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CN113480598B - Preparation method and application of bioactive tetrapeptides - Google Patents

Preparation method and application of bioactive tetrapeptides Download PDF

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CN113480598B
CN113480598B CN202110909484.1A CN202110909484A CN113480598B CN 113480598 B CN113480598 B CN 113480598B CN 202110909484 A CN202110909484 A CN 202110909484A CN 113480598 B CN113480598 B CN 113480598B
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bioactive
ace
tetrapeptide
buffer solution
borate buffer
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CN113480598A (en
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苏永昌
刘智禹
刘淑集
陈贝
乔琨
许旻
陈晓婷
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Fisheries Research Institute Of Fujian (fujian Aquatic Disease Prevention Center)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention discloses a bioactive tetrapeptide, a preparation method and application thereof, belonging to the technical field of bioactive peptides. A method for preparing bioactive tetrapeptides, comprising the following steps: s0: preparation of immobilized ACE gel affinity adsorption column, S1: preparation of peptide component, S2: affinity adsorption and elution of peptide components, S3: peptide sequence identification, S4: and verifying ACE inhibition activity of the synthesized bioactive tetrapeptide. The invention discloses a bioactive tetrapeptide and a preparation method and application thereof, and the prepared immobilized ACE gel affinity adsorption column is used for screening the bioactive tetrapeptide, has the characteristics of rapid and efficient screening, and simple and easy operation, can be used for rapid large-scale screening of the bioactive tetrapeptide, and adopts puffer fish skin as a raw material to obtain the amino acid sequence of the bioactive tetrapeptide, and the prepared bioactive tetrapeptide has ACE inhibitory activity and DPP-IV inhibitory activity.

Description

Preparation method and application of bioactive tetrapeptides
Technical Field
The invention belongs to the technical field of active peptides, and particularly relates to a preparation method and application of bioactive tetrapeptides.
Background
With the progress and development of society, the living standard of people is gradually improved, the proportion of fat and heat in diet is gradually increased, and the incidence of cardiovascular diseases in China is rapidly increased due to the reduction of exercise quantity, wherein hypertension is dominant.
Under normal conditions, the human body can ensure that the source and the way of blood sugar are kept balanced through two large regulating systems, namely hormone regulation and nerve regulation, so that the blood sugar is maintained at a certain level. However, under the combined action of genetic factors (such as family history of diabetes) and environmental factors (such as unreasonable diet, obesity, etc.), the two major regulatory functions are disturbed, and rise of blood glucose level occurs.
The puffer fish is a fish in a warm zone and a tropical offshore bottom layer, inhabits the middle and lower layers of the ocean, a small number of species enter fresh water rivers, and when the fish encounters external danger, the whole body is enabled to float on the water surface in a spherical shape, and small thorns on the skin are erected at the same time, so that the fish is self-defended.
Angiotensin Converting Enzyme (ACE) inhibitory peptide is a small molecular polypeptide formed after proteolysis, has remarkable blood pressure reducing effect, and is more effective than other common blood pressure reducing medicines, and the ACE inhibitory peptide has no toxic or side effect and has no influence on normal blood pressure. ACE plays an important role in blood pressure regulation, and through excision of two amino acids (His-Leu) at the carbon end, the originally inactive angiotensin I can be converted into active angiotensin II, so that vasoconstriction is caused, and blood pressure is increased; ACE can deactivate skin soothing and activating functions with vasodilation, and also causes blood pressure rising, and ACE inhibitory peptide can block two biochemical reaction processes caused by ACE, so as to play a role in reducing blood pressure.
The existing preparation of bioactive tetrapeptides requires adsorption and elution treatment of peptide components after enzymolysis, wherein an adsorption column plays a vital role, and particularly, the packing of the adsorption column influences the elution efficiency.
The existing technology for preparing bioactive tetrapeptides from foods such as hazelnuts, yak milk, shellfish meat and medlar is in need of technology for preparing bioactive tetrapeptides by using puffer fish as a raw material.
