v), containing 0.1% trifluoroacetic acid) flow rate 1mL / min isocratic elution, detection wavelength 228nm. The activity of inhibiting ACE was calculated as follows:

[0052] ACE inhibitory activity(%)=(A control -A inhibitor ) / A control

[0053] Among them, A inhibitor is the relative area of ​​the HA peak obtained by the reaction of ACE and HHL in the presence of an inhibitor, A control IC is the relative area of ​​the HA peak obtained by the reaction of ACE and HHL without inhibitor. 50 The value was defined as the peptide concentration that inhibited ACE activity by half.

[0054] Alkaline protease and other proteases were combined for step-by-step enzymatic hydrolysis, and the results of ACE inhibitory activity were as follows: Figure 2As shown in the figure, after the step-by-step enzymatic hydrolysis of flavor protease, trypsin and papain with alkaline protease, the ACE inhibition rate increased by 6.94%, 10.14% and 11.41% respectively compared with the single enzyme hydrolysis. The combination of alkaline protease and chymotrypsin had the largest increase, and the ACE inhibition rate increased from 48.32% to 67.98%. Therefore, the Alca l as-Chymotrypsin two-step hydrolysis method was used to gradually degrade proteins according to the specific preferences of different enzymes to obtain peptides with more antihypertensive activity.

[0055] Example 4 Ultrafiltration and gel chromatography purification of soybean peptides

[0056] In order to further analyze the ACE inhibitory activity of peptides in different molecular weight ranges, we used ultrafiltration technology to further separate the protein hydrolysate. The enzymatic hydrolysate after two-step hydrolysis by alkaline protease and chymotrypsin was separated into different components by ultrafiltration membrane: <3kDa, 3-10kDa, 10-30kDa and greater than 30kDa. The ACE inhibitory activity of peptides in different molecular weight ranges was measured, and the results are shown in Figure 3 As shown in the figure, components with different molecular weights have different ACE inhibitory activities. When the sample concentration is 50 μg / mL, as the molecular weight of the hydrolyzed component decreases, the ACE inhibitory activity gradually increases. When the molecular weight of the peptide segment is 3-10 kDa and 10-30 kDa, the ACE inhibitory activity is 65.72% (IC 50 =40.98±1.40μg / mL) and 50.89% (IC 50 =65.91±1.51μg / mL), and the inhibition rate of the component <3kDa was the highest, which was 72.57% (IC 50 =32.59±1.22μg / mL). After separation by ultrafiltration membrane, these low molecular weight peptides showed stronger ACE inhibitory activity, confirming that peptides with molecular weight <3kDa are the source of ideal ACE inhibitory peptides.

[0057] Then, the peptides were further enriched and purified by gel chromatography column Sephadex G-25. Gel chromatography is based on the molecular exclusion effect for separation. During the elution process, low molecular weight peptides have a long flow path into the gel pores and elute slowly; high molecular weight peptides flow through the gaps, have a short path and elute faster. Figure 4 As shown in Figure 2, within the retention time of 50-200 min, three components, G1, G2, and G3, were collected. The ACE inhibitory activity of the three components was determined, and G3 showed the best ACE inhibitory ability (69.65±0.62%), with an IC 50 The value was 33.73 ± 0.89 μg / mL. Therefore, LC-MS / MS was performed on the G3 fraction to identify ACE IPs in SPI.

[0058] Example 5 LC-MS / MS Identification and Analysis of Peptides and Distribution Characteristics of G3 Components, Peptide Screening

[0059] In order to further clarify the ACE inhibitory effect of G3 component, its molecular weight and amino acid sequence were identified by UHPLC-Q-Orbitrap-MS / MS and Peaks Studio 7.5, respectively. G3 component was identified by UHPLC-Q-Orbitrap-MS / MS, and a total of 991 peptides with ALC>90% were screened out. The number of peptides and peak area are shown in Figure 5 As shown in the figure, the outer pie chart represents the peptide MS area, and the inner pie chart represents the content of the number of peptides. Figure 5 It can be seen that among these peptides, tetrapeptides to nonapeptides are distributed, among which the MS peak areas of heptapeptides and hexapeptides are relatively high, accounting for 28.4% and 27.6% of the total MS area, respectively. Hexapeptides have the largest number of peptides, with a total of 288, accounting for 29.1% of the total number of peptides. On this basis, PeptideRanker (with a scoring threshold of >0.5) and BIOPEP-UWM (with a scoring threshold of >0.5) were used to screen out 252 peptides with potential ACE inhibitory effects.

