CN113480607B - Active small molecule peptide and preparation method and application thereof - Google Patents

Active small molecule peptide and preparation method and application thereof Download PDF

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Publication number
CN113480607B
CN113480607B CN202110909486.0A CN202110909486A CN113480607B CN 113480607 B CN113480607 B CN 113480607B CN 202110909486 A CN202110909486 A CN 202110909486A CN 113480607 B CN113480607 B CN 113480607B
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peptide
buffer solution
ace
active small
borate buffer
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CN113480607A (en
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刘智禹
苏永昌
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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Fisheries Research Institute Of Fujian (fujian Aquatic Disease Prevention Center)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Abstract

The invention discloses an active small molecule peptide, a preparation method and an application thereof, belonging to the technical field of active small molecule peptides, and the active small molecule peptide is a pentapeptide, and has an amino acid sequence as follows: TLFGL. A preparation method of active small molecule peptide comprises 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: sequence identification of the peptide, S4: and (3) verifying the ACE inhibitory activity of the synthesized active small molecule peptide. The invention discloses an active small molecular peptide, a preparation method and application thereof, wherein the prepared immobilized ACE gel affinity adsorption column used for screening the active small molecular peptide has the characteristics of fast and efficient screening and simple and convenient operation, and can be used for fast large-scale screening of the active small molecular peptide.

Description

Active small molecule peptide and preparation method and application thereof
Technical Field
The invention belongs to the technical field of active small molecular peptides, and particularly relates to an active small molecular peptide and a preparation method and application thereof.
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 exercise amount is reduced, so that the incidence of cardiovascular diseases in China is rapidly increased, wherein hypertension is the main factor.
Under normal conditions, a human body can ensure the balance of the source and the route of blood sugar through two major regulating systems of 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 and the like), the two major regulatory functions are disordered, and the blood sugar level is increased.
The puffer fish is a fish in the warm temperate zone and the tropical near-sea bottom layer, inhabits the middle and lower layers of the sea, has few species entering the fresh water river, and makes the whole body float on the water surface in a spherical shape when encountering external danger, and small thorns on the skin are erected to defend the puffer fish.
Angiotensin Converting Enzyme (ACE) inhibitory peptide is a small molecular polypeptide formed by proteolysis, has a remarkable blood pressure lowering effect, and more effectively has no toxic or side effect and has no influence on normal blood pressure compared with other common blood pressure lowering medicaments. ACE plays an important role in blood pressure regulation, and through the removal of two amino acids (His-Leu) at the carbon terminal, the originally inactive angiotensin I can be converted into active angiotensin II, so that vasoconstriction is caused, and the blood pressure is increased; the ACE also can lead the relaxing skin with the vasodilatation function to be inactivated and also can cause the situation of blood pressure rise, and the ACE inhibitory peptide can block two biochemical reaction processes caused by the ACE to play the role of reducing the blood pressure.
The existing prepared active small molecular peptide needs to carry out adsorption and elution treatment on the peptide component after enzymolysis, wherein an adsorption column plays a vital role, and especially a filler of the adsorption column influences elution efficiency.
The existing technology for preparing the active small molecular peptide with the blood pressure reducing effect from foods such as hazelnuts, yak milk, shellfish meat, medlar and the like needs a technology for preparing the active small molecular peptide by taking the puffer fish as a raw material.
Disclosure of Invention
The invention aims to provide an active small molecular peptide, a preparation method and application thereof, wherein the prepared immobilized ACE gel affinity adsorption column used for screening the active small molecular peptide has the characteristics of high screening speed and efficiency and simple and convenient operation, and can be used for quick large-scale screening of the active small molecular peptide.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides an active small molecule peptide, which is pentapeptide, and has an amino acid sequence as follows: TLFGL.
Preferably, the IC of ACE inhibition ratio of the active small molecule peptide TLFGL 50 The value is 0.05-0.06umol/mL.
Preferably, the IC of DPP-IV inhibition ratio of the active small molecule peptide TLFGL 50 The value is 21.0-22.0mg/mL.
