Uric acid reducing polypeptide as well as application and preparation method thereof

Abstract

The invention discloses a uric acid reducing polypeptide, the polypeptide is derived from a peptide fragment, the'bindingerge 'value of the peptide fragment in molecular docking with xanthine oxidase is less than-1.5 kcal / mol, and the peptide fragment, in molecular docking with the xanthine oxidase, of the polypeptide is derived from a Peptide Ranker biological activity score gt; and 0.7 of a polypeptide. The uric acid reducing polypeptide is obtained by screening natto powder subjected to pepsin enzymolysis, natto powder enzymatic hydrolysate obtained through enzymolysis has the activity of inhibiting xanthine oxidase, the screened uric acid reducing polypeptide can be developed into a uric acid reducing medicine, and active ingredients are added into functional food and health food, so that the uric acid reducing polypeptide has a good application prospect. Good market prospects are realized in medicine and food industries.

Description

Technical Field

[0001] The present invention relates to the technical field of functional polypeptides, and particularly to uric acid-lowering polypeptides and their applications and preparation methods. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease caused by excessive production or impaired excretion of uric acid in the body, and its clinical manifestation is an increase in serum uric acid level. Uric acid is the end product of purine metabolism in the human body, which is mainly formed by a series of enzymatic reactions of nucleic acids and other purine substances decomposed by cell metabolism and dietary purines. Patients with hyperuricemia are usually prone to gout. At the same time, hyperuricemia can also trigger various metabolic syndromes, such as renal failure, coronary heart disease, diabetes, hypertension and other diseases.

[0003] In this context, finding safe and effective methods for lowering uric acid has become a research hotspot in the fields of medicine and nutrition. Natto, as a traditional fermented food, is widely recognized as a food with various health care functions in countries such as Japan and Europe. In recent years, global scientists have conducted in-depth research on the nutritional value and health effects of natto, and found that it has various physiological activities such as thrombolysis and lipid-lowering. However, although natto and its products have shown significant effects in regulating cardiovascular health, there is no systematic research report on their effects on uric acid metabolism. Therefore, exploring the potential role of natto and its products in lowering uric acid not only has important theoretical significance, but may also provide new ideas and methods for the prevention and treatment of hyperuricemia. Summary of the Invention

[0004] In order to achieve these and other advantages according to the present invention, on the one hand, a preferred embodiment of the present invention provides an uric acid-lowering polypeptide, which is derived from a peptide segment with a "binding_energy" value <

[0005] -1.5 kcal / mol when docked with xanthine oxidase, and the peptide segment of this polypeptide docked with xanthine oxidase is derived from a polypeptide with a Peptide Ranker bioactivity score > 0.7.

[0006] According to a preferred embodiment of the present invention, the amino acid sequence of this polypeptide is any one or more of FGPVAP, PGGF, VFPF, and PFL.

[0007] On the other hand, a preferred embodiment of the present invention provides the application of the above-mentioned uric acid-lowering polypeptide in the preparation of uric acid-lowering drugs.

[0008] On the other hand, a preferred embodiment of the present invention provides the application of the above-mentioned uric acid-lowering polypeptide in the preparation of foods or health products suitable for people with hyperuricemia.

[0009] On the other hand, a preferred embodiment of the present invention provides a method for preparing the uric acid-lowering polypeptide, comprising the following steps:

[0010] S1. Prepare a nattokinase hydrolysate

[0011] Use a protease to hydrolyze nattokinase powder to obtain a nattokinase hydrolysate;

[0012] S2. Determine the xanthine oxidase inhibition rate

[0013] Determine the inhibitory activity against xanthine oxidase;

[0014] S3. Identify the sequence of the nattokinase hydrolysate

[0015] Perform LC-MS / MS determination on the nattokinase hydrolysate to analyze its polypeptide composition;

[0016] S4. Screen the uric acid-lowering polypeptide

[0017] First, screen out potential bioactive peptides with Peptide Ranker > 0.7 from the identified polypeptide fragments, and then dock them through AutoDock 4.2.6 software to screen out the most potential uric acid-lowering polypeptide.

[0018] According to a preferred embodiment of the present invention, the protease is pepsin.

[0019] According to a preferred embodiment of the present invention, the conditions for hydrolysis are as follows: use a pepsin solution with a concentration of 0.1% - 1.5%, mix it according to the ratio of nattokinase activity to pepsin solution of 5000 - 10000 IU / ml, the pH value is 1.6 ± 0.2, carry out at 37 ± 2°C, and the hydrolysis time is 1 - 2 h; after the hydrolysis is completed, raise the temperature to above 95°C to inactivate the enzyme for 10 - 15 min. After the hydrolysis is completed, centrifuge to take the supernatant, which is the nattokinase hydrolysate.

