A turtle peptide with angiotensin-converting enzyme inhibitory activity, its preparation and application

Abstract

This invention discloses a turtle peptide with angiotensin-converting enzyme (ACE) inhibitory activity, its preparation, and its application, belonging to the field of biotechnology. Using turtle as raw material, this invention obtains peptide components with a molecular weight less than 5 kDa through enzymatic hydrolysis and ultrafiltration. Sequencing reveals three peptide sequences with ACE inhibitory activity: AAGPAGGNLN, EAGAQGPPGA, and GARGDK. In vitro activity experiments verified the ACE inhibitory function of the three turtle peptides. The turtle peptide combination prepared by this invention exhibits significant ACE inhibitory activity and can be used to develop functional foods or antihypertensive drugs. Furthermore, the preparation method is environmentally friendly, efficient, and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a turtle peptide with angiotensin-converting enzyme (ACE) inhibitory activity, its preparation, and its application. Background Technology

[0002] Hypertension is one of the major risk factors for cardiovascular and cerebrovascular diseases, and its prevention and treatment have become a key issue in global public health. Angiotensin-converting enzyme (ACE) plays a crucial role in the human blood pressure regulation mechanism, catalyzing the conversion of angiotensin I into angiotensin II, which has a strong vasoconstrictive effect, and degrading bradykinin, which relaxes blood vessels, thereby leading to elevated blood pressure. Therefore, inhibiting ACE activity has become one of the important strategies for controlling hypertension. Currently, commonly used synthetic ACE inhibitors (such as captopril and enalapril) have significant antihypertensive effects, but long-term use may be accompanied by side effects such as cough, rash, and kidney damage. Therefore, exploring safe and low-toxicity ACE-inhibiting peptides from natural food resources has become a hot topic in functional food and drug research and development in recent years. Natural bioactive peptides usually have the characteristics of wide availability, high biocompatibility, and easy metabolism and absorption, and have good development potential.

[0003] As a traditional food and medicine source, soft-shelled turtle is rich in high-quality protein and various bioactive components. Studies have reported that its extracts possess certain immunomodulatory and antioxidant functions; however, the systematic exploration and efficient utilization of bioactive peptides from its protein resources remain insufficient. Existing methods for preparing bioactive peptides mostly employ single enzymatic hydrolysis processes and lack efficient screening and validation systems targeting ACE inhibitory activity, resulting in unstable product activity, low screening efficiency, and difficulty in achieving industrial-scale targeted production. Furthermore, conventional bioactive peptide screening methods often rely on repeated experiments involving in vitro activity assays and separation and purification, which are time-consuming and labor-intensive, and make it difficult to accurately obtain peptide sequences with high binding activity. With the development of liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology and molecular docking simulation, rapid identification and screening of bioactive peptides using bioinformatics methods has become possible; however, this strategy has not yet been fully applied in the development of bioactive peptides from soft-shelled turtle protein.

[0004] Therefore, developing an efficient and environmentally friendly method for preparing turtle ACE inhibitory peptides, and using modern analytical techniques and computational simulations to achieve precise screening and verification of active peptides, is of great significance for enriching the sources of natural antihypertensive products and increasing the added value of turtle protein. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a turtle peptide with angiotensin-converting enzyme inhibitory activity, as well as its preparation and application. This invention combines modern peptidomics technology to screen and verify specific active peptide sequences, providing raw materials and technical support for the development of turtle-derived antihypertensive functional foods or drugs.

[0006] According to a first aspect of the present invention, the present invention first provides a turtle peptide having angiotensin-converting enzyme inhibitory activity, wherein the amino acid sequence of the turtle peptide is any of the following: a. AAGPAGGNLN (SEQ ID NO:1); b. EAGAQGPPGA (SEQ ID NO:2); c. GARGDK (SEQ ID NO: 3).

[0007] According to some specific embodiments of the present invention, the turtle peptide is chemically synthesized or obtained by enzymatic hydrolysis and purification of turtle protein.

