Polypeptides having angiotensin converting enzyme inhibitory activity and uses thereof

Abstract

The present application relates to the technical field of polypeptide, and discloses a polypeptide with angiotensin converting enzyme inhibitory activity and application thereof.The polypeptide with angiotensin converting enzyme inhibitory activity is derived from almond protein, and the amino acid sequence of the polypeptide from N-terminal to C-terminal is Lys-Phe-Pro-Leu-Trp, as shown in SEQ ID NO.1, the molecular weight is 689.84Da, the IC 50 is 5.882ug / mL.The polypeptide screened by the polypeptide with angiotensin converting enzyme inhibitory activity and application thereof has angiotensin converting enzyme inhibitory activity, can be widely used for preparing blood pressure lowering products, and meanwhile, the almond polypeptide prepared by enzymolysis from almond protein provides a theoretical basis for efficient utilization of almond protein, and has important significance for sustainable development of almond resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, and particularly relates to polypeptides with angiotensin converting enzyme inhibitory activity and application thereof. BACKGROUND

[0002] Hypertension is a major risk factor for cardiovascular disease, known as the "silent killer", which can easily cause myocardial infarction and stroke, and even endanger life. It has been reported that by 2025, 1.6 billion people will suffer from hypertension. Angiotensin converting enzyme (ACE) is involved in blood pressure regulation through the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). In the RAS, renin produced in the kidney converts angiotensinogen to angiotensin I (ANG I), which can be further converted to biologically active ANG II by ACE. ANG II can release aldosterone, which in turn increases Na + concentration, vasoconstriction, and ultimately elevated blood pressure. In the KKS, bradykinin is a vasodilatory peptide that can bind to β receptors in various tissues and organs such as bronchial smooth muscle and vascular smooth muscle, increasing intracellular Ca 2+ concentration and stimulating endothelial nitric oxide synthase to convert L-arginine to NO. NO can maintain normal vasodilation, thereby maintaining blood pressure. ACE can degrade and inactivate bradykinin, leading to insufficient NO levels, resulting in insufficient vasodilation and elevated blood pressure. ACE inhibitors can reduce the conversion of ANG I and inactivate bradykinin, thereby lowering blood pressure. Common antihypertensive drugs include captopril and lisinopril, but it is still important to find natural ACE inhibitors with fewer side effects for blood pressure reduction.

[0003] Prunus armeniaca L., a deciduous tree in the Rosaceae family, is known as "golden fruit" due to its nutritional value and medicinal properties. It can be propagated from small-scale sowing to large-scale sowing and is one of the important commercial fruits. Almond is the seed of apricot fruit, a fruit with medicinal and edible properties, and has high nutritional value and health functions, rich in fat, protein, and carbohydrates, polyphenols, and various trace elements. The protein content in almond is 23% to 27%, and the essential amino acids are complete. Almond has various physiological activities such as antioxidant, blood pressure lowering, and blood sugar lowering, and almond protein hydrolysate and almond protein peptides have been proven to have blood pressure lowering effects. In addition to direct consumption, almond can also be pressed into almond oil or prepared into plant beverages. Almond cake after pressing almond oil is usually used as fertilizer or feed, sold at a low price, or even discarded directly, resulting in waste of resources. If the discarded almond cake can be effectively utilized and further processed to create higher value-added products, it will achieve the benign development of the almond industry and is of great significance for the sustainable development of almond resources. SUMMARY

[0004] The application aims to provide a polypeptide with angiotensin converting enzyme inhibitory activity and application thereof, the screened polypeptide has angiotensin converting enzyme inhibitory activity, can be widely used for preparing a blood pressure reducing product, and is prepared from almond protein by enzymolysis, thereby providing a theoretical basis for efficient utilization of almond protein and having important significance for sustainable development of almond resources.

[0005] To achieve the above-mentioned object, in a first aspect, the application provides a polypeptide with angiotensin converting enzyme inhibitory activity, which is derived from almond protein, and the amino acid sequence of the polypeptide from N-terminal to C-terminal is Lys-Phe-Pro-Leu-Trp, as shown in SEQ ID NO. 1, and the molecular weight is 689.84 Da.

[0006] Further, the polypeptide has angiotensin converting enzyme inhibitory activity, and the inhibition type of the polypeptide on angiotensin converting enzyme is mixed type inhibition.

[0007] Further, the IC 50 of the polypeptide is 5.882 μg / mL.

[0008] In a second aspect, the application provides application of the above-mentioned polypeptide in preparation of an angiotensin converting enzyme inhibitor.

[0009] In a third aspect, the application provides application of the above-mentioned polypeptide in preparation of a blood pressure reducing product.

[0010] Further, the product includes health products or pharmaceutical products.

