ACE inhibitory peptide as well as screening method and application thereof

Abstract

The invention relates to an ACE inhibitory peptide as well as a screening method and application thereof, and belongs to the technical field of bioactive peptides. The invention provides an ACE inhibitory peptide. The amino acid sequence of the ACE inhibitory peptide is shown as RF, RW or KF. According to the method disclosed by the invention, turbot muscle protein is taken as a research object, virtual enzymolysis is utilized to assist activity prediction, preliminary screening is carried out by combining a bioinformatics method, polypeptide and an ACE three-dimensional structure are docked by adopting a semi-flexible molecular docking method, further screening is carried out, and the ACE inhibitory peptide is obtained. The ACE inhibition rates of the ACE inhibitory peptide obtained through screening are 76.61%, 80.71% and 79.18%, and compared with a method for extracting the ACE inhibitory peptide in the prior art, the ACE inhibitory peptide does not need enzymolysis, consumes less time and is lower in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to an ACE inhibitory peptide and a screening method and application thereof. Background Art

[0002] ACE, also known as kinase II, is a zinc ion (Zn 2+ ) is a dipeptidyl carboxypeptidase that plays a key role in blood pressure regulation. ACE inhibitory peptides are considered to be effective ingredients for assisting in the prevention of hypertension. Compared with synthetic peptides, food-derived ACE inhibitory peptides have the advantages of no side effects, a wide variety, and easy preparation. In 2005, my country isolated high-purity ACE inhibitory peptides from fish protein hydrolysates for the first time. At present, ACE inhibitory peptides from hundreds of marine organisms (clams, sardines, flounder, etc.) have been reported internationally. Food-derived marine bioactive peptides are a healthier and more natural alternative for preventing / treating lifestyle diseases. Turbot, also known as turbot, has tender meat and high nutritional value. my country's turbot industry ranks first in the world's turbot farming industry and is one of the pillar industries of my country's marine fish. It is rich in protein, and fish protein hydrolysates (FPHs) have a high digestibility and have a high development and utilization value.

[0003] The extraction methods of food-derived active peptides include hydrolysis, enzymatic hydrolysis, microbial fermentation, ultrasound-assisted extraction and microwave-assisted extraction. Among them, enzymatic hydrolysis is one of the most commonly used methods. The peptides prepared by enzymatic hydrolysis can efficiently and selectively cut specific peptide bonds to obtain target peptides with higher purity. Moreover, the enzymatic hydrolysis process will not destroy the core structure of its biological activity, and the enzymatic hydrolysis products have stronger activity. However, the time-consuming and high-cost enzymatic hydrolysis process is a key problem that needs to be solved urgently.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an ACE inhibitory peptide and a screening method and application thereof, so as to solve the problems of time-consuming and high cost of the ACE inhibitory peptide extraction method in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an ACE inhibitory peptide, wherein the amino acid sequence of the ACE inhibitory peptide is shown as RF, RW or KF.

[0008] The present invention provides a method for screening ACE inhibitory peptides, comprising the following steps:

[0009] (1) performing virtual enzymatic hydrolysis on the protein at a temperature of 23 to 27°C to obtain a polypeptide;

[0010] (2) comparing the peptides obtained in step (1) with a database to screen out peptides with ACE inhibitory activity;

[0011] (3) predicting the biological activity of the peptides screened in step (2), and screening the peptides with activity scores greater than 0.5 as active peptides;

[0012] (4) The water solubility and toxicity of the active peptide obtained in step (3) are analyzed. At the same time, a three-dimensional structure of the active peptide obtained in step (3) is constructed, and a semi-flexible molecular docking is performed with the three-dimensional structure of ACE. The active peptide with good water solubility, non-toxicity, -CDOCKERInteraction Energy docking value and -CDOCKER Energy docking value both greater than 60 is an ACE inhibitory peptide.

[0013] Preferably, the protein in step (1) is turbot myosin.

[0014] Preferably, the enzyme for the virtual enzymatic hydrolysis in step (1) is alkaline protease, and the pH value of the virtual enzymatic hydrolysis is 8-10.

[0015] Preferably, the database in step (2) is BIOPEP-UWM and AHTPDB.

[0016] Preferably, the water solubility analysis in step (4) includes analysis of the net charge and isoelectric point of the active peptide.

[0017] Preferably, the method for preparing the ACE three-dimensional structure in step (4) is to remove water molecules in the three-dimensional structure of the natural ACE crystal, retain Zn 2+ and Cl - .