Disclosure of Invention
The invention aims to provide a preparation method and application of bioactive tetrapeptides, wherein the prepared immobilized ACE gel affinity adsorption column is used for screening bioactive tetrapeptides, has the characteristics of rapid and efficient screening, simple and easy operation, can be used for rapid large-scale screening of bioactive tetrapeptides, adopts puffer fish skin as a raw material to obtain bioactive tetrapeptides amino acid sequences, and has ACE inhibitory activity and DPP-IV inhibitory activity, can be used for preparing antihypertensive drugs and dipeptidyl peptidase IV inhibitors, and has good blood pressure and blood glucose reducing effects.
To achieve the purpose, the invention adopts the following technical scheme:
the amino acid sequence of the bioactive tetrapeptide is YVLL.
Preferably, the biologically active tetrapeptide YVLL is ACE inhibition IC 50 The value is 0.25-0.26 umol/mL.
Preferably, the DPP-IV inhibition rate IC of bioactive tetrapeptide YVLL 50 The value is 1.25-1.35 mg/mL.
The invention also provides a preparation method of the bioactive tetrapeptide, which comprises the following steps: s0: preparation of immobilized ACE gel affinity adsorption column, S1: preparing a peptide component, namely taking fresh fish skin of the puffer fish, mincing, adding water, homogenizing to form fish skin slurry liquid, adding protease for enzymolysis, performing enzyme deactivation treatment after enzymolysis, cooling, regulating the pH value to be neutral to obtain an enzymolysis liquid, performing microfiltration on the enzymolysis liquid, and performing ultrafiltration treatment to obtain the peptide component with the molecular weight less than 1kDa, wherein S2: affinity adsorption and elution of peptide components, carrying out affinity adsorption and elution on the peptide components with molecular weight smaller than 1kDa obtained in the step S1 by using an immobilized ACE gel affinity adsorption column prepared in the step S0, carrying out ultraviolet detection, collecting elution peak components with the wavelength of 220nm, respectively named as 1 st to 24 th components, then carrying out ACE activity measurement, selecting 16 th components for desalination, concentrating under reduced pressure for later use, and carrying out S3: and (3) identifying the sequence of the peptide, carrying out mass spectrometry on the 16 th component after the decompression concentration in the step S2, and analyzing the mass spectrometry result to obtain the bioactive tetrapeptide YVLL, S4: and (3) performing solid-phase synthesis on the bioactive tetrapeptide YVLL according to the amino acid sequence determined in the step S3, and verifying ACE inhibition activity of the synthesized bioactive tetrapeptide.
Preferably, the specific step of step S0 is: s01: ACE is dissolved in borate buffer to prepare an ACE chelating ligand solution, S02: adding dilute hydrochloric acid into cyanogen bromide activated agarose gel 4B for swelling, filtering, washing with borate buffer solution, filtering, and removing residual HCl to obtain cyanogen bromide activated agarose 4B filler, S03: mixing the ACE chelating ligand solution with cyanogen bromide activated agarose 4B filler, uniformly stirring, chelating overnight, and after chelating, cleaning and filtering with 4-6 times of borate buffer solution with the volume of the cyanogen bromide activated agarose 4B filler to remove redundant ACE, S04: uniformly mixing the filtered cyanogen bromide activated agarose 4B filler with Tris-HCl buffer solution, carrying out gentle shaking, and filtering, wherein S05: after filtration, acetate buffer and Tris-HCl buffer are adopted to alternately wash and filter for 3-5 times to obtain ACE immobilized gel filler, the ACE immobilized gel filler is suspended in borate buffer and filled into a chromatographic column, and the immobilized ACE gel affinity adsorption column is obtained.
Preferably, the enzyme activity unit of ACE is 4.5-5.5U/ml, the pH of the borate buffer solution is 8.1-8.4, the molar concentration is 0.08-0.12M, the borate buffer solutions of the rest steps except the borate buffer solution of the step S05 all contain 0.45-0.55M NaCl, and the mass volume ratio of the cyanogen bromide activated agarose gel 4B to the diluted hydrochloric acid is 0.8-1.2g:5mL, 0.9-1.1mM of dilute hydrochloric acid, 0-4 ℃ of chelation overnight, 7.9-8.1 of Tris-HCl buffer solution, 0.08-0.12M of molar concentration, 0.45-0.55M of NaCl of Tris-HCl buffer solution in step S05, 23-27 ℃ of gentle shaking temperature, 1.8-2.2h of shaking time, 3.8-4.2 of acetate buffer solution, 0.08-0.12M of molar concentration and 0.45-0.55M of NaCl.
