Dpp-iv inhibiting peptide from sea cucumber gonad, preparation method and application in preparation of blood sugar regulating product

Abstract

This invention belongs to the field of biotechnology and discloses a sea cucumber gonad-derived DPP-IV inhibitory peptide, its preparation method, and its application in the preparation of blood glucose regulation products. This invention obtains four DPP-IV inhibitory peptides through enzymatic hydrolysis and molecular docking screening of sea cucumber gonads, with amino acid sequences shown in SEQ.ID.NO.1~4. The docking energies with the receptor DPP-IV are -12.2 kcal / mol, -9 kcal / mol, -8.5 kcal / mol, and -8.2 kcal / mol, respectively. Experimental verification shows that the above four sea cucumber gonadal peptides have high DPP-IV inhibitory activity and can be applied to the development of blood glucose regulation products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide, its preparation method, and its application in the preparation of blood glucose regulation products. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease caused by abnormally high blood glucose levels, seriously affecting human health. Its pathogenesis involves insufficient insulin secretion or impaired insulin action. In the body's sophisticated blood glucose regulation system, GLP-1, as an important incretin hormone, effectively promotes insulin secretion from pancreatic β-cells, thereby exerting a hypoglycemic effect. However, DPP-IV (dipeptidyl peptidase IV) can rapidly degrade GLP-1, significantly shortening its physiological activity and duration of action, making DPP-IV a highly valuable key target in current diabetes drug development. Based on this mechanism, DPP-IV inhibitors can significantly prolong the duration of GLP-1's activity in vivo by blocking the hydrolysis of GLP-1 by DPP-IV, thereby enhancing its ability to promote insulin secretion. Therefore, the development of DPP-IV inhibitors has become one of the important directions in the current research and development of health foods and hypoglycemic drugs.

[0003] Among numerous DPP-IV inhibitors, DPP-IV inhibitory peptides derived from bioactive proteins exhibit unique advantages. These bioactive peptides are naturally sourced protein fragments, possessing not only excellent biocompatibility and high safety, but also easy absorption by the human body and well-defined targeting effects, thus showing broad development prospects in the pharmaceutical and health food fields. In recent years, homology modeling and molecular docking techniques have been applied to elucidate the binding modes and mechanisms of action between DPP-IV inhibitory peptides and targets such as DPP-IV, providing a powerful technical pathway for the efficient, targeted screening and functional elucidation of DPP-IV inhibitory peptides.

[0004] Sea cucumber gonads, as an visceral byproduct generated during sea cucumber processing, are often treated as waste in the production process and fail to be rationally utilized. In fact, sea cucumber gonads are rich in high-quality protein and various bioactive components, possessing enormous resource potential. Using modern enzymatic hydrolysis technology, active peptides with DPP-IV inhibitory function in sea cucumber gonadal proteins can be selectively released, thereby developing highly effective and safe hypoglycemic factors.

[0005] Therefore, using sea cucumber gonads as raw material and employing enzymatic hydrolysis to mine potential DPP-IV inhibitory peptides from their protein resources not only helps provide novel, naturally derived, and clearly defined glucose-regulating functional factors, but also effectively enhances the added value and resource utilization of sea cucumber processing byproducts, aligning with the concept of sustainable development. Investigating the interaction between sea cucumber-derived DPP-IV inhibitory peptides and DPP-IV receptors, and identifying their glucose-regulating mechanism, can provide a foundation for the application of sea cucumber-derived DPP-IV inhibitory peptides. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides a sea cucumber gonad-derived DPP-IV inhibitory peptide, its preparation method, and its application in the preparation of blood glucose regulation products. The sea cucumber-derived DPP-IV inhibitory peptide of this invention is prepared and screened from sea cucumber gonadal tissue, exhibits significant DPP-IV inhibitory activity, and can be used for blood glucose regulation.

[0007] The specific technical solution is as follows:

[0008] One objective of this invention is to provide a sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide, wherein the DPP-Ⅳ inhibitory peptide is selected from at least one peptide with an amino acid sequence as shown in SEQ.ID.NO.1~4.

[0009] Among them, SEQ.ID.NO.1 is YYPLK, whose docking energy with the receptor DPP-Ⅳ is -12.2 kcal / mol.

[0010] Among them, SEQ.ID.NO.2 is GPFGQ, which has a docking energy of -9 kcal / mol with the receptor DPP-Ⅳ.

[0011] Among them, SEQ.ID.NO.3 is AGQRGPAGPTGPTGP, which has a docking energy of -8.5 kcal / mol with the receptor DPP-Ⅳ.

[0012] Among them, SEQ.ID.NO.4 is AGPTGPTGP, which has a docking energy of -8.2 kcal / mol with the receptor DPP-Ⅳ.