[0018] Preferably, the radius of the semi-flexible molecule docking in step (4) is The center coordinates of the semi-flexible molecule docking are X: 38.977; Y: 38.645; Z: 50.183.

[0019] The present invention also provides the use of the ACE inhibitory peptide or the ACE inhibitory peptide screened by the screening method in preparing a product for treating and / or preventing hypertension.

[0020] The present invention has the following technical effects and advantages:

[0021] The present invention provides an ACE inhibitory peptide and a screening method and application thereof. Turbot muscle protein is taken as a research object, virtual enzymatic hydrolysis is used to assist activity prediction, and a preliminary screening is carried out in combination with a bioinformatics method. A semi-flexible molecular docking method is used to dock the polypeptide with the ACE three-dimensional structure to further screen and obtain the ACE inhibitory peptide. The ACE inhibitory peptides screened by the present invention have ACE inhibition rates of 76.61%, 80.71%, and 79.18%. Compared with the prior art method for extracting ACE inhibitory peptides, no enzymatic hydrolysis is required, so it is less time-consuming and has lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the 3D structure of turbot myosin;

[0023] Figure 2 This is the natural crystal structure of ACE;

[0024] Figure 3 The docking conformation diagram of ACE inhibitory peptide KF, wherein Figure A represents the overall diagram of the optimal docking conformation, and Figure B represents the 2D diagram of the optimal docking conformation;

[0025] Figure 4 It is the docking conformation diagram of ACE inhibitory peptide RF, wherein Figure A represents the overall diagram of the optimal docking conformation, and Figure B represents the 2D diagram of the optimal docking conformation;

[0026] Figure 5 Figure 3 is the docking conformation diagram of ACE inhibitory peptide RW, where Figure A represents the overall diagram of the optimal docking conformation, and Figure B represents the 2D diagram of the optimal docking conformation. DETAILED DESCRIPTION

[0027] The present invention provides an ACE inhibitory peptide, wherein the amino acid sequence of the ACE inhibitory peptide is shown as RF, RW or KF.

[0028] The present invention provides a method for screening ACE inhibitory peptides, comprising the following steps:

[0029] (1) performing virtual enzymatic hydrolysis on the protein at a temperature of 23 to 27° C. to obtain a polypeptide, wherein the temperature is preferably 25° C.;

[0030] (2) comparing the peptides obtained in step (1) with a database to screen out peptides with ACE inhibitory activity;

[0031] (3) predicting the biological activity of the peptides screened in step (2), and screening the peptides with activity scores greater than 0.5 as active peptides;

[0032] (4) The water solubility and toxicity of the active peptide obtained in step (3) are analyzed. At the same time, a three-dimensional structure of the active peptide obtained in step (3) is constructed, and a semi-flexible molecular docking is performed with the three-dimensional structure of ACE. The active peptide with good water solubility, non-toxicity, -CDOCKERInteraction Energy docking value and -CDOCKER Energy docking value both greater than 60 is an ACE inhibitory peptide.

[0033] In the present invention, the protein in step (1) is turbot myosin.

[0034] In the present invention, the enzyme for the virtual enzymatic hydrolysis in step (1) is alkaline protease, preferably alkaline protease, and the pH value of the virtual enzymatic hydrolysis is 8-10, preferably 9.

[0035] In the present invention, the databases in step (2) are BIOPEP-UWM and AHTPDB.

[0036] In the present invention, the water solubility analysis in step (4) includes the analysis of the net charge and isoelectric point of the active peptide.

[0037] In the present invention, the preparation method of the ACE three-dimensional structure in step (4) is to remove the water molecules in the three-dimensional structure of the natural ACE crystal, retain the Zn 2+ and Cl - .

[0038] In the present invention, the radius of the semi-flexible molecule docking in step (4) is The center coordinates of the semi-flexible molecule docking are X: 38.977; Y: 38.645; Z: 50.183.

[0039] The present invention also provides the use of the ACE inhibitory peptide or the ACE inhibitory peptide screened by the screening method in preparing a product for treating and / or preventing hypertension.

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] 1. Materials and Methods

[0043] 1.1 Experimental Materials

[0044] The UniProt protein database was used to query the turbot myosin, whose UniProtKB ID number is A0A8D3A461, the sequence is in FAST format, and the number of amino acids is 1954. The 3D structure was obtained by querying the AlphaFold2 database. The results are as follows: Figure 1shown.

[0045] The native crystal structure of ACE was obtained from the PDB protein database, and its PDB code is 1O8A. Figure 2 shown.