[0020] The present invention has at least the following beneficial effects: The uric acid-lowering polypeptide of the present invention is obtained by screening nattokinase powder after pepsin enzymolysis. The enzymolysis solution of nattokinase powder obtained by enzymolysis has the activity of inhibiting xanthine oxidase. 1554 polypeptides are identified by sequencing the enzymolysis solution. There are 20 peptide segments with Peptide Ranker > 0.7. After docking the 20 peptide segments with the xanthine oxidase molecule, the "binding_energy" values of 4 nattokinase peptides FGPVAP, PGGF, VFPF, and PFL are below -1.5 Kcal, indicating that these peptide segments have the best docking effect with the xanthine oxidase molecule, and have been verified by animal experiments to have a significant uric acid-lowering function. The uric acid-lowering polypeptide screened this time can be developed into a uric acid-lowering drug, and the active ingredient can be added to functional foods and health foods, which has good market prospects in the pharmaceutical and food industries.

[0021] Other advantages, objectives, and features of the present invention will be partially reflected by the following description, and partially will be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the interaction mode of polypeptide FGPVAP with the xanthine oxidase molecule respectively;

[0023] Figure 2 It is a schematic diagram of the interaction mode of polypeptide PGGF with the xanthine oxidase molecule respectively;

[0024] Figure 3 It is a schematic diagram of the interaction mode of polypeptide VFPF with the xanthine oxidase molecule respectively;

[0025] Figure 4 It is a schematic diagram of the interaction mode of polypeptide PFL with the xanthine oxidase molecule respectively. DETAILED DESCRIPTION OF THE INVENTION

[0026] The following further describes the present invention in detail with reference to the drawings, so that those skilled in the art can implement it according to the description in the specification.

[0027] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious deformations. The basic principles defined in the following description can be applied to other implementation schemes, deformation schemes, improvement schemes, equivalent schemes, and other technical schemes that do not deviate from the spirit and scope of the present invention.

[0028] Example 1 Preparation of Nattokinase Powder

[0029] 1. Preparation of Nattokinase Powder

[0030] (1) Preparation method of liquid fermentation natto powder: (1) Preparation of fermentation broth: Mix 2% g / 100 ml of soy peptone, 3% of glucose, 2% of Na2HPO4·12H2O, 0.5% of NaH2PO4·H2O, 0.02% of MgSO4·7H2O, and 0.02% of CaCl2 in proportion to prepare the fermentation broth; (2) Take the cryopreserved Bacillus subtilis seeds, thaw and refreeze, and inoculate them into the liquid medium in a shake flask at a ratio of 0.1%. Incubate in a shaking incubator at 180 rpm and 37 °C for 16 - 18 hours to obtain the seed liquid. Among them, the liquid medium contains: 15 g / L of glucose, 15 g / L of peptone, 10 g / L of yeast extract, 5 g / L of disodium hydrogen phosphate dodecahydrate, 1 g / L of sodium dihydrogen phosphate monohydrate, 0.2 g / L of magnesium sulfate, and the pH is 7.0 - 7.6. The Bacillus subtilis seed liquid is inoculated into the fermentation broth at a ratio of 3% ml / 100 ml, and cultured at a dissolved oxygen of 30%, 35 - 40 °C, and 300 rpm for 20 h to obtain the original fermentation broth; then the original fermentation broth is centrifuged and separated, and the supernatant is concentrated through a filter membrane. Dry to obtain natto powder. The source of Bacillus subtilis is: China Center for Type Culture Collection, preservation number CCTCC NO: M2022132. Specifically, Bacillus subtilis can use the strain with the preservation number CCTCC NO: M2022132. The Bacillus subtilis with the preservation number CCTCC NO: M2022132 is preserved in China Center for Type Culture Collection, named Bacillus subtilis ZF01, classified as Bacillus subtilis, the preservation address is Wuhan, China, and the preservation date is February 18, 2022.

