An abalone bioactive peptide, its preparation method and application
By preparing an abalone bioactive peptide with the amino acid sequence DYPRPW and binding it to HMGR protein, the problems of wasted abalone by-products and side effects of traditional lipid-lowering drugs were solved, achieving a highly efficient and low-toxicity lipid-lowering effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FISHERIES RESEARCH INSTITURE OF FUJIAN
- Filing Date
- 2023-06-08
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies have failed to effectively utilize abalone by-products to prepare peptides with lipid-lowering effects, and traditional lipid-lowering drugs have side effects, leading to resource waste and health risks.
Using abalone viscera as raw material, an abalone bioactive peptide with the amino acid sequence DYPRPW was prepared by alkaline protease hydrolysis, filtration, sequence identification, and virtual screening. This peptide was then combined with HMGR protein to form a complex, which was used to assist in lowering blood lipids.
Abalone active peptides can effectively inhibit the accumulation of total cholesterol and total triglycerides in cells, thus having a lipid-lowering effect with few toxic side effects, providing a new approach for the high-value utilization of abalone by-products.
Smart Images
Figure CN116535465B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of polypeptide technology, and in particular to an abalone active peptide, its preparation method, and its application. Background Technology
[0002] Hyperlipidemia refers to a condition where the level of cholesterol or triglycerides in the blood exceeds the normal range. Its causes include abnormal fat metabolism or transport, such as low levels of high-density lipoprotein (HDL) and high levels of low-density lipoprotein (LDL) in the blood. It easily leads to cardiovascular and cerebrovascular diseases such as coronary heart disease, stroke, and hypertension, seriously threatening human health. Currently, statins are the most commonly used lipid-lowering drugs in clinical practice. Reliable studies have shown that statin use has two main side effects: elevated transaminase levels and the risk of muscle pain. Therefore, developing highly effective and low-toxicity adjunctive lipid-lowering products is a new direction for the industry. However, current processing and utilization of abalone mainly focuses on canning, freezing, and fresh sales, with low utilization of deep processing methods and significant waste of by-products.
[0003] The raw materials for preparing lipid-lowering peptides are abundant and diverse. Continuously developing novel, high-value animal-derived proteins with rich sources for preparing bioactive peptides with lipid-lowering effects is of practical significance. Existing research on abalone bioactivity mainly focuses on the antioxidant activity of gonads, viscera, shells, and gills (byproducts). No technology has been found for preparing peptides with lipid-lowering effects using abalone byproducts as raw materials. Summary of the Invention
[0004] The purpose of this invention is to provide an abalone active peptide, its preparation method and application. It uses abalone by-products as raw materials to obtain polypeptides with lipid-lowering effects, providing a new idea for the high-value processing and utilization of abalone by-products, and also expanding resources for the development of natural lipid-lowering products.
[0005] To achieve the above objectives, this invention discloses an abalone active peptide with the amino acid sequence: DYPRPW.
[0006] The preparation method of the above-mentioned abalone active peptide includes the following steps:
[0007] S1. Raw Material Preparation
[0008] The viscera of abalone are selected as the raw material;
[0009] S2. Enzymatic hydrolysis
[0010] The viscera of abalone were hydrolyzed using alkaline protease to obtain an enzymatic hydrolysate, and the enzyme was inactivated after the hydrolysis was completed.
[0011] S3. Filtering
[0012] The enzymatic hydrolysate was centrifuged, coarsely filtered, and ultrafiltered to collect multiple polypeptides with a molecular weight of less than 1 kDa, which were then freeze-dried into powder.
[0013] S4. Sequence Identification
[0014] The lyophilized peptide powder was subjected to sequence identification, and multiple peptide sequences were obtained. Repeated sequences were deleted, and multiple non-repeating peptides were obtained by screening.
[0015] S5. Virtual Filtering
[0016] Using the protein HMGR as the receptor, multiple peptides were virtually screened using docking software. The peptides with the highest docking scores were then synthesized in a solid phase to obtain the aforementioned abalone active peptide.
