CN116589533A - Euphausia superba small molecule active peptide, and preparation method and application thereof - Google Patents
Euphausia superba small molecule active peptide, and preparation method and application thereof Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 71
- 241000239370 Euphausia superba Species 0.000 title claims abstract description 34
- 150000003384 small molecules Chemical class 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
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- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 33
- 241000239366 Euphausiacea Species 0.000 claims abstract description 31
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- 238000000034 method Methods 0.000 claims abstract description 21
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 19
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 6
- 108010093894 Xanthine oxidase Proteins 0.000 claims description 50
- 102100033220 Xanthine oxidase Human genes 0.000 claims description 50
- 102000004190 Enzymes Human genes 0.000 claims description 35
- 108090000790 Enzymes Proteins 0.000 claims description 35
- 229940088598 enzyme Drugs 0.000 claims description 35
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- 239000000463 material Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
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- 201000001431 Hyperuricemia Diseases 0.000 claims description 10
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- BQSJTQLCZDPROO-UHFFFAOYSA-N febuxostat Chemical compound C1=C(C#N)C(OCC(C)C)=CC=C1C1=NC(C)=C(C(O)=O)S1 BQSJTQLCZDPROO-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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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- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Pain & Pain Management (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
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- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a euphausia superba small molecule active peptide, a preparation method and application thereof. According to the invention, the defatted euphausia superba powder is used for preparing the XOD inhibitory peptide, and the obtained polypeptide has a high XOD inhibition rate. Through LC-MS/MS identification, 5 small molecular polypeptides with XOD inhibition activity are obtained from the defatted antarctic krill powder enzymatic hydrolysate by screening: LPPYSKE, EDVEGAVR, LDLPGWVK, LDDAFNHL and WDRPLVE. These 5 polypeptides all have varying degrees of XOD inhibitory activity. The euphausia superba peptide prepared by the method has good XOD inhibition capability, and can be applied to the deep processing fields of health products, medicines and the like. The invention can utilize the byproducts of aquatic product processing in a high value, and can effectively improve the resource utilization rate and the industrial economic value.
Description
Technical Field
The invention relates to the technical field of bioactive peptides, in particular to a euphausia superba small molecule bioactive peptide, a preparation method and application thereof.
Background
Hyperuricemia (HUA) is a metabolic disease caused by abnormal purine metabolism or hypouric acid excretion in humans, and is mainly manifested by abnormally elevated serum uric acid levels (male blood uric acid > 7 mg/dl, female blood uric acid > 6 mg/dl). With the development of socioeconomic performance, lifestyle and dietary structure changes, HUA has been increasingly attracting public attention as a common disease. The related data show that the combined prevalence rate of the hyperuricemia of the adult in China is as high as 13.3 percent, and the trend of the hyperuricemia of the adult in China is younger. Furthermore, HUA plays an important role in related metabolic diseases, and hyperuricemia is generally regarded as a prognostic indicator of chronic kidney disease, diabetes, cardiovascular disease and inflammation. The uric acid reducing drugs such as allopurinol, febuxostat, benzbromarone and the like which are clinically used at present have obvious treatment effects, but have serious adverse reactions such as anaphylactic reaction, liver and kidney damage and the like and are high in price. Therefore, the development of safe, efficient and economical uric acid lowering drugs has become an important and hot spot of current research.
The bioactive peptide is polypeptide with special physiological functions of resisting tumor, regulating immunity, resisting bacteria, relieving pain, resisting inflammation, etc. obtained through fermentation, enzymolysis, chemical hydrolysis, etc. In recent years, bioactive peptides become hot spots in the field of uric acid reduction research due to the characteristics of easy absorption, low preparation cost, high safety and the like.