The invention also provides a preparation method of the active small molecule peptide, which comprises the following steps: s0: preparation of 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, performing enzyme deactivation treatment after enzymolysis, cooling, adjusting the pH value to be neutral to obtain enzymolysis liquid, performing microfiltration on the enzymolysis liquid, and performing ultrafiltration treatment to obtain the peptide components with the molecular weight of less than 1kDa, S2: affinity adsorption and elution of peptide components, performing affinity adsorption and elution on the peptide components with the molecular weight of less than 1kDa obtained in the step S1 by using the immobilized ACE gel affinity adsorption column prepared in the step S0, performing ultraviolet detection, wherein the ultraviolet detection wavelength is 220nm, collecting elution peak components, respectively naming the elution peak components as 1-24 th components, then performing ACE activity determination, selecting the 17 th component for desalination, and performing reduced pressure concentration for later use, wherein the step S3: and (3) identifying the sequence of the peptide, performing mass spectrometry on the 17 th component subjected to reduced pressure concentration in the step S2, and analyzing the mass spectrometry result to obtain the active small molecule peptide TLFGL, S4: and (4) carrying out solid phase synthesis on the active small molecular peptide TLFGL according to the amino acid sequence determined in the step S3, and verifying the ACE inhibitory activity of the synthesized active small molecular peptide.
Preferably, the specific step of step S0: s01: dissolving ACE in borate buffer solution to prepare an ACE chelating ligand solution, S02: adding dilute hydrochloric acid into the cyanogen bromide activated agarose gel 4B for swelling, filtering, washing by using borate buffer solution, filtering to remove residual HCl, obtaining the cyanogen bromide activated agarose gel 4B filler, and S03: mixing an ACE chelating ligand solution with cyanogen bromide activated agarose 4B filler, uniformly stirring, chelating overnight, after chelating, cleaning and filtering by using borate buffer solution with the volume of 4-6 times that of the cyanogen bromide activated agarose 4B filler, removing redundant ACE, and S04: and (3) uniformly mixing the filtered cyanogen bromide activated agarose 4B filler with a Tris-HCl buffer solution, carrying out soft oscillation and then filtering, wherein the step S05: and after filtering, alternately cleaning and filtering for 3-5 times by adopting acetate buffer solution and Tris-HCl buffer solution to obtain the ACE immobilized gel filler, suspending the ACE immobilized gel filler in borate buffer solution, and filling the borate buffer solution into a chromatographic column to obtain the immobilized ACE gel affinity adsorption column.
Preferably, the enzyme activity unit of ACE is 4.5-5.5U/ml, the pH of borate buffer solution is 8.1-8.4, the molar concentration is 0.08-0.12M, the borate buffer solutions in the other steps except the borate buffer solution in the step S05 all contain 0.45-0.55M NaCl, and the mass-to-volume ratio of cyanogen bromide activated sepharose 4B to dilute hydrochloric acid is 0.8-1.2g:5mL, the molar concentration of the dilute hydrochloric acid is 0.9-1.1mM, the temperature condition of chelating 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, the Tris-HCl buffer solution of the step S05 contains 0.45-0.55M NaCl, the oscillation temperature of gentle oscillation is 23-27 ℃, the oscillation time is 1.8-2.2h, the pH of the acetate buffer solution is 3.8-4.2, the molar concentration is 0.08-0.12M, and the NaCl contains 0.45-0.55M.
Preferably, in step S1, alkali with the enzyme activity unit of 5 ten thousand U/g is adoptedAnd (2) carrying out enzymolysis by using the alkaline protease under the conditions 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, the water added after mincing is distilled water, the mass volume ratio of the fresh fish skin of the puffer fish to the distilled water is 0.9-1.10L, homogenizing is carried out by using a tissue homogenizer with the rotating speed of 11000-13000rpm/min, carrying out enzyme deactivation treatment by boiling and heating for 9-11min after the enzymolysis, adjusting the pH value to be neutral by using HCl, a ceramic membrane element with the diameter of 30mm multiplied by 800mm is adopted for microfiltration, the filtration precision is 200nm, and the membrane flux is 600-1000L/M 2 And H, ultrafiltration adopts a polyether sulfone ultrafiltration membrane with the molecular weight cutoff of 1 kDa.