[0046] 1.2 Screening method

[0047] 1.2.1 Computer virtual enzymolysis

[0048] Using computer simulation and bioinformatics methods, the turbot myosin A0A8D3A461 sequence was taken as the research object. Through the "enzyme action" module in BIOPEP-UWM, subtilisin (EC 3.4.21.62) was used for virtual hydrolysis at pH 9 and temperature 25°C to obtain 78 active peptides. The results are shown in Table 1.

[0049] Table 1 Subtilisin virtual enzymolysis results

[0050]

[0051]

[0052] 1.2.2 Peptide screening

[0053] The 78 virtually enzymatically hydrolyzed peptides were compared with the BIOPEP-UWM and AHTPDB databases to screen out peptides with ACE inhibitory activity. The results are shown in Table 2.

[0054] Table 2 Peptides with ACE inhibitory activity

[0055]

[0056]

[0057] 1.2.3 Prediction of biological activity of ACE inhibitory peptides

[0058] The repeated sequences of the peptides with ACE inhibitory activity in Table 2 were removed, and their biological activities were predicted by the Petide Ranker program of bioware (http: / / bioware.ucd.ie / ~compass / biowareweb / ), and they were ranked according to the activity scores. The cleavage site and molecular weight of each peptide were counted. The results are shown in Table 3.

[0059] Table 3 Biological activity of ACE inhibitory peptides

[0060]

[0061]

[0062] Generally, the higher the activity score, the stronger its biological activity, and a peptide with an activity score of >0.5 can be identified as a peptide with high activity. According to the results in Table 3, peptides with an activity score of >0.5 were screened as active peptides, a total of 10, and the amino acid sequences were GF, RF, RW, AF, NF, KF, VF, GL, VW, and GIL.

[0063] 1.2.4 Water solubility and toxicity analysis

[0064] 1.2.4.1 Water solubility analysis

[0065] The isoelectric point, net charge number and hydrophobicity of the 10 active peptides were measured and their water solubility was predicted using the solubility prediction website Innovagen (http: / / www.innovagen.com / ). The measurement results are shown in Table 4.

[0066] Table 4 Water solubility of active peptides

[0067] Active Peptides Isoelectric point Net charge Hydrophobicity Water Solubility GF 5.53 0 7.34 Difference RF 10.9 1 8.00 good R W 10.95 1 7.62 good AF 5.53 0 6.69 Difference NF 5.36 0 7.04 Difference KF 9.93 1 9.93 good VF 5.56 0 5.56 Difference GL 5.6 0 7.80 Difference VW 5.58 0 5.35 Difference GIL 5.6 0 6.68 Difference

[0068] 1.2.4.2 Toxicity analysis

[0069] ToxinPred is a unique computer toxicity simulation tool suitable for predicting the toxicity of peptides / proteins. The probability value range from 0 to 1 indicates the possibility that a protein or peptide is toxic. According to its toxicity probability value, it is divided into different toxicity levels: non-toxic, low toxicity, moderate toxicity and high toxicity. Enter the sequence of each active peptide to be tested in turn on the ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpre) website. If the result is Non-Toxin, it means it is not toxic. The toxicity of the 10 active peptides screened out was predicted by the ToxinPred tool. The results of the 10 active peptides were all Non-Toxin, indicating that the 10 active peptides were non-toxic and could be used as foodborne active peptides.

[0070] 1.2.5 Semi-flexible molecular docking

[0071] 1.2.5.1 Construction of three-dimensional structure of active peptide

[0072] In Discovery Studio 2019Client software, the Macromolecules module was used to draw and construct the structures of active peptides GF, RF, RW, AF, NF, KF, VF, GL, VW, and GIL. The "Prepare Ligands" and "FullMinimization" modules were used to define oligopeptides as ligands and perform structural optimization to obtain the three-dimensional structures of active peptides GF, RF, RW, AF, NF, KF, VF, GL, VW, and GIL, respectively. After removing redundant conformations, the structures were saved in PDB format.

[0073] 1.2.5.2 Construction of ACE three-dimensional structure

[0074] ACE natural crystals usually contain impurities such as water and heteroatoms. Removing impurities through pretreatment can ensure the purity of the crystal structure, thereby reducing the impact of impurities on molecular docking.

[0075] Use Discovery Studio 2019 Client software to open the PDB file of ACE natural crystal, delete the water molecules and heteroatoms in the 3D crystal structure, and keep the Zn 2+ and Cl - The protein structure was optimized by the “CleanProtein” function in the Macromolecules module, multiple protein conformations were eliminated, and then hydrogen atoms and complete amino acid residues were completed to obtain the three-dimensional structure of ACE, which was saved in PDB format after processing.