[0013] Specifically, the gonads of the sea cucumber are the mixed male and female gonads of the spiny sea cucumber (Apostichopus japonicu).

[0014] A second objective of this invention is to provide a method for preparing the above-mentioned sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide, comprising the following steps:

[0015] S1. Obtain sea cucumber gonadal peptides;

[0016] S2. Sequence identification of sea cucumber gonadal peptides;

[0017] S3. Molecular docking of sea cucumber gonadal peptides with receptor DPP-Ⅳ was performed to screen for DPP-Ⅳ inhibitory peptides.

[0018] Furthermore, in step S1: sea cucumber gonadal peptides are obtained by enzymatic hydrolysis of sea cucumber gonads.

[0019] Specifically, in step S1, the preferred working conditions for enzymatic hydrolysis include: adding pepsin to the raw material to be treated for enzymatic hydrolysis, and then adding flavor protease for enzymatic hydrolysis.

[0020] More specifically, in step S1, the preferred working conditions for enzymatic hydrolysis include: adding pepsin to the raw material to be treated, adjusting the pH to 2.0~4.0, and hydrolyzing at 35~37℃ for 3~5 h; then adding flavor protease, adjusting the pH to 6.0~7.0, and hydrolyzing at 40~55℃ for 2~3 h; and inactivating the enzyme.

[0021] The preferred dosage of pepsin is 1000~3000 U / g based on the raw material to be treated.

[0022] The preferred amount of flavor protease is 1000~2000 U / g based on the raw material to be treated.

[0023] Specifically, in step S1: Pretreatment is preferred before enzymatically hydrolyzing the sea cucumber gonads. The pretreatment includes homogenizing the sea cucumber gonads and then heating them in a boiling water bath for 10-30 minutes.

[0024] Furthermore, in step S1: after enzymatic hydrolysis, the hydrolysate is separated and purified.

[0025] Furthermore, in step S1: the separation and purification includes fractionating the enzymatic hydrolysate using nanofiltration and ultrafiltration. Nanofiltration removes salts and free amino acids, while ultrafiltration removes macromolecules. Specifically, it is preferable to obtain a fraction with a molecular weight of 200-3000 Da by performing nanofiltration and ultrafiltration on the enzymatic hydrolysate.

[0026] Furthermore, in step S1: after obtaining sea cucumber gonadal peptide, the inhibitory activity of the compound can be evaluated using an in vitro model based on the crystal structure of DPP-Ⅳ.

[0027] Further, in step S2: peptide sequence analysis was performed using LC-MS / MS, and the sequences were compared and analyzed against a database to obtain the complete peptide sequences. The mass spectrometry database search software used was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database.

[0028] Specifically, in step S2: it is preferable to desalt the product obtained in step S1 before performing peptide sequence analysis. A C18 StageTip column is preferably used for desalting.

[0029] Further, in step S3: molecular docking is performed using Vina-2.0 within the PyRX software to screen for DPP-Ⅳ repressive peptides. The affinity value represents the binding ability between the two; the lower the docking energy, the more stable the binding between the ligand and receptor.

[0030] The third objective of this invention is to provide the application of the above-mentioned sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide in the preparation of blood glucose regulation products.

[0031] Furthermore, the aforementioned sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide is applied to the preparation of products that help maintain healthy blood sugar levels.

[0032] This invention also provides the application of the above-mentioned sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide in the preparation of DPP-Ⅳ inhibitors.

[0033] Compared with existing technologies, the present invention has the following beneficial effects: The present invention obtains four DPP-Ⅳ inhibitory peptides through enzymatic hydrolysis and molecular docking screening of sea cucumber gonads. Specifically, the docking energy of SEQ.ID.NO.1 with the DPP-Ⅳ receptor is -12.2 kcal / mol, SEQ.ID.NO.2 with -9 kcal / mol, SEQ.ID.NO.3 with -8.5 kcal / mol, and SEQ.ID.NO.4 with -8.2 kcal / mol. Experimental verification shows that the above-mentioned DPP-Ⅳ inhibitory peptides have high DPP-Ⅳ inhibitory activity and hypoglycemic effects, and can be applied to the development of blood glucose regulation products. Attached Figure Description

[0034] Figure 1 The diagram shows the molecular docking of the peptide with the amino acid sequence shown in SEQ.ID.NO.1 with the receptor DPP-IV.

[0035] Figure 2 The diagram shows the molecular docking of the peptide with the amino acid sequence shown in SEQ.ID.NO.2 with the receptor DPP-IV.

[0036] Figure 3 The diagram shows the molecular docking of the peptide with the amino acid sequence shown in SEQ.ID.NO.3 with the receptor DPP-IV.