Preferably, step S2 specifically includes: s21: and (2) mounting the immobilized ACE gel affinity adsorption column prepared in the step S0 on a medium-pressure protein purifier, balancing for 18-22min by using borate buffer solution with the molar concentration of 0.08-0.12M and the pH value of 7.9-8.1, wherein the balancing flow rate is 0.45-0.55mL/min, detecting by using an ultraviolet detector, the ultraviolet detection wavelength is 220nm, and the pH value is S22: after balancing, carrying out loading adsorption on the peptide component with the molecular weight of less than 1kDa obtained in the step S1, wherein the loading amount is 4.5-5.5mL, the mobile phase uses borate buffer solution with the molar concentration of 0.08-0.12M and the pH of 7.9-8.1, the flow rate is 0.45-0.55mL/min, observing until the detection baseline is stable, 14-16min, eluting with borate buffer solution with the molar concentration of 0.08-0.12M and the pH of 7.9-8.1 as the elution mobile phase, wherein the borate buffer solution contains 0.8-1.2M NaCl, the elution flow rate is 0.45-0.55mL/min, collecting the elution peak components, and S23, wherein after carrying out balance again on the borate buffer solution with the molar concentration of 0.08-0.12M and the pH of 7.9-8.1 by using the borate buffer solution with the molar concentration of 0.08-0.12M and the pH of 7.9-8.1 for 18-22min, carrying out loading and elution on the peptide component for many times according to the step S22, combining the elution peak components, respectively, carrying out desalination and carrying out selective separation on the peptide component after carrying out desalination, and carrying out the first active ingredient, and carrying out the second concentration, and carrying out the second determination on the second active ingredient, and carrying out the second determination.
Preferably, in step S3, mass spectrometry is performed by LC-MS/MS under the following conditions: the chromatographic column is PepMap RPLC C 18 ,75μm i.d.×150mm,3μm，
Figure BDA0003202977340000041
Positive ion mode, scan range: m/z is 300-1500Da, the spraying voltage of an emitter is 2-kV, the de novo Sequencing analysis is carried out on the result of mass spectrometry by using PEAKS STUDIO software, and the high-credible activity small molecular peptide amino acid sequence is obtained.
The invention also provides application of the active small molecule peptide in preparing a medicine for reducing blood pressure.
The invention also provides application of the active small molecule peptide in preparation of a dipeptidyl peptidase IV inhibitor.
The invention has the beneficial effects that:
1. the puffer fish skin is adopted as a raw material to obtain an active small molecular peptide amino acid sequence, and the prepared active small molecular peptide TLFGL has ACE inhibitory activity, can be applied to blood pressure lowering medicines, and has a good blood pressure lowering effect.
2. The prepared immobilized ACE gel affinity adsorption column is used for screening active small molecular peptides, has the characteristics of high screening speed and high efficiency and simple and easy operation, and can be used for quick large-scale screening of the active small molecular peptides.
3. The prepared active small molecular peptide TLFGL has DPP-IV inhibitory activity, can be applied to dipeptidyl peptidase IV inhibitors, and has good blood sugar reducing effect.
4. The active small molecule peptide TLFGL and ACE form a hydrogen bond network, and the two have 4 hydrogen bond interactions in total, so that the stability is high.
5. The active small molecule peptide TLFGL Gly4 skeleton amide oxygen atom has electronegativity and can be combined with Zn of a binding site 2+ Forming stronger polar action, thereby destroying the original functions of Zn ions.
6. The active small molecule peptide TLFGL is a hydrophobic residue that can bind to a pocket residue to form strong hydrophobic interactions and van der waals potentials.
Drawings
FIG. 1 is a graph showing the ACE inhibitory activity assay of fractions 1 to 24 collected in step S23 of the present invention.
