A polypeptide, its preparation and use

Abstract

The application discloses a kind of polypeptide and its preparation and application, belong to active peptide technical field.The application is prepared from walnut meal protein and obtained 3 kinds of active peptides, the amino acid sequences of 3 kinds of polypeptides are respectively Leu-Pro-Gln-Phe (LPQF), Leu-Pro-Ser-Phe (LPSF) or Vla-Pro-Phe-Pro (VPFP), and the inhibition activity of dipeptidyl peptidase-IV (DPP-IV) is detected to 3 kinds of polypeptides, therefore, the 3 kinds of polypeptides described in the application can be used for preparing the drug for treating hypoglycemia.

Description

TECHNICAL FIELD

[0001] The present application relates to a polypeptide and its preparation and application, belonging to the technical field of active peptides. BACKGROUND

[0002] Diabetes is one of the most fatal chronic diseases with the highest incidence in the world. It is reported that 537 million adults suffered from diabetes in 2021, and it is expected to increase to 643 million in 2030 and 783 million in 2045, of which type II diabetes accounts for about 90% of diabetes cases. The synthetic hypoglycemic drugs currently used in clinical practice (such as saxagliptin and sitagliptin, etc.) have been proven to have many negative effects, such as weight gain, cardiovascular problems, hypoglycemia and other adverse reactions. Dipeptidyl peptidase-IV (DPP-IV) inhibitors are the latest therapy for type II diabetes.

[0003] DPP-IV is a metabolic enzyme distributed in human tissues, which is responsible for cleaving and inactivating the intestinal insulin-promoting hormone glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), resulting in a decrease in endogenous GLP-1 and GIP levels, inhibiting the release of insulin from pancreatic beta cells, while promoting the secretion of glucagon from pancreatic alpha cells, i.e. inhibiting insulin levels and increasing blood glucose levels in the body. Therefore, inhibiting the activity of DPP-IV can prevent the degradation of GLP-1 and GIP to some extent, thereby better regulating blood glucose. In this context, some food-derived protein bioactive peptides with strong DPP-IV inhibitory activity are considered as a safe means to prevent and control the occurrence and development of diabetes. Therefore, the development of food-derived protein bioactive peptides to inhibit DPP-IV and prevent and control the occurrence and development of diabetes has become a global research hotspot.

[0004] Walnut is one of the world's four major nuts, with very high nutritional value, and has been considered as the best food material for warming the lungs and kidneys, promoting heart and brain health, and strengthening the body. Walnut kernels are rich in oil, and the content of unsaturated fatty acids in the oil is high, which is an important material basis for the industrial economic benefits of walnuts. A large amount of walnut meal is produced in walnut oil production, and the walnut meal has high protein content, which is a rich source of bioactive peptides. A small part of it is developed as walnut protein powder, but most of it is still used as animal feed. Whether walnut meal can be effectively developed is a new economic benefit point for the walnut industry.

[0005] Peptides are defined as fragments of proteins, usually consisting of 2-20 amino acid residues, with a variety of biological activities. The potential of bioactive peptides in the prevention of chronic diseases, especially diabetes, is particularly recognized. Bioactive peptides in food sources refer to two categories, one is bioactive peptides that can effectively promote the function or state of the body to improve human health, and the other is bioactive peptides that can impart umami and rich flavor to food. Therefore, walnut meal, a by-product of walnut oil production, can be used to develop walnut meal protein peptides with stable and simple preparation technology and good blood glucose lowering effect, which not only provides new raw materials for preventing and assisting in controlling blood glucose levels of diabetic patients, but also finds a new benefit growth point for the walnut industry. SUMMARY

[0006] One of the purposes of the present application is to provide a kind of polypeptide, the sequence of the polypeptide is Leu-Pro-Gln-Phe (LPQF), Leu-Pro-Ser-Phe (LPSF) or Vla-Pro-Phe-Pro (VPFP).

[0007] Preferably, the polypeptide in the present application can be obtained by artificial synthesis in addition to being isolated from defatted walnut meal.