[0017] Preferably, in step S2, the amount of alkaline protease added is 8000-12000 U / g substrate, the pH value is 9.5-10.5, the ratio of substrate to ultrapure water is 1:40-60, the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 3-6h.
[0018] Preferably, in step S2, the amount of alkaline protease added is 10000 U / g substrate, the pH value is 10.0, the ratio of substrate to ultrapure water is 1:50, the enzymatic hydrolysis temperature is 55℃, and the enzymatic hydrolysis time is 4h.
[0019] Preferably, in step S3, the enzymatic hydrolysate is centrifuged using a plate centrifuge, coarsely filtered using a ceramic membrane, and the filtrate is ultrafiltered using a membrane with a molecular weight of <1 kDa to collect multiple polypeptides with a molecular weight of less than 1 kDa.
[0020] Preferably, in step S4, after the lyophilized peptide powder is desalted by a desalting column, mass spectrometry analysis is performed using a nano-spray ion source mass spectrometer to obtain the peptide sequence.
[0021] Preferably, in step S5, the HMGR protein molecule is hydrogenated and dehydrated using the docking software Discovery Studio to minimize energy. The polypeptide molecule is drawn as a small molecule using Discovery Studio Client as a ligand. Finally, the active pocket of the HMGR protein is determined, and virtual screening is performed under the conditions of pocket range 8 and box edge 4.
[0022] The present invention also discloses a protein-peptide complex in which the peptide and the protein are bound together, wherein the amino acid sequence of the peptide is DYPRPW and the protein is HMGR protein.
[0023] Preferably, the crystal structure of the HMGR protein is 1HWK.
[0024] Furthermore, this invention also discloses the application of the above-mentioned abalone active peptide in the preparation of adjuvant lipid-lowering drugs.
[0025] The present invention has the following beneficial effects:
[0026] 1. The active peptide of this invention can effectively inhibit the accumulation of total cholesterol and total triglycerides in cells, and has a high inhibition rate on cholesterol micelle solubility. It has the effect of lowering blood lipids and can be used to prepare adjuvant lipid-lowering drugs. Compared with traditional lipid-lowering drugs, it has the advantages of high specificity and low toxicity and side effects.
[0027] 2. The abalone active peptide of this invention is prepared from abalone viscera, which makes use of waste and does not waste the precious abalone meat. It provides a new idea for the high-value processing and utilization of abalone by-products and expands resources for the development of natural lipid-lowering products. Attached Figure Description
[0028] Figure 1 This is the HPLC chromatogram of abalone active peptides.
[0029] Figure 2 This is the mass spectrum of abalone active peptides.
[0030] Figure 3 The effect of different concentrations of DYPRPW on the viability of Hep-G2 cells.
[0031] Figure 4 The effect of different concentrations of DYPRPW on total cholesterol (TC) in Hep-G2 cells.
[0032] Figure 5 The effect of different concentrations of DYPRPW on total triglycerides (TG) in Hep-G2 cells.
[0033] Figure 6 The cholesterol micelle solubility inhibition rate of different concentrations of DYPRPW.
[0034] Figure 7 The results show the molecular docking between 1HWK and DYPRPW.
[0035] Figure 8 for Figure 7 Enlarged schematic diagram of part A in the middle.
[0036] Figure 9 This is a graph showing the root mean square deviation (RMSD) of all atoms of a protein in a protein-ligand complex relative to its initial structure.
[0037] Figure 10 This is a graph showing the root mean square deviation (RMSD) of all atoms of the ligand in the protein-ligand complex relative to its initial structure.
[0038] Figure 11 The RMSF value represents the change in the amino acid backbone atoms of the protein-ligand complex over time.
[0039] Figure 12 This is a statistical diagram of protein-ligand complex interactions (categorized by amino acid participation).
[0040] Figure 13 A statistical graph of the interaction forces between proteins and small molecules (categorized by time). Detailed Implementation
[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.
[0042] This invention discloses an abalone bioactive peptide with the amino acid sequence DYPRPW, as shown in SEQ ID NO:1. This abalone bioactive peptide can be used in the preparation of adjuvant lipid-lowering drugs.