[0076] 1.2.5.3 Semi-flexible molecular docking

[0077] The three-dimensional structure of ACE was defined as a rigid molecule, and the three-dimensional structures of the 10 active peptides were defined as flexible molecules. The semi-flexible molecular docking method was used to perform semi-flexible molecular docking using the "CDOCKER" module of Discovery Studio 2019 Client software. 2+ The active coordinates of the active center are (X: 38.977; Y: 38.645; Z: 50.183), and the docking radius is set to The docking parameters are shown in Table 5.

[0078] Table 5 Semi-flexible molecular docking parameters

[0079]

[0080]

[0081] Through the Dock Ligands (CDOCKER) module of Discovery Studio 2019Client, semi-flexible molecular docking was performed with “-CDOCKER Energy” value and “-CDOCKER Interaction Energy” value as quantitative evaluation indicators. The semi-flexible molecular docking score results are shown in Table 6.

[0082] Table 6 Semi-flexible molecular docking value results

[0083]

[0084] The active peptides were semi-flexible molecular docking with the three-dimensional structure of ACE. Successful docking indicated that the active peptides had ACE inhibitory activity, and failed docking indicated that the active peptides did not have ACE inhibitory activity. The higher the docking value, the higher the ACE inhibitory activity of the active peptides. Table 6 shows that the active peptides GF, RF, RW, AF, NF, KF, VF, GL, and VW can be semi-flexible molecular docking with the three-dimensional structure of ACE, and the docking values ​​are between 46.2597 and 71.9677, indicating that the active peptides GF, RF, RW, AF, NF, KF, VF, GL, and VW have ACE inhibitory activity, among which the scores of the active peptides RF, RW, and KF are significantly higher than those of other active peptides, indicating that the active peptides RF, RW, and KF have higher ACE inhibitory activity; the active peptide GIL cannot be semi-flexible molecular docking with the three-dimensional structure of ACE, indicating that the active peptide GIL does not have ACE inhibitory activity.

[0085] Combined with water solubility, toxicity analysis and semi-flexible molecular docking scores, it was found that the active peptides RF, RW and KF had good water solubility, were non-toxic and could be docked with the three-dimensional structure of ACE, and the -CDOCKER Interaction Energy docking values ​​and -CDOCKER Energy docking values ​​were both >60. Therefore, the RF, RW and KF active peptides were screened as ACE inhibitory peptides.

[0086] Example 2

[0087] The ACE inhibitory peptides KF, RF, and RW obtained by screening in Example 1 were semi-flexibly docked with the three-dimensional structure of ACE. The docking conformation diagram is shown in FIG. Figures 3 to 5 As shown, Figure 3 The docking conformation diagram of ACE inhibitory peptide KF, Figure A shows the overall diagram of the optimal docking conformation, and Figure B shows the 2D diagram of the optimal docking conformation;

[0088] Figure 4 The docking conformation diagram of ACE inhibitory peptide RF, Figure A shows the overall diagram of the optimal docking conformation, and Figure B shows the 2D diagram of the optimal docking conformation; Figure 5Figure 1 is the docking conformation diagram of ACE inhibitory peptide RW. Figure A shows the overall diagram of the optimal docking conformation, and Figure B shows the 2D diagram of the optimal docking conformation.

[0089] Hydrogen bonds are the main force that maintains the binding of active peptides to ACE. The interaction of multiple hydrogen bonds can significantly improve the stability of the binding between ACE and active peptides. ACE is a tetrahedral enzyme that depends on Zn701. It binds to the Zn in the metal center active site of ACE. 2+ The ions interact to inhibit the hydrophobic amino acids at the N-terminus of the peptide, thereby inactivating the ACE active site and inhibiting the activity of ACE. Figures 3 to 5 It can be seen that KF forms 4 hydrogen bonds with TYR394, ARG402, HIS410, and GLU411; RF forms 5 hydrogen bonds with ALA356, GLU411, and PRO407; RW forms 5 hydrogen bonds with ALA356, TYR394, ARG402, and HIS410, and all of them form hydrogen bonds with ZN 2+ There is a π bond with VAL518, which indicates that RF, RW and KF have the function of inhibiting ACE activity.

[0090] Example 3

[0091] The ACE inhibition rates of the three ACE inhibitory peptides RF, RW and KF screened in Example 1 were determined.

[0092] Principle: ACE is a dipeptidase that can hydrolyze HHL into hippuric acid (Hip) and histidylleucine (HL). Hippuric acid has a maximum absorption wavelength at 228nm.