[0008] The second purpose of the present application is to provide a preparation method of the polypeptide, which is prepared from defatted walnut meal, and the specific steps are as follows:

[0009] (1) defatted walnut meal is subjected to enzymatic modification treatment with food industry protease to obtain protein enzymatic product.

[0010] (2) the protein enzymatic product obtained in step (1) is separated by ultrafiltration membrane to obtain separation product.

[0011] (3) the separation product obtained in step (2) is determined for DPP-Ⅳ inhibitory activity, and the component with the highest activity is screened.

[0012] (4) the component with the highest activity is purified by reverse phase high performance liquid chromatography system to obtain purified product.

[0013] (5) the purified product is determined for DPP-Ⅳ inhibitory activity, and the component with the highest activity is screened, i.e. three kinds of polypeptides are obtained.

[0014] Preferably, the food industry protease in step (1) is bromelain.

[0015] Preferably, the ultrafiltration membrane used in step (2) is 10 kDa and 3 kDa.

[0016] Preferably, the separation product obtained in step (2) has three parts with molecular weight less than 3 kDa, molecular weight between 3-10 kDa and molecular weight greater than 10 kDa.

[0017] Preferably, the walnut meal is a by-product after walnut oil is pressed.

[0018] The third object of the present application is to provide an application of the polypeptide, which has the following two aspects:

[0019] (1) the application of the polypeptide in the preparation of a hypoglycemic drug.

[0020] (2) the application of the polypeptide in the preparation of a health product for helping to maintain a healthy level of blood glucose.

[0021] Advantages of the present application

[0022] (1) the half inhibitory concentration (IC 50 ) of the polypeptides LPQF, LPSF and VPFP to DPP-IV is 50.03, 53.06 and 52.09 μg / mL (i.e. 99.34, 114.70 and 113.58 μmol / L) respectively, and they have good water solubility and can be absorbed by the human intestinal tract, thus having little side effects on the human body.

[0023] (2) the polypeptides can be obtained not only by artificial synthesis, but also from defatted walnut meal, thus providing a new way for the resource utilization of defatted walnut meal. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 DPP-IV inhibitory activity of walnut meal protein hydrolysate hydrolyzed by different proteases for different food industries.

[0025] Figure 2 DPP-IV inhibitory activity of different components of bromelain hydrolysate after ultrafiltration separation.

[0026] Figure 3 DPP-IV inhibitory activity of different components after purification by a liquid chromatography system.

[0027] Figure 4 Half inhibitory concentration of LPQF, LPSF and VPFP to DPP-IV. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be further described below by combining with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application, and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0029] Unless otherwise specified, all are conventional methods, and the materials, reagents, etc. are obtained from commercial channels.

[0030] Example 1

[0031] Preparation of polypeptides from defatted walnut meal

[0032] The defatted walnut meal used in this example is a by-product obtained from the production of walnut oil by aqueous extraction, and the specific steps are as follows

[0033] (1) The defatted walnut meal was dried at 60°C by conventional air blowing, crushed, passed through a 40-mesh silk screen, and the undersize material was collected for enzymatic hydrolysis.

[0034] (2) The undersize material from step (1) was subjected to enzymatic hydrolysis under the optimal working conditions of bromelain, alkaline protease, papain, protease, flavor protease, neutral protease, trypsin, pancreatin, animal hydrolyzed proteinase, and pepsin, respectively. After the enzymatic hydrolysis was completed, the enzyme activity was inactivated by boiling in water for 20 min to obtain an enzyme hydrolysate.

[0035] (3) The enzyme hydrolysate obtained in step (2) was cooled to room temperature and centrifuged at 5000 rpm for 20 min to obtain the supernatant, which was the enzyme hydrolysate. An appropriate amount of the enzyme hydrolysate was subjected to vacuum freeze-drying to obtain a dried material, and the remaining enzyme hydrolysate was reserved for use.