[0031] (2) Preparation method of solid fermentation natto powder: Wash and remove impurities from soybeans, mix soybeans and nutrient solution at a mass - volume ratio of 1:3, soak at 8 °C for 12 hours and then drain; among them, the nutrient solution contains: 30 g / L of glucose, 8 g / L of disodium hydrogen phosphate dodecahydrate, 0.5 g / L of sodium dihydrogen phosphate monohydrate, 0.5 g / L of magnesium sulfate, and the pH is 7.0. Then steam at 105 °C and 0.02 MPa for 40 minutes. After steaming and cooling, inoculate the Bacillus subtilis seed liquid at 5% ml / 100 g of the raw material weight, and ferment in a fermentation device for 36 h to obtain the fresh natto after fermentation, where the temperature in the fermentation device is controlled at 37 - 41 °C and the humidity is 75 - 85%; dry the fresh natto and crush it to obtain natto powder. The source of Bacillus subtilis is: China Center for Type Culture Collection, preservation number CCTCC NO: M2022132.

[0032] 2. Preparation of Nattokinase Hydrolysate: Use a 0.5% pepsin solution and mix it according to the ratio of nattokinase activity to 6000 IU / ml of pepsin solution, with a pH value of 1.6, and carry out enzymatic hydrolysis at 37°C for 1.5 h; after the enzymatic hydrolysis, raise the temperature to above 95°C and inactivate the enzyme for 10 min; after the enzymatic hydrolysis, centrifuge and take the supernatant, which is the nattokinase hydrolysate.

[0033] 3. Determination of Xanthine Oxidase Inhibition Rate

[0034] Determination of the inhibitory activity against xanthine oxidase;

[0035] The double-enzyme method is used to determine the inhibition rate of the sample against xanthine oxidase. In the double-enzyme coupling method, xanthine is decomposed by xanthine oxidase to produce H2O2, and horseradish peroxidase catalyzes the formation of a red compound from 4-aminoantipyrine and phenol during the process of catalyzing H2O2, and its absorbance value will be generated at 508 nm, from which the inhibitory activity of xanthine oxidase can be calculated.

[0036] The specific method is as follows: Add 200 μL of the sample solution, 50 μL of 0.5 U / mL xanthine oxidase solution to a 10 mL colorimetric tube in sequence, vortex and mix well, and incubate at 37°C for 10 min. Then add 400 μL of 2.0 mmol / L xanthine solution, and start the reaction with 3 mL of the chromogenic solution; incubate at 37°C for 20 min; boil for 5 min, and cool to room temperature; measure the absorbance value at 508 nm. Use the group without adding xanthine as the blank group A0; the group without adding the sample as the positive blank group Ac; the group without adding xanthine oxidase as the zero-adjustment group; the measurement group as As; calculation formula: the inhibition rate (%) of the sample's XOD (xanthine oxidase) = (Ac - (As - A0)) / Ac * 100%.

[0037] Test the solid nattokinase hydrolysate and the liquid nattokinase hydrolysate, and set up the positive drug allopurinol. The results are shown in Table 1.

[0038] Table 1 Determination of the inhibitory activity of nattokinase hydrolysate against XOD

[0039] Grouping Xanthine Oxidase Inhibition Rate % Liquid Natto Powder 78.25±0.34 Solid Natto Powder 75.08±0.53 Allopurinol 81.24±0.37

[0040] 4. Identification of the Sequence of Nattokinase Hydrolysate

[0041] Perform LC-MS / MS determination on the nattokinase hydrolysate to analyze its polypeptide composition;

[0042] The specific operation is as follows:

[0043] Transfer the nattokinase hydrolyzate to a 10KD ultrafiltration centrifuge tube, centrifuge at 12,000g for 10 min; desalt the ultrafiltered solution using a C18 desalting column; elute the sample with Eltion buffer (0.1% FA, 60% ACN), and transfer the elution solution to a new EP tube; centrifuge and concentrate the eluted sample to dryness for mass spectrometry analysis; after desalting, the sample is centrifuged to dryness and then redissolved in 100 μL of Nano-LC mobile phase A (0.1% formic acid / water), bottled and loaded for online LCMS analysis. The dissolved sample is loaded onto a nanoViper C18 pre-column (3 μm, ), and then desalted with a 20 μL volume rinse. The liquid phase is an UltiMate 3000 RSLC nano-nanoliter liquid phase system (ThermoFisher, USA). After the sample is desalted and retained on the pre-column, it is separated by the analytical column. The specifications of the analytical column are a C18 reversed-phase chromatography column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm ) The gradient used in the experiment was that mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 60 min. The mass spectrometry was performed using a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a Nano Flex nano-spray ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the heating temperature of the ion transfer tube was 320 °C. The mass spectrometry scanning mode was data-dependent acquisition (DDA, Data Dependent Analysis). The resolution of the first-order mass spectrometry scan was 70000, the scanning range was 350 - 1500 m / z, and the maximum injection time was 100 ms. In each DDA cycle, at most 20 second-order spectra with charges from 2+ to 5+ were collected, and the maximum injection time of the second-order mass spectrometry ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 25 seconds. The original raw spectral files collected by the mass spectrometry were processed and analyzed for data using PEAKS Online 11 (Bioinformatics Solutions Inc., Waterloo, Canada) software. The database was the Bacillus subtilis database downloaded from Uniprot, and the retrieval parameters were set as follows: the mass tolerance of the first-order mass spectrometry was 10 ppm, the second-order mass spectrometry was 0.03 Da, and the enzyme was set to none. Variable modifications: protein N-terminal acetylation ((Protein N-term)), asparagine / glutamine deamidation (Deamidation(NQ)), methionine oxidation (Oxidation(M)), glutamate pyroglutamation (Pyro-glu), glutamine pyroglutamyl cyclization (Pyro-Gln).

