A method for screening and synthesizing oyster ACE inhibitory peptides based on ligand fishing

Abstract

The present invention discloses a method for screening and synthesizing oyster ACE inhibitory peptides based on ligand fishing, which belongs to the technical field of bioactive peptides. The method includes the preparation of oyster meat hydrolysate, ultrafiltration separation, ligand fishing, polypeptide sequence identification and synthesis. The method utilizes the specific binding between ACE ligand and ACE inhibitory peptide to specifically screen oyster meat hydrolysate with a molecular weight of 3-8kDa with a high signal value, thereby greatly narrowing the screening range. At the same time, based on the oyster meat hydrolysate with a molecular weight of 3-8kDa, UPLC-MS is used to identify the polypeptide amino acid sequence, and solid phase synthesis and ACE inhibition rate determination are performed according to the polypeptide amino acid sequence to screen out a highly active ACE inhibitory peptide, the amino acid sequence of which is SLPWPMKPMNLIE, and the ACE inhibition rate is 70.1%, IC 50 The value is 0.177mmol / L. The method disclosed in the present invention is based on ligand fishing with high specificity and sensitivity, can provide a new option for the preparation of ACE inhibitory peptides, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and in particular relates to a method for screening and synthesizing oyster ACE inhibitory peptides based on ligand fishing. Background Art

[0002] With the rapid development of science and technology, people's quality of life has been greatly improved, and the proportion of high-calorie and high-fat in the diet has gradually increased, resulting in a significant increase in the incidence of cardiovascular diseases, chronic kidney disease, arteriosclerosis and stroke, among which hypertension is a recognized risk factor. Hypertension is a disease characterized by increased systolic and diastolic blood pressure in arterial blood vessels, accompanied by a variety of complications. Studies have shown that it affects 15-20% of the adult population worldwide and is showing an upward trend. The treatment and prevention of hypertension has become a major problem facing people.

[0003] In the development of drugs to control hypertension, ACE inhibitors and angiotensin receptor blockers are now clinically used to treat various cardiovascular diseases, such as captopril, lisinopril, enalapril, benazepril, etc. Although these drugs have been widely used in clinical treatment, long-term use will produce some adverse effects, such as cough, rash, angioedema, loss of taste, etc. Food-derived ACE inhibitory peptides have the characteristics of high biological activity, low toxicity and side effects, natural safety, and easy absorption, making them ideal preparation raw materials. Therefore, the development of natural and safe antihypertensive active substances has become a research hotspot today.

[0004] Oysters are a general term for mollusks of the order Pectinata and family Ostrea, commonly known as sea oysters, oysters, etc. They are one of the important marine biological resources available to humans and one of the most common shellfish along the coast of China. Oysters are rich in protein, amino acids, glycogen and other ingredients. They are not only of high nutritional value, but also have multiple physiological activities such as lowering blood pressure, lowering cholesterol, reducing inflammation, and anti-oxidation in their protein hydrolysates. They are an important source of marine active peptides and are widely used in the fields of special medical foods and health foods.

[0005] At present, obtaining high-purity ACE inhibitory peptides requires extraction, separation and purification, structural identification and other processes. The extraction methods include enzymatic hydrolysis, microbial fermentation, recombinant DNA, chemical synthesis and the like. The separation and purification methods include membrane separation, gel chromatography, ion exchange chromatography, reverse HPLC. For example, patent CN113024633A separates and purifies the enzymatic hydrolysate by a series of methods such as ultrafiltration, gel chromatography, and high-performance liquid chromatography, and combines bioinformatics screening to obtain a high-purity ACE inhibitory tetrapeptide. The existing separation system and pretreatment methods are relatively complex.