[0036] (4) The dried material obtained in step (3) was subjected to DPP-IV inhibition activity determination, and the determination results are shown in Table 1. Figure 1 According to the determination results, the product of bromelain hydrolysis of walnut meal protein was selected for further experiments because it had the strongest DPP-IV inhibition activity. Figure 1

[0037] (5) The enzyme hydrolysate obtained in step (3) was separated by ultrafiltration membranes with molecular weight cut-offs of 10 kDa and 3 kDa to obtain three components with molecular weights less than 3 kDa, between 3-10 kDa, and greater than 10 kDa. The DPP-IV inhibition activities of the three components were determined, and the determination results are shown in Table 2. Figure 2 According to the determination results, the component with a molecular weight less than 3 kDa was selected for further experiments because it had the strongest DPP-IV inhibition activity. Figure 2

[0038] ​​(6) The components with molecular weight less than 3 kDa were purified by reversed-phase high performance liquid chromatography system under the following conditions: mobile phase A was water containing 0.1% trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was 10 mL / min, and the elution gradient was 8-60% B for 35 min. The detection wavelength was 220 nm. The purified component PI was obtained at 4.0-6.4 min, the purified component P2 was obtained at 15.0-16.4 min, the purified component P3 was obtained at 17.7-18.6 min, the purified component P4 was obtained at 19.7-20.6 min, and the purified component P5 was obtained at 23.9-25.4 min. The five components were determined for DPP-IV inhibitory activity, and the determination results are shown in Table 2. Figure 3 According to the determination results, the component 4 (P4) was screened for the highest inhibitory activity, and P4 was vacuum freeze-dried to obtain walnut meal protein peptide. Figure 3 According to the determination results, the component 4 (P4) was screened for the highest inhibitory activity, and P4 was vacuum freeze-dried to obtain walnut meal protein peptide.

[0039] (7) The peptide mass spectrometry information of P4 obtained in step (6) was analyzed by high-throughput ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS 2 ). Chromatographic conditions: Infinity-lab Poroshell 120 EC-C18 column (1.9 μm, 2.1 x 100 mm), column temperature 30°C, mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was water containing 0.1% formic acid, flow rate 0.20 mL / min. The elution gradient was as follows: 0-1 min (5% A), 1-2.5 min (5-10% A), 2.5-12.5 min (10-25% A), 12.5-20 min (25-52.5% A), 20-22 min (52.5-95% A), 22-24 min (95% A), 24-25 min (95-5% A). Mass spectrometry conditions: positive ion mode (ESI+), spray voltage 3.2 kV, capillary temperature 350°C, and dry temperature 350°C. The data acquisition range was 200-2000 m / z, and the scanning mode included full mass spectrum and dd-MS2. The resolution of full mass spectrum was 70000, and the resolution of dd-MS2 was 17500.

[0040] The mass spectrometry data were analyzed by Peaks Studio 8.0 software, and P4 component was analyzed by de novo sequencing. 57 peptides with average confidence level (ALC%) greater than 85% were screened, and the peptide component in P4 is shown in Table 1.

[0041] Table 1 Peptide information identified in P4 component

[0042]

[0043]

[0044] (8) The biological activity of the 57 peptides in Table 4 was evaluated using the Peptide Ranker program (http: / / disilldeep.ucd.ie / PeptideRankert). According to the activity score, 7 peptides with potential biological activity were screened, and the results are shown in Table 2. The DPP-IV inhibition activity potential of the 7 peptides was evaluated using the BIOPEP-UWM platform (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), and 3 peptides with inhibitory activity were screened, namely LPQF, LPSF, and VPFP. The amino acid sites of the three peptides for DPP-IV inhibition were predicted, and the results are shown in Table 3. According to Table 3, the amino acid sites of the three peptides for DPP-IV inhibition were rich.