Xanthine oxidase (XOD, EC 1.17.3.2) is a key enzyme regulating uric acid production during the metabolic process in humans. XOD is mainly found in the liver and small intestine of humans, and can continuously oxidize hypoxanthine and xanthine to produce uric acid, while also converting purine substances taken in food into uric acid. In addition, in the process of catalyzing purine metabolism by XOD, active oxygen molecules such as superoxide anion free radicals and hydrogen peroxide are generated to participate in oxidative stress of organisms, so that physical health is further damaged. Thus, lowering uric acid levels by inhibiting XOD activity is an important method for treating hyperuricemia.
Antarctic krill (Antarctic krill) has a huge biological reserve, being the largest animal protein resource on earth. Defatted antarctic krill powder is an economic byproduct produced in the krill oil production process, contains high-quality protein and has high protein content, but the products are mainly used as food, feed additives and the like, and the resource utilization rate is low. How to effectively utilize the high-quality protein in the defatted euphausia superba powder becomes a problem to be solved.
Disclosure of Invention
The invention aims to provide euphausia superba small molecule active peptide and a preparation method thereof, and another aim is to provide application thereof.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a euphausia superba small molecule active peptide, the amino acid sequence of which is: LPPYSKE, or EDVEGAVR, or LDLPGWVK, or LDDAFNHL, or WDRPLVE.
Wherein LPPYSKE specifically comprises: leu-Pro-Pro-Tyr-Ser-Lys-Glu;
the EDVEGAVR is specifically: glu-Asp-Val-Glu-Gly-Ala-Val-Arg;
the LDLPGWVK is specifically: leu-Asp-Leu-Pro-Gly-Trp-Val-Lys;
the LDDAFNHL is specifically: leu-Asp-Asp-Ala-Phe-Asn-His-Leu;
the WDRPLVE is specifically: trp-Asp-Arg-Pro-Leu-Val-Glu.
The preparation method of the euphausia superba small molecule active peptide comprises the following steps:
(1) Enzymatic hydrolysis is carried out on defatted euphausia superba powder to obtain enzymatic hydrolysate;
(2) High-temperature enzyme deactivation;
(3) Centrifugal separation is carried out to obtain euphausia superba enzymolysis supernatant;
(4) Desalting and freeze-drying the euphausia superba enzymolysis supernatant to obtain euphausia superba enzyme defrosting dry powder, namely euphausia superba micromolecular active polypeptide mixture.
Further, the preparation method further comprises the steps of further purifying by adopting an affinity ultrafiltration method: dissolving the antarctic krill enzyme thawing dry powder, incubating with the XOD enzyme together, ultrafiltering and centrifuging after incubation to obtain trapped fluid, eluting to enable the XOD enzyme to denature and release the combination with the small molecular peptide, collecting the obtained permeate, and drying to obtain the purified antarctic krill active polypeptide mixture.
Preferably, the concentration of the enzymolysis liquid of the freeze-dried powder in the step (5) is 50-250mg/ml, the concentration of the XOD enzyme is 0.1-0.5U/ml, and the volume ratio of the enzymolysis liquid to the XOD enzyme is 1:1-1:5, endowing the material for 20-60min. Preferably, the concentration of the enzymolysis liquid is 200 mg/mL, the concentration of the XOD enzyme is 0.2U/mL, and the volume ratio of the enzymolysis liquid to the XOD enzyme is 1:2, incubation temperature is 37 ℃ and incubation time is 30 min.
Further, identifying euphausia superba small molecular active peptides based on LC-MS/MS mass spectrum, and then screening to finally obtain the 5 euphausia superba small molecular active peptides.
Preferably, in the step (1), the defatted antarctic krill powder and water are uniformly mixed according to a certain mass ratio (1:1-10), the optimal enzymolysis pH value is adjusted, enzyme is added to start the enzymolysis reaction, and the pH value of the solution is kept constant in the reaction process; the protease is selected from alkaline protease, neutral protease, papain and flavourzyme.
Preferably, the protease is alkaline protease with enzymolysis pH of 9.5 and enzymolysis temperature of 55deg.C, and protease activity of 2×10 5 U/g; the mass fraction of the alkaline protease used is 1.6%; the enzymolysis time is 4.6 h.
