CN108929370B

CN108929370B - Three egg source active peptides with ACE (angiotensin converting enzyme) inhibitory activity

Info

Publication number: CN108929370B
Application number: CN201810808833.9A
Authority: CN
Inventors: 于志鹏; 樊玥; 赵文竹; 曲艾钰
Original assignee: Bohai University
Current assignee: Bohai University
Priority date: 2018-07-15
Filing date: 2018-07-15
Publication date: 2021-06-25
Anticipated expiration: 2038-07-15
Also published as: CN108929370A

Abstract

The invention belongs to the field of bioactive peptides, and particularly relates to three tripeptides capable of being combined with angiotensin converting enzyme and inhibiting the activity of the angiotensin converting enzyme. The amino acid sequences of the characteristic tripeptides provided by the invention are FMK, DCL and DWK respectively. The invention obtains the ACE inhibitory tripeptide sequence by means of a molecular docking technology and multiple rounds of virtual screening, and the method has the advantages of strong characteristics, high sensitivity, simple operation and the like, and also provides an identification method of the active peptide.

Description

Three egg source active peptides with ACE (angiotensin converting enzyme) inhibitory activity

Technical Field

The invention belongs to the field of bioactive peptides, and mainly relates to three egg source bioactive peptides with ACE (angiotensin converting enzyme) inhibitory activity.

Background

Bioactive peptides are specific protein fragments that improve human health by effectively promoting body function or status. These bioactive peptides are inactive in the parent protein, but can be released for activity by proteolysis or fermentation. In vivo, these bioactive peptides can exhibit a variety of activities that not only improve nutrient utilization, but also affect the cardiovascular, endocrine, immune, and nervous systems. At present, a plurality of active peptides are industrialized, soybean active peptide developed by Japan company has the function of reducing cholesterol, glutamine peptide has the function of immunoregulation, and ovotransferrin peptide and the like for treating diabetes mellitus. Eggs have long been considered as an ideal source of protein, vitamins and minerals. Researches find that compared with milk protein active peptide, egg protein is a better material for preparing the active peptide, and the egg-derived bioactive peptide plays an important role in preventing diseases and guaranteeing human health, thereby promoting the research of human beings on egg deep processing technology.

angiotensin-I Converting Enzyme (ACE, EC3.4.15.1) is a zinc-containing dicarboxy peptidase widely distributed in organism, and can be activated by chloride ion to treat blood pressure of human body and mammalsAnd cardiovascular function play important regulatory roles and are considered to be an important target for the treatment of hypertension. The ACE inhibitory peptide is a small peptide substance with ACE inhibitory activity, and is often combined with ACE in different modes to inhibit the effect of hydrolyzing angiotensin I without activity catalyzed by ACE into angiotensin II, so that the purpose of reducing blood pressure is achieved. Egg-derived enzymolysis products have been reported to have ACE inhibitory activity, and a patent (publication No. CN101696233A, application date 2009.10.29) discloses an egg white protein angiotensin converting enzyme inhibitory peptide and a preparation method thereof, wherein the egg white protein active peptide RVPSL, a semi-Inhibitory Concentration (IC) is identified by using a traditional classical method₅₀) It was 0.020 mM. Although the conventional processing method can clarify the sequence of the peptide, the conventional processing method has the defects of long experimental period, high investment, possibility of neglecting some high-activity peptide fragments and the like.

The invention utilizes ACE as a target point to carry out screening of tripeptide with ACE inhibitory activity and obtain an amino acid sequence, thereby not only saving time and improving economic benefit, but also providing a foundation for research of functional products and medicines of antihypertensive active peptide, and obviously improving the industrialized production and application potential of the ACE inhibitory peptide.