[0043] Based on the same inventive concept, this invention also discloses a protein-peptide complex in which the peptide and the protein are bound together, wherein the amino acid sequence of the peptide is DYPRPW, the protein is HMGR protein, and the crystal structure of the HMGR protein is 1HWK.
[0044] The preparation method of the above-mentioned abalone active peptide includes the following steps:
[0045] S1. Raw Material Preparation
[0046] The viscera of abalone are selected as the raw material.
[0047] S2. Enzymatic hydrolysis
[0048] Alkaline protease was used to enzymatically hydrolyze the viscera of abalone. The alkaline protease dosage was 8000–12000 U / g substrate, the pH was 9.5–10.5, the substrate-to-ultrapure water ratio was 1:40–60, the hydrolysis temperature was 40–60℃, and the hydrolysis time was 3–6 h. As a preferred embodiment, the alkaline protease dosage was 10000 U / g substrate, the pH was 10.0, the substrate-to-ultrapure water ratio was 1:50, the hydrolysis temperature was 55℃, and the hydrolysis time was 4 h. After hydrolysis, the enzyme was inactivated to obtain the hydrolysate.
[0049] S3. Filtering
[0050] The enzymatic hydrolysate was centrifuged using a plate centrifuge. After centrifugation, it was coarsely filtered through a ceramic membrane. The filtrate was then ultrafiltered through a membrane with a molecular weight of <1 kDa to collect multiple peptides with a molecular weight of less than 1 kDa, which were then freeze-dried into powder.
[0051] S4. Sequence Identification
[0052] The lyophilized peptide powder was sequenced, yielding multiple peptide sequences. Repeated sequences were removed, and multiple non-repeating peptides were selected. Specifically, the lyophilized peptide powder was desalted using a C18 desalting column, followed by mass spectrometry analysis using a nanospray ionization source to obtain the peptide sequences. The entire system was a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, MA, USA) with a tandem EASY-nanoLC 1200. A total of 1 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm). The sample was separated using a gradient at a rate of 60 min, with the column flow rate controlled at 300 nL / min, the column temperature at 40 °C, and the electrospray voltage at 2 kV. The gradient started at 2% B phase, increased non-linearly to 35% at 47 min, increased to 100% within 1 min, and maintained for 12 min. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows:
[0053] MS: Scan range (m / z): 200-2000; resolution: 70000; AGC target: 3e6; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 17500; AGC target: 1e5; maximum injection time: 45ms; collision energy: 28; dynamic exclusion time: 30s.
[0054] S5. Virtual Filtering
[0055] Using the protein HMGR with a crystal structure of 1HWK as the receptor, the crystal structure (PDB ID: 1HWK) of 3-hydroxy-3-methylglutaryl-CoA reductase (natural) was downloaded from the RCSB protein database (http: / / www.rcsb.org). The HMGR protein molecule was hydrogenated and dehydrated using the docking software Discovery Studio to minimize energy usage. Small molecules were plotted using Discovery Studio Client as ligands. Finally, the active pocket of the HMGR protein was determined, and virtual screening was performed with a pocket range of 8 and a box edge of 4.
[0056] The results of the virtual screening were ranked using Grid Score scores. Based on the ranking and interaction, peptides with stronger binding interactions with the pocket (i.e., peptides with higher scores) were selected for solid-phase synthesis to obtain the desired abalone active peptides.
[0057] The abalone active peptide of this invention was synthesized using a solid-phase method, and the purity and molecular weight of the synthesized peptide were identified using HPLC and LC / MS. Figure 1As shown, HPLC analysis confirmed that the purity of DYPRPW reached 99.53%. Figure 2 As shown, the relative molecular mass of DYPRPW was determined to be 832.9 by LC / MS. The physicochemical parameters of DYPRPW were analyzed using ProtParam, as shown in Table 1.
[0058] Table 1 Physicochemical parameters of abalone active peptides
[0059]
[0060] The toxicity and efficacy of the above-mentioned abalone bioactive peptides were determined as follows:
[0061] 1. Cytotoxicity assay
[0062] Hep-G2 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin. When the cells reached 80-90% confluence (logarithmic growth phase), they were digested, gently pipetted to mix, centrifuged to collect the cells, and diluted to 1*102. 5 Cells / mL. Add 100 μL of cell suspension to a 96-well plate and incubate at 37°C for 14-16 h in a CO2 incubator. When the cells reach 80-90% confluence, incubate the cells with different concentrations (0, 50, 100, 200, 400 μM) of peptide DYPRPW for 24 h. Determine cell viability according to the CCK8 kit method.