Preferably, in the step S1, alkaline protease with the unit of enzyme activity of 5 ten thousand U/g is adopted for enzymolysis, the enzymolysis condition is that the addition amount of the alkaline protease is 0.8-1.2 percent of the mass of the fish skin slurry liquid, the pH value is 7.8-8.2, the enzymolysis temperature is 58-62 ℃, the enzymolysis time is 5.8-6.2 hours, and the fish skin slurry liquid is mincedThe added water is distilled water, the mass volume ratio of fresh fish skin of the puffer fish to the distilled water is 0.9-1.1kg:10L, a tissue homogenizer with the rotating speed of 11000-13000rpm/min is adopted for homogenization, enzyme deactivation treatment is carried out by boiling and heating for 9-11min after enzymolysis, the pH value is regulated to be neutral by HCl, the microfiltration is carried out by adopting a ceramic membrane element with the diameter of 30mm multiplied by 800mm, the filtering precision is 200nm, and the membrane flux is 600-1000L/M 2 And H, ultrafiltering by adopting a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa.
Preferably, step S2 specifically includes: s21: on a medium-pressure protein purification instrument, an immobilized ACE gel affinity adsorption column prepared in the step S0 is installed, borate buffer with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1 is used for balancing for 18-22min, the balancing flow rate is 0.45-0.55mL/min, and the immobilized ACE gel affinity adsorption column is detected by an ultraviolet detector, the ultraviolet detection wavelength is 220nm, S22: and (3) carrying out loading adsorption of the peptide component with the molecular weight smaller than 1kDa, which is obtained in the step S1, wherein the loading amount is 4.5-5.5mL, the mobile phase is balanced by using borate buffer with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1, the flow rate is 0.45-0.55mL/min, the peptide component is loaded and eluted for 14-16min after the detection baseline is stable, the borate buffer with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1 is used as an eluting mobile phase, the borate buffer contains 0.8-1.2M NaCl, the eluting flow rate is 0.45-0.55mL/min, eluting peak components are collected, S23 is carried out by using borate buffer with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1 again, the peptide component is loaded and eluted for multiple times according to the step S22, the eluting peak components are combined, the eluting peak components are respectively designated as 1 st-24 th components, the active components are subjected to the step S22, the concentration is carried out, and the concentration is carried out, and the molecular weight is 100D is determined, and the concentration is carried out.
Preferably, in step S3, mass spectrometry is performed using LC-MS/MS under the following conditions: the chromatographic column is PepMap RPLC C 18 75 μm i.d. ×150mm, 3 μm,100 a, cationic mode, scan range: m/z 300-1500 Da, emitter spray voltage 2-kV, and analysis of the result of mass spectrometry by PEAKS STUDIO software from the head sequencing de novo Sequencing to obtain highly reliable tetrapeptide amino acid sequence.
The invention also provides application of the bioactive tetrapeptides in preparation of antihypertensive drugs.
The invention also provides application of the bioactive tetrapeptide in preparation of dipeptidyl peptidase IV inhibitor.
The beneficial effects of the invention are as follows:
1. the bioactive tetrapeptide YVLL prepared by using the puffer fish skin as a raw material has ACE inhibition activity, can be applied to antihypertensive drugs, and has good antihypertensive effect.
2. The prepared immobilized ACE gel affinity adsorption column is used for screening bioactive tetrapeptides, has the characteristics of rapid and efficient screening, and simple and easy operation, and can be used for rapid large-scale screening of bioactive tetrapeptides.
3. The prepared bioactive tetrapeptide YVLL has DPP-IV inhibitory activity, can be applied to dipeptidyl peptidase IV inhibitors, and has good blood glucose reducing effect.
