CN108997477A - Tripeptides with ACE inhibitory activity - Google Patents
Tripeptides with ACE inhibitory activity Download PDFInfo
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- CN108997477A CN108997477A CN201810815190.0A CN201810815190A CN108997477A CN 108997477 A CN108997477 A CN 108997477A CN 201810815190 A CN201810815190 A CN 201810815190A CN 108997477 A CN108997477 A CN 108997477A
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- inhibitory activity
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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
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
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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
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- Life Sciences & Earth Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention mainly relates to the tripeptides with ACE inhibitory activity, peptide sequence is respectively WGK and FQK, belongs to field of biotechnology.The present invention is by online database, by virtually being digested to protein, predict active peptide potential activity, water solubility, ADMET (absorption, metabolism and toxicity) property, and through multi-turns screens such as molecular dockings, two kinds are obtained with the inhibition active tripeptides of ACE after verifying external ACE inhibitory activity using high performance liquid chromatography.Its sequence of two kinds of active peptides of the present invention is short, safety and is easily obtained.
Description
Technical field
The invention belongs to field of biotechnology, the tripeptides with ACE inhibitory activity is related generally to.
Background technique
Angiotensin converting enzyme (Antiotensin-I Converting Enzyme, ACE, EC3.4.15.1) is one
Dipeptides carboxylic acid of the kind containing zinc, can be activated by chloride ion, and have wider substrate specificity.ACE is to body blood pressure and angiocarpy
Function plays important adjustment effect.The activity for inhibiting ACE is considered as treating the important and effective method of one kind of hypertension.
It is demonstrated experimentally that the ace inhibitory peptide of some food protein sources has no toxic side effect, safety is high, and to normotensive without
Hypotensive activity.As what the development of bioinformatics and biologically active peptide were studied gos deep into, the level-one of more and more protein
The amino acid sequence of structure and active peptide is illustrated, and the restriction enzyme site of protease is also more and more clear.For from material protein
The biologically active peptide that unknown nucleotide sequence is found in matter provides the foundation, and provides the foundation of science.More protein tridimensional knots
Structure is confirmed, and a large amount of albumen of generation have been chosen as potential treatment target and have been studied, thus have expanded molecular docking technology
With application range of the virtual screening method in small molecule is designed and developed.For the virtual screening method of food-borne biologically active peptide
It is a kind of effective research means, is to screen by the booster action of computer with potential source biomolecule active amino acid sequence
Key technology.Using online data, not only reduces proof strength and shorten the R&D cycle, also improve the successful probability of screening.
By this approach application in virtual screening method of the ace inhibitory peptide based on molecular docking technology, can largely save research at
Originally the period of identification ace inhibitory peptide and is effectively reduced.In the method that tradition obtains ace inhibitory peptide, food-borne albumen
Enzymolysis product complicated components contain into hundred to thousands of peptide fragments, and the peptide fragment that molecular weight is close or charge is equal is difficult to carry out
It isolates and purifies, huge problem is brought for subsequent Structural Identification.Not only to take a significant amount of time for isolating and purifying, and
And be difficult to obtain high-purity, single substance, the application of virtual screening can effectively solve ace inhibitory peptide face in screening process
The problem faced.
Summary of the invention
Method of the present invention by utilizing bioinformatics utilizes the molecule of active peptide by online active peptide database
The properties such as amount, bioactivity scoring, water-soluble, ADMET (absorption, metabolism and toxicity) carry out multi-turns screen, binding molecule docking
Obtain with two kinds of biologically active peptides of ACE (PDB ID:1O86) stable bond, and by high performance liquid chromatography confirm its in vitro
ACE inhibitory activity, tripeptide sequence are respectively WGK and FQK, corresponding IC50Value is respectively 222.74 μM and 250 μM.Finally utilize
Molecular docking software illustrates ACE-WGK and ACE-FQK interaction and binding site.
Technical solution of the present invention:
Tripeptides with ACE inhibitory activity, amino acid sequence are respectively WGK and FQK.
The tripeptides comes from Chicken Albumin, any to carry out to it including using the peptide sequence of the ACE inhibitory activity as core
It is corresponding adjustment or modification.
