Polypeptide with ACE inhibitory activity and application thereof

Abstract

The invention discloses a polypeptide with ACE inhibitory activity and application thereof, and relates to the technical field of biology. The amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1. The invention further provides application of the polypeptide in preparation of antihypertensive drugs, products beneficial to maintaining the healthy level of blood pressure and angiotensin converting enzyme inhibitors. The ACE inhibitory peptide in tuna blood is efficiently screened through technologies of pepsin and pancreatin enzymolysis, ultrafiltration, liquid chromatography-tandem mass spectrum sequence identification and the like, and the polypeptide with the amino acid sequence being YPWTQR is finally obtained through screening. The polypeptide has good ACE inhibition activity, and the IC50 value of the polypeptide to ACE can reach 46.29 [mu] mol / L. The polypeptide has the potential of being developed into antihypertensive drugs and products beneficial to maintaining the health level of blood pressure, and has high market application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide with ACE inhibitory activity and application thereof. Background Art

[0002] Hypertension is a common cardiovascular and cerebrovascular disease worldwide, which seriously threatens human health. Angiotensin converting enzyme (ACE) plays a key role in regulating blood pressure. It maintains blood pressure balance in the body by catalyzing the conversion of angiotensin I into angiotensin II, which has a blood pressure-raising effect, and degrading bradykinin, which has a blood pressure-lowering effect. Food-derived ACE inhibitory peptides, as a natural polypeptide substance, have become a research hotspot in recent years because of their safety and easy absorption. ACE inhibitory peptides can effectively regulate blood pressure, prevent and treat hypertension and related diseases by inhibiting ACE activity. However, ACE inhibitory peptides in the prior art have problems such as low activity, poor stability, and low bioavailability, which limit their wide application.

[0003] Tuna is recommended by international nutrition organizations as one of the three most nutritious fishes for its tender meat, high content of protein, fat, vitamins, minerals, and rich unsaturated fatty acids and taurine, and is loved by consumers all over the world. The tuna resources in my country's South China Sea are rich, and the resources occupy an important position in the world's fisheries. Among them, the resources of small tuna such as flat rudder skipjack tuna, round rudder skipjack tuna, and tuna are 850,000 tons, and the annual catch is 230,000 tons; the annual catch of large tuna such as yellowfin tuna is about 17,000 tons. However, in recent years, the catch of small tuna in my country has been only 5,000 tons, and the amount of unused resources is huge, with huge industrial development potential. As one of the main species caught in tuna fisheries, yellowfin tuna needs to be bled quickly after being caught to maintain the quality of its fish meat. Although blood is a source of high-quality protein, most animal blood is discharged in the form of waste and has basically not been developed and utilized, resulting in a serious waste of resources. The present invention intends to utilize tuna blood to explore functional active peptides with ACE inhibitory activity, thereby providing technical support for the development of products that help maintain healthy blood pressure levels. Summary of the invention

[0004] The purpose of the present invention is to provide a polypeptide having ACE inhibitory activity and its application to solve the problems existing in the above-mentioned prior art. The polypeptide has good ACE inhibitory activity, IC 50 The value can reach 46.29μmol / L, providing technical support for the development of products that help maintain healthy blood pressure levels.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a polypeptide having ACE inhibitory activity, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for preparing the above polypeptide, comprising the step of synthesizing the polypeptide by solid phase synthesis.

[0008] The present invention also provides the use of the above polypeptide in the preparation of blood pressure lowering drugs.

[0009] The present invention also provides the use of the above polypeptide in preparing a product that helps maintain a healthy blood pressure level.

[0010] Furthermore, the product is a health product or a functional food.

[0011] The present invention also provides the use of the above polypeptide in the preparation of angiotensin converting enzyme inhibitors.

[0012] The present invention also provides a blood pressure lowering drug, the active ingredient of which includes the above polypeptide.

[0013] Furthermore, the blood pressure lowering drug also includes pharmaceutically acceptable excipients.

[0014] The present invention also provides a product that helps maintain a healthy blood pressure level, the active ingredient of which includes the above-mentioned polypeptide;

[0015] The product is a health product or a functional food.

[0016] The present invention also provides an angiotensin converting enzyme inhibitor, the active ingredient of which includes the above polypeptide.

