CN116284220B - Cholesterol esterase inhibition polypeptide combination and preparation method and application thereof - Google Patents
Cholesterol esterase inhibition polypeptide combination and preparation method and application thereof Download PDFInfo
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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
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- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- 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
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a preparation method of a cholesterol esterase inhibition polypeptide combination, which comprises the following steps: 1) Adding bonito red meat into double distilled water for homogenate for later use; 2) Adding alkaline protease into the bonito red pulp homogenate obtained in the step 1) for enzymolysis treatment; 3) Freeze-drying the enzymolysis liquid obtained in the step 2); 4) Performing polypeptide de novo sequencing on the enzymolysis liquid frozen powder obtained in the step 3); 5) Based on the polypeptide sequence results, cholesterol esterase molecules are used for docking, resulting in cholesterol esterase inhibitory polypeptide combinations (FFPE and/or VLMF). The bonito cholesterol esterase inhibitory polypeptide combination (FFPE and/or VLMF) provided by the invention has an inhibitory effect on cholesterol esterase and has obvious lipid-lowering activity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cholesterol esterase inhibition polypeptide combination and a preparation method and application thereof.
Background
The bonito belongs to one kind of tuna, is also called as small tuna, has the characteristics of high protein, low fat and the like, is rich in polyunsaturated fatty acids with biological activity such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and the like, and meanwhile, is rich in methionine, taurine, minerals and vitamins, and is a green pollution-free healthy food recommended by the International nutrient Association. The tuna products mainly comprise raw fillets and canned fish, are favored by consumers, but the processing process can generate more byproducts, such as fish skin, viscera, minced meat, fish bones and the like, and account for 50-70% of the weight of the tuna. Currently, byproducts are processed mainly as fishing lures or animal feeds, with lower commercial value. Bonito has a strong market potential as a large-scale and inexpensive tuna. However, the meat quality is sour, the taste is poor, the proportion of dark meat is large, and the fishy smell is heavy, so that the meat is not well developed and utilized, and is generally only sold as a can or a wooden fish essence seasoning, so that the research on related products is very little, and the economic benefit is low. How to effectively utilize the dark meat of bonito to develop high added value products has attracted attention from aquatic processing enterprises.
In recent years, a great deal of research shows that the food protein source bioactive peptide is a potential efficacy factor for regulating the metabolism of organisms and promoting the health of human bodies. Bioactive peptides are usually composed of 2-20 (and from a perspective 2-50) amino acids, and these sequences often exhibit biological activity by being embedded in a parent protein sequence, and generally exhibit stronger activity after being released by hydrolysis by fermentation, enzymolysis, gastrointestinal digestion, and the like. Compared with protein, the peptide has the characteristics of low molecular weight, easy absorption, high stability and the like. In recent years, scientific researchers have carried out a series of research work on bonito red meat enzymolysis peptides, li Shufan and the like, so as to prepare the tuna red meat enzymolysis peptides, wherein the enzymolysis peptides play a positive role in resisting fatigue and regulating intestinal flora of mice, and through analysis, the relative content of polypeptide sequences of the former ten in enzymolysis liquid contains 4-11 amino acids (205-1199 Da), and the sequences are respectively: KEFT, EESAS, RYDD, VEKE, TIRM, FPRM, PVALSCHC, MLGGFGNW, MIGGFGNW, YRDFYYKT and PPCQLINQTVS. Pu Yuehua and other researches show that the long-fin tuna head polypeptide TIP3C (200-800 Da) has a strong scavenging effect on 4 free radicals, the scavenging rate is obviously increased along with the increase of mass concentration, and the dose effect relationship is obvious. Chen Yue and the like can increase the memory of mice and increase the skin elasticity of the mice by increasing the content of SOD and GSH-Px in brain tissues of the mice, reducing the content of MDA and up-regulating the expression level of memory related genes; improving the sleep quality of mice. Sun Tingting and the like can improve the body mass, serum insulin, glycosylated hemoglobin and blood lipid level of db/db diabetic mice, and the action effect of Sun Tingting and the like can be related to the expression level of CYP11B1 which is the gene related to the downregulation of aldosterone synthesis. The enzyme hydrolysis of bonito with Xing-Wei Xiang to obtain the polypeptide <1 kDa can improve colitis in mice, mainly by increasing SOD and GSH-Px and reducing expression of inflammatory factors LPS, IL-6 and TNF-alpha. The pentapeptide (ACECD) is obtained by separating and purifying enzymatic hydrolysate of bonito fish meat by H Zhong, etc., has molecular weight of 600-1000 Da, and has antioxidant and xanthine oxidase activity inhibiting effects.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to design and provide a cholesterol esterase inhibition polypeptide combination and a preparation method and an application technical scheme thereof.