[0008] Typically, but not limited to, the protein obtained by enzymatic hydrolysis and purification from turtle protein can be: S1. Remove the head and bleed the turtle, cut it into equal parts, remove impurities and defatting; S2. Perform enzymatic hydrolysis by adding alkaline protease, and ensure the hydrolysis temperature is consistent with the optimal temperature of the enzyme used. S3. Take the enzyme hydrolysate from S2 to inactivate the enzyme, then centrifuge at high speed and take the supernatant. S4. The supernatant is separated by ultrafiltration and spray-dried to obtain turtle peptide powder; S5. Separate, purify, and screen to obtain the turtle peptide.

[0009] Preferably, in step S1, the turtle is decapitated and bled, and then divided into equal pieces of 0.5cm x 0.5cm. The pieces are placed in a 2.5-3% NaCl solution (1:20-1:30, w / v) and stirred continuously at 4°C for 12-24 hours to remove water-soluble and salt-soluble impurities. After washing repeatedly with distilled water and draining thoroughly, the sample is placed in a 10% isopropanol solution of the same volume as the NaCl solution and soaked at 4°C for 12-24 hours to remove fat. After washing repeatedly with distilled water and draining thoroughly, the sample is ready for use.

[0010] Preferably, in step S2, enzymatic hydrolysis is performed, wherein the amount of alkaline protease added is 0.2-5% of the weight of the turtle raw material, the hydrolysis time is 1-10 hours, and the hydrolysis temperature is consistent with the optimal temperature of the enzyme used. Preferably, in step S3, the enzyme hydrolysate from S2 is taken for enzyme inactivation. The enzyme inactivation conditions are: heating to 85-120℃ and maintaining for 10-60 minutes, followed by high-speed centrifugation and collection of the supernatant. Preferably, in step S4, the supernatant is separated by ultrafiltration to collect components with a molecular weight of less than 5 kDa. The ultrafiltration conditions are: operating pressure 30 psi, operating temperature 20-25℃, and feed concentration 10-15 g / L. The supernatant is then spray-dried to obtain turtle peptide powder.

[0011] Preferably, in step S5, the amino acid sequence of the obtained turtle peptide component is identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS); separation, purification, and screening are then performed. Based on the mass spectrometry fragment ion (b-ion and y-ion) information, the peptide sequence is deduced using database search and de novo sequencing algorithms; stable isotope labeling or chemical modification methods are used to enhance the mass spectrometry fragment ion signal and improve sequencing accuracy.

[0012] Preferably, in step S5, molecular docking technology is used to simulate docking of the sequencing-obtained peptides with the active site of ACE; the active site of ACE includes amino acid residues Gln281, Lys511, His353, and His513 in the S1 pocket; peptides that form hydrogen bonds and hydrophobic interactions with ACE are screened.

[0013] According to a second aspect of the present invention, the present invention provides a turtle peptide composition having angiotensin-converting enzyme inhibitory activity, said turtle peptide composition comprising at least one of the turtle peptides shown in SEQ ID NO:1~3.

[0014] According to some specific embodiments of the present invention, the turtle peptide composition comprises three turtle peptides shown in SEQ ID NO:1 to SEQ ID NO:3.

[0015] According to some specific embodiments of the present invention, the turtle peptide composition further includes pharmaceutical excipients; the pharmaceutical excipients include solvents, fillers, dispersants, stabilizers, plasticizers or flavoring agents; the selection of the type and amount of excipients should conform to pharmaceutically acceptable types and dosages.

[0016] According to a third aspect of the invention, the invention also provides the use of the aforementioned turtle peptide composition in the preparation of antihypertensive drugs.

[0017] According to some specific embodiments of the present invention, the antihypertensive drug is an angiotensin-converting enzyme (ACE) inhibitor.