[0006] Ligand fishing technology is based on the affinity between molecules, and uses the interaction between unpurified mother liquor and active ligands to screen and determine the bioactive molecules bound to the ligands. It has the characteristics of high sensitivity, high specificity, high sensitivity, high precision, high throughput screening, simple operation, and rapid analysis. For example, patent CN104211765B uses the fishing method to screen peptides that specifically bind to TRB3 protein, a potential target for tumor treatment, with the characteristics of short detection time and simple operation. Therefore, the present invention discloses a method for screening and synthesizing oyster ACE inhibitory peptides based on ligand fishing, which not only expands new ideas for the research and development of ACE peptide antihypertensive products and the high-value utilization of production by-products, has good application value, but also provides scientific references for the in-depth research and development and utilization of oysters. Summary of the invention

[0007] Technical problems to be solved: In response to the above technical problems, the purpose of the present invention is to disclose a method for screening and synthesizing oyster ACE inhibitory peptides based on ligand fishing, which belongs to the field of bioactive peptide technology. The method includes the preparation of oyster meat hydrolysate, ultrafiltration separation, ligand fishing, polypeptide sequence identification and synthesis. This method utilizes the specific binding between ACE ligands and ACE inhibitory peptides to specifically screen oyster meat hydrolysates with molecular weights of 3-8kDa with high signal values, greatly narrowing the screening range. At the same time, based on the molecular weight of oyster meat hydrolysates of 3-8kDa, UPLC-MS is used to identify the amino acid sequence of the polypeptide, and solid phase synthesis and ACE inhibition rate are determined based on the polypeptide amino acid sequence to screen out a highly active ACE inhibitory peptide, the amino acid sequence of which is SLPWPMKPMNLIE, and the ACE inhibition rate is 70.1%, IC 50 The value is 0.177mmol / L. The method disclosed in the present invention is based on ligand fishing with high specificity and sensitivity, can provide a new option for the preparation of ACE inhibitory peptides, and has broad application prospects.

[0008] Technical solution: An oyster ACE inhibitory peptide synthesized based on ligand fishing screening, the amino acid sequence of the oyster ACE inhibitory peptide is: SLPWPMKPMNLIE.

[0009] A method for preparing an oyster ACE inhibitory peptide synthesized based on ligand fishing screening comprises the following steps:

[0010] S1. pre-treating the oysters and then performing enzymatic hydrolysis to obtain oyster meat enzymatic hydrolysate;

[0011] S2. separating the oyster meat hydrolysate by ultrafiltration to obtain oyster meat hydrolysates with molecular weights of <3 kDa, 3-8 kDa, and >8 kDa;

[0012] S3. The ACE ligand was amino-coupled and fixed on the CM5 chip, and then the oyster meat hydrolysates with molecular weights <3 kDa, 3-8 kDa and >8 kDa were injected into the CM5 chip channel coupled with ACE ligand at a flow rate of 30 μL / min for 180 s;

[0013] S4. Recover the components bound to ACE on the sensor recovery surface by using the Biacore T200 system to obtain samples recovered from the fishing test;

[0014] S5. Take an appropriate amount of the sample recovered from the fishing experiment and dissolve it in 0.1% formic acid aqueous solution, and use UPLC-MS to identify the amino acid sequence of the oyster ACE inhibitory peptide;

[0015] S6. Evaluate the potential activity of oyster ACE inhibitory peptides, screen peptides with scores > 0.5 for solid phase synthesis, and obtain synthetic peptides;

[0016] S7 used RP-HPLC to determine the purity of the synthetic peptides, and used ESI-MS mass spectrometry to identify the molecular weight and theoretical value of the synthetic peptides;

[0017] S8. Determine the ACE inhibition rate of the synthetic peptide and screen out ACE inhibitory peptides.

[0018] Furthermore, the specific enzymatic hydrolysis method of the oyster meat hydrolysate in step S1 is: fresh eviscerated oyster meat, add double distilled water at a material-liquid ratio of 1:3 to homogenize, then adjust the pH to 7.0, heat to 55°C in a constant temperature water bath, first add papain for constant temperature enzymatic hydrolysis for 4 h, then add neutral protease for constant temperature enzymatic hydrolysis for 3.5 h, inactivate the enzyme in a boiling water bath for 10 min, cool, centrifuge at 4°C and 4000rpm for 10 min, and take the supernatant for later use.