The amino acid residue of the ACE-WGK catalytic site is respectively Gln281, Tyr520, His353, His513,
Tyr523, Glu384 and Lys511.The amino acid residue of ACE-FQK catalytic site is respectively Ala354, His353, His513,
Tyr523, Tyr520 and Lys511.
Items experimental method involved in the present invention is specific as follows:
1, the screening and assessment of material protein
Egg is searched from National Center for Biotechnology Information (NCBI) database
The relevant protein sequence of albumen is analyzed, and the sequence letter according to the important component and parent protein that are Chicken Albumin is selected
The availability of breath.According to " potential source biomolecule activity profile " tool, BIOPEP-UWM (http://www.uwm.edu.pl/ is used
Biochemia/index.php/en/biopep Chicken Albumin sequence) is analyzed.In this process, the Chicken Albumin of release is living
Property peptide is compared with the peptide sequence registered, and the peptide sequence shows high ACE rejection ability.Egg protein these
The frequency of ace inhibitory peptide segment shows potentiality of these egg proteins as ace inhibitory peptide candidate.
2, virtual screening ace inhibitory peptide
Using pepsin (pH 1.3) and trypsase by ExPASy PeptideCutter (http: //
Web.expasy.org/peptide_cutter/) Chicken Albumin is virtually hydrolyzed, selects all tripeptides,
PeptideRanker (http://bioware.ucd.ie/~compass/biowareweb/Server_pages/
Peptideranker.php Activity Score is carried out in), is selected the high tripeptides of scoring, is used online tool Innovagen
(http://www.innovagen.com/proteomics-tools) and admetSAR (http: //
Lmmd.ecust.edu.cn/admetsar1/re dict/) water-soluble and ADMET (absorption, metabolism and toxicity) property is predicted respectively
Matter.
Screen obtained active peptide in 2017 R2 Client software of Discovery Studio with ACE (PDB ID:
Molecular docking 1O86) is carried out, stable tripeptides in conjunction with ACE is selected and carries out solid-state chemical reaction method.
3, using the external ACE inhibitory activity of high performance liquid chromatography verifying active peptide
Hippuroyl histamine acyl leucine (HHL) substrate solution is taken, addition inhibitor mixed is uniform, in 37 DEG C of waters bath with thermostatic control
3~5min is preheated, ACE liquid is then added and is sufficiently mixed, after 37 DEG C of heat preservation 30min, the 1mol/L HCl added terminates reaction,
Obtain reaction solution.Borate buffer is used to substitute inhibitor solution as blank control group simultaneously.The reaction solution is directly with HPLC system
System is analyzed.Chromatographic condition: 25 DEG C of column temperature, flow velocity 0.5mL/min, mobile phase acetonitrile: 25: 75 isocratic elution of water, detection
Wavelength 228nm.
Active kyrine WGK and FQK of the invention significantly inhibits effect, IC to ACE50Respectively 222.74 and 250 μ
M.Therefore, the tripeptides WGK and FQK have external ACE inhibitory activity.
4, molecular docking prediction ACE and active peptide interaction
The molecular docking of ACE and tripeptides are carried out using 2017 R2 Client software of Discovery Studio, wherein
ACE (PDB ID:1O86, Resolution:) crystal structure be from RCSB Protein Data Bank
(Https: //www.rcsb.org/) obtain, the 3D structure of active peptide is minimized using the field of force CHARMm, and using steady
Fixed energy conformer carries out docking research.Use the CDOCKER module of 2017 R2 Client software of Discovery studio
Molecular docking is carried out in binding site semi-flexible.Docking operation is with coordinate x:40.4974, y:34.8829, z:44.3814, docking
Radius isAccording to obtained Interworking Data, the CDOCKER energy of calculated ACE-WGK and ACE-FQK.
Positive beneficial effect of the invention:
(1) amino acid sequence according to the present invention, ACE of the present invention can be obtained by chemical synthesis and is inhibited
Peptide (WGK and FQK).
(2) present invention is oriented the virtual screening in conjunction with polypeptide by Computer-aided Design Technology, pure saving
Change, under the premise of the time of identification peptide sequence and cost, reduces the number of the compound of actual test in biological activity determination
Amount improves the successful probability of screening.