[0018] According to a third aspect of the present invention, the present invention also provides the use of the aforementioned turtle peptide in the preparation of antihypertensive drugs or functional foods.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) Clearly Defined Activity: This invention combines modern peptidomics, de novo sequencing, and molecular docking technology to achieve precise and efficient screening of ACE-inhibiting peptides. This invention confirms that the selected turtle peptides (AAGPAGGNLN, EAGAQGPPGA, GARGDK) possess significant ACE-inhibiting activity, laying the foundation for their application in functional foods or pharmaceuticals.

[0020] 2) The turtle peptides of this invention can be obtained through chemical synthesis or by enzymatic hydrolysis and purification of turtle protein. Using turtle as the raw material enhances the added value of turtle processing byproducts. The enzymatic hydrolysis method is mild and environmentally friendly, and ultrafiltration effectively enriches the active components. Attached Figure Description

[0021] Figure 1 Flowchart of turtle peptide preparation and activity evaluation process; Figure 2 The HPLC purity chromatogram of AAGPAGGNLN is shown. Figure 3 The mass spectrum of AAGPAGGNLN; Figure 4 The HPLC purity chromatogram for EAGAQGPPGA is shown. Figure 5 The mass spectrum of EAGAQGPPGA; Figure 6 The HPLC purity chromatogram for GARGDK is shown below. Figure 7 This is the mass spectrum of GARGDK. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments.

[0023] All raw materials used in this invention are commercially available.

[0024] Steps S1-S4 in the following examples illustrate the process of separating and purifying turtle peptide powder from turtle protein via enzymatic hydrolysis: S1. Remove the head and bleed the turtle, then cut it into equal pieces of 0.5cm x 0.5cm. Place the pieces in a 2.5-3% NaCl solution (1:20-1:30, w / v) and stir continuously at 4℃ for 12-24 hours to remove water-soluble and salt-soluble impurities. Then wash repeatedly with distilled water and drain thoroughly. Place the sample in a 10% isopropanol solution (equal to the amount of NaCl solution) and soak at 4℃ for 12-24 hours to remove fat. Wash repeatedly with distilled water and drain thoroughly before use.

[0025] S2. Perform enzymatic hydrolysis, wherein the amount of alkaline protease added is 0.2-5% of the weight of the turtle raw material, the hydrolysis time is 1-10 hours, and the hydrolysis temperature is consistent with the optimal temperature of the enzyme used. S3. Take the enzyme hydrolysate from S2 and inactivate the enzyme. The inactivation conditions are: heat to 85-120℃ and maintain for 10-60 minutes, then centrifuge at high speed and take the supernatant. S4. The supernatant is separated by ultrafiltration to collect components with a molecular weight of less than 5 kDa. The ultrafiltration conditions are: operating pressure 30 psi, operating temperature 20-25℃, and feed concentration 10-15 g / L. The supernatant is then spray-dried to obtain turtle peptide powder.

[0026] To obtain peptides with angiotensin-converting enzyme (ACE) inhibitory activity, this embodiment provides methods for further sequencing, identification, purification, and screening of turtle peptides, as detailed in S5 and S6.

[0027] S5. Sequencing of turtle peptides and identification of amino acid sequences of the obtained turtle peptide components by liquid chromatography-tandem mass spectrometry (LC-MS / MS); separation, purification, and screening. Peptide sequencing and mass spectrometry identification: Analysis of ultrafiltered turtle peptide components using LC-MS / MS. Mass spectrometry conditions: Electrospray ionization source, positive ion mode, acquisition of secondary mass spectra. De novo sequencing: Using stable isotope labeling (e.g., 2 H, 13 C 15 Strategies involving N-terminus (N) or chemical modification (such as modifying the N-terminus or C-terminus of a peptide with a positively charged reagent) are used to create paired fragment ions from the same peptide. By comparing the mass spectra of different labeled samples, the b-ion and y-ion series can be accurately identified, thereby deriving the complete amino acid sequence. Bioinformatics analysis: Software (such as MaxQuant and Pep-MRMer) is used to search libraries and perform de novo sequencing analysis on the mass spectrometry data.