[0019] Furthermore, the amount of papain added is 4000 U / g, and the amount of neutral protease added is 4000 U / g, both based on the weight of oyster meat.

[0020] Furthermore, the ultrafiltration separation conditions of the oyster meat hydrolysate in step S2 are: under ice bath conditions, the oyster meat hydrolysate is ultrafiltered step by step through 8 kDa and 3 kDa ultrafiltration membranes to retain the oyster meat hydrolysate ultrafiltration components with molecular weights <3 kDa, 3-8 kDa and >8 kDa, and then stored for later use after evaporation concentration and freeze-drying.

[0021] Furthermore, the amino coupling method in step S3 is as follows: ACE with a concentration of 20 μg / mL is fixed on a CM5 chip, the running buffer is a phosphate buffer with a pH of 7.4, the ligand protein, sodium hydroxide, EDC, NHS, an empty test tube, and ethanolamine reagent are placed according to the instrument requirements, and the coupling ligand program is run.

[0022] Further, the determination conditions of UPLC-MS in step S5 are as follows: chromatographic column: Acclaim PePmap 100 chromatographic column with an inner diameter of 75 μm, a length of 2 cm, and a filler particle size of 3 μm, a flow rate of 0.4 μL / min, mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid, elution gradient: 0 min 5% B, 0-6 min 8% B, 6-40 min 30% B, 40-45 min 60% B, 45-56 min 80% B, positive ion mode, ESI spray voltage is 2.0 kV, primary mass spectrometry resolution is set to 70000, secondary scanning resolution is 17500, mass spectrometry accuracy: primary ±15 ppm, secondary ±20 ppm, primary scanning range is 350-2000 Da, and the ion quantity captured by the ion trap mass analyzer Orbitrap is 1e 5 -3e 6 The substances were identified by secondary scanning, and the amino acid sequences of the analyzed samples were retrieved from the oyster database using Mascot.

[0023] Furthermore, the oyster ACE inhibitory peptide is used in the preparation of medicines or health products for preventing hypertension and assisting in lowering blood pressure. Beneficial Effects

[0024] 1. The oyster ACE inhibitory peptide screened by the present invention has an amino acid sequence of SLPWPMKPMNLIE, which has good ACE inhibitory activity, with an ACE inhibition rate of 70.1% and an IC 50 The value is 0.177mmol / L, and it also exhibits a non-competitive inhibition relationship. It can bind to the active center of ACE to form a stable complex, inhibit its catalytic activity, and play a better spatial hindrance role in the binding of angiotensin I and ACE, thereby reducing the generation of angiotensin II and increasing the level of bradykinin.

[0025] 2. The oyster ACE inhibitory peptide screened out in the present invention is a substrate of CYP3A4, participates in the metabolism of organisms, exhibits the characteristics of non-toxicity and easy absorption during the metabolic process, can be absorbed or assimilated through the intestine, and can cross the blood-brain barrier without causing harmful effects on the brain.

[0026] 3. The method of the present invention utilizes ligand fishing technology to specifically bind ACE ligands to relevant bioactive peptides in oyster hydrolysates on the chip, which greatly reduces the preparation steps and time of existing preparation methods. It also has the characteristics of high specificity, high sensitivity, high precision, high throughput screening, simple operation, and rapid analysis. It can be efficiently and quickly compatible with subsequent biochemical analysis, structural identification and other related research technologies, making the research on bioactive peptide molecules tend to be integrated and automated.