[0011] In a fourth aspect, the application provides an angiotensin converting enzyme inhibitor, and the effective component is the polypeptide as shown in SEQ ID NO. 1.

[0012] In a fifth aspect, the application provides a blood pressure reducing product, and the effective component is the polypeptide as shown in SEQ ID NO. 1.

[0013] The polypeptide with angiotensin converting enzyme inhibitory activity and application thereof have the following advantages and positive effects:

[0014] 1. The application uses almond protein as raw material, adopts neutral protease and alkaline protease for step-by-step enzymolysis of the almond protein to obtain almond polypeptide. The polypeptide sequence is determined by HPLC-MS / MS, and a fragment with angiotensin converting enzyme inhibitory activity is screened out through bioinformatics, molecular docking technology and in-vitro activity experiment, and one almond polypeptide KFPLW with the best angiotensin converting enzyme inhibitory activity is screened out. According to the in-vitro activity experiment result (IC 50 = 5.882 μg / mL), the potential of KFPLW in inhibiting ACE activity is verified, and it is indicated that KFPLW can be widely used for preparing a blood pressure reducing product.

[0015] 2、The application lays a theoretical foundation for the deep processing of almond protein by exploring the interaction site and interaction force of KFPLW and ACE through molecular docking technology. The molecular docking results show that the interaction between KFPLW and ACE is mainly mediated by hydrogen bond and hydrophobic interaction, and the binding sites are mainly Glu143, Ser516, Asn70, Asn66, Ala356, Leu139, Leu81, Leu140, Val518 and Phe391. KFPLW changes the conformation of ACE by combining with these amino acid residues, hinders the substrate from entering, and thus inhibits the activity of ACE.

[0016] 3、The application prepares almond polypeptide from almond protein powder by enzymatic hydrolysis, which provides a theoretical basis for efficient utilization of almond protein and has important significance for sustainable development of almond resources.

[0017] The technical solutions of the application will be further described in detail below with the help of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a virtual screening flowchart of almond peptide;

[0019] Figure 2 is a docking result diagram of KFPLW and ACE, wherein A is an overall diagram, and B is an enlarged image in the black box in A;

[0020] Figure 3 is a three-dimensional and two-dimensional view of the interaction between KFPLW and ACE, wherein A is a three-dimensional view, and B is a two-dimensional view;

[0021] Figure 4 is the in vitro ACE inhibitory activity of synthetic peptide;

[0022] Figure 5 is a diagram of the relationship between angiotensin converting enzyme concentration and enzymatic reaction rate;

[0023] Figure 6 is a Lineweaver-Burk diagram of KFPLW inhibiting ACE. DETAILED DESCRIPTION

[0024] The technical solutions of the application will be further described in detail below with the help of the accompanying drawings and examples.

[0025] Unless otherwise defined, the technical terms or scientific terms used in the application shall have the usual meanings understood by those skilled in the art to which the application belongs.

[0026] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0027] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0028] Example

[0029] (1) Enzymatic hydrolysis of almond protein:

[0030] Dissolve 4g of almond protein in 100mL of distilled water and stir in an 85℃ water bath for 20min. Then cool to room temperature and adjust the pH and temperature of the protein solution to 6.9 and 45℃, respectively. Then add 2000U / g of neutral protease for enzymatic hydrolysis. After 2.5h of hydrolysis, place the protein solution in a 100℃ water bath for 10min to inactivate the enzyme. After the hydrolysis, cool to room temperature, adjust the pH of the protein solution to 9.0, and heat to 55℃. Then add 2000U / g of alkaline protease for 2.5h of enzymatic hydrolysis. During the hydrolysis, use 0.5M NaOH aqueous solution to adjust the protein solution to the initial pH of the two-stage hydrolysis. After the hydrolysis, boil in a water bath for 10min to inactivate the enzyme. After cooling to room temperature, add 1M HCl aqueous solution to adjust the pH to 7.0. Centrifuge at 10000r / min for 10min, collect the supernatant, freeze-dry and store at -20℃.

[0031] (2) Ultrafiltration:

[0032] The almond protein hydrolysate was dissolved in 0.01M PBS buffer and ultrafiltered using ultrafiltration membranes with molecular cutoffs of 10kDa and 1kDa to obtain three fractions with molecular cutoffs of MW>10kDa, 1–10kDa, and <1kDa, respectively. All fractions were lyophilized and stored at -20°C.