Preferably, the enzyme deactivation temperature in the step (2) is 100 ℃, and the enzyme deactivation time is 10-20 min, preferably 15min.
Preferably, the centrifugation speed in the step (3) is 2000-5000r/min, the centrifugation time is 20-60min, preferably the centrifugation speed is 4000 r/min, and the centrifugation time is 30 min.
Preferably, the desalination method in the step (4) is dialysis desalination, wherein a dialysis bag of 200 Da is selected to desalt 48 h in pure water, the freezing temperature is-80 ℃, and the drying time is 24 h.
The application of the euphausia superba small molecule active peptide in inhibiting Xanthine Oxidase (XOD).
Furthermore, the application of the euphausia superba small molecule active peptide in preparing products for reducing high uric acid is provided.
The invention has the advantages and beneficial effects that:
according to the invention, the defatted euphausia superba powder is used for preparing the XOD inhibitory peptide, and the obtained polypeptide has a high XOD inhibition rate. Through LC-MS/MS identification, 5 small molecular polypeptides with XOD inhibition activity are obtained from the defatted antarctic krill powder enzymatic hydrolysate by screening: LPPYSKE, EDVEGAVR, LDLPGWVK, LDDAFNHL and WDRPLVE. These 5 polypeptides all have varying degrees of XOD inhibitory activity.
The euphausia superba peptide prepared by the method has good XOD inhibition capability, and can be applied to the deep processing fields of health products, medicines and the like. The invention can utilize the byproducts of aquatic product processing in a high value, and can effectively improve the resource utilization rate and the industrial economic value.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
FIG. 1 is a mass spectrum Baseak diagram of a sample in example 6.
FIG. 2 is a secondary mass spectrum of a small molecule active polypeptide with the amino acid sequence LPPYSKE.
FIG. 3 is a secondary mass spectrum of a small molecule active polypeptide with an amino acid sequence of EDVEGFAVR.
FIG. 4 is a secondary mass spectrum of a small molecule active polypeptide with an amino acid sequence of LDLPGWVK.
FIG. 5 is a secondary mass spectrum of a small molecule active polypeptide with an amino acid sequence of LDDAFNHL.
FIG. 6 is a secondary mass spectrum of a small molecule active polypeptide with an amino acid sequence WDRPLVE.
FIG. 7 is a graph showing the result of molecular docking of the peptide LPPYSKE with XOD.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are provided to illustrate the invention and not to limit the invention.
In the following examples, the experimental methods for measuring XOD inhibition rates of each sample were as follows:
(1) Preparing a solution:
xanthine oxidase solution (0.05U/mL): taking 77 mu L of enzyme solution, and using 1 XPBS buffer solution to fix the volume to 10 mL;
xanthine solution (0.40 mmol/L): 6.08 mg xanthine powder was weighed out and dissolved in 400. Mu.L 1 mol/L NaOH and then buffered to a volume of 100 mL.
(2) The experimental method comprises the following steps:
50. Mu.L of the sample to be measured (diluted to 9 mg/mL) and 50. Mu.L of a xanthine oxidase solution having a concentration of 0.05U/mL were added to each well of a 96-well plate, the mixture was shaken for 30 s, incubated at 25℃for 5min, 150. Mu.L of a xanthine solution of 0.40 mM was added, and after 30 s shaking, incubation was carried out at 25℃for 25 min, and the absorbance at 290 nm was measured.
(3) The calculation formula is as follows:
the xanthine oxidase inhibitory activity was calculated as follows:
wherein A is 1 Representing the absorbance of the sample solution plus enzyme; a is that 2 Indicating that the sample solution was not enzymatically absorbance; a is that 3 Absorbance of a blank group representing buffer solution instead of sample solution; a is that 4 The absorbance without enzyme was shown for the blank.