4. The bioactive tetrapeptide YVLL and ACE form a hydrogen bond network, and the two have 8 hydrogen bond interactions and high stability.
5. Bioactive tetrapeptide YVLL Val2 skeleton amide oxygen atom has electronegativity and can be combined with Zn of site 2+ A stronger polar effect is formed, thereby destroying the functions of Zn ions.
6. Bioactive tetrapeptides YVLL are all hydrophobic residues that can bind pocket residues to form strong hydrophobic interactions and van der waals potential energy.
Drawings
FIG. 1 is a graph showing ACE inhibitory activity of the 1 st to 24 th fractions collected in step S23 of the present invention.
FIG. 2 is an affinity elution profile of an immobilized ACE gel affinity adsorption column of the present invention for a peptide component.
FIG. 3 is a graph showing the concentration of the bioactive tetrapeptide YVLL of the present invention versus ACE inhibition.
FIG. 4 is a graph showing the binding pattern of the bioactive tetrapeptide YVLL of the present invention to ACE.
FIG. 5 shows the inhibition pattern and mechanism of action of the bioactive tetrapeptides YVLL and ACE of the present invention.
FIG. 6 is a graph showing the antihypertensive effect of the bioactive tetrapeptide YVLL of the present invention on SHR rats.
FIG. 7 is a graph showing the DPP-IV inhibitory effect of the bioactive tetrapeptide YVLL of the present invention.
Detailed Description
The invention will now be further described with reference to the drawings and detailed description.
The amino acid sequence of the bioactive tetrapeptide provided in this example is YVLL.
The embodiment also provides a preparation method of the bioactive tetrapeptide, which comprises the following steps:
s0: preparation of immobilized ACE gel affinity adsorption column
An ACE chelating ligand solution was prepared by dissolving ACE having an enzyme activity unit of 5U/ml in 50ml of 0.1M borate buffer (pH 8.3, containing 0.5M NaCl). 10g of cyanogen bromide activated agarose 4B filler was weighed into a 100ml beaker, 50ml of 1mM HCl was added to swell, and after filtration, the mixture was washed with 0.1M borate buffer (pH 8.3, containing 0.5M NaCl) and dried by filtration to remove residual HCl, thereby obtaining cyanogen bromide activated agarose 4B filler. The ACE chelating ligand solution was mixed with cyanogen bromide activated agarose 4B filler, stirred well, and then chelated overnight at 4 ℃, after which the chelating was washed and filtered with 5 volumes of cyanogen bromide activated agarose 4B filler in 0.1M borate buffer (pH 8.3, containing 0.5M NaCl) to remove excess ACE. The dried cyanogen bromide activated agarose 4B filler was mixed with 50mL of 0.1M Tris-HCl buffer (pH 8.0) and gently shaken at 25℃for 2h. After filtration, 0.1M acetate buffer (pH 4.0, containing 0.5M NaCl) and 0.1M Tris-HCl buffer (pH 8.0, containing 0.5M NaCl) were alternately washed and filtered 3 times to obtain ACE immobilized gel filler. The ACE immobilized gel filler is suspended in 0.1M borate buffer (pH 8.3) and filled into a chromatographic column (phi 0.9X1.57 cm) with the column volume of 5ml, namely the immobilized ACE gel affinity adsorption column.
S1: preparation of peptide component
Taking 1000g of fresh fish skin of puffer fish, mincing, adding 10L of distilled water, homogenizing at 12000rpm/min in a tissue homogenizer to form fish skin slurry liquid, and adding alkaline solutionThe addition amount of protease (5U/g) is 1%, the pH value is 8.0, the enzymolysis is carried out for 6 hours at the temperature of 60 ℃, the enzymolysis is carried out, the boiling and the heating are carried out for 10 minutes, the enzyme deactivation treatment is carried out, and the pH value is regulated to be neutral by HCl after cooling. The enzymolysis liquid is applied to ceramic membrane elements with phi 30mm multiplied by 800mm (the filtering precision is 200nm, 600-1000L/M) 2 H) After microfiltration, ultrafiltration treatment is carried out by using a polyethersulfone ultrafiltration membrane (with a molecular weight cut-off of 1 kDa) to obtain a peptide component with a molecular weight of less than 1 kDa.