[0044] 5. Screening of uric acid-lowering polypeptides

[0045] First, potential bioactive peptides with Peptide Ranker > 0.7 were screened out from the identified polypeptide fragments, and then they were docked using AutoDock 4.2.6 software to screen out the most potential uric acid-lowering polypeptides.

[0046] First, the 1554 peptide segments identified by LC-MS / MS above were preliminarily screened according to Peptide Ranker, and the peptide segments with a bioactivity prediction score > 0.7 were screened out and molecularly docked with xanthine oxidase. The smaller the "binding_energy" value, the better the docking effect and the higher the potential uric acid-lowering ability. The peptide segments with a bioactivity prediction score > 0.7 are shown in Table 2:[[]]

[0047] Table 2 Prediction scores of polypeptide bioactivity

[0048] Polypeptide Bioactivity Prediction Score FGPVAP 0.727581 PGGF 0.980656 VFPF 0.977466 PFL 0.977357 FQFPDGF 0.971693 FFDPFNG 0.953303 LIGFLLL 0.867945 LIGFLLL 0.867945 FDPFNGYNSGF 0.823594 FYYGKGL 0.776089 GSGQYSWIING 0.765256 LIGFLLI 0.759761 FYYGKG 0.756249 LAKAPLF 0.753795 FIIGGG 0.72541 SWIING 0.706022 YSWIING 0.703246 FPAGTL 0.800063 IFIGGG 0.738351 ALNIVFM 0.732743

[0049] The crystal structure of xanthine oxidase (PDB ID: 1N5X) was obtained from the RCSB database (http: / / www.rcsb.org / ). The molecular docking scores of the polypeptides with a bioactivity prediction score higher than 0.70 and xanthine oxidase were as shown in Table 3 below using AutoDock 4.2.6 software.

[0050] Table 3 Molecular docking scores of potential uric acid-lowering polypeptides and xanthine oxidase

[0051] Polypeptide binding_energy / Kcal FGPVAP -3.44 PGGF -3.08 VFPF -2.05 PFL -2.12 FQFPDGF 0.42 FFDPFNG 2.21 LIGFLLL 1.2 LIGFLLL 2.34 FDPFNGYNSGF No classification FYYGKGL 3.01 GSGQYSWIING No classification LIGFLLI 2.41 FYYGKG 2.91 LAKAPLF 3.49 FIIGGG 0.5 SWIING 3.26 YSWIING 3.95 FPAGTL -0.06 IFIGGG 0.81 ALNIVFM 1.69

[0052] As Figures 1 - 4 , the schematic diagrams of the interaction modes between the polypeptides FGPVAP, PGGF, VFPF, PFL and xanthine oxidase. When the polypeptides were docked with xanthine oxidase, the key amino acid residues interacting with xanthine oxidase were GLU879, ASP1246, GLU1092, ASN1137, LYS1251, LYS165, ARG161, and the interaction forces were hydrogen bonds or electrostatic interactions, etc. These polypeptides were considered potential uric acid-lowering polypeptides.

[0053] Animal experiments on experimental uric acid-lowering polypeptides

[0054] The uric acid-lowering polypeptides FGPVAP, PGGF, VFPF and PFL were prepared by Fmoc solid-phase synthesis method, and their peptide purity was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry analysis.

[0055] Establishment of hyperuricemia mouse model: Adult Kunming mice were administered with potassium oxonate 400 mg / kg combined with hypoxanthine 500 mg / kg by gavage every day for 14 consecutive days. Then, the mouse serum was collected, and the uric acid level in the mouse serum was measured using a kit and an automatic biochemical analyzer. The uric acid level in the model group (215.31 μmol / L) was significantly higher than that in the normal group (102.84 μmol / L), indicating successful modeling.