FIG. 2 is an affinity elution profile of the immobilized ACE gel affinity adsorption column of the present invention for peptide components.
FIG. 3 is a graph showing the measurement of the concentration of the active small molecule peptide TLFGL of the present invention on the ACE inhibitory rate.
Fig. 4 shows the binding pattern of the active small molecule peptide TLFGL of the present invention with ACE.
FIG. 5 shows the inhibition pattern and mechanism of action of the active small molecule peptides TLFGL and ACE.
FIG. 6 is a graph showing the effect of the active small molecule peptide TLFGL of the present invention on lowering blood pressure in SHR rats.
FIG. 7 is a graph showing the effect of DPP-IV inhibition of the active small molecule peptide TLFGL of the present invention.
Detailed Description
The invention will now be further described with reference to the drawings and the detailed description.
In the active small molecule peptide provided in this embodiment, the active small molecule peptide is a pentapeptide, and the amino acid sequence: TLFGL.
The embodiment also provides a preparation method of the active small molecule peptide, which comprises the following steps:
s0: preparation of immobilized ACE gel affinity adsorption column
ACE chelating ligand solution was prepared by dissolving ACE with 5U/ml enzyme activity in 50ml of 0.1M borate buffer (pH 8.3, containing 0.5M NaCl). Weighing 10g cyanogen bromide activated agarose 4B filler into a 100ml beaker, adding 50mL 1mM HCl for swelling, filtering, washing with 0.1M borate buffer solution (pH 8.3, containing 0.5M NaCl), filtering, removing residual HCl, and obtaining the cyanogen bromide activated agarose 4B filler. Mixing an ACE chelating ligand solution and a cyanogen bromide activated agarose 4B filler, uniformly stirring, chelating overnight at 4 ℃, after chelating, washing and filtering with 0.1M borate buffer (pH 8.3, containing 0.5M NaCl) with the volume 5 times of that of the cyanogen bromide activated agarose 4B filler, and removing redundant ACE. The filtered cyanogen bromide activated Sepharose 4B filler was mixed well with 50mL of 0.1M Tris-HCl buffer (pH 8.0), and gently shaken at 25 ℃ for 2h. After filtration, 0.1M acetate buffer (pH4.0, containing 0.5M NaCl) and 0.1M Tris-HCl buffer (pH8.0, containing 0.5M NaCl) were washed alternately and filtered 3 times to obtain the ACE immobilized gel filler. Suspending the ACE immobilized gel filler in 0.1M borate buffer solution (pH 8.3), and filling into a chromatographic column (phi 0.9 × 1.57 cm) with a column volume of 5ml to obtain the immobilized ACE gel affinity adsorption column.
S1: preparation of peptide Components
Taking 1000g fresh fish skin of puffer fish, mincing, adding 10L of distilled water, homogenizing in a tissue homogenizer at a rotating speed of 12000rpm/min to form fish skin slurry liquid, carrying out enzymolysis for 6h under the conditions that the addition amount of alkaline protease (5 ten thousand U/g) is 1%, the pH value is 8.0 and the enzymolysis temperature is 60 ℃, boiling and heating for 10min for enzyme deactivation after enzymolysis, cooling and adjusting the pH value to be neutral by using HCl. The enzymolysis solution is applied to ceramic membrane element (filtration precision 200nm, 600-1000L/M) with diameter of 30mm × 800mm 2 H) After microfiltration, a polyether sulfone ultrafiltration membrane (with the molecular weight cutoff of 1 kDa) is adopted for ultrafiltration treatment, and a component with the molecular weight of less than 1kDa is obtained.
S2: affinity adsorption and elution of peptide components
S21: the immobilized ACE gel affinity adsorption column prepared in step S0 is well installed on an intermediate pressure protein purifier (AKTA pure 25), and is balanced with 0.1M borate buffer (pH 8.3) for 20min at a balanced flow rate of 0.5mL/min, and is detected by an ultraviolet detector with an ultraviolet detection wavelength of 220nm.