[0093] Determination method: Under 37°C water bath conditions, 5 μL of sample was drawn, 15 μL of ACE solution with an enzyme activity of 60 mU / ml was added, 25 μL of substrate HHL was added after 5 min of incubation, 10 μL of 10% TFA was added after 25 min to terminate the reaction, 200 μL of ultrapure water was added, and the membrane was passed (0.22 μm). The content of hippuric acid was determined by high performance liquid chromatography, wherein the liquid phase conditions were as follows: Hypersil BDS C18 column (4.6 mm*250 mm, 5 μm), the mobile phase was 30% methanol (containing 0.1% TFA, 0.05% sodium acetate buffer solution pH 3.3), the detection wavelength was 228 nm, the mobile phase flow rate was 1 mL / min, the peak area of ​​hippuric acid in the chromatogram was recorded as A, the sample was replaced with ultrapure water, and the other conditions remained unchanged, the peak area of ​​hippuric acid in the chromatogram was recorded as A0, and the determination results of the three ACE inhibitory peptides RF, RW, and KF are shown in Table 7.

[0094] The calculation formula of ACE inhibition rate is as follows:

[0095] ACE inhibition rate (%) = (A0-A) / A0×100

[0096] Table 7 Determination results of three ACE inhibitory peptides: RF, RW, and KF

[0097] A0(mAU) A(mAU) RF 1107.308 258.969 R W 1847.3 356.4 KF 483.393 100.654

[0098] After calculation, it was found that the ACE inhibition rates of ACE inhibitory peptides RF, RW, and KF were 76.61%, 80.71%, and 79.18%, respectively.

[0099] It can be seen from the above embodiments that the present invention provides an ACE inhibitory peptide, the amino acid sequence of the ACE inhibitory peptide is shown as RF or RW. According to the method of the present invention, turbot muscle protein is used as the research object, virtual enzymatic hydrolysis is used to assist activity prediction, and a preliminary screening is carried out in combination with a bioinformatics method. The polypeptide is docked with the ACE three-dimensional structure by a semi-flexible molecular docking method to further screen and obtain ACE inhibitory peptides. The ACE inhibitory peptides screened by the present invention have ACE inhibition rates of 76.61%, 80.71%, and 79.18%, respectively. Compared with the prior art method for extracting ACE inhibitory peptides, no enzymatic hydrolysis is required, which is less time-consuming and has lower cost.

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ACE inhibitory peptide, characterized in that The amino acid sequence of the ACE inhibitory peptide is shown as RF, RW or KF.

2. The method for screening ACE inhibitory peptides according to claim 1, characterized in that: The steps include: (1) performing virtual enzymatic hydrolysis on the protein at a temperature of 23 to 27°C to obtain a polypeptide; (2) comparing the peptides obtained in step (1) with a database to screen out peptides with ACE inhibitory activity; (3) predicting the biological activity of the peptides screened in step (2), and screening the peptides with activity scores greater than 0.5 as active peptides; (4) The water solubility and toxicity of the active peptide obtained in step (3) are analyzed. At the same time, a three-dimensional structure of the active peptide obtained in step (3) is constructed, and a semi-flexible molecular docking is performed with the three-dimensional structure of ACE. The active peptide with good water solubility, non-toxicity, -CDOCKERInteraction Energy docking value and -CDOCKER Energy docking value both greater than 60 is an ACE inhibitory peptide.

3. The screening method according to claim 2, characterized in that The protein in step (1) is turbot myosin.

4. The screening method according to claim 2, characterized in that The enzyme for the virtual enzymatic hydrolysis in step (1) is alkaline protease, and the pH value of the virtual enzymatic hydrolysis is 8-10.

5. The screening method according to claim 2, characterized in that The databases in step (2) are BIOPEP-UWM and AHTPDB.

6. The screening method according to claim 2, characterized in that The water solubility analysis in step (4) includes analysis of the net charge and isoelectric point of the active peptide.

7. The screening method according to claim 2, characterized in that The preparation method of the ACE three-dimensional structure in step (4) is to remove the water molecules in the three-dimensional structure of the natural ACE crystal and retain the Zn 2+ and Cl - .

8. The screening method according to claim 2, characterized in that The radius of the semi-flexible molecule docking in step (4) is The center coordinates of the semi-flexible molecule docking are X: 38.977; Y: 38.645; Z: 50.

183.

9. Use of the ACE inhibitory peptide according to claim 1 or the ACE inhibitory peptide screened by the screening method according to any one of claims 2 to 8 in the preparation of a product for treating and / or preventing hypertension.

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