[0028] S6. Molecular docking screening of ACE repressor peptides: Receptor preparation: Obtain the three-dimensional structure of ACE (e.g., 1 UZF) from a protein database (PDB). Preprocessing includes hydrogenation, charge distribution, and energy minimization. Peptide preparation: Construct three-dimensional structures from the sequencing-obtained peptides and optimize them. Molecular docking: Use software such as AutoDock Vina to dock the peptides to the active site of ACE. Focus on the interaction forces (hydrogen bonds, hydrophobic interactions, etc.) between the peptides and key residues in the ACE active site (e.g., Gln281, Lys511, His353, His513 in the S1 pocket). Screening criteria: Peptides with low binding free energy and stable interactions with key residues are selected as candidate ACE repressor peptides.

[0029] In vitro synthesis of bioactive peptides and evaluation of ACE inhibitory activity: Peptide synthesis: The three target peptides were chemically synthesized using a solid-phase peptide synthesis method (Fmoc strategy), and purified by HPLC. Purity and structure were identified by mass spectrometry. ACE inhibitory activity assay: Method 1 (Sodium trinitrobenzenesulfonate colorimetric method): The target peptide was added to the reaction system of ACE and the substrate hippuryl-histyl-leucine. The product content was determined by colorimetric reaction, and the ACE inhibition rate was calculated. Method 2 (Enzyme coupling method): Using GGCN as a substrate, a yellow product was generated by GGT coupling reaction. The absorbance change was monitored at 405 nm, and the inhibition rate was calculated. Method 3 (Fluorescence resonance energy transfer method): Using fluorescently labeled peptides as substrates, the fluorescence resonance energy transfer signal change caused by ACE enzyme cleavage was utilized. The fluorescence intensity change was monitored at an excitation wavelength of 325 nm and an emission wavelength of 393 nm, and the inhibition rate was calculated.

[0030] Example 1: Enzymatic hydrolysis and ultrafiltration separation of turtle peptides 1. Take 1 kg of fresh turtle paste, add 3 L of deionized water and mix well. Adjust the pH to 7.0 with 1 M NaOH.

[0031] 2. Add alkaline protease (2% of the weight of the turtle slurry) and enzymatically hydrolyze in a 50℃ water bath for 5 hours.

[0032] 3. After the enzymatic hydrolysis is complete, heat the hydrolysate to 95°C and maintain it for 15 minutes to inactivate the enzyme.

[0033] 4. After the enzyme inactivation solution is cooled, it is separated using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa at an operating pressure of 30 psi and a temperature of 25°C. The permeate with a molecular weight of <5 kDa is collected and freeze-dried to obtain turtle peptide powder (yield of about 12%).

[0034] Example 2: LC-MS / MS sequencing and de novo sequencing of peptides 1. The turtle peptide powder obtained in Example 1 was dissolved in 0.1% formic acid aqueous solution and desalted by C18 desalting column.

[0035] 2. Analysis was performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry. Column: C18 column (2.1 mm × 150 mm, 1.7 μm); Mobile phase: A was 0.1% formic acid in water, B was 0.1% formic acid in acetonitrile; gradient elution.

[0036] 3. Mass spectrometry conditions: electrospray ionization source, positive ion mode; secondary mass spectrometry uses data-dependent acquisition mode.

[0037] 4. The obtained mass spectrometry data were used to search the database using software, and the unknown sequences were sequenced from scratch using a stable isotope-labeled method: the turtle peptide sample was divided into two parts, one part was used... 12C-labeling reagent (light labeling) modifies the N-terminus of the peptide, and another part uses... 13 C-labeled reagent (relabeled) is used to modify the N-terminus of peptides, and the mixture is injected afterward. By comparing the paired b and y ions generated by the lightly and heavily labeled peptides, the complete sequences of peptides AAGPAGGNLN, EAGAQGPPGA, and GARGDK are deduced.