S2: affinity adsorption and elution of peptide components
S21: the immobilized ACE gel affinity adsorption column prepared in the step S0 is mounted on a medium-pressure protein purification instrument (AKTA pure 25), balanced for 20min by using 0.1M borate buffer (pH 8.3) and the balanced flow rate is 0.5mL/min, and detected by an ultraviolet detector, wherein the ultraviolet detection wavelength is 220nm.
S22: after equilibration, the peptide fractions having a molecular weight of less than 1kDa obtained in step S1 were subjected to adsorption by 5mL of a mobile phase of 0.1M borate buffer (pH 8.3), a flow rate of 0.5mL/min, elution was performed 15min after a stationary detection baseline was observed, with 0.1M borate buffer (pH 8.3 containing 1M NaCl) as the eluting mobile phase, an elution flow rate of 0.5mL/min, and the eluting peak fractions were collected.
S23, after the immobilized ACE gel affinity adsorption column is re-equilibrated for 20min by using 0.1M borate buffer (pH 8.3), peptide components are loaded and eluted for a plurality of times according to the step S22, eluting peak components for a plurality of times are combined and respectively named as 1 st to 24 th components, and then ACE activity measurement is carried out, as shown in figure 1, the 3 rd, 16 th and 17 th components have higher ACE inhibition activity.
The affinity elution profile of the immobilized ACE gel affinity adsorption column for the peptide component is shown in fig. 2.
S3: and (3) identifying the sequence of the peptide, carrying out mass spectrometry on the 16 th component after the decompression concentration in the step S2, analyzing the result of the mass spectrometry to obtain the bioactive tetrapeptide YVLL, and specifically, carrying out mass spectrometry by adopting LC-MS/MS under the following measurement conditions: the chromatographic column is PepMap RPLC C 18 , 75μm i.dX 150mm, 3 μm,100 a, cationic mode, scan range: m/z 300-1500 Da, emitter spray voltage 2-kV, and analysis of the result of mass spectrometry by PEAKS STUDIO software from the head sequencing de novo Sequencing to obtain highly reliable tetrapeptide amino acid sequence.
S4: and (3) performing solid-phase synthesis on the bioactive tetrapeptide YVLL according to the amino acid sequence determined in the step S3, and verifying ACE inhibition activity of the synthesized bioactive tetrapeptide. The results show that the bioactive tetrapeptide YVLL has good ACE inhibition activity and IC of ACE inhibition rate of the bioactive tetrapeptide YVLL 50 The value was 0.2560umol/mL (0.1297 mg/mL).
The concentration of bioactive tetrapeptide YVLL versus ACE inhibition is shown in figure 3.
Mechanism research:
simulation analysis of the docking mechanism of bioactive tetrapeptides YVLL and ACE was performed using Discovery Studio software, with the following results:
as shown in fig. 4-5, a hydrogen bond network is formed between YVLL and ACE. The two share 8 hydrogen bond interactions. These hydrogen bonding interactions are critical for the stable binding of polypeptides to ACE. Binding pocket residues that form hydrogen bonding with polypeptides include: ala356, tyr523, ala354, ala354, gln281, lys511, tyr520, his513 and the like. The polypeptide Val2 skeleton amide oxygen atom has electronegativity and can be combined with Zn of a binding site 2+ A stronger polar effect is formed, thereby destroying the functions of Zn ions. The polypeptides are all hydrophobic residues, and the hydrophobic residues can combine with pocket residues to form stronger hydrophobic effect and Van der Waals potential energy.
The invention also provides application of the bioactive tetrapeptides in preparation of antihypertensive drugs.
Animal hypotension experiments:
using SHR rats as animal models, YVLL was tested at a dose of 20 mg/kg (rat body weight) by a one-time tail intravenous injection, with a captopril tail intravenous injection dose of 5 mg/kg (rat body weight) as a drug control group.