Example 1:
a method for preparing antarctic krill active peptide with XOD inhibitory activity, comprising the following steps:
(1) Enzymolysis: the defatted antarctic krill powder and water are mixed according to the proportion of 1:5, uniformly mixing the materials according to the mass ratio, uniformly dispersing the materials by a high-speed dispersing machine, preheating the materials to 50 ℃, adjusting the pH to 9, adding alkaline protease to start enzymolysis reaction, oscillating in a water bath, and maintaining the pH value of the solution constant in the reaction process;
(2) High-temperature enzyme deactivation: inactivating enzyme in boiling water bath at 100deg.C for 15min;
(3) And (3) centrifugal separation: 4000 Centrifuging for 30 min at r/min, and filtering to obtain enzymolysis liquid;
(4) Desalting and freeze-drying: desalting and freeze-drying the supernatant obtained in the step (3) to obtain antarctic krill enzyme thawing dry powder.
Example 2
A method for preparing antarctic krill peptide with XOD inhibitory activity, the method comprising the steps of:
(1) Enzymolysis: the defatted antarctic krill powder and water are mixed according to the proportion of 1:5, uniformly mixing the materials according to the mass ratio, uniformly dispersing the materials by a high-speed dispersing machine, preheating the materials to 50 ℃, adjusting the pH to 7, adding neutral protease to start enzymolysis reaction, oscillating in a water bath, and maintaining the pH value of the solution constant in the reaction process;
(2) High-temperature enzyme deactivation: inactivating enzyme in boiling water bath at 100deg.C for 15min;
(3) And (3) centrifugal separation: 4000 Centrifuging for 30 min at r/min, and filtering to obtain enzymolysis liquid;
(4) Desalting and freeze-drying: desalting and freeze-drying the supernatant obtained in the step (3) to obtain antarctic krill enzyme thawing dry powder.
Example 3
A method for preparing antarctic krill peptide with XOD inhibitory activity, the method comprising the steps of:
(1) Enzymolysis: the defatted antarctic krill powder and water are mixed according to the proportion of 1:5, uniformly mixing the materials according to the mass ratio, uniformly dispersing the materials by a high-speed dispersing machine, preheating the materials to 55 ℃, adjusting the pH to 6.5, adding papain to start enzymolysis reaction, oscillating in a water bath, and maintaining the pH value of the solution constant in the reaction process;
(2) High-temperature enzyme deactivation: inactivating enzyme in boiling water bath at 100deg.C for 15min;
(3) And (3) centrifugal separation: 4000 Centrifuging for 30 min at r/min, and filtering to obtain enzymolysis liquid;
(4) Desalting and freeze-drying: desalting and freeze-drying the supernatant obtained in the step (3) to obtain antarctic krill enzyme thawing dry powder.
Example 4
A method for preparing antarctic krill peptide with XOD inhibitory activity, the method comprising the steps of:
(1) Enzymolysis: the defatted antarctic krill powder and water are mixed according to the proportion of 1:5, uniformly mixing the materials according to the mass ratio, uniformly dispersing the materials by a high-speed dispersing machine, preheating the materials to 50 ℃, adjusting the pH to 7, adding flavourzyme to start enzymolysis reaction, oscillating in a water bath, and maintaining the pH value of the solution constant in the reaction process;
(2) High-temperature enzyme deactivation: inactivating enzyme in boiling water bath at 100deg.C for 15min;
(3) And (3) centrifugal separation: 4000 Centrifuging for 30 min at r/min, and filtering to obtain enzymolysis liquid;
(4) Desalting and freeze-drying: desalting and freeze-drying the supernatant obtained in the step (3) to obtain antarctic krill enzyme thawing dry powder.
Example 5
The invention adopts a UV method to measure the XOD inhibition activity of the enzyme extract.