[0037] Figure 4 The diagram shows the molecular docking of the peptide with the amino acid sequence shown in SEQ.ID.NO.4 with the receptor DPP-IV.

[0038] Figure 5 This is a graph showing the inhibition rate of peptide powder at different concentrations against DPP-IV in step S1 of the example.

[0039] Figure 6 This is a graph showing the inhibition rate of DPP-IV by the positive control sitagliptin in step S1 of the example.

[0040] Figure 7 The graph shows the inhibition rate of DPP-IV by the synthetic DPP-IV inhibitory peptide at a concentration of 2 mg / mL. Detailed Implementation

[0041] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] In the specific implementation: the pepsin used was purchased from Sangon Biotech; the flavor protease used was purchased from Solarbio.

[0043] Example

[0044] The steps for preparing sea cucumber gonad-derived DPP-Ⅳ inhibitory peptide are as follows:

[0045] S1. Preparation of sea cucumber gonadal peptides:

[0046] (1) Raw material pretreatment: After homogenizing the sea cucumber gonadal tissue (mixed male and female gonads), heat it in a boiling water bath for 15 minutes to obtain a homogenate, and let it cool before use.

[0047] (2) Raw material enzymatic hydrolysis: Add 2000 U / g of pepsin to the homogenate obtained in step (1), adjust the pH to 2.5, and enzymatically hydrolyze at 37℃ for 4 h; then add 1200 U / g of flavor protease to the homogenate, adjust the pH to 7.0, and enzymatically hydrolyze at 50℃ for 2 h; boil at 100℃ to inactivate the enzyme; then centrifuge at 5000 r / min for 15 min, take the supernatant, and obtain the enzymatic hydrolysate.

[0048] (3) Purification: The enzymatic hydrolysate obtained in step (2) is subjected to nanofiltration and ultrafiltration fractionation. First, a 200 Da nanofiltration membrane is used to remove salt and free amino acids, and then a 3000 Da spiral wound membrane is selected for ultrafiltration. The 200~3000 Da fraction is freeze-dried to obtain sea cucumber gonadal peptide powder, which is stored at -20℃ for later use.

[0049] (4) Assay for DPP-IV enzyme inhibitory activity: The peptide powder obtained in step (3) was prepared into a solution with deionized water, and the mass concentration gradient was set to 1, 2, 3, 4, and 5 mg / mL. Tris-HCl (0.1 mol / L, pH 8.0) buffer was used for solution preparation. The sample solution and Gly-Pro-pNA (1.6 mM, 25 μL) were added to a 96-well microplate and incubated with shaking at 37°C for 10 min. Then DPP-IV (10 ng / mL, 50 μL) was added and incubated with shaking at 37°C for 60 min to carry out the reaction. After the reaction, HAc-NaAc (pH 4.0, 1 mol / mL, 100 μL) buffer was added, and the absorbance value was measured at 405 nm. Sitagliptin, a first-line clinical drug, was used as the positive control. The inhibition rate of DPP-IV activity was calculated using the following formula:

[0050] DPP-Ⅳ activity inhibition rate (%) =

[0051] In the above formula, A represents the absorbance value. Test results are shown below. Figure 5 . Figure 5 In the figure, the horizontal axis represents peptide concentration. Figure 6 The inhibition rate of DPP-IV by the positive control sitagliptin is shown. Figure 5 It can be seen that, like the positive control drug, sea cucumber gonadal peptide has a significant inhibitory effect on DPP-IV, and the inhibitory efficiency shows a dose-response relationship, with its IC50 value being [missing information]. 50 It is 5.11 mg / mL.

[0052] S2. Sequence identification of sea cucumber gonadal peptides:

[0053] The peptide powder obtained in step S1 was desalted using a C18 StageTip column and subjected to peptide sequence analysis using LC-MS / MS. The complete peptide sequence was obtained by comparison analysis with a database. The mass spectrometry database search software was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database.

[0054] S3. Sea cucumber gonadal peptides were used for molecular docking with DPP-IV as the target, utilizing its crystal structure (PDB ID: 1PFQ):

[0055] SDF format files of the main active ingredients of the core drug were obtained from the PubChem database. Key target protein structures were collected from the PDB database. Pymol-2.1.0 software was used to optimize the targets by removing water molecules and small molecule ligands, and AutoDock Tools-1.5.6 was used for hydrogenation and charge processing. The results were then saved as pdbqt format. Using the key targets as receptors and their corresponding active ingredients as ligands, molecular docking was performed using Vina-2.0 within PyRX software. The binding energy was calculated, and the results were output. Finally, PyMol software was used for result visualization. The affinity (kcal / mol) value represents the binding ability; the lower the docking energy, the more stable the binding between the ligand and receptor. PyMol was used for visualization analysis, and 2D visualization was performed using Discovery Studio 2020 Client. Four DPP-IV repressive peptides and their docking energies were screened. The amino acid sequences of the four DPP-IV repressive peptides are shown in SEQ.ID.NO.1~4. The amino acid sequences and corresponding docking energies of the four DPP-Ⅳ inhibitory peptides are shown in Table 1.