As shown in fig. 6, the results showed that YVLL decreased 30.5%, 21.1%, 27.2%, 28.4% and 20.2% after tail vein injection by systolic blood pressure of 140.7, 159.9, 147.5, 145.1 and 161.6mmHg, respectively, and had better antihypertensive effect than captopril control group after 2, 4, 6, 12 and 24 hours. After one injection of YVLL, the blood pressure reducing effect can last for more than 24 hours. Experiments show that the YVLL has good blood pressure reducing effect.
The invention also provides application of the bioactive tetrapeptide in preparation of dipeptidyl peptidase IV inhibitor.
Determination of polypeptide inhibition DPP-IV Activity:
DPP-IV inhibitor, namely dipeptidyl peptidase IV inhibitor, is a medicament for treating type 2 diabetes, can inhibit inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), improve endogenous GLP-1 and GIP levels, promote insulin release by islet beta cells, and inhibit glucagon secretion by islet alpha cells, thereby improving insulin levels, reducing blood glucose, and being difficult to induce hypoglycemia and increase body weight.
As shown in FIG. 7, the results of measuring DPP-IV inhibition activity of bioactive tetrapeptide YVLL show that bioactive tetrapeptide YVLL has good DPP-IV inhibition activity, good blood sugar reducing effect and IC of DPP-IV inhibition rate of bioactive tetrapeptide YVLL 50 The value was 1.3mg/mL.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand that; the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> institute for aquatic products in Fujian province (center for controlling diseases in Fujian aquatic products)
<120> a bioactive tetrapeptide, its preparation method and application
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 4
<212> PRT
<213> Fugu (Takiugu)
<400> 1
Tyr Val Leu Leu
1

Claims (7)

1. A preparation method of bioactive tetrapeptides is characterized in that,
the method comprises the following steps:
s0: preparing an immobilized ACE gel affinity adsorption column;
s1: preparing peptide components, namely taking fresh fish skin of puffer fish, mincing, adding water, homogenizing to form fish skin slurry liquid, adding protease for enzymolysis, adopting alkaline protease with the enzyme activity unit of 5 ten thousand U/g for enzymolysis, wherein the enzymolysis condition is that the addition amount of the alkaline protease is 0.8-1.2% of the mass of the fish skin slurry liquid, the pH value is 7.8-8.2, the enzymolysis temperature is 58-62 ℃, the enzymolysis time is 5.8-6.2h, carrying out enzyme deactivation treatment after enzymolysis, cooling, regulating the pH value to be neutral, obtaining enzymolysis liquid, carrying out microfiltration on the enzymolysis liquid, and then carrying out ultrafiltration treatment, thus obtaining peptide components with the molecular weight less than 1 kDa;
s21: installing the immobilized ACE gel affinity adsorption column prepared in the step S0 on a medium-pressure protein purification instrument, balancing with borate buffer solution with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1 for 18-22min, balancing the flow rate of 0.45-0.55mL/min, and detecting in an ultraviolet detector, wherein the ultraviolet detection wavelength is 220nm;
s22: performing loading adsorption of the peptide component with the molecular weight smaller than 1kDa obtained in the step S1 after balancing, wherein the loading amount is 4.5-5.5mL, the mobile phase is subjected to borate buffer solution with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1, the flow rate is 0.45-0.55mL/min, 14-16min after the detection baseline is stable is observed, the borate buffer solution with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1 is used as an elution mobile phase for eluting, the borate buffer solution contains 0.8-1.2M NaCl, the elution flow rate is 0.45-0.55mL/min, and the elution peak component is collected;
s23, re-balancing the immobilized ACE gel affinity adsorption column with borate buffer solution with the molar concentration of 0.08-0.12 and M, pH of 7.9-8.1 for 18-22min, loading and eluting peptide components for multiple times according to the step S22, combining the eluting peak components for multiple times, desalting the eluting peak components by using a dialysis bag with the molecular weight cut-off of 100D, and concentrating under reduced pressure for later use;
s3: and (3) identifying the sequence of the peptide, carrying out mass spectrometry on the elution peak component after the decompression concentration in the step (S2), and carrying out mass spectrometry by adopting LC-MS/MS under the following conditions: the chromatographic column is PepMap RPLC C 18 75 μm i.d. ×150mm, 3 μm,100 a, cationic mode, scan range: m/z 300-1500 Da, emitter spraying voltage 2-kV, analyzing the result of the mass spectrometry by PEAKS STUDIO software to obtain the amino acid sequence of one bioactive tetrapeptide which is YVLL;
s4: and (3) performing solid-phase synthesis on the bioactive tetrapeptide YVLL according to the amino acid sequence determined in the step S3, and verifying ACE inhibition activity of the synthesized bioactive tetrapeptide.