The invention is realized by the following technical scheme:
In a first aspect the invention provides a cholesterol esterase inhibiting polypeptide combination comprising a polypeptide as described in SEQ ID NO.1 and/or a polypeptide as described in SEQ ID NO. 2.
In a second aspect, the invention provides the use of a combination of cholesterol esterase inhibitory polypeptides as described above as a cholesterol esterase inhibitor.
The third aspect of the invention provides an application of the cholesterol esterase inhibition polypeptide combination in preparing lipid-lowering medicaments.
According to a fourth aspect of the present invention, there is provided a method for preparing the above cholesterol esterase inhibitory polypeptide combination, characterized by comprising the steps of:
1) Adding bonito red meat into double distilled water for homogenate for later use;
2) Adding alkaline protease into the bonito red pulp homogenate obtained in the step 1) for enzymolysis treatment;
3) Freeze-drying the enzymolysis liquid obtained in the step 2) into powder;
4) Performing polypeptide de novo sequencing on the enzymolysis liquid frozen powder obtained in the step 3);
5) And according to the polypeptide sequence result, butting with cholesterol esterase molecules to obtain a cholesterol esterase inhibition polypeptide combination.
Further, the enzymolysis conditions in the step 2) are as follows: the enzyme adding amount is 5000U/g, the pH value is 8, and the feed liquid ratio is 1:5, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 10 hours.
Compared with the prior art, the bonito cholesterol esterase inhibitory polypeptide combination (FFPE and/or VLMF) provided by the invention has an inhibitory effect on cholesterol esterase and has obvious lipid-lowering activity.
Drawings
FIG. 1 is a flow chart of screening for cholesterol esterase inhibitory polypeptide combinations of the invention.
Description of the embodiments
The invention is further illustrated by the following examples.
Example 1: preparation of cholesterol esterase inhibiting polypeptide combinations
1) Adding bonito red meat into double distilled water for homogenate for later use;
2) Adding alkaline protease into the bonito red pulp homogenate obtained in the step 1) for enzymolysis treatment, wherein the enzymolysis conditions are as follows: the enzyme adding amount is 5000U/g, the pH value is 8, and the feed liquid ratio is 1:5, the enzymolysis temperature is 45 ℃, the enzymolysis time is 10 hours, the enzymolysis is finished, the enzyme is inactivated in a boiling water bath for 15 minutes, after cooling, 10000r/min is centrifuged for 30 minutes, and the supernatant, namely the bonito protein enzymolysis liquid, is collected;
3) Freeze-drying the bonito proteolytic liquid obtained in the step 2) into powder, and preserving at-20 ℃;
4) Performing polypeptide de novo sequencing on the enzymolysis liquid frozen powder obtained in the step 3);
5) According to the polypeptide sequence result, the cholesterol esterase molecules are used for butt joint to obtain a cholesterol esterase inhibition polypeptide combination (FFPE and/or VLMF peptides).
Example 2: screening of cholesterol esterase inhibiting polypeptide combinations
As shown in fig. 1, the specific screening steps include:
(1) At this stage of screening, only polypeptide sequences with an Average Local Confidence (ALC) value of at least 60% were selected, as this value gives a high degree of confidence that a particular amino acid is accurately present in the sequence, creating a de novo predicted peptide library comprising 48684 sequences.
(2) The PEPTIDERANKER server based on the N-to-1 neural network returns a predicted probability report of biological activity from the peptide library, and over 80% of the 1984 polypeptides were labeled as having potential biological activity.
(3) Prior to exploring the potential biological activity that might be exhibited, it was necessary to predict the impact of polypeptide toxic properties on the organism, and ToxinPred servers were used to predict the toxicity of a given peptide to help identify a particular peptide without development limitations, SVM scores below 0 were considered non-toxic, a total of 1939 peptides.
(4) 1939 Nontoxic peptides are further butted with cholesterol esterase (PDB ID:1 AQL) through Pepsite, and the combined significance P value is less than 0.05, so that the peptide has certain inhibition potential; since Pepsite2 is the maximum input of 10 peptides, peptides higher than 10 amino acids are input in compromise, the average P value is calculated; of 1939 peptides, 1808 peptides can have significant interaction with cholesterol esterase 1AQL, intersection polypeptides which can have significant binding with target enzymes in a peptide library can be generated, and 1808 peptides are subjected to virtual screening; the potential protein-peptide interactions (P < 0.05) and their P-values between peptides (length <10 AA) and cholesterol esterases (PDB code: 1 AQL) as determined for Pepsite server in table 1.