Disclosure of Invention

The object of the present invention is to provide: an isolated biologically active tripeptide FMK comprising (a) a tripeptide consisting of the amino acids Phe-Met-Lys; (b) or a polypeptide which is derived from the (a) and has the same activity by substituting, deleting or adding one or more amino acids in an amino acid sequence shown by Phe-Met-Lys;

an isolated biologically active tripeptide DCL comprising (c) a tripeptide consisting of amino acids represented by Asp-Cys-Leu; (d) or (c) a polypeptide which is derived from the amino acid sequence shown by Asp-Cys-Leu by substituting, deleting or adding one or more amino acids and has the same activity;

an isolated bioactive tripeptide DWK comprising (e) a tripeptide consisting of amino acids represented by Asp-Trp-Lys; (f) or (e) a polypeptide having equivalent activity and derived by substituting, deleting or adding one or more amino acids in the amino acid sequence represented by Asp-Trp-Lys.

The bioactive tripeptide is derived from chicken eggs. The tripeptide derivative refers to a polypeptide derivative obtained by modifying the tripeptide at the amino acid side chain group, the amino terminal or the carboxyl terminal by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation.

The invention utilizes the bioinformatics method, utilizes the properties of molecular weight, biological activity score, water solubility, ADMET and the like of active peptides to carry out multi-round screening by means of an online active peptide database, combines a molecular docking experiment to obtain three active peptides stably combined with ACE, and verifies the in vitro ACE inhibitory activity by high performance liquid chromatography, wherein the amino acid sequences of the three active peptides are FMK, DCL and DWK and corresponding IC₅₀The values were 36.60. mu.M, 95.39. mu.M and 74.49. mu.M, respectively. And finally, the amino acid residues of the catalytic sites of the interaction of FMK, DCL and DWK with ACE respectively are clarified by utilizing molecular docking software.

The ACE inhibitory peptide has the beneficial effects that: the ACE inhibitory peptide derived from the eggs has good in-vitro ACE inhibitory activity; the three ACE inhibitory peptides are products obtained after enzymolysis of main protease in the gastrointestinal tract in vivo, are stable under the digestion condition in vivo, can be completely and directly absorbed and utilized through the gastrointestinal tract to play the biological activity function of the ACE inhibitory peptides, and have very important significance and application value for developing health care products for improving hypertension or preparing medicines for reducing blood pressure.

Drawings

The invention is illustrated in figure 3, wherein:

FIG. 1ACE and FMK interaction 2D profiles;

FIG. 2ACE and DCL interaction 2D profiles;

fig. 3ACE and DWK interaction 2D map.

Detailed Description

The experimental steps adopted by the invention are as follows:

EXAMPLE 1 screening of the starting Material

The invention searches a protein sequence related to the egg protein from an NCBI database for analysis, and selects an egg mucin precursor as a parent protein of ACE inhibitory peptide. The accession number is NP-989992, and the amino acid number is 2108.

Example 2 screening of the tripeptides FMK, DCL and DWK

Egg protein (access: NP-989992) is virtually hydrolyzed by Pepsin (pH 1.3) and Trypsin in ExPASY PeptideCutter (https:// web. expass. org/peptide _ cutter), peptide sequences containing different amino acids are obtained by hydrolysis, all peptide sequences containing three amino acids are selected, activity scores of tripeptides are carried out in PeptideRanker (http:// biovar. ucd. ie/. about. glass/biovar. web/Server _ pages/PeptideRanker. php), the score interval is 0.01-0.99, tripeptides with the score higher than 0.65 are selected, and 14 tripeptides with the activity score higher than 0.65 are selected. Meanwhile, the 14 peptides are respectively predicted in water-solubility Innovagen (http:// www.innovagen.com/proteomics-tools) and toxicity ToxinPred (http:// crdd. osdd. net/raghava// toxincred /), and tripeptides with good water-solubility and no toxicity are screened out. The peptide sequences, activity scores, water solubility and toxicity predictions for these 14 tripeptides are shown in table 1. ADMET prediction is carried out on the tripeptide screened in the last step by using admETSAR (http:// lmmd. environment. edu. cn/admissarl/predict /), the tripeptide with good gastrointestinal absorption (HIA +) is determined in an emphasized mode, and seven tripeptides with prediction results of HIA + are screened and obtained, wherein the tripeptides are GWR, FMK, MCK, EGF, DCL, DWK and CGD. Seven tripeptides were molecularly interfaced with ACE (PDB ID: 1O86) as potential highly potent ACE inhibitory peptide candidates. The results show that three active peptides are stably combined with ACE, the CDOCKER values are shown in table 2, and selected three active peptides FMK, DCL and DWK are tripeptides identified by the invention.