The freeze-dried peptide powder of Euphausia superba obtained in examples 1-4 was taken for XOD inhibition analysis, respectively. The XOD inhibition rates of the four product lyophilized peptide powders are shown in table 1. The measurement result shows that the product obtained by enzymolysis of alkaline protease has significantly higher XOD inhibitory activity than the enzymolysis products obtained under other three conditions, and the use of alkaline protease for enzymolysis of defatted antarctic krill powder is more beneficial to the preparation of XOD inhibitory peptide. Therefore, the XOD inhibitory peptide prepared by the method realizes the high-efficiency utilization of the byproducts of aquatic product processing, can be used as a medicine or health food raw material for treating and preventing HUA, effectively improves the utilization efficiency of defatted antarctic krill powder, and obtains active peptide powder by using a protease enzymolysis method, and has the advantages of low price, economy, environmental protection, high safety and good application prospect.
TABLE 1 Antarctic krill peptide XOD inhibition by different proteases
The antarctic krill enzyme thawed dry powder prepared in example 1 above was purified: dissolving the antarctic krill enzyme thawing dry powder, incubating with the XOD enzyme together, ultrafiltering and centrifuging after incubation to obtain a trapped fluid, eluting with 70% (v/v) methanol solution to enable the XOD enzyme denaturation to release the combination with the small molecular peptide, collecting the obtained permeate, and drying to obtain the purified antarctic krill small molecular active polypeptide mixture. The concentration of the enzymolysis liquid of the freeze-dried powder is 200 mg/mL, the concentration of the XOD enzyme is 0.2U/mL, and the volume ratio of the enzymolysis liquid to the XOD enzyme is 1:2, incubation temperature is 37 ℃ and incubation time is 30 min.
Example 6
LC-MS/MS mass spectrum identification of euphausia superba XOD small molecule inhibitory peptide comprises the following steps:
(1) Solution quantification, taking the sample obtained after purification of example 1, desalting with C18 StageTip and vacuum drying. After drying the peptide was reconstituted with 0.1% FA and OD280 was used to determine peptide concentration for LC-MS analysis.
(2) The XO-inhibiting peptide sequence was determined using the LC-MS/MS method. The detection is based on previous methods. Buffer A was 0.1% formic acid (v/v) in water and buffer B was 0.1% formic acid (v/v) in acetonitrile (v/v) in water. The column was equilibrated with 100% solution a. Samples were introduced into a C18 capillary capture column (100 μm x 20 mm, 5 μm, dr. Maisch GmbH) and then subjected to gradient separation through a C18 separation column (75 μm x 150 mm,3 μm, dr. Maisch GmbH) at a flow rate of 300 nl/min. The liquid phase separation gradient is as follows:
2-5% of B phase lasts for 0-2 min,5-28% of B phase lasts for 2-44 min,28-40% of B phase lasts for 44-51 min,40-100% of B phase lasts for 51-53 min, and B phase keeps 100% of B phase lasts for 53-60 min.
After separation of the peptide fragments, DDA (data dependent acquisition) mass spectrometry was performed using a Q-exact mass spectrometer. Analysis duration was 60min, detection mode: positive ion, parent ion scan range: 350-1800 m/z; first order mass spectrum resolution: 60000 @m/z 200; AGC target:3e6; first order maximumit: 50 ms, a primary mass spectrum obtained by mass spectrometry of the sample is shown in figure 1.
Peptide fragment secondary mass spectrometry was collected as follows: the secondary mass spectrum (MS 2 scan) of 20 highest intensity parent ions was acquired triggered after each full scan (full scan). Secondary mass spectrum resolution: 15000 @m/z 200; AGC target 1e5; second order maximumit: 50 ms; MS2 Activity Type: HCD; isolation window:1.6m/z; normalized collision energy:28.
(3) The Uniprot protein database was analyzed using PEAKS Studio 10.6 software to finally obtain the identified peptides.