[0033] (3) Screening of bioactive peptides (screening process as follows) Figure 1 As shown):

[0034] The <1 kDa component obtained by ultrafiltration of the almond protein enzymatic hydrolysate was first desalted by C18 desalting column, and then analyzed by HPLC-MS / MS equipped with online nanospray ion source. A total of 19485 polypeptide sequences were identified in the <1 kDa component of the almond protein enzymatic hydrolysate, and duplicate peptides were removed. Peptides with an average local confidence (ALC) of ≥80, Area ≠ 0, ppm = -5-5, Mass <1000 were screened, and their biological activity was predicted according to the Peptide Ranker online website, with a biological activity score >0.5. A total of 1609 peptide sequences were obtained. Then, the antihypertensive activity of the peptides was predicted by AHTpin, and 12 peptides with ALC >95, Peptide Ranker activity score >0.95, and AHTpin SVM ≥1.2 were obtained. The toxicity and allergenicity of the 12 peptides were predicted, and 6 peptides with no potential toxicity and allergenicity were subjected to molecular docking with ACE to simulate the interaction of the polypeptides with angiotensin-converting enzyme. The results are shown in Table 1. The lower the docking score, the stronger the interaction of the polypeptide with angiotensin-converting enzyme. Five polypeptide sequences with the best docking effect were selected.

[0035] Table 1 Polypeptide sequences with potential ACE inhibitory activity and activity prediction

[0036]

[0037] (4) Active peptide synthesis:

[0038] The five active peptides KFPLW, FPWLQ, FRWPQ, YFPF, and FGPY with no potential allergenicity and toxicity and the smallest docking binding energy were selected for synthesis using the Fmoc solid-phase method (purity ≥95%). The purity and molecular weight of the synthesized peptides were identified by HPLC-MS / MS. The samples were stored at -20°C for further analysis.

[0039] (5) Molecular docking of polypeptides with ACE:

[0040] The crystal structure of angiotensin-converting enzyme (PDB:1O8A) was downloaded from the RCSB protein database (https: / / www.rcsb.org / ), and the ligand and water molecules in the protein structure were removed using PyMOL software. AutoDock Vina added polar hydrogen, and the molecular structure file was saved in pdbqt format. Then the structure of the ligand polypeptide was drawn using ChemDraw 20.0 software, and geometric optimization (energy minimization) was performed using Chem 3D 20.0 software, and saved as a PDB format file. The docking box was set at coordinates (x:43.439, y:34.009, z:47.652), and the active center was covered by an 80x70x74 grid box with a grid point distance of The search parameters for molecular docking were set to 20. The docking model with the lowest binding energy value in each docking attempt was selected, representing the most favorable combination mode. Finally, the protein-peptide interaction was analyzed using Discovery Studio 2017.

[0041] Molecular docking results as follows Figure 2 As shown, Figure 2 This indicates that KFPLW forms hydrogen bonds with amino acid residues Glu143, Ser516, Asn70, Asn66, and Ala356 on the ACE side chain, and forms hydrophobic interactions with Leu139, Leu81, Leu140, Val518, and Phe391. This demonstrates that hydrogen bonding and hydrophobic interactions play a crucial role in the inhibition of ACE activity by KFPLW. KFPLW can inhibit ACE activity by altering the conformation of the ACE active site. Figure 3 Three-dimensional and two-dimensional views of the interaction between KFPLW and ACE.

[0042] (6) In vitro determination of the inhibitory effect of peptides on ACE activity:

[0043] The peptide sample, hippuryl-histyl-leucine hydrate (HHL), and angiotensin-converting enzyme were dissolved in 0.01M pH 8.3 (containing 0.3M NaCl) borate buffer solution. 4.7mM 25μL HHL and 12.5μL peptide sample (0.00313~5mg / mL) were mixed and incubated at 37℃ for 5 min. 25μL ACE was added and the reaction was carried out at 37℃ for 30 min. 375μL 0.3M NaOH solution was used to terminate the reaction and maintain the reaction system alkaline. 25μL 2% phthalaldehyde methanol solution was added, and the reaction was carried out at room temperature in the dark for 10 min. 50μL 6M HCl was then added to terminate the reaction. Finally, 200μL of the liquid was transferred to a completely black 96-well plate, and its fluorescence intensity was measured at an excitation wavelength of 340nm and an emission wavelength of 455nm. The formula for calculating the ACE inhibition rate is shown in equation (1):

[0044]

[0045] Among them, F a The fluorescence intensity of the control group (without peptides) is F. b F represents the fluorescence intensity of the sample group. c The fluorescence intensity is the value of the blank group (containing no peptides and ACE).

[0046] In vitro activity test results are as follows Figure 4 As shown, Figure 4 This indicates that KFPLW's IC 50 Lowest value (IC) 50=5.882 μg / mL).