TABLE 1 Activity score, Water solubility and toxicity prediction results for selected tripeptides

TABLE 2 binding energy of tripeptides to ACE in molecular docking

Example 3 in vitro Activity verification

ACE inhibitory peptides are obtained by virtual screening and molecular docking of egg proteins, and in order to verify the ACE inhibitory activity of FMK, DCL and DWK, high performance liquid chromatography is adopted.

1. Instruments and chemical articles: c18 liquid chromatography column, Shimadzu LC-2030 high Performance liquid chromatography, equyl-histidyl-leucine (HHL), Hippuric Acid (HA), Angiotensin Converting Enzyme (ACE) was purchased from Sigma, and acetonitrile, methanol and trifluoroacetic acid (TFA) was purchased from Fisher Scientific. All reagents were chromatographically pure.

2. High performance liquid chromatography analysis: taking HHL substrate solution, adding an inhibitor, uniformly mixing, preheating in a 37 ℃ constant-temperature water bath for 3-5 min, then adding ACE (angiotensin converting enzyme) solution, fully mixing, preserving heat at 37 ℃ for 30min, immediately reacting, and simultaneously using boric acid buffer solution to replace the inhibitor solution as a blank control group. The reaction solution was directly analyzed by an HPLC system.

Chromatographic conditions are as follows: column temperature 25 ℃, flow rate 0.5mL/min, sample size 10 μ L, mobile phase acetonitrile: the water is eluted at a ratio of 25: 75 isocratic, and the detection wavelength is 228 nm.

The inhibition of tripeptides at different concentrations was calculated using the following formula:

ACE inhibitory Activity (%) - (A-B)/Ax 100%

Wherein A is the peak area of the reaction blank, the reaction blank mixture containing the same volume of buffer solution instead of the sample; b is the peak area of the reaction in the presence of ACE and of an enzymatic peptide sample, IC₅₀Values are defined as the concentration of inhibitor that inhibits 50% of ACE activity under the assay conditions.

Definition of ACE activity: one unit (U) of ACE activity is defined as the amount of enzyme required to form 1mol HA per minute from catalytic HHL at 37 ℃.

The results of experiments on inhibiting the activity of ACE enzyme by tripeptides show (Table 3) that three tripeptides of the invention have remarkable ACE inhibiting properties on ACE.

TABLE 3 inhibitory Activity of tripeptides on ACE enzyme

Example 4 virtual docking analysis of tripeptides with ACE

FMK, DCL and DWK were interfaced with AEC (PDB ID: 1O86), respectively, using Discovery Studio 2017 software under CHARmm force field conditions. Maps 1, 2, 3 show the interaction of the tripeptides FMK, DCL and DWK, respectively, with specific inhibitory sites on the ACE enzyme.

The interaction between tripeptide and ACE mainly comprises Van der Waals force, hydrogen bonding force, hydrophobic interaction and electrostatic interaction, wherein the amino acid residues of FMK and ACE catalytic sites are Val518, His513, Lys511, Tyr520, Tyr523, His353, Ala354, Glu384, Thr282, Val380 and Glu 376; the amino acid residues of the catalytic sites of DCL and ACE are His353, Ala354, Glu384, His513, Lys511, Tyr520, His353, Val380 and Gln281, respectively; the amino acid residues of the catalytic sites of DWK and ACE are Glu411, Tyr523, His357, Phe457, His513, Gln281, Lys511, Trp279, Glu376, Ala354, Glu384 and His353, respectively.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. An egg source active peptide with ACE inhibitory activity is characterized in that the amino acid sequence of the egg source active peptide is DCL.

2. The use of the egg-derived active peptide with ACE inhibitory activity of claim 1 in the preparation of a health product for improving hypertension or a medicament for lowering blood pressure.