(3) by online database BIOPEP-IUM (Http:// www.uwm.edu.pl/biochemia/ index.php/en/biopep) and AHTPDB (http://crdd.osdd.net/raghava/ahtpdb/) search, this hair
Two kinds of tripeptides with ACE inhibitory activity involved in bright are new food-borne ace inhibitory peptide sequence.
Detailed description of the invention
2 width of attached drawing of the present invention, in which:
Fig. 1 ACE-WGK (PDB ID:1O86) molecular docking map；
Fig. 2 ACE-FQK (PDB ID:1O86) molecular docking map.
Specific embodiment
The invention will be further elaborated in a manner of specific embodiment below.
The screening and assessment of 1 raw material of embodiment
It searches the relevant protein sequence of Chicken Albumin from ncbi database to be analyzed, selecting foundation is Chicken Albumin
Important component and the sequence information of parent protein availability.Finally selected albumen ovalbumin (Accession:1OVA_
A), -1 precursor of vitellogenin (Accession:NP_001004408 XP_422384), -2 precursor of vitellogenin
(Accession:NP_001026447 XP_422370), ovum iron transfer (Accession:1AIV_A), amino acid number difference
It is 386,1912,1850 and 686.
According to " potential source biomolecule activity profile " tool, is analyzed using BIOPEP-UWM and carry out four kinds of egg protein sequences, the work
Tool can be obtained in http://www.uwm.edu.pl/biochemia/index.php/en/biopep.In this process, it will release
The Chicken Albumin active peptide put is compared with peptide sequence, what the peptide sequence showed to report in BIOPEP-UWM database
ACE rejection ability.The frequency of these ace inhibitory peptide segments of protein shows that these egg proteins are waited as ace inhibitory peptide
Select the potentiality of object.It is generated using the percentage that following formula calculates ace inhibitory peptide:
M=A/N
Wherein A is the quantity of ace inhibitory peptide segment in protein sequence, and N is the quantity of Amino Acids in Proteins residue.Institute
Having segment is the ace inhibitory peptide generated by theoretical measurement.
Computer analyzes ovalbumin, -1 precursor of vitellogenin, -2 precursor of vitellogenin, the M value point of ovum iron transfer
It Wei 0.251,0.085,0.101 and 0.172.
The screening and determination of activity of 2 tripeptides of embodiment
Chicken Albumin is carried out by ExPASy PeptideCutter using pepsin (pH 1.3) and trypsase
Virtual hydrolysis, selects all tripeptides, Activity Score is carried out in PeptideRanker, selectes the high tripeptides of scoring.
Water-soluble and ADMET property is predicted respectively using online tool Innovagen and admetSAR.Filter out water solubility
Good tripeptides.In ADMET (absorption, metabolism and toxicity) prediction, the good (good of measurement gastrointestinal absorption emphatically
Intestinal absorption, HIA+) and can penetrate tripeptides (the could penetrate the Blood- of blood-brain barrier
Brain Barrier, BBB+), as potential efficient ace inhibitory peptide candidate.
The molecular docking of ACE and tripeptides, active peptide are carried out using 2017 R2 Client software of Discovery Studio
3D structure minimized using the field of force CHARMm, and docking research is carried out using stable energy conformer.In CDOCKER
Docking semi-flexible is carried out under module.Docking operation is with coordinate x:40.4974, y:34.8829 and z:44.3814 progress.According to institute
Obtained Interworking Data selectes docking energy value relatively minimum two kinds of conjugates ACE-WGK and ACE-FQK, CDOCKER value point
It Wei not -96.449 and -111.976kcal/mol.Solid-state chemical reaction method is finally carried out, is carried out using high performance liquid chromatography external
The verifying of ACE activity.
The verifying of 3 external activity of embodiment
Using the ACE inhibitory activity of high performance liquid chromatography verifying WGK and FQK.Take hippuroyl histamine acyl leucine (HHL)
Substrate solution, addition inhibitor mixed is uniform, and 3~5min is preheated in 37 DEG C of waters bath with thermostatic control, and it is sufficiently mixed that ACE liquid is then added
It closes, after 37 DEG C of heat preservation 30min, the 1mol/L HCl added terminates reaction, obtains reaction solution.It is substituted simultaneously with borate buffer
Inhibitor solution is as blank control group.The reaction solution is directly analyzed with HPLC system.