[0027] 4. The screening method disclosed in the present invention has the characteristics of short detection time, convenient operation, high efficiency, etc., and is suitable for large-scale industrial production. It not only expands new ideas for the research and development of ACE peptide antihypertensive products and the high-value utilization of production by-products, has good application value, but also provides scientific reference for the in-depth research and development and utilization of oysters. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the enrichment of ACE with different ligands in Example 1;

[0029] Figure 2 It is the signal value of the ligand amino coupling and surface test in Example 2;

[0030] Figure 3 is the secondary mass spectrum of the specific peptide segment in Example 2;

[0031] Figure 4 is the purity analysis of the synthetic peptide in Example 2;

[0032] Figure 5 It is the mass spectrometry identification of the synthetic peptide in Example 2;

[0033] Figure 6 is the effect of different synthetic peptides on ACE inhibitory activity in Example 2;

[0034] Figure 7 is the effect of different synthetic peptide concentrations on ACE inhibition rate in Example 2;

[0035] Figure 8 It is the Lineweaver-Burk plot of T2 peptide against ACE in Example 2. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments:

[0037] Example 1 Determination of ACE concentration

[0038] Response values ​​at different pH and different ACE ligand concentrations

[0039] 100 μL PBS buffer was added to fully dissolve the ACE recombinant protein, which was 500 μg / mL ACE stock solution. 10 μg / mL ACE solutions with different pH values ​​(4.0, 4.5, 5.0, 5.5) were prepared using 10 mM sodium acetate buffer as diluent and tested on the instrument (flow rate 5 μL / mL, injection time 120 s, 50 mM sodium hydroxide solution as regeneration solution) to explore the effect of sodium acetate solutions with different pH values ​​on the response value.

[0040] 100 μL PBS buffer was added to fully dissolve the ACE recombinant protein, which was 500 μg / mL ACE stock solution. 10 mM sodium acetate buffer at pH 5.0 was used as the diluent to prepare ACE solutions of different concentrations (0, 10, 20, 40, 60 μg / mL), and the ACE solution was tested on the machine (flow rate 5 μL / mL, injection time 120 s, 50 mM sodium hydroxide solution as the regeneration solution) to explore the effect of different ACE concentrations on the response value.

[0041] Depend on Figure 1 A shows that the response value of the ligand solution changes the most when the pH is 5.0. When the pH is <5.0, the ACE ligand cannot be electrostatically adsorbed on the surface of the CM5 chip, so pH 5.0 sodium acetate buffer is selected as the diluent. Figure 1 B shows that when the ACE concentration is 10-20 μg / mL, the response value increases with the increase of concentration. When it is 20-60 μg / mL, the phenomenon of no enrichment occurs. It may be that the ligand original solution contains substances similar to surfactants. When the content is high, it affects the electrostatic adsorption between the ligand amino group and the glucose hydroxyl group on the chip surface. Therefore, the ACE concentration is selected as 20 μg / mL. Example 2

[0042] A method for preparing an oyster ACE inhibitory peptide synthesized based on ligand fishing screening comprises the following steps:

[0043] Step 1: Prepare oyster meat hydrolysate

[0044] The oysters were pretreated and double distilled water was added at a solid-liquid ratio of 1:3 to prepare a homogenate. The pH was then adjusted to 7.0 and heated to 55°C in a constant temperature water bath. Papain (4000 U / g oyster meat) was first added for constant temperature enzymolysis for 4 h, and then neutral protease (4000 U / g oyster meat) was added for constant temperature enzymolysis for 3.5 h. The enzyme was inactivated in a boiling water bath for 10 min, cooled, and centrifuged at 4°C and 4000 rpm for 10 min. The supernatant was taken for later use.

[0045] Step 2: Ultrafiltration separation of oyster meat hydrolysate

[0046] The oyster meat hydrolysate was separated step by step through 8 kDa and 3 kDa ultrafiltration membranes to obtain oyster meat hydrolysates with molecular weights of <3 kDa, 3-8 kDa and >8 kDa.

[0047] Step 3: ACE ligand coupling and fishing test

[0048] The ACE ligand with a concentration of 20 μg / mL was amino-coupled and fixed on the CM5 chip. Then, the oyster meat hydrolysate with molecular weight <3 kDa, 3-8 kDa and >8 kDa was injected into the CM5 chip channel coupled with ACE ligand at a flow rate of 30 μL / min for 180 s. The running buffer was a pH 7.4 phosphate buffer. The ligand protein, sodium hydroxide, EDC, NHS, empty test tube, and ethanolamine reagent were placed according to the instrument requirements, and the coupling ligand program was run.