TABLE 2 PEAKS Studio analysis parameter set-up
After mass spectrum data retrieval, PSM FDR is less than or equal to 0.01 and Protein FDR is less than or equal to 0.01 and is respectively peptide fragment, site and egg
Screening criteria for white identification. And finally, identifying the sample to obtain 943 peptide fragments and 13 protein sequences.
Example 7
Screening peptide fragments by molecular docking, comprising the steps of:
(1) The polypeptide from example 6 was 3D modeled in ChemDraw 21.0.0 software and energy minimized as a ligand.
(2) The XOD (PDB ID:1N 5X) containing the ligand TEI was obtained by downloading from the protein database PDB (http:// www.rcsb.org /). The resulting XOD crystal structure was imported into Discovery Studio 2019 Client (DS 2019) and subjected to some preparatory operations such as dehydration hydrogenation atoms prior to molecular docking.
(3) The molecular docking is carried out by taking 1N5X as a receptor protein, taking the polypeptide of virtual enzymolysis as a ligand, taking (X: 96.574, Y:55.310, Z: 39.332) as a docking center, and taking 15A as a docking radius. And carrying out molecular docking by adopting a semi-flexible docking LibDock in the Dock liquids, setting parameters for rigid optimization and rapid screening, and setting the rest parameters as default values for molecular docking. And (5) performing the next operation on the screened polypeptide according to the score.
Results: 82 peptide fragments were selected from 943 peptide fragments in example 6 and subjected to DS2019 simulation docking with XOD, and 5 peptide fragments were obtained by screening according to LibDock Score for synthesis; the secondary mass spectrograms of the 5 peptide fragments are shown in fig. 2-6, and the butt joint schematic diagram is shown in fig. 7.
Example 8
The peptide fragment obtained by screening in example 7 is subjected to solid phase synthesis and activity verification, and comprises the following steps:
(1) Peptide LPPYSKE, EDVEGAVR, LDLPGWVK, LDDAFNHL and WDRPLVE were synthesized by Fmoc solid phase synthesis method from biological engineering (Shanghai) Co., ltd.
(2) Preparing the polypeptide into 0.1-5mg/mL solution, and measuring the in vitro XO inhibition rate of the synthesized polypeptide by adopting a UV method.
TABLE 3 peptide fragment information and XOD inhibition
Note that: (1) The LibDock Score shown in the table is the average of the highest values of the 5 docking results, due to the randomness of the molecular docking results.
(2) The toxicity of the polypeptide was calculated by http:// crdd. Osdd. Net/raghava// toxinpred.
As shown in Table 3, the 5 peptide fragments have higher XOD inhibitory activity, wherein the peptide fragment with the amino acid sequence LPPYSKE shows the highest XOD inhibitory activity. And the prepared antarctic krill enzyme thawed dry powder IC 50 The value is 3.11 mg/mL, namely the XOD inhibition activity of the 5 small molecule active peptides prepared by the invention can be up to 10 times compared with the XOD inhibition activity of the 5 small molecule active peptides, and the 5 small molecule active peptides have extremely obvious XOD inhibition activity.
The above examples are only preferred embodiments of the present invention and do not limit the scope of the claims, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the scope of the invention.
Claims (8)
1. The euphausia superba small molecule active peptide is characterized in that the amino acid sequence of the active peptide is as follows: LPPYSKE, or EDVEGAVR, or LDLPGWVK, or LDDAFNHL, or WDRPLVE; wherein LPPYSKE specifically comprises: leu-Pro-Pro-Tyr-Ser-Lys-Glu;
the EDVEGAVR is specifically: glu-Asp-Val-Glu-Gly-Ala-Val-Arg;
the LDLPGWVK is specifically: leu-Asp-Leu-Pro-Gly-Trp-Val-Lys;
the LDDAFNHL is specifically: leu-Asp-Asp-Ala-Phe-Asn-His-Leu;
the WDRPLVE is specifically: trp-Asp-Arg-Pro-Leu-Val-Glu.