Chromatographic condition: 25 DEG C of column temperature, flow velocity 0.5mL/min, mobile phase acetonitrile: 25: 75 isocratic elution of water, Detection wavelength
228nm。
By the external ACE inhibitory activity of experimental verification WGK and FQK, 222.74 μM and 250 μM of difference.
4 tripeptides of embodiment virtually docks analysis with ACE's
Using 2017 R2 Client software of Discovery Studio, under the conditions of the field of force CHARMm, by WGK and FQK
It is docked respectively with AEC (PDB ID:1O86), illustrates tripeptides and the specific inhibition site interaction type of ACE enzyme.
Interaction between tripeptides and ACE (PDB ID:1O86) mainly include Van der Waals force, Hydrogenbond active force,
Two kinds of forms of hydrophobic effect and electrostatic interaction, wherein the amino acid residue of ACE-WGK catalytic site is respectively Gln281,
Tyr520, His353, His513, Tyr523, Glu384 and Lys511 (Fig. 1).The amino acid residue of the catalytic site of ACE-FQK
Respectively Ala354, His353, His513, Tyr523, Tyr520 and Lys511 (Fig. 2).
Claims (3)
1. the tripeptides with ACE inhibitory activity, characterized in that the amino acid sequence of the tripeptides is respectively WGK and FQK.
2. as described in claim 1 with the tripeptides of ACE inhibitory activity, characterized in that WQK and ACE (PDB ID:1O86) phase
The amino acid residue for the catalytic site that interaction is related to is respectively Gln281, Tyr520, His353, His513, Tyr523,
Glu384 and Lys511；The amino acid residue of catalytic site that FQK and ACE (PDB ID:1O86) interaction is related to is respectively
Ala354, His353, His513, Tyr523, Tyr520 and Lys511.
3. as described in claim 1 with the tripeptides of ACE inhibitory activity, characterized in that tripeptides WGK and FQK are all from egg,
Including using the tripeptide sequence with ACE inhibitory activity as core, any corresponding adjustment or modification that it is carried out.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317240A (en) * | 2019-07-02 | 2019-10-11 | 渤海大学 | A kind of tripeptides DMG and its application |
| CN110628856A (en) * | 2019-10-17 | 2019-12-31 | 武汉普诺金生物科技股份有限公司 | Antihypertensive small molecular peptide, and preparation method and application thereof |
| CN113480598A (en) * | 2021-08-09 | 2021-10-08 | 福建省水产研究所（福建水产病害防治中心） | Bioactive tetrapeptide and preparation method and application thereof |
| CN113801193A (en) * | 2021-09-16 | 2021-12-17 | 北京工商大学 | Wheat germ protein polypeptide with alpha-glucosidase inhibitory activity and preparation thereof |
| CN115010784A (en) * | 2022-05-16 | 2022-09-06 | 江南大学 | ACE inhibitory peptide and application thereof |
Citations (1)
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| CN108033995A (en) * | 2017-12-19 | 2018-05-15 | 渤海大学 | Two kinds of ace inhibitory peptides for deriving from Larimichthys crocea titin |
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2018
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| CN108033995A (en) * | 2017-12-19 | 2018-05-15 | 渤海大学 | Two kinds of ace inhibitory peptides for deriving from Larimichthys crocea titin |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317240A (en) * | 2019-07-02 | 2019-10-11 | 渤海大学 | A kind of tripeptides DMG and its application |
| CN110628856A (en) * | 2019-10-17 | 2019-12-31 | 武汉普诺金生物科技股份有限公司 | Antihypertensive small molecular peptide, and preparation method and application thereof |
| CN113480598A (en) * | 2021-08-09 | 2021-10-08 | 福建省水产研究所（福建水产病害防治中心） | Bioactive tetrapeptide and preparation method and application thereof |
| CN113801193A (en) * | 2021-09-16 | 2021-12-17 | 北京工商大学 | Wheat germ protein polypeptide with alpha-glucosidase inhibitory activity and preparation thereof |
| CN115010784A (en) * | 2022-05-16 | 2022-09-06 | 江南大学 | ACE inhibitory peptide and application thereof |
| CN115010784B (en) * | 2022-05-16 | 2023-03-31 | 江南大学 | ACE inhibitory peptide and application thereof |
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