[0063] Effects of different concentrations of DYPRPW on the viability of Hep-G2 cells, such as Figure 3 As shown in the figure. The results indicate that treatment with DYPRPW at low concentrations of 50 μM to high concentrations of 400 μM had no significant effect on the cell viability of Hep-G2 cells, indicating that the 400 μM peptide treatment was non-toxic to Hep-G2 cells.
[0064] 2. Determination of TC and TG content
[0065] When cells reach the logarithmic growth phase, digest the cells, gently pipette to mix, centrifuge to collect the cells, add culture medium, pipette to mix again, count the cells using a hemocytometer, and dilute the cell count to 4 x 10⁹ cells with complete culture medium. 5Cells / mL. Add 250 μL of cell suspension to a 48-well plate and incubate at 37°C for 14-16 h in a CO2 incubator. When the cells reach 80-90% confluence, add different concentrations (0, 50, 100, 200, 400 μM) of peptides and free fatty acids (10% oleic acid and palmitic acid) and incubate for 24 h. A blank control group without added free fatty acids and peptides is also included. Wash cells with DPBS and then lyse them on ice for 45 min using 1.5% Triton-X100. Measure the TC and TG content of the cells using the Nanjing Jiancheng Triglyceride (TG) assay kit and the Total Cholesterol (TC / TCH) assay kit.
[0066] Prepare protein standards of different concentrations. Mix reagent A and reagent B in a 50:1 ratio (prepare fresh for immediate use). Each well contains 25 μL of sample and 200 μL of reaction solution. Incubate in the dark for 30 min before measuring the OD562 nm of the reaction wells.
[0067] Total cholesterol (TC) content (mmol / g prot) = (Sample A - Blank A) / (Standard A - Blank A) * Standard C (5.17) ÷ Cpr
[0068] Total triglyceride (TG) content (mmol / g prot) = (Sample A - Blank A) / (Standard A - Blank A) * Standard C (2.26) ÷ Cpr
[0069] The effects of different concentrations of DYPRPW treatment on total cholesterol (TC) accumulation in Hep-G2 cells are as follows: Figure 4 As shown. By Figure 4 It can be seen that treatment with different concentrations of DYPRPW significantly inhibited the accumulation of total cholesterol in Hep-G2 cells, and the intracellular cholesterol accumulation level decreased with increasing treatment concentration.
[0070] The effects of different concentrations of DYPRPW treatment on total triglyceride (TG) accumulation in Hep-G2 cells are as follows: Figure 5 As shown. By Figure 5 It can be seen that treatment with different concentrations of DYPRPW inhibited the accumulation of total triglycerides in Hep-G2 cells. The accumulation of triglycerides in cells of each group gradually decreased with the increase of treatment concentration, and there were significant differences between the treatment concentrations of 100, 200 and 400 μM and the model group.
[0071] 3. Determination of the in vitro cholesterol micelle solubility inhibition rate
[0072] The inhibition rate of peptides on cholesterol micelle solubility was determined using the method of Nagaoka et al., with slight modifications. The micelle solution (1 mL) contained 10 mM sodium taurine, 0.4 mM cholesterol, 1 mM oleic acid, 132 mM NaCl, and 15 mM sodium phosphate (pH 7.4). The peptide powder was dissolved in PBS to a final concentration of 10 mM, and then mixed with the cholesterol micelle solution to achieve final peptide concentrations of 50, 100, 200, and 400 μM. The mixed samples were ultrasonically emulsified at 37°C for 30 min and incubated with shaking at 37°C for 24 h. After centrifugation at 10000 r for 20 min at room temperature, the supernatant was used to determine the cholesterol content using a Nanjing Jiancheng total cholesterol test kit. A blank experiment was also performed, using an equal volume of PBS instead of the peptide solution. Finally, the inhibition rate of the peptide solution on cholesterol micelle solubility was calculated. The calculation formula is as follows:
[0073] Total cholesterol (TC) content (mmol / L) = Standard (5.17)
[0074] Cholesterol micelle solubility inhibition rate (%) =
[0075] The effect of different concentrations of DYPRPW on the inhibition rate of cholesterol micelle solubility is as follows: Figure 6 As shown. By Figure 6 It can be seen that the inhibition rate of peptide treatment on cholesterol micelle solubility is enhanced with increasing treatment concentration, and the effect is most obvious when the treatment concentration is 400 μM.