S22: and (2) after balancing, carrying out sample loading adsorption on the peptide component with the molecular weight of less than 1kDa obtained in the step S1, wherein the sample loading amount is 5mL, the mobile phase is 0.1M borate buffer solution (pH 8.3), the flow rate is 0.5mL/min, observing that the detection baseline is stable for 15min, eluting by using 0.1M borate buffer solution (pH 8.3 containing 1M NaCl) as an elution mobile phase, and collecting the elution peak component at the elution flow rate of 0.5 mL/min.
S23, after the immobilized ACE gel affinity adsorption column is balanced for 20min again by 0.1M borate buffer solution (pH 8.3), peptide component sampling and elution are carried out for multiple times according to the step S22, peak components of multiple times of elution are combined and are respectively named as 1 st to 24 th components, then ACE activity determination is carried out, as shown in figure 1, 3 rd, 16 th and 17 th components are shown to have higher ACE inhibitory activity, the 17 th component is selected in the method, a dialysis bag with the molecular weight cutoff of 100D is adopted to desalt the 17 th component, and the component is subjected to reduced pressure concentration for later use.
The affinity elution pattern of the immobilized ACE gel affinity adsorption column for active small molecule peptides is shown in FIG. 2.
S3: and (3) identifying the sequence of the peptide, performing mass spectrometry on the 17 th component after the decompression concentration in the step S2, and analyzing the mass spectrometry result to obtain the active small molecule peptide TLFGL, specifically, performing mass spectrometry by adopting LC-MS/MS, wherein the detection conditions are as follows: the chromatographic column is PepMap RPLC C 18 ,75μm i.d.×150mm,3μm，
Figure BDA0003202977340000071
Positive ion mode, scan range: m/z is 300-1500Da, the spraying voltage of an emitter is 2-kV, the de novo Sequencing analysis is carried out on the result of mass spectrometry by using PEAKS STUDIO software, and the high-credible activity small molecular peptide amino acid sequence is obtained.
S4: and (3) carrying out solid phase synthesis on the active small molecular peptide TLFGL according to the amino acid sequence determined in the step S3, and verifying the ACE inhibitory activity of the synthesized active small molecular peptide. The result shows that the active small molecular peptide TLFGL has good ACE inhibitory activity, and the ACE inhibitory rate IC of the active small molecular peptide TLFGL 50 The value was 0.0575umol/mL (0.0316 mg/mL).
The graph of the determination of the concentration of the active small molecule peptide TLFGL on ACE inhibition is shown in fig. 3.
Mechanism research:
the Discovery Studio software is used for carrying out butt joint mechanism simulation analysis on the active small molecule peptide TLFGL and ACE, and the results are as follows:
as shown in fig. 4-5, a hydrogen bonding network is formed between the TLFGL and ACE. Both share 4 hydrogen bonding interactions. These hydrogen bonding effects are critical for stable binding of the active small molecule peptide to ACE. Binding pocket residues that hydrogen bond with the active small molecule peptide include: tyr523, his353, and the like. The active small molecular peptide Gly4 skeleton amide oxygen atom has electronegativity and can be combined with Zn of a binding site 2+ Forming stronger polar action, thereby destroying the original functions of Zn ions. The active small molecule peptides are all hydrophobic residues. These hydrophobic residues, in combination with pocket residues, form strong hydrophobic interactions and van der waals potentials.
The invention also provides application of the active small molecule peptide in preparing a medicine for reducing blood pressure.
Animal blood pressure lowering experiment:
using SHR rat as animal model, TLFGL was administered at 10mg/kg (rat body weight) for one-time tail vein injection experiment, and captopril tail vein injection at 5mg/kg (rat body weight) as drug control group.
As shown in FIG. 6, the results showed that TLFGL showed systolic blood pressures of 164.4, 149.4, 154.8, 131.8, 158.8mmHg after 2, 4, 6, 12, 24h, respectively, after tail vein injection, which were reduced by 18.4%, 25.9%, 23.2%, respectively,
34.6 percent and 21.3 percent, and the blood pressure reducing effect is better than that of a captopril control group. Wherein, the systolic blood pressure value of 12h is close to the normal blood pressure, and the blood pressure reducing effect can last for more than 24h after one-time injection of the active small molecular peptide TLFGL. Experiments show that TLFGL has good effect of reducing blood pressure.