[0060] In order to further analyze the characteristics of these peptides, Seq log was used to analyze the amino acid distribution of the peptides. The analysis results of the amino acid distribution of peptides 4-9 are shown in the following figure. Figure 6 As shown, the amino acid characters represent the proportion. The larger the amino acid character, the higher the proportion. The results showed that aromatic amino acids (F) and hydrophobic amino acids (L, P) appeared most frequently at the C-terminus and N-terminus, polar uncharged amino acids (S, G) accounted for a higher proportion at the C-terminus, and polar positively charged amino acids (R, H, K) accounted for a higher proportion at the N-terminus. As the peptide chain extends, the proportion of acidic amino acids (D, E) at the C-terminus and the second position from the C-terminus increases. In addition, aromatic and cyclic amino acids (such as Trp W and Phe F) are the most common C-terminal residues in the structures of highly active ACE inhibitors. For example, the dipeptide VW (IC 50 =0.1mg / mL),VF(IC 50 =0.135 mg / mL). The hexapeptide VVCVPW (IC 50 =4.1 μM).

[0061] Based on this, the screening criteria are peptides containing W and F residues at the C-terminus. The peptides with a peptide ranking score > 0.5 are predicted for allergens, toxicity, and water solubility. The prediction results are shown in the following table:

[0062]

[0063]

[0064] Combined with the water solubility, toxicity and sensitization shown in the table, four peptides with Trp at the C-terminus and one with Phe at the C-terminus were finally selected and synthesized, namely DHPPASW, GDGLKW, GKGLW, EWEGF and LGKW.

[0065] The IC50 values ​​of these peptides were distributed between 33.98 μM and 78.23 μM by measuring the ACE inhibitory activity in vitro. Among them, the IC50 values ​​of GKGLW and GDGLKW were 50 The values ​​were 34.96 and 33.98 μM, respectively, indicating that they are strong inhibitors of ACE. 50 The molecular docking affinity values ​​of ACE and the measured data are shown in the following table:

[0066]

[0067] Example 6 Analysis of the molecular interaction mechanism between polypeptide and ACE

[0068] In order to explore the interaction mechanism between these five bioactive peptides and ACE receptors, molecular docking was performed using MOE 2019 software to evaluate the stability of the binding conformation. Molecular docking method of potential bioactive peptides and ACE receptors: Using the MOE2019 molecular docking tool, the three-dimensional structure of the screened potential bioactive peptides was successfully constructed and energy minimized. The ACE (PDB ID: 1086) crystal structure data comes from the RCSB protein database (https: / / www.rcsb.org). The DoGSiteScorer online tool (https: / / prote i ns.pl us / ) was used to predict the protein receptor binding cavity and site of ACE. The three-dimensional diagram of the interaction between the five peptides and the ACE receptor is shown in Figure 2. Figure 7 As shown in A, B is a three-dimensional diagram of molecular docking of GKGLW and ACE molecule, C is a three-dimensional diagram of molecular docking of GDGLKW and ACE molecule, and D is a three-dimensional diagram of molecular docking of EWEGF and ACE molecule. Figure 8 This is a two-dimensional diagram of the molecular docking of GKGLW and ACE molecules. Fig. 9 This is a two-dimensional diagram of molecular docking of GDGLKW and ACE molecules. Fig.10 This is a two-dimensional diagram of molecular docking of EWEGF and ACE molecules.

[0069] The docking results are shown in the molecular docking affinity data of synthetic peptides and ACE in Table 1. All five peptides can enter the binding pocket of ACE, and the docking binding energy is between -10.91 kcal / mol and -12.31 kcal / mol, with an average binding energy of -11.36 kcal / mol. The stability of the receptor-ligand complex can be judged by the docking energy. The lower the docking energy, the more stable the binding. From the binding energy point of view, GKGLW and GDGLKW have good ACE inhibitory activity, which is basically consistent with the ACE inhibition experiment.

[0070] Peptide-ACE complex 3D ( Figure 7 BD) and 2D ( Figure 8 , 9 , 10) The structural diagram shows that the peptide and ACE residues interact through multiple mechanisms, including van der Waals forces, hydrophobic forces, electrostatic forces, etc., and hydrogen bonds are the main interaction forces for forming stable peptide-ACE complexes. Among them, GKGLW (-12.31 kcal / mol) with the lowest binding energy forms 5 hydrogen bonds with ACE residues A1 a354, Tyr523, His387, Ser284 and Gl n281. GKGLW forms 2 hydrogen bonds with the S1 pocket (A1 a354, Tyr523) and 1 hydrogen bond with the S2 pocket (Gl n281). GDGLKW (-11.04 kcal / mol) forms 4 hydrogen bonds with ACE, involving residues including Gl n403, Arg522, His353 and His387. However, EWEGF (-10.91 kcal / mol) only formed one hydrogen bond with ACE residue His387.