[0076] 4. Molecular docking verification
[0077] The X-ray crystal structure of 1 HWK was obtained from a protein database. The protonation state of the small molecule was set to pH = 7.4, and the compound was expanded into a 3D structure using Open Babel. A series of preparations were performed on the receptor protein and ligand using the AutoDock tool (ADT3). The docking box was generated using the AutoGrid program, followed by molecular docking using Autodock Vina (1.2.0). The optimal binding conformation was selected to analyze interactions. Finally, a protein-ligand interaction map was generated using PyMOL.
[0078] The key amino acids for the docking of the protein HMGR with DYPRPW in the 1HWK crystal structure are shown in Table 2.
[0079] Table 21 Key amino acids for HWK-DYPRPW docking
[0080]
[0081] like Figure 7 ,8 As shown, the 1HWK protein is represented by a dark blue cartoon model, the ligands by cyan stick models, and their binding sites by magenta stick structures. Nonpolar hydrogen atoms are omitted. Hydrogen bonds, ionic interactions, and hydrophobic interactions are depicted as yellow, magenta, and green dashed lines, respectively.
[0082] The discovered interactions were analyzed and categorized based on their interactions. Multiple sets of interactions were found between receptor proteins and small molecules, such as the hydrogen bond formed between LYS691 of 1HWK and its ligand. Under the influence of these interactions, the binding energy of the protein-small molecule complex was -8.2 kcal / mol, exhibiting excellent overall performance.
[0083] 5. Molecular dynamics simulation
[0084] Desmond / Maestro noncommercial version 2022.1 was selected as the kinetic simulation software. The complex system was equipped with TIP3P using the TIP3P water model, and a 0.15M sodium chloride solution was added to equilibrate the system. After minimizing and relaxing the system, a 100 ns molecular dynamics simulation was performed in an isothermal and isobaric system at 300 K and 1 bar. Trajectory coordinates were recorded every 100 ps. Molecular dynamics analysis was performed using Desmond's simulation interaction plot.
[0085] like Figure 9 , 10 As shown, the protein exhibited significant fluctuations during the simulation, and its RMSD remained unstable. Conversely, the ligand's RMSD stabilized at approximately 3.5 Å after the initial fluctuations. This indicates that both proteins possess a degree of initial conformational stability. The large protein fluctuations may be due to the larger size of the tetrameric protein, which contains other components with relatively poor stability, thus leading to greater volatility.
[0086] like Figure 11 As shown, the overall RMSF values of the amino acid residues that form the binding forces in small molecules are relatively small, but some individual amino acids have relatively large RMSF values, indicating that a significant conformational change occurs during the binding process. The RMSF of some atoms within the small molecule itself is also relatively large, confirming the above viewpoint.
[0087] like Figure 12 , 13As shown, the simulation revealed a rich array of interactions between the protein and the small molecule, resulting in multiple hydrogen bonds. For instance, the hydrogen bond formation frequency for ASP690 reached 99%, while for LYS735 it reached 98%, indicating that these amino acids played a crucial role in their binding. Furthermore, multiple hydrophobic interactions and water bridges were formed to further facilitate the binding. In conclusion, we can conclude that the small molecule and protein, through kinetic simulation, optimized their original binding conformation, making it more stable and exhibiting higher affinity.
[0088] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.
Claims
1. An abalone bioactive peptide, characterized in that: Its amino acid sequence is: DYPRPW.
2. The use of the abalone active peptide according to claim 1 in the preparation of adjuvant lipid-lowering drugs.