The invention also provides application of the active small molecule peptide in preparation of a dipeptidyl peptidase IV inhibitor.
And (3) determining the activity of the active small molecule peptide for inhibiting DPP-IV:
the DPP-IV inhibitor, namely a dipeptidyl peptidase IV inhibitor, is a medicament for treating type 2 diabetes, and the medicaments can inhibit the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic secretion active small molecule peptide (GIP), improve the levels of endogenous GLP-1 and GIP, promote insulin release of pancreatic beta cells, and inhibit glucagon secretion of pancreatic alpha cells, thereby improving insulin levels, reducing blood sugar, and being difficult to induce hypoglycemia and increase body weight.
The DPP-IV inhibitory activity of the active small molecule peptide TLFGL is determined, as shown in figure 7, the result shows that the active small molecule peptide TLFGL has certain DPP-IV inhibitory activity, and the DPP-IV inhibitory rate IC of the active small molecule peptide TLFGL 50 The value was 21.5mg/mL.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art; the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> research institute for aquatic products of Fujian province (center for preventing and treating diseases of Fujian aquatic products)
<120> active small molecule peptide, preparation method and application thereof
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 5
<212> PRT
<213> Fugu ocellatus (Takifugu)
<400> 1
Thr Leu Phe Gly Leu
1 5

Claims (3)

1. An active small molecule peptide, which is characterized in that,
the active small molecular peptide is pentapeptide, and the amino acid sequence is as follows: TLFGL.
2. A method for preparing active small molecule peptide is characterized in that,
the method comprises the following steps:
s0: preparing an immobilized ACE gel affinity adsorption column;
s01: dissolving ACE in borate buffer solution to prepare an ACE chelating ligand solution;
s02: adding dilute hydrochloric acid into the cyanogen bromide activated agarose gel 4B for swelling, filtering, cleaning by using borate buffer solution, filtering to remove residual HCl, and obtaining cyanogen bromide activated agarose 4B filler;
s03: mixing an ACE chelating ligand solution and a cyanogen bromide activated agarose 4B filler, uniformly stirring, chelating overnight, and after chelating, cleaning and filtering by using a borate buffer solution with the volume 4-6 times that of the cyanogen bromide activated agarose 4B filler to remove redundant ACE;
s04: uniformly mixing the filtered cyanogen bromide activated agarose 4B filler with a Tris-HCl buffer solution, carrying out soft oscillation and filtering;
s05: after filtering, alternately cleaning and filtering for 3-5 times by adopting acetate buffer solution and Tris-HCl buffer solution to obtain an ACE immobilized gel filler, suspending the ACE immobilized gel filler in borate buffer solution, and filling into a chromatographic column to obtain an immobilized ACE gel affinity adsorption column;
the enzyme activity unit of ACE is 4.5-5.5U/ml;
the pH value of the borate buffer solution is 8.1-8.4, the molar concentration is 0.08-0.12M, and the borate buffer solutions in the other steps except the borate buffer solution in the step S05 all contain 0.45-0.55M NaCl;
the mass-volume ratio of the cyanogen bromide activated sepharose 4B to the dilute hydrochloric acid is 0.8-1.2g:5mL, the molar concentration of the dilute hydrochloric acid is 0.9-1.1mM;
the temperature condition of chelation overnight is 0-4 ℃;
the pH value of the Tris-HCl buffer solution is 7.9-8.1, the molar concentration is 0.08-0.12M, and the borate buffer solutions in the other steps except the borate buffer solution in the step S05 all contain 0.45-0.55M NaCl;
the oscillating temperature of the soft oscillation is 23-27 ℃, and the oscillating time is 1.8-2.2h;
the pH of the acetate buffer solution is 3.8-4.2, the molar concentration is 0.08-0.12M, and the acetate buffer solution contains 0.45-0.55M NaCl;