[0047] (7) Determination of the type of ACE inhibition by amygdalin:

[0048] Reversibility assessment: Under conditions of no inhibitor and with different inhibitor concentrations (3 and 12 μg / mL), and with a fixed substrate concentration, fluorescence intensity was measured at excitation wavelengths of 340 nm and emission wavelengths of 455 nm, respectively, according to step (8). The reversibility was assessed by linearly fitting Origin2022 with enzyme concentration [S] as the x-axis and enzyme reaction rate V as the y-axis.

[0049] Inhibition type study: The kinetics of ACE activity inhibition at different HHL concentrations (0.282–9.4 mM) were studied in the presence of peptide samples (0 μg / mL) and at two different concentrations (3 and 80 μg / mL).

[0050] ACE inhibition patterns were determined by Lineweaver-Burk plots. Mixed inhibition was calculated using the following formula:

[0051]

[0052] In equations (2), (3), and (4), V represents the enzymatic reaction rate with or without KFPLW; V max [S] represents the maximum enzyme reaction rate; [I] represents the HHL concentration; [I] represents the KFPLW mass concentration; K m K is the Michaelis constant; i K is the equilibrium constant for the binding of inhibitors to enzymes; is This is the equilibrium constant for the binding of the inhibitor to the enzyme-substrate complex.

[0053] The results of the suppression reversibility judgment are as follows Figure 5 As shown, Figure 5 This indicates that as the peptide concentration increases, the slope of the linear curve decreases, and the reaction rate decreases. The reaction rate without inhibitors is higher than that with inhibitors, and both rates increase with increasing ACE enzyme concentration. The intersection of the inhibitor-free group and the group with inhibitors at the origin indicates that the inhibition of ACE by peptide KFPLW is reversible, meaning the inhibition of enzyme activity is not permanent, and KFPLW binds to ACE via non-covalent bonds. Figure 6 To create a Lineweaver-Burk double reciprocal plot, use... Figure 6 Determine the type of ACE inhibition caused by KFPLW, K m It increases with increasing peptide concentration, V maxdecreased with the increase of peptide concentration, all the fitting straight lines intersected in the second quadrant, which indicated that it belonged to the mixed type of competitive and non-competitive inhibition among the reversible inhibition. Among them, K i and K is were 28.49 mM and 53.87 mM, respectively. The K m value increased when the peptide concentration increased, which meant that more peptides combined with the enzyme to reduce the formation of enzyme-substrate complex, and K i was less than K is , which indicated that the affinity of KFPLW to free enzyme was stronger than that of ACE-substrate complex. KFPLW could combine to the active site to interfere with the binding ability of substrate to ACE. At the same time, KFPLW had the ability to combine with the non-active site on ACE, which could cause the change of molecular conformation, thus twisting the molecular arrangement of the active site to prevent the substrate from binding, so the substrate binding ability was reduced.

[0054] The present application adopts neutral protease and alkaline protease to obtain enzyme hydrolysate by stepwise enzymatic hydrolysis of almond protein, separates by using an ultrafiltration membrane with a cut-off of 1 kDa, and identifies the composition of polypeptides less than 1 kDa by HPLC-MS / MS. By using bioinformatics and molecular docking, polypeptides with angiotensin converting enzyme inhibitory activity are screened, and the obtained almond polypeptides are verified by in vitro activity experiments to have good angiotensin converting enzyme inhibitory activity, with an IC 50 of 5.882 μg / mL. The inhibition type of the almond polypeptides on angiotensin converting enzyme is mixed type by inhibition kinetics. The present application prepares almond polypeptides by enzymatic hydrolysis from almond protein, which provides a theoretical basis for efficient utilization of almond protein and has important significance for sustainable development of almond resources.

[0055] Therefore, the polypeptides with angiotensin converting enzyme inhibitory activity and the application thereof are used in the present application, the screened polypeptides have angiotensin converting enzyme inhibitory activity, can be widely used for preparing blood pressure lowering products, and the present application prepares almond polypeptides by enzymatic hydrolysis from almond protein, which provides a theoretical basis for efficient utilization of almond protein and has important significance for sustainable development of almond resources.

[0056] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application rather than limit them, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A polypeptide with angiotensin-converting enzyme inhibitory activity, characterized in that: It is derived from almond protein, and the amino acid sequence of the polypeptide from the N-terminus to the C-terminus is Lys-Phe-Pro-Leu-Trp, as shown in SEQ ID NO.1, with a molecular weight of 689.84 Da.

2. The application of the polypeptide as described in claim 1 in the preparation of a blood pressure lowering product, wherein the product is a health product or a pharmaceutical product.

3. An angiotensin-converting enzyme inhibitor, characterized in that: The active ingredient is a polypeptide as shown in SEQ ID NO.

1.

4. A blood pressure lowering product, characterized in that: The active ingredient is a polypeptide as shown in SEQ ID NO.1, and the product is a health supplement or a medicine.

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