[0049] The components bound to ACE on the sensor recovery surface were recovered by the Biacore T200 system to obtain samples recovered from the fishing test;

[0050] Determination of fishing signal value:

[0051] PBS was used as the negative control group, and the binding of samples of different molecular weights to ACE on the chip was analyzed from the response changes of the sensorgram.

[0052] Depend on Figure 2 It can be seen that pH 7.4 PBS buffer was used as the mobile phase, pH 5.0 sodium acetate buffer was used to prepare 20 μg / mL ACE2 ligand solution, and the ACE protein was fixed on the second channel of the CM5 chip through the amino coupling procedure. The final ligand coupling amount was 6078.4 RU. When PBS buffer and oyster hydrolysate of different ultrafiltration components flowed through the chip surface, the signal value corresponding to the 3-8 kDa component solution increased significantly, indicating that this component may contain components that bind to ACE ligands.

[0053] Step 4: Peptide sequence identification

[0054] An appropriate amount of samples recovered from the fishing experiment were dissolved in 0.1% formic acid aqueous solution, and the amino acid sequence of the oyster ACE inhibitory peptide was identified by UPLC-MS and tested separately.

[0055] Instrument conditions were as follows: chromatographic column: Acclaim PePmap 100 (75 μm×2 cm, 3 μm); flow rate: 0.4 μL / min; mobile phase A: water (containing 0.1% formic acid); mobile phase B: acetonitrile (containing 0.1% formic acid); elution gradient: 0 min 5% B, 0-6 min 8% B, 6-40 min 30% B, 40-45 min 60% B, 45-56 min 80% B; positive ion mode; ESI spray voltage was 2.0 kV; primary mass spectrometry resolution was set to 70000; secondary scanning resolution was 17500; mass spectrometry accuracy: primary ±15 ppm, secondary ±20 ppm; primary scanning range was 350-2000 Da, and the ion quantity captured by the ion trap mass analyzer Orbitrap was 1e 5-3e 6 The substances were identified by secondary scanning, and the amino acid sequences of the analyzed samples were retrieved from the oyster database using Mascot.

[0056] Table 1 Amino acid sequence identification and activity scoring

[0057] serial number sequence Protein Source Mass-to-charge ratio m / z Molecular weight SVM Score T1 LPMGWDKF Toll-like protein 497.24 992.5 -0.02 T2 SLPWPMKPMNLIE Toll-like protein 794.92 1586.8 0.14 T3 AIPGPPSPISTP Suppressor of hairless 567.32 1132.6 0.8 T4 DPVSIAAPSGLNI Suppressor of hairless 627.34 1252.7 -1

[0058] As shown in Table 1, PeptideRanker was used to predict the probability of biological activity of all peptides in the mass spectrometry identification results. The predicted scores were between 0 and 1. The closer the peptides were to 1, the more likely they were to have biological activity. There were four peptides with scores greater than 0.5. The secondary mass spectra of their peptides are shown in Figure 1. Figure 3 , Figure 3 A represents T1 peptide, B represents T2 peptide, C represents T3 peptide, and D represents T4 peptide. The SVM Score of T2 peptide and T3 peptide in AHTpin prediction is greater than 0, indicating that they have anti-hypertensive potential.

[0059] Step 5: Peptide synthesis and activity determination

[0060] The potential activity of oyster ACE inhibitory peptides was evaluated, and peptides with a screening score > 0.5 were subjected to solid phase synthesis to obtain synthetic peptides;

[0061] The purity of the synthetic peptide was determined by RP-HPLC. Analytical conditions: chromatographic column: Kromasil C18 (4.6×150 mm, 5 μm); mobile phase A: acetonitrile (containing 1% trifluoroacetic acid); mobile phase B: water (containing 1% trifluoroacetic acid); elution gradient: 0-0.01 min 5% A, 0.01-25 min 50% A, 25-30 min 90% A; flow rate: 1 mL / min; detection wavelength: 214 nm; injection volume: 20 μL, chromatographic peak area was calculated by automatic integration, and the purity calculation formula was: peptide purity (%) = (peptide chromatographic peak area / total chromatographic peak area) × 100.