[0017] The present invention discloses the following technical effects:

[0018] The present invention uses pepsin and pancreatic enzyme hydrolysis, ultrafiltration, liquid chromatography-tandem mass spectrometry sequence identification and other technologies to efficiently screen ACE inhibitory peptides in tuna blood, and finally screens and obtains a polypeptide with an amino acid sequence of YPWTQR (Tyr-Pro-Trp-Thr-Gln-Arg). The polypeptide has good ACE inhibitory activity, IC 50 The value can reach 46.29μmol / L. The peptide has the potential to be developed into a blood pressure-lowering drug and a product that helps maintain a healthy blood pressure level, and has a high market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the inhibition curve of peptide YPWTQR on ACE;

[0021] Figure 2 The three-dimensional structure diagram of the molecular docking of YPWTQR and ACE;

[0022] Figure 3 Schematic diagram of the molecular docking planar interaction between YPWTQR and ACE. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] Preparation of ACE inhibitory peptides:

[0030] 1. Extract tuna blood protein using Solebow EX1200 kit. Dilute the extracted protein to a concentration of 5% (W / V), adjust the pH to 3.0 with 1 mol / L hydrochloric acid, add 3% (W / W) pepsin (250U / mg), and perform enzymolysis for 3 hours at 37°C and 400rpm on a shaker, then adjust the solution to pH 8.0 with 1 mol / L NaOH, add 4% (W / W) pancreatin (8×USP), and perform enzymolysis for 8 hours at 37°C. After the enzymolysis is completed, inactivate the enzyme in a water bath for 10 minutes, cool the enzymolysis solution, centrifuge at 6500g at 4°C for 25 minutes, and retain the supernatant.

[0031] 2. The supernatant was filtered through an ultrafiltration centrifuge tube with a molecular weight of less than 3 KDa at 5000g and centrifuged at 4°C for 30 min to obtain a crude polypeptide solution with a molecular weight of less than 3 KDa. The crude polypeptide solution was desalted with a C18 desalting column and then freeze-dried.

[0032] 3. The crude peptide freeze-dried powder was reconstituted with 20 μL 0.1% formic acid solution and analyzed by LC-MS / MS. 0.1% formic acid solution was used as solution A, 0.1% formic acid acetonitrile aqueous solution (84% acetonitrile) was used as solution B, 95% solution A was balanced, and the C18 reverse phase analytical column was separated at a flow rate of 300 nL / min. The mass spectrometry scanning range was 300-1800 m / z, the resolution of the primary mass spectrometry was 60000, the AGC (Automatic gain control) target was 1e6, the maximum injection time was 50 ms, and the dynamic exclusion time was 30.0 s. The mass-to-charge ratio of the peptide and peptide fragments was collected according to the following method: 20 fragment spectra (MS2 scan) were collected after each full scan (fullscan), the MS2 Activation Type was HCD, the Isolation window was 1.5 m / z, and the secondary mass spectrometry resolution was 15000. The peptides were identified and quantified using the UniProt database (FDR < 1%).

[0033] 4. Among the numerous peptides, a peptide YPWTQR (Tyr-Pro-Trp-Thr-Gln-Arg) with ACE inhibitory activity was identified.

[0034] 5. ToxinPrep and Swiss-Port were used to predict the toxicity of peptides; PeptideRanker and PeptideCutter were used to predict the potential biological activity and gastrointestinal digestibility of peptides, and admetSAR was used to predict the human intestinal absorbability of peptides. The specific prediction information of peptides is shown in Table 1.

[0035] Table 1 Peptide prediction information

[0036]

[0037]

[0038] Example 2

[0039] The company was commissioned to carry out solid phase synthesis of the peptide YPWTQR and verify its ACE inhibitory activity in vitro.

[0040] 1. In vitro ACE inhibition assay

[0041] Using high performance liquid chromatography, ACE and hippurylhistidylleucine (HHL) were dissolved in 0.1 mol / L sodium borate solution (0.3 mol / L NaCl, pH 8.3). After 15 μL of peptide solution (distilled water as a control) was mixed with 15 μL of ACE (100 U / L) solution, the mixture was kept at 37°C for 5 min, and then 30 μL of HHL substrate was added. After reacting at 37°C for 1 h, 60 μL of 1 mol / L hydrochloric acid solution was added to terminate the reaction, and 200 μL of water was added for dilution. The mixture was filtered through a 0.22 μM filter membrane and analyzed on an HPLC machine. The liquid chromatography was performed on a C18 column, with 10 μL of sample injected, mobile phase A was water, mobile phase B was acetonitrile, flow rate was 0.5 mL / min, and detection wavelength was 228 nm. The activity inhibition rate of ACE inhibitory peptides was determined. When the ACE inhibition rate was 50%, the concentration of the sample was the IC 50 Half inhibitory concentration value. Each experiment was performed in triplicate.