[0071] Because the number of hydrogen bonds can directly reflect or determine the affinity and stability between the ACE receptor and the peptide, it explains that GKGLW and GDGLKW have a strong inhibitory effect on ACE. In addition, GKGLW and GDGLKW also form non-bonded interactions with the metal ion Zn2+ (an important component of the ACE active catalytic site), interfering with the binding of ACE to Zn2+, thereby inhibiting its biological activity. From the docking results, it can be seen that GKGLW and GDGLKW mainly achieve strong ACE inhibitory activity by forming hydrogen bonds with the active pocket of ACE, and when the C-terminus is tryptophan, it is more conducive to binding with ACE to enhance its inhibitory activity. Therefore, the ACE inhibitory activity of these peptides may be due to: on the one hand, they compete with the substrate by completely embedding into the catalytic cavity of ACE, and on the other hand, they hinder the entry of the substrate by occupying the substrate recognition surface.

[0072] Example 7 Molecular dynamics simulation of polypeptide and ACE receptor

[0073] To further verify the binding of ligand and receptor at the atomic level, three parallel 100 ns MD simulations were performed for GKGLW, GDGLKW and EWEGF. Specifically, the protein parameters were generated by AMBER14SB force field, sodium ions were added to the solvent environment of TIP3P tank, and The buffer solution maintains a neutral pH. On the premise of ensuring the normal structure of the system, the fastest descent method and conjugate gradient method are used to minimize the energy of the solvent and solute structure. The whole system was heated to 300K under the constraint of . All chemical bonds involving hydrogen atoms were suitable for SHAKE algorithm. After the system energy and density reached equilibrium, 100ns molecular dynamics simulations were performed on the ACE receptor complex of potential bioactive peptides.

[0074] The simulation results are as follows Fig.11 As shown in the figure, A is the curve of the radius of rotation (Rg), B is the root mean square deviation (RMSD) graph, C is the root mean square fluctuation (RMSF) graph, and D is the binding free energy graph of the peptide and ACE calculated by the MMPBSA method. The Rg value can be used to evaluate the structural compactness of the protein system. The simulation results are shown in Fig.11 As shown in A. The average Rg values ​​of GKGLW, GDGLKW and EWEGF are 23.80, 23.78 and This indicates that GKGLW and GDGLKW bind more tightly to ACE. The root mean square deviation (RMSD) is an important indicator for evaluating the stability of the ACE-peptide system, reflecting the degree of deviation of the protein molecule from the initial structure during the dynamic simulation. Generally speaking, peptides with higher activity are usually accompanied by higher RMSD values. The simulation results are shown in Figure 2. Fig.11 As shown in B. Complex and GDGLKW-ACE complex The average RMSD of This indicates that the conformational changes of GKGLW-ACE and GDGLKW-ACE complexes are more frequent, resulting in higher ACE inhibitory activity. The root mean square fluctuation (RMSF) is an indicator used to describe the average movement amplitude of a single amino acid residue within a specific time range. The simulation results are shown in Fig.11 As shown in C. The results show that the average RMSF value of the GKGLW-ACE complex Lower than EWEGF-ACE complex This indicates that the structure of the GKGLW-ACE complex is relatively stable. The binding energy reflects the strength of the interaction between the ligand and the protein. The binding free energy of the three peptides was calculated using the MMPBSA method. The calculation results are shown in Fig.11As shown in D. Compared with the binding free energy of EWEGF (-43.4832 kJ / mol), the binding energies of GKGLW (-100.3606 kJ / mol) and GDGLKW (-93.6422 kJ / mol) are more conducive to binding with ACE. The MMPBSA results showed that GKGLW and GDGLKW have higher ACE inhibitory activity, which is consistent with the ACE inhibitory activity study.

[0075] Finally, the Gibbs free energy was calculated based on the RMSD and Rg values, and the conformational stability of the complex was characterized. Fig.12 As shown in Figures EF, E is the ACE-GKGLW Gibbs free energy diagram (2D, 3D), and F is the ACE-GDGLKW Gibbs free energy diagram (2D, 3D). It can be seen from the figure that there is only one energy well in the two-dimensional free energy landscape of GKGLW and GDGLKW. This indicates that the system exists in a single stable conformation and is unlikely to undergo significant conformational transitions or polymorphisms under given conditions. These findings provide further supporting evidence for the binding mechanism of peptide-protein interactions, indicating that both peptides can rapidly bind to ACE to form a stable and favorable complex structure.