[0062] ESI-MS mass spectrometry was used to identify the molecular weight and theoretical value of the synthetic peptide. Instrument conditions: ESI ion source; positive ion mode; scanning range: 0-2000 Da; mobile phase A: water (containing 1% formic acid); mobile phase B: acetonitrile (containing 1% formic acid); flow rate: 0.2 mL / min; the ACE inhibition rate of the synthetic peptide was determined, and ACE inhibitory peptides were screened.

[0063] Peptide synthesis activity prediction

[0064] The potential activity of peptides was scored using the Peptide Ranker tool, and peptides with a screening score > 0.5 were subjected to solid-phase synthesis (purity ≥ 95%), their in vitro ACE inhibition rate was determined, and the inhibitory activity was predicted using AHTpin.

[0065] Depend on Figure 4 It can be seen that Figure 4 A represents T1 peptide, B represents T2 peptide, C represents T3 peptide, and D represents T4 peptide. The RP-HPLC purification results show that the main peak accounts for the majority, with only very few impurity peaks appearing. The purity of T1 peptide, T2 peptide, T3 peptide, and T4 peptide are calculated to be 95.80%, 98.19%, 97.30%, and 99.73%, respectively, and the purity of each peptide segment is greater than 95%.

[0066] Depend on Figure 5 It can be seen that Figure 5 A represents T1 peptide, B represents T2 peptide, C represents T3 peptide, and D represents T4 peptide. The synthetic peptides were identified by ESI-MS mass spectrometry. The molecular weight and theoretical value on the mass spectrum were used to determine whether they were the target products. The main ion peak mass-to-charge ratio of T1 peptide was 497.64 m / z, with two charges, which was Figure 3 The ion peaks of the T1 peptide in A are close, so it can be considered that the synthetic peptide is consistent with the peptide sequence LPMGWDKF identified by the mass spectrometry of the recovered samples of the experimental group. After comparison, the main ion peaks of the T3 peptide and the T4 peptide are similar to the mass spectrometry results of the recovered samples; the mass-to-charge ratio of the main ion peak of the T2 peptide is 794.92m / z, and the mass-to-charge ratio of the synthetic peptide is 778.79m / z. Because the former has a variable modification O at the methionine (M) in the peptide, the molecular weight difference is 32.

[0067] Determination of inhibitory activity of synthetic ACE inhibitory peptides

[0068] 1mM FAPGG solution, 0.1U / mL ACE solution and 0.5mg / mL peptide solution were prepared with 80mM HEPES buffer (containing 300mM NaCl, pH8.2). 40μL 1mM FAPGG solution, 50μL 0.5mg / mL peptide solution, 10μL 0.1U / mL ACE solution were mixed with 100μL HEPES buffer, and HEPES buffer was used instead of peptide solution as blank control. After preheating at 37°C for 5min, the absorbance of sample well (A1) and blank well (B1) was measured at 340nm, and the absorbance of A2 and B2 was measured after continuing to react at 37°C for 30min. The ACE inhibition rate was calculated using the formula: ACE inhibition rate (%) = 100×(ΔB-ΔA) / ΔB, where ΔA = A1-A2, ΔB = B1-B2.

[0069] According to the above method, the ACE inhibition rates of synthetic peptides T1 (0.6, 0.8, 1.0, 1.2, 1.4 mg / mL), T2 (0.05, 0.10, 0.15, 0.20, 0.40 mg / mL), T3 (0.5, 0.75, 1.0, 1.25, 1.5 mg / mL), and T4 (0.1, 0.2, 0.4, 0.6, 0.8 mg / mL) were determined, and GraphPadPrism9 was used to plot the ACE inhibition rate (%) against lg (sample concentration) to calculate the half inhibitory concentration IC 50 .