[0038] Example 3: Molecular docking screening of ACE inhibitory peptides 1. Download the ACE crystal structure (1UZF) from the PDB database, process it with AutoDock Tools software, and define the active center as the region containing residues Gln281, Lys511, His353, and His513.

[0039] 2. The three-dimensional structures of peptides AAGPAGGNLN, EAGAQGPPGA, and GARGDK were introduced for semi-flexible molecular docking.

[0040] 3. The docking results showed that all three peptides could stably bind to the ACE active site: AAGPAGGNLN: Hydrogen bonds are formed between Ala1, Gly3 and Gln281, and hydrophobic interactions are formed between Leu9 and His353.

[0041] EAGAQGPPGA: Glu1 forms a salt bridge with Lys511, and Pro6 and Pro7 form a hydrophobic interaction with His513.

[0042] GARGDK: Arg3 forms hydrogen bonds with Gln281, and Asp5 forms hydrogen bonds with Lys511.

[0043] The predicted binding energies of all three peptides are below -7 kcal / mol, indicating strong affinity.

[0044] Example 4: In vitro synthesis of bioactive peptides 1. Peptide synthesis: ① Synthesis order: from the C end to the N end of the sequence, the steps are as follows: a. Weigh n equivalents of resin and place it in the reactor. Add DCM (dichloromethane) to swell for half an hour. Then remove the DCM, add 2n equivalents of the first amino acid in the sequence, 2n equivalents of DIEA, an appropriate amount of DMF, DCM (appropriate amount means enough to fully agitate the resin), DIEA (diisopropylethylamine), DMF (dimethylformamide), and DCM. Bubble the reaction under nitrogen for 60 minutes. Then add approximately 5n equivalents of methanol and react for half an hour. Remove the reaction solution and wash with DMF and MEOH. b. Add the second amino acid in the sequence (also a 2n equivalent), 2n equivalents of HBTU (1-hydroxy, benzo[3], trichlorfon tetramethylhexafluorophosphate), and DIEA to the reactor. Bubble the reaction with N2 for half an hour, wash off the liquid, and detect ninhydrin. Then, cap the reaction with pyridine and acetic anhydride. Finally, wash thoroughly, add an appropriate amount of decapping solution to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash again, and detect ninhydrin. c. Add the different amino acids in the sequence sequentially, following the method in step b; d. After drying the resin with nitrogen, remove it from the reaction column and pour it into a flask. Then add a certain amount of cutting fluid (composition is 95% TFA, 2% ethylenedithiol, 2% triisopropylsilane, 1% water) to the flask (the ratio of cutting fluid to resin is about 10 ml / g), shake, and filter out the resin. e. Obtain the filtrate, then add a large amount of diethyl ether to the filtrate to precipitate the crude product, then centrifuge and wash to obtain the crude product of the sequence; ② Peptide purification: The crude product was purified to the required purity using high-performance liquid chromatography (HPLC). For example... Figures 2-7 The figures show the HPLC purity chromatograms and mass spectra of the three peptides.

[0045] ③ Lyophilized peptides: The purified liquid was concentrated in a freeze dryer and freeze-dried into a white powder to obtain AAGPAGGNLN (Ala-Ala-Gly-Pro-Ala-Gly-Gly-Asn-Leu-Asn), EAGAQGPPGA (Glu-Ala-Gly-Ala-Gin-Gly-Pro-Pro-Gly-Ala), and GARGDK (Gly-Ala-Arg-Gly-Asp-Lys).

[0046] 2. Assay for ACE inhibitory activity (fluorescence resonance energy transfer method): The method described in this invention is based on the principle of fluorescence resonance energy transfer (FRET), and utilizes the characteristic that ACE enzymes cleave fluorescently labeled substrates, resulting in changes in fluorescence signals, to quantitatively assess the activity of inhibitors.