[0042] ACE inhibition rate calculation formula:

[0043] ACE inhibition rate (%) = (AB) / A × 100%;

[0044] Among them, A is the peak area of ​​HHL in the control group, and B is the peak area of ​​HHL in the synthetic peptide.

[0045] In vitro ACE inhibition experiments showed that the IC 50 The value was 46.29 μmol / L ( Figure 1), indicating that the polypeptide YPWTQR screened by the present invention has good ACE inhibitory activity and has the potential to be developed into a blood pressure-lowering drug and a product that helps maintain a healthy blood pressure level.

[0046] 2. Molecular docking experiment

[0047] The peptide YPWTQR was molecularly docked with ACE to clarify its target. The crystal structure of ACE (PDB number: 1O8A) was obtained from the RCSB protein database (http: / / www.rcsb.org / ), and the peptide YPWTQR was semi-flexibly docked with ACE using Autodockvina. Zn was retained before molecular docking. 2+ and Cl - , remove water molecules and other ligands. Molecular docking is centered on the S1 active pocket of ACE (Ala354, Glu384 and Tyr523), that is, the coordinates are X: 44.831, Y: 34.109, Z: 46.363. The binding energy represents the interaction strength between the ligand and the macromolecule, and a lower score indicates a strong affinity between the target macromolecule and the ligand. Usually a binding energy less than -5kcal / mol indicates a better binding force. The binding energies of the 9 docking results are all less than -5kcal / mol. According to the size of the binding energy, the best binding mode with ACE is selected to determine the interaction mode between the peptide and ACE. Figure 2-Figure 3 The optimal docking posture of peptide YPWTQR and ACE is shown, and the binding energy in this case is -9.9 kcal / mol (Table 2). Figure 3 It also showed that the binding of polypeptide YPWTQR to ACE amino acid residues mainly depends on hydrogen bonds, that is, other forces. Specifically, polypeptide YPWTQR forms hydrogen bonds with amino acid residues (HIS353, SER355, HIS410, ALA356, ASN66, PRO515, LEU139, SER516), forms hydrophobic forces with amino acid residues (HIS358, ALA63, PHE391, TYP360), and forms electrostatic interactions with GLU143 and ZN701.

[0048] 3. Hydrophilicity and isoelectric point evaluation and prediction

[0049] The total average hydrophilicity and isoelectric point of peptide YPWTQR were evaluated and predicted by ExPasy and Pepdraw, and the results are shown in Table 2. The total average hydrophilicity can be used to characterize the hydrophilicity and hydrophobicity of proteins, where the larger the negative value, the stronger the hydrophilicity. The results show that peptide YPWTQR has good hydrophilicity. The isoelectric point of peptide YPWTQR is greater than 7, indicating that it is alkaline.

[0050] Table 2 Binding energy, overall average hydrophilicity and isoelectric point of peptide YPWTQR

[0051]

[0052] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide having ACE inhibitory activity, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A method for preparing the polypeptide according to claim 1, characterized in that: The method comprises the step of synthesizing the polypeptide by adopting a solid phase synthesis method.

3. Use of the polypeptide according to claim 1 in the preparation of antihypertensive drugs.

4. Use of the polypeptide according to claim 1 in preparing a product that helps maintain healthy blood pressure levels.

5. The use according to claim 4, characterized in that: The product is a health product or a functional food.

6. Use of the polypeptide according to claim 1 in the preparation of angiotensin converting enzyme inhibitors.

7. A blood pressure lowering drug, characterized in that: The active ingredient comprises the polypeptide according to claim 1.

8. The antihypertensive drug according to claim 7, characterized in that: The blood pressure lowering drug also includes pharmaceutically acceptable excipients.

9. A product that helps maintain healthy blood pressure levels, characterized in that: The active ingredient comprises the polypeptide according to claim 1; The product is a health product or a functional food.

10. An angiotensin converting enzyme inhibitor, characterized in that The active ingredient comprises the polypeptide according to claim 1.

Images