[0070] To further clarify the in vitro ACE inhibitory activity of synthetic peptides, the inhibition rate of different synthetic peptides on ACE enzyme at the same concentration (C = 0.5 mg / mL) was tested.

[0071] Depend on Figure 6 It can be seen that the inhibition rates of T1, T2, T3 and T4 peptides were 24.2±2.9%, 70.1±1.0%, 25.5±0.4% and 32.5±1.2%, respectively.

[0072] Depend on Figure 7 It can be seen that A, B, C, and D represent the fitting curves of different concentrations of T1 peptide, T2 peptide, T3 peptide, and T4 peptide and ACE inhibition rate, respectively. The IC 50 The values ​​were 2.919mmol / L, 0.177mmol / L, 4.372mmol / L, and 1.190mmol / L, respectively. The IC 50 At the lowest level, it is a potential ACE inhibitory peptide.

[0073] Kinetic analysis of enzyme inhibition by T2 peptide

[0074] The reaction rate of FAPGG solution (0.1, 0.125, 0.16, 0.25, 0.5 mmol / L) containing T2 peptide and ACE was measured, and a Lineweaver-Burk double reciprocal plot was prepared with the reciprocal of the enzymatic reaction rate (1 / v) as the ordinate and the reciprocal of the substrate concentration (1 / S) as the abscissa to calculate the Michaelis constant K m and the maximum enzymatic reaction rate V max .

[0075] Table 2 V of different concentrations of T2 peptide max and K m value

[0076] parameter 0 mg / mL 0.2 mg / mL 0.4 mg / mL <![CDATA[V max (mmol / L / min)]]> 1.23 0.032 0.034 <![CDATA[K m (mmol)]]> 0.01 0.01 0.01

[0077] From Table 2 and Figure 8 It can be seen that with the increase of T2 peptide concentration, V max Reduce, Km Its inhibition mode to ACE is non-competitive.

[0078] Analysis of the absorption, distribution, metabolism, excretion and toxicity of T2 peptide

[0079] The AdmetSAR online tool was used to analyze and predict the absorption, distribution, metabolism, and excretion characteristics of T2 peptides, and the online tool ToxinPred was used to predict peptide toxicity.

[0080] Table 3 Prediction results of absorption, distribution, metabolism, excretion and toxicity (ADMET) analysis of T2 peptide

[0081] Peptide sequence SLPWPMKPMNLIE Blood-brain barrier permeability 0.9919 Human intestinal absorption value 0.9226 CYP2C9 substrates Non-substrate CYP2D6 substrates Non-substrate CYP3A4 substrates Substrate CYP1A2 inhibitors Non-inhibitor CYP2C9 inhibitors Non-inhibitor CYP2D6 inhibitors Non-inhibitor CYP2C19 inhibitors Non-inhibitor CYP3A4 inhibitors Non-inhibitor Water Solubility -3.2163 toxicity Non-toxic

[0082] As shown in Table 3, the human intestinal absorption value of T2 peptide is positive, indicating that the peptide can be absorbed or assimilated through the intestine, and its blood-brain barrier is positive, indicating that it can pass through the blood-brain barrier during metabolism, but will not have a harmful effect on the brain; cytochrome P450 is an important biosynthetic enzyme for drug metabolism, especially CYP1, CYP2, and CYP3 families are the most important enzyme systems involved in the metabolism of different substances, and each cytochrome P450 enzyme has a unique substrate specificity. The peptide is predicted to be a substrate of CYP3A4 and participates in biological metabolism. In addition, the peptide is predicted to be a soluble protein, and the ToxinPred results show that it is non-toxic, and can be further used in mouse in vivo experiments.

[0083] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. An oyster ACE inhibitory peptide synthesized based on ligand fishing screening, characterized in that: The amino acid sequence of the oyster ACE inhibitory peptide is: SLPWPMKPMNLIE.

2. Use of the oyster ACE inhibitory peptide synthesized based on ligand fishing screening according to claim 1 in the preparation of drugs for treating hypertension.

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