[0047] Reaction system: Dilute the peptide sample to be tested to a final concentration of 0.5 mg / ml. Mix the diluted inhibitor sample, ACE enzyme, and a fluorescently labeled substrate in a suitable buffer system and incubate at 37°C in the dark for 30-60 minutes. After incubation, measure the fluorescence intensity of the reaction system using a fluorescence detection device at an excitation wavelength of 325 nm and an emission wavelength of 393 nm. Each sample was measured in triplicate, and the average of the three parallel measurements was used as the final result.

[0048] Table 1 - Methods for determining ACE inhibitory activity Note: Assay Buffer: Detection buffer; Assay Reagent: Reaction reagent containing ACE enzyme; MLN-4760 solution: Positive control inhibitor solution; Blank control: Contains only buffer and substrate, without ACE enzyme.

[0049] Inhibition rate calculation: Based on the measured fluorescence intensity value, the inhibition rate of the inhibitor sample to be tested is calculated according to the following formula: Inhibition rate (%) = (RFU) / (Further fluorescence intensity value) 100%酶活性对照 - RFU 样品 ) / (RFU 100%酶活性对照 - RFU 空白对照 () × 100%. Where RFU represents relative fluorescence units.

[0050] Table 2 - Results of Inhibitory Activity Measurement Note: Case 1: AAGPAGGNLN; Case 2: EAGAQGPPGA; Case 3: GARGDK; Case 4: AAGPAGGNLN and EAGAQGPPGA mixed in equal mass; Case 5: AAGPAGGNLN and GARGDK mixed in equal mass; Case 6: EAGAQGPPGA and GARGDK mixed in equal mass; Case 7: AAGPAGGNLN, EAGAQGPPGA, and GARGDK mixed in a mass ratio of 1:1:1; the total peptide mass is the same in all cases.

[0051] The experimental results are shown in Table 2. All three peptides showed ACE inhibitory activity, with values ​​of AAGPAGGNLN (33.54%), EAGAQGPPGA (13.67%), and GARGDK (60.24%). When the three peptides were combined in a mass ratio (1:1:1), the ACE inhibition rate was measured to be 74.20%, which was significantly higher than that of the individual monomers, proving that the combination showed a good inhibitory effect on ACE.

[0052] The scope of protection of this invention is not limited to the above embodiments. Any modifications or alterations made by those skilled in the art without departing from the scope of the technical solution of this invention using the disclosed technical content are equivalent implementation cases and all fall within the scope of protection of this invention.

Claims

1. An angiotensin converting enzyme inhibitory activity peptide of fish mucus, characterized in that, The amino acid sequence of the turtle peptide is any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

2. The soft-shelled turtle peptide according to claim 1, characterized in that, The turtle peptide is chemically synthesized or obtained by enzymatic hydrolysis and purification of turtle protein.

3. A composition of anguiphankin having angiotensin converting enzyme inhibitory activity, characterized in that, The turtle peptide composition comprises at least one of the turtle peptides shown in SEQ ID NO:1~3.

4. The soft-shelled turtle peptide composition of claim 3, characterized in that, The turtle peptide composition comprises the three turtle peptides shown in SEQ ID NO:1 to SEQ ID NO:

3.

5. The soft-shelled turtle peptide composition of claim 3, wherein, It also includes pharmaceutical excipients.

6. The turtle peptide composition according to claim 5, characterized in that, The pharmaceutical excipients include solvents, fillers, dispersants, stabilizers, plasticizers, or flavoring agents.

7. The use of the turtle peptide composition according to any one of claims 3-6 in the preparation of antihypertensive drugs.

8. The application according to claim 7, characterized in that, The blood pressure-lowering drug is an angiotensin-converting enzyme (ACE) inhibitor.

9. The use of the turtle peptide according to claim 1 in the preparation of antihypertensive drugs or functional foods.

Images