2. The method for preparing bioactive tetrapeptides according to claim 1, wherein,
the specific steps of the step S0 are as follows:
s01: dissolving ACE in borate buffer solution to prepare ACE chelating ligand solution;
s02: adding dilute hydrochloric acid into cyanogen bromide activated agarose gel 4B for swelling, filtering, washing with borate buffer solution, and filtering to remove residual HCl to obtain cyanogen bromide activated agarose 4B filler;
s03: mixing the ACE chelating ligand solution with cyanogen bromide activated agarose 4B filler, uniformly stirring, chelating overnight, and after chelating, cleaning and filtering with 4-6 times of borate buffer solution with the volume of the cyanogen bromide activated agarose 4B filler to remove redundant ACE;
s04: uniformly mixing the filtered cyanogen bromide activated agarose 4B filler with Tris-HCl buffer solution, and filtering after gentle shaking;
s05: after filtration, acetate buffer and Tris-HCl buffer are adopted to alternately wash and filter for 3-5 times to obtain ACE immobilized gel filler, the ACE immobilized gel filler is suspended in borate buffer and filled into a chromatographic column, and the immobilized ACE gel affinity adsorption column is obtained.
3. The method for preparing bioactive tetrapeptides according to claim 1, wherein,
the unit of the enzyme activity of ACE is 4.5-5.5U/ml;
the pH of the borate buffer solution is 8.1-8.4, the molar concentration is 0.08-0.12M, and the borate buffer solutions of the rest steps except the borate buffer solution of the step S05 all contain 0.45-0.55M NaCl;
the mass volume ratio of the cyanogen bromide activated agarose gel 4B to the dilute hydrochloric acid is 0.8-1.2g:5mL of diluted hydrochloric acid with a molar concentration of 0.9-1.1mM;
the temperature condition for chelation overnight is 0-4 ℃;
the pH of the Tris-HCl buffer solution is 7.9-8.1, the molar concentration is 0.08-0.12M, and the Tris-HCl buffer solution in the step S05 contains 0.45-0.55M NaCl;
the oscillation temperature of the gentle oscillation is 23-27 ℃ and the oscillation time is 1.8-2.2h;
the acetate buffer has a pH of 3.8-4.2 and a molar concentration of 0.08-0.12M and contains 0.45-0.55M NaCl.
4. The method for preparing bioactive tetrapeptides according to claim 1, wherein,
in the step S1 of the above-mentioned process,
the water added after mincing is distilled water, and the mass volume ratio of the fresh fish skin of the puffer fish to the distilled water is 0.9-1.1kg:10L;
homogenizing by using a tissue homogenizer with the rotating speed of 11000-13000 rpm/min;
performing enzymolysis, boiling and heating for 9-11min, inactivating enzyme, and adjusting pH to neutrality with HCl;
the microfiltration adopts ceramic membrane elements with phi 30mm multiplied by 800mm, the filtration precision is 200nm, and the membrane flux is 600-1000L/M 2 And H, ultrafiltering by adopting a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 1 kDa.
5. The method for preparing bioactive tetrapeptides according to claim 1, wherein,
IC of ACE inhibition of the bioactive tetrapeptide YVLL 50 The value is 0.25-0.26 umol/mL.
6. The method for preparing bioactive tetrapeptides according to claim 1, wherein,
IC of DPP-IV inhibition rate of bioactive tetrapeptide YVLL 50 The value is 1.25-1.35 mg/mL.
7. Use of a bioactive tetrapeptide prepared according to the method for preparing a bioactive tetrapeptide of any one of claims 1-6 in the preparation of a antihypertensive drug or a dipeptidyl peptidase iv inhibitor.
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