s1: preparing a peptide component, namely taking fresh fish skin of puffer fish, mincing, adding water, homogenizing to form fish skin slurry liquid, adding protease for enzymolysis, performing enzyme deactivation treatment after enzymolysis, cooling, adjusting 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 of less than 1 kDa; carrying out enzymolysis by adopting alkaline protease with the enzyme activity unit of 5 ten thousand U/g, 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, the pH value is 7.8-8.2, the enzymolysis temperature is 58-62 ℃, and the enzymolysis time is 5.8-6.2h;
adding distilled water as water after mincing, wherein the mass volume ratio of fresh puffer fish skin to distilled water is 0.9-1.1kg;
adopting a tissue homogenizer with the rotating speed of 11000-13000rpm/min for homogenate;
boiling and heating for 9-11min after enzymolysis for enzyme deactivation, and adjusting the pH value to be neutral by adopting HCl;
the microfiltration adopts ceramic membrane elements with the diameter of phi 30mm multiplied by 800mm, the filtration precision is 200nm, and the membrane flux is 600-1000L/M 2 H, performing ultrafiltration by using a polyether sulfone ultrafiltration membrane with the molecular weight cutoff of 1 kDa;
s2: performing affinity adsorption and elution on the peptide component, performing affinity adsorption and elution on the peptide component with the molecular weight of less than 1kDa obtained in the step S1 by using the immobilized ACE gel affinity adsorption column prepared in the step S0, performing ultraviolet detection with the wavelength of 220nm, collecting an elution peak component, desalting the elution peak component, and performing reduced pressure concentration for later use;
s21: on a medium-pressure protein purifier, the immobilized ACE gel affinity adsorption column prepared in the step S0 is well installed, borate buffer solution with the molar concentration of 0.08-0.12M and the pH value 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, wherein the ultraviolet detection wavelength is 220nm;
s22: after balancing, carrying out loading adsorption on the peptide component with the molecular weight of less than 1kDa obtained in the step S1, wherein the loading amount is 4.5-5.5mL, the mobile phase uses borate buffer solution with the molar concentration of 0.08-0.12M and the pH of 7.9-8.1, the flow rate is 0.45-0.55mL/min, 14-16min after the detection baseline is observed to be stable, the borate buffer solution with the molar concentration of 0.08-0.12M and the pH of 7.9-8.1 is used as an elution mobile phase for elution, the borate buffer solution contains 0.8-1.2M NaCl, the elution flow rate is 0.45-0.55mL/min, and collecting the elution peak component;
s23, balancing the immobilized ACE gel affinity adsorption column for 18-22min by using borate buffer solution with the molar concentration of 0.08-0.12M and the pH value of 7.9-8.1 again, then carrying out multiple times of loading and elution of peptide components according to the step S22, combining the multiple times of elution peak components, desalting the elution peak components by using a dialysis bag with the molecular weight cutoff of 100D, and concentrating under reduced pressure for later use;
s3: performing peptide sequence identification, performing mass spectrometry on the elution peak component subjected to reduced pressure concentration in the step S2, and analyzing the mass spectrometry result to obtain an amino acid sequence TLFGL of one of the active small molecule peptides;
performing mass spectrometry by using LC-MS/MS under the following measurement conditions: the chromatographic column is PepMap RPLC C 18 ,75μm i.d.×150mm,3μm，
Figure FDA0003942593830000031
Positive ion mode, scan range: m/z is 300-1500Da, the spraying voltage of an emitter is 2-kV, and the result of mass spectrometry is analyzed by PEAKS STUDIO software to obtain the high-credibility active small molecular peptide amino acid sequence;
s4: and (3) carrying out solid phase synthesis on the active small molecular peptide TLFGL according to the amino acid sequence determined in the step S3, and verifying the ACE inhibitory activity of the synthesized active small molecular peptide.
3. Use of the active small molecule peptide of claim 1 for the preparation of a hypotensive drug or a dipeptidyl peptidase iv inhibitor.
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