(5) LeDOCK and AutoDock Vina.1.2 were used for virtual screening and mutual validation to determine promising peptide fragments. In the virtual screening of the above strategy, the following coordinate definitions are used: the coordinates of cholesterol esterase (PDB ID: 1 AQL) docking are: for LeDock, xmin=14.5, xmax=59.5, ymin=13.1, ymax=68.1, zmin=17.5 and zmax=62.5, for Vina, center_x=37.0, center_y=40.6, center_z=40.0, size_x=45, size y=55 and size_z=45. The above active site regions contain catalytic triads (i.e., SER194, D320, and HIS 435) and substrate binding sites (i.e., G107, a108, and a 195). Consensus scoring of the target enzyme-peptide complex with the lowest binding energy was performed using X-Score 1.3 software, which showed that the dissociation constant of-logKd, the μΜ affinity, i.e. -logKd =6.0, of the peptide fragment was retained. Following a virtual screening assay in 560 peptides, the promising peptide fragments were further docked with the target enzyme to demonstrate a possible binding pattern.
(6) Global sampling server HPEPDOCK is used to generate the 100 constellations and relatively rank by the docking score. Visualization and annotation of the binding sites of promising peptides on the target enzyme active site using UCSF chimera1.15, ligPlot +v.2.2.4 listing the non-binding interactions of target enzyme-peptide docking, MM/GBSA binding free energy calculation using MM/GBSA module of HawkDock server for binding structure, final screening of 560 peptides resulted: a bonito cholesterol esterase inhibitory polypeptide combination (FFPE and/or VLMF).
Example 3: functional verification of cholesterol esterase inhibiting polypeptide combinations
Cholesterol esterase inhibitory polypeptide combinations (FFPE and/or VLMF) on cholesterol esterase activity inhibition:
1) Dissolving 0.25 mg pig pancreatic cholesterol esterase in 10mL deionized water, centrifuging (8000 r/min,10 min), and collecting supernatant;
2) Substrate buffer: preparing 0.1mol/L phosphate buffer solution (pH 7.04) (containing 0.1mol/L NaCL,0.02 mol/L p-nitrophenyl butyrate and 6 mmol/L sodium taurocholate);
3) Respectively taking substrate buffer solution 925 [ mu ] L, and FFPE and VLM with different concentrations of 50 [ mu ] L and 25 [ mu ] L of enzyme solution in a centrifuge tube, incubating for 5min at 25 ℃, measuring a light absorption value (405 nm), and adding 25 [ mu ] L of deionized water into a control group; the inhibition ratio was calculated according to the following formula:
Cholesterol esterase= (control absorbance-sample absorbance)/control absorbance, as shown in table 2 for inhibition of cholesterol esterase activity at different concentrations of FFPE and VLM.
Table 2:
| concentration (mg/mL) peptide sequence | 0.1 | 0.5 | 1.0 | 2.5 |
| FFPE | 45.23%±2.6 | 55.53%±4.6 | 67.28%±2.9 | 89.21%±4.9 |
| VLMF | 35.56%±2.6 | 42.13%±5.2 | 57.68%±4.1 | 69.11%±3.2 |
Claims (2)
1. A cholesterol esterase inhibitory polypeptide combination comprising a polypeptide as set forth in SEQ ID No.1 and a polypeptide as set forth in SEQ ID No. 2.
2. A method of preparing a cholesterol esterase inhibitory polypeptide combination according to claim 1, comprising the steps of:
1) Adding bonito red meat into double distilled water for homogenate for later use;
2) Adding alkaline protease into the bonito red pulp homogenate obtained in the step 1) for enzymolysis treatment;
3) Freeze-drying the enzymolysis liquid obtained in the step 2) into powder;
4) Performing polypeptide de novo sequencing on the enzymolysis liquid frozen powder obtained in the step 3);
5) According to the polypeptide sequence result, using cholesterol esterase molecules to dock so as to obtain a cholesterol esterase inhibition polypeptide combination;
The enzymolysis conditions in the step 2) are as follows: the enzyme adding amount is 5000U/g, the pH value is 8, and the feed liquid ratio is 1:5, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 10 hours.
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| AR086250A1 (en) * | 2011-05-05 | 2013-11-27 | Hoffmann La Roche | FUSION POLIPEPTIDE PRESENTING A SEQUENCE OF AMINO ACIDS AND USING THE SAME |
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| Quantitative Structure-Activity Relationship Study of Bitter Peptides;HYUN-OCK KIM等;《J. Agric. Food Chem.》;20061231;第54卷;表1 * |
| 益生菌降胆固醇的机制及其评价策略研究进展;高媛等;《食品科学》;20231231;第44卷(第21期);第322-329页 * |
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