2. The preparation method of the euphausia superba small molecule active peptide is characterized by comprising the following steps:
(1) Enzymatic hydrolysis is carried out on defatted euphausia superba powder to obtain enzymatic hydrolysate;
(2) High-temperature enzyme deactivation;
(3) Centrifugal separation is carried out to obtain euphausia superba enzymolysis supernatant;
(4) Desalting and freeze-drying the euphausia superba enzymolysis supernatant to obtain euphausia superba enzyme defrosting dry powder, namely euphausia superba micromolecular active polypeptide mixture.
3. The method of claim 2, further comprising further purifying by affinity ultrafiltration: dissolving the antarctic krill enzyme thawing dry powder, incubating with the XOD enzyme together, ultrafiltering and centrifuging after incubation to obtain trapped fluid, eluting to enable the XOD enzyme to denature and release the combination with the small molecular peptide, collecting the obtained permeate, and drying to obtain the purified antarctic krill active polypeptide mixture.
4. The method of claim 3, wherein the concentration of the antarctic krill enzyme thawing dry powder enzymatic hydrolysate is 50-250mg/ml, the concentration of the XOD enzyme is 0.1-0.5U/ml, and the volume ratio of enzymatic hydrolysate to XOD enzyme is 1:1-1:5, endowing the material for 20-60min.
5. The preparation method of claim 2, wherein the euphausia superba small molecule active peptides are identified based on LC-MS/MS mass spectrometry, and then screened to obtain 5 euphausia superba small molecule active peptides with the amino acid sequences: LPPYSKE, or EDVEGAVR, or LDLPGWVK, or LDDAFNHL, or WDRPLVE.
6. The preparation method of claim 2, wherein in the step (1), defatted antarctic krill powder and water are uniformly mixed, the optimal enzymolysis pH value and temperature are adjusted, enzyme is added to start the enzymolysis reaction, and the pH value of the solution is kept constant during the reaction; the protease is one or a combination of more of alkaline protease, neutral protease, papain and flavourzyme.
7. Use of the euphausia superba small molecule active peptide of claim 1 for inhibiting xanthine oxidase.
8. The use of the euphausia superba small molecule active peptide of claim 1 in the preparation of a reduced hyperuricemia product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143949A (en) * | 2023-08-31 | 2023-12-01 | 青岛海洋食品营养与健康创新研究院 | Euphausia superba source high F value oligopeptide and application thereof in liver protection |
| CN117567562A (en) * | 2023-11-24 | 2024-02-20 | 中国水产科学研究院黄海水产研究所 | Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide as well as preparation method and application thereof |
| CN117568430A (en) * | 2023-11-07 | 2024-02-20 | 中国海洋大学 | Antarctic krill peptide with uric acid reducing activity and application thereof |
| CN117568430B (en) * | 2023-11-07 | 2024-05-31 | 中国海洋大学 | Antarctic krill peptide with uric acid reducing activity and application thereof |
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2023
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143949A (en) * | 2023-08-31 | 2023-12-01 | 青岛海洋食品营养与健康创新研究院 | Euphausia superba source high F value oligopeptide and application thereof in liver protection |
| CN117143949B (en) * | 2023-08-31 | 2024-04-05 | 青岛海洋食品营养与健康创新研究院 | Euphausia superba source high F value oligopeptide and application thereof in liver protection |
| CN117568430A (en) * | 2023-11-07 | 2024-02-20 | 中国海洋大学 | Antarctic krill peptide with uric acid reducing activity and application thereof |
| CN117568430B (en) * | 2023-11-07 | 2024-05-31 | 中国海洋大学 | Antarctic krill peptide with uric acid reducing activity and application thereof |
| CN117567562A (en) * | 2023-11-24 | 2024-02-20 | 中国水产科学研究院黄海水产研究所 | Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide as well as preparation method and application thereof |
| CN117567562B (en) * | 2023-11-24 | 2024-06-04 | 中国水产科学研究院黄海水产研究所 | Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide as well as preparation method and application thereof |
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