[0056] Table 1. Amino acid sequence and docking energy of sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide

[0057]

[0058] The molecular docking results of the peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 with the receptor DPP-Ⅳ are shown in the following figures. Figures 1-4 .

[0059] like Figure 1 As shown, the peptide represented by the amino acid sequence SEQ.ID.NO.1 forms hydrogen bonds with the target protein DPP-IV at THR251, LYS122, GLN123, ASP709, ASP739, TRP201, and TYR195; a carbon-hydrogen bond with LYS122; an alkyl group interaction with ALA707, PHE240, and VAL121; a π-acceptor hydrogen bond interaction with ARG253 and TRP124; a π-alkyl group interaction with VAL254; and a π-σ group interaction with VAL252. Figure 2 As shown, the peptide represented by the amino acid sequence SEQ.ID.NO.2 forms hydrogen bonds with the target protein DPP-IV at TYR195, TRP201, ASP192, TYR211, GLN153, and GLN123, a π-alkyl interaction with ALA707, and a T-type π-π bond interaction with PHE240. Figure 3As shown, the peptide represented by the amino acid sequence SEQ.ID.NO.3 forms hydrogen bonds with GLN308, PHE364, SER462, GLU464, TRP305, VAL303, SER212, and THR156 of the target protein DPP-IV; forms alkyl interactions with HIS363 and PRO218; forms C-H bonds with THR307 and TRP215; forms π-alkyl interactions with PRO159 and TRP216; and forms π-σ interactions with TRP215. Figure 4 As shown, the peptide represented by the amino acid sequence SEQ.ID.NO.4 forms hydrogen bonds with VAL303, TRP215, TRP154, SER212, SER106, ARG61, TRP157, and SER158 of the target protein DPP-IV, an alkyl group with PRO159, and a carbon-hydrogen bond with THR156. Figures 1-4 It is known that the binding of DPP-Ⅳ inhibitory peptide to DPP-Ⅳ is mainly through hydrogen bonding, van der Waals forces, and non-covalent interactions.

[0060] test

[0061] Based on the mass spectrometry sequencing results, Jiangsu Genscript Biotech Co., Ltd. was commissioned to chemically synthesize the above four peptide sequences. Then, the DPP-IV inhibition method was used to verify their in vitro glycemic regulatory activity. The test concentration was 2 mg / mL, and the test method was as described in step S1 (4) of the example. The inhibition rates of the four peptides against DPP-IV are as follows: Figure 7 As shown. Figure 7 The horizontal axis represents the sequence of the corresponding peptide.

[0062] The test results showed that the peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 all had certain DPP-IV inhibitory activity, indicating that all four peptides had significant effects in lowering blood sugar levels and had potential applications in food, especially in the preparation of health foods that help maintain healthy blood sugar levels.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DPP-IV inhibiting peptide from the gonad of sea cucumber, characterized in that, It is selected from at least one peptide whose amino acid sequence is shown in SEQ ID NO. 1 to 4.

2. The method for preparing the sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide as described in claim 1, characterized in that, Includes the following steps: S1. Obtain sea cucumber gonadal peptides; S2. Sequence identification of sea cucumber gonadal peptides; S3. Molecular docking of sea cucumber gonadal peptides with receptor DPP-Ⅳ was performed to screen for DPP-Ⅳ inhibitory peptides.

3. The production method according to claim 2, characterized by, In step S1: Sea cucumber gonadal peptides are obtained by enzymatic hydrolysis of sea cucumber gonads.

4. The production method according to claim 3, characterized by, In step S1, the working conditions for enzymatic hydrolysis include: adding pepsin to the raw material to be treated for enzymatic hydrolysis, and then adding flavor protease for enzymatic hydrolysis.

5. The preparation method according to claim 3, characterized in that, In step S1: After enzymatic hydrolysis, the hydrolysate is separated and purified.

6. The preparation method according to claim 5, characterized in that, In step S1: the separation and purification includes fractionating the enzymatic hydrolysate using nanofiltration and ultrafiltration.

7. The preparation method according to claim 2, characterized in that, In step S2: Peptide sequence analysis was performed using LC-MS / MS.

8. The application of the sea cucumber gonadal-derived DPP-Ⅳ inhibitory peptide as described in claim 1 in the preparation of blood glucose regulation products.

9. The application according to claim 8, characterized in that, It is used in the preparation of products that help maintain healthy blood sugar levels.

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