[0076] Example 8 Stability test of two peptides in gastrointestinal digestion

[0077] Before bioactive peptides can pass through the intestinal epithelium and exert their antihypertensive effects, they must resist digestion in the gastrointestinal tract and maintain their integrity. Although digestive enzymes break down macromolecules into small molecules that are more easily absorbed by the body, these decomposition products may lose their original biological activity. The fate of peptides under oral administration conditions can be simulated by in vitro simulation of gastrointestinal digestion, referring to the INFODEST digestion method (e.g. Fig.13 Simulated salivary fluid (SSF), gastric fluid (SGF), and simulated intestinal fluid (SI F) were prepared as shown in the flow chart of in vitro digestion of food (Figure A). SSF consisted of salivary amylase (75 U / mL) and 1.5 mM CaCl2. SGF consisted of pepsin (2000 U / mL), gastric lipase (60 U / mL), and 0.15 mM CaCl2, pH 3.0. SI F consisted of trypsin (100 U / mL) and 10 mM bile salts in 0.6 mM CaCl2. Digestion was performed at 37°C with constant shaking. In vitro incubation with pepsin at pH 1.2 for 1 h and with trypsin and chymotrypsin at pH 7.8 for 3.5 h. Gastric digests were removed after 0.5 and 1 h, respectively, and intestinal digests were removed after 1.5, 2.5, and 4 h of digestion, respectively. Enzymes were inactivated by heating at 90°C.

[0078] The results of the digestive tract stability test of GKGLW and GDGLKW, such as Fig.13 As shown in B, IC at the end of GKGLW digestion 50 The value changed from 34.96 μM to 35.08 μM. Similarly, the IC 50 The value also increased from 33.98 μM to 34.21 μM. There was no significant difference in the ACE inhibitory effect between the undigested peptide and the treated peptide, so GKGLW and GDGLKW have good gastrointestinal stability during gastrointestinal digestion, and they can still maintain good activity after entering the blood.

[0079] In summary, the present invention provides a new targeted dual enzymatic hydrolysis technology for preparing soybean ACE inhibitory peptides with characteristic structures. The soybean enzymatic hydrolysis products were prepared by targeting alkaline protease and chymotrypsin, which achieved rapid screening of ACE inhibitory peptides and provided experimental basis for structure-activity relationship. The inhibitory peptide GKGLW (IC 50 =34.96 μM) and GDGLKW (IC 50 =33.98 μM) showed good ACE inhibition ability, high safety, no toxic side effects, good gastrointestinal stability during gastrointestinal digestion, and had good market value and application prospects.

[0080] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also belong to the scope of the present invention and should be defined by the claims.

Claims

1. An angiotensin converting enzyme inhibitory peptide, characterized in that: Its amino acid sequence is GKGLW and / or GDGLKW.

2. An angiotensin converting enzyme inhibitory peptide composition, characterized in that: The composition comprises the angiotensin converting enzyme inhibitory peptide according to claim 1 and excipients acceptable in medicine, food or health care products.

3. Use of the angiotensin converting enzyme inhibitor peptide according to claim 1 or the composition according to claim 2 in the preparation of angiotensin converting enzyme inhibitor drugs and / or auxiliary antihypertensive health products or foods.

4. A method for screening angiotensin converting enzyme inhibitory peptides as claimed in claim 1, characterized in that: The following steps are involved: (1) using alkaline protease and chymotrypsin to hydrolyze soybean protein step by step to obtain a protein peptide solution; (2) ultrafiltration of the protein peptide solution to obtain components with different molecular weights, and screening out peptides with a molecular weight of <3 kDa; (3) enriching and purifying the peptide fragment obtained in step (2) by gel chromatography column, collecting the components and determining the ACE inhibitory activity of different components, and screening the component with the strongest ACE inhibitory ability; (4) The components obtained in step (3) were identified by LC-MS / MS, and peptides with ALC>90% were screened out. PeptideRanker and BIOPEP-UWM were used to further screen out peptides with potential ACE inhibitory effects. Seqlogo was used to analyze the amino acid distribution of the peptides, and peptides containing W and F residues at the C-terminus were screened out. (5) Among the peptides containing W and F residues at the C-terminus obtained in step (4), peptides with good water solubility, non-toxicity and non-sensitizing properties are selected, and the ACE inhibitory activity of the peptides is measured to screen out the peptides GKGLW and GDGLKW with the strongest ACE inhibitory ability.

5. The screening method according to claim 4, characterized in that The screened peptide sequences were virtually verified and evaluated, and molecular docking was performed using MOE 2019 software to evaluate the stability of the binding conformation between the peptide and the ACE molecule.

6. The screening method according to claim 4, characterized in that The screened peptide sequences were subjected to molecular dynamics simulation with the ACE receptor, and based on the Rg value, RMSD, RMSF, binding free energy, and Gibbs free energy data, it was verified that GKGLW and GDGLKW formed more stable complexes with ACE.

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