[0056] Animal grouping and administration method: Seventy adult Kunming mice were selected and randomly divided into 2 groups. There were 10 mice in the normal group. After the model was successfully established in the remaining 60 mice, they were randomly divided into 6 groups. The grouping is shown in Table 5, including the model group, the positive control group, and the uric acid-lowering polypeptide groups (FGPVAP, PGGF, VFPF, and PFL). Then, the test samples were administered orally to each group. The normal group and the model group were given an equal volume of normal saline by gavage. The administration dose of allopurinol in the positive group was 50 mg / Kg, and the daily administration dose of the test samples in each uric acid-lowering polypeptide group was 50 mg / Kg, once a day, for 28 consecutive days. After that, kits and an automatic biochemical analyzer were used to measure the levels of uric acid, urea nitrogen, and creatinine in the serum of each group.

[0057] Results: The measurement results of serum uric acid, urea nitrogen, and creatinine in each group of animals are shown in Tables 4 - 5.

[0058] Table 4 Measurement results of serum uric acid in animals before and after modeling

[0059] Grouping Uric Acid Level (μmol / L) Normal Group 102.84±4.28 Model Group 215.31±6.07*

[0060] Table 5 Measurement results of uric acid and renal function in each group of animals

[0061]

[0062]

[0063] Note: * Compared with the normal group, p < 0.05. # Compared with the model group, p < 0.05.

[0064] As can be seen from Table 4, the modeling was successful this time.

[0065] As can be seen from Table 5, compared with the model group, the levels of uric acid, urea nitrogen, and creatinine in the uric acid-lowering polypeptide groups were significantly decreased; compared with the positive control group, there were no significant differences in each index of the mouse serum, but compared with the modeling control group, there were significant changes. The serum creatinine and urea nitrogen concentrations are often used to reflect the renal function status. The higher the value, the worse the renal function. As shown in the table, the levels of urea nitrogen and creatinine in the uric acid-lowering polypeptide groups were significantly lower than those in the model group, showing a protective effect on the kidneys. It shows that the uric acid-lowering polypeptides screened in the present invention can effectively reduce the uric acid level, and at the same time have a protective effect on the kidneys, and the effect is the same as that of allopurinol, having a good uric acid-lowering effect.

[0066] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated and described examples here.

Claims

1. A uric acid-lowering polypeptide, characterized in that: The peptide is derived from docking with the xanthine oxidase molecule The peptides with "binding_energy" value < -1.5 kcal / mol, the peptides for molecular docking with xanthine oxidase are derived from peptides with Peptide Ranker biological activity score > 0.

7.

2. The uric acid-lowering polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is any one or more of FGPVAP, PGGF, VFPF, and PFL.

3. Use of the uric acid-lowering polypeptide according to any one of claims 1 to 2 in the preparation of a uric acid-lowering drug.

4. Use of the uric acid-lowering polypeptide according to claim 3 in the preparation of food or health products suitable for people with hyperuricemia.

5. The method for preparing the uric acid-lowering polypeptide according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Preparation of natto powder enzymatic hydrolysate Using protease to enzymatically hydrolyze natto powder to obtain natto powder enzymatic hydrolyzate; S2. Determination of xanthine oxidase inhibition rate Determination of xanthine oxidase inhibitory activity; S3. Sequence identification of natto powder hydrolysate The natto powder hydrolysate was subjected to LC-MS / MS to analyze its peptide composition; S4. Screening of uric acid-lowering peptides The identified peptide fragments were first screened for potential bioactive peptides with Peptide Ranker>0.7, and then docked using AutoDock 4.2.6 software to screen out the most potential uric acid-lowering peptides.

6. The method for preparing the uric acid-lowering polypeptide according to claim 5, characterized in that: The protease is pepsin.

7. The method for preparing the uric acid-lowering polypeptide according to claim 5, characterized in that: The conditions for the enzymolysis are: using a pepsin solution with a concentration of 0.1%-1.5%, mixing according to the ratio of nattokinase activity and pepsin solution 5000-10000 IU / ml, pH value of 1.6±0.2, carrying out the enzymolysis at 37±2°C, and the enzymolysis time of 1-2h; after the enzymolysis is completed, the temperature is raised to above 95°C, the enzyme is inactivated for 10-15min, and after the enzymolysis is completed, the supernatant is centrifuged to obtain the natto powder enzymolysis solution.

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