[0045] Table 2 Peptides with potential biological activity in P4 components

[0046] Sequence number Peptide sequence Predicted score of biological activity 1 WPLR 0.95708 2 TFFHFR 0.938165 3 WVAF 0.912957 4 LPQF 0.905117 5 LPSF 0.897727 6 VPFP 0.897371 7 HFR 0.883585

[0047] Table 3 Three peptides with DPP-IV inhibitory activity in walnut meal protein peptides screened using computer bioinformatics tools and their amino acid sites for DPP-IV inhibition

[0048]

[0049] Example 2

[0050] Prediction of the properties of the three peptides obtained in Example 1 for absorption, distribution, metabolism, excretion, and toxicity (ADMET)

[0051] First, the amino acid sequences of the polypeptides shown in Table 3 were converted into simplified molecular input line entry specifications (SMILES). The ADME properties of the three peptides screened above were predicted using AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1). Peptides with good solubility, easy absorption, good metabolic performance, and low toxicity (or non-toxic) were further analyzed for molecular docking research, and the screening results are shown in Table 4. According to Table 4, the three peptides screened above all have good solubility, can be absorbed by the human intestinal tract, have good metabolic performance, and are non-toxic.

[0052] Table 4 Prediction of ADME properties of screened peptides using SMILES code

[0053]

[0054] Example 3

[0055] Molecular docking of the three polypeptides prepared in Example 1 with DPP-IV

[0056] The crystal structure of DPP-IV as a template for molecular docking studies was obtained from the RCSB Protein Data Bank (http: / / www.rcsb.org) with PDB ID 4PNZ. The three-dimensional structure of each polypeptide was constructed using the "Construct a Protein" tool provided by SYBYL-X 2.0 software. Specifically, molecular docking was performed using the Surflex-Dock tool in SYBYL-X 2.0 software. The best binding conformation of the polypeptides with DPP-IV was predicted according to the consistency score (C-score) and total score (T-score), the interactions between the peptides and the active sites of DPP-IV were constructed, and the peptide segments LPQF, LPSF and VPFP that stably bind to DPP-IV (PDB ID: 4PNZ) were obtained.

[0057] DPP-IV contains three active pockets in total, namely S1, S2 and the key S3. The S1 pocket is composed of amino acid residues Ser630, Asn710, His740, Tyr631, Val656, Trp629, Tyr666 and Val711; the S2 pocket is composed of Arg125, Glu205, Glu206 and Ser209; and the S3 pocket is composed of Tyr547, Arg358 and Phe357. As shown in Table 5, all three peptides interact with the S1, S2 or key S3 pocket of DPP-IV. LPQF, VPFP and LPSF establish 7 (Tyr666 Glu205 His126 Arg125 Tyr547 Pro550 ), 4 (Tyr666 Arg669 Glu206 ) and 6 (Tyr662 Glu206 Tyr547 Arg125 ) hydrogen bond interactions. These three peptides also form 5, 9, and 7 hydrophobic interactions with DPP-IV, respectively. Interestingly, two key amino acid residues, Phe357 and Ser209, are common in all three peptides, suggesting that this may be an important mechanism for their DPP-IV inhibitory activity. In summary, these three peptides can alter DPP-IV conformation to exert their DPP-IV inhibitory activity.

[0058] Table 5 Interactions of LPQF, LPSF and VPFP with DPP-IV (PDB ID: 4PNZ)

[0059]

[0060] Example 4

[0061] The half inhibition rate of the three-segment polypeptide prepared in Example 1 in inhibiting DPP-IV activity

[0062] The walnut meal protein active peptides LPQF, LPSF, and VPFP prepared in Example 1 were synthesized at Shanghai Jiepeptide Biotechnology Co., Ltd. to obtain products with a purity greater than 98%. Active peptide solutions with concentrations of 250, 125, 62.5, 31.25, and 15.62 μg / mL were prepared using DPP assay buffer (No. 700211; Cayman Chemical, USA). The DPP-IV inhibitor activity assay kit (No. 700210; Cayman Chemical, USA) was used to determine the ability of the three peptides to inhibit DPP-IV. The experimental procedures were performed according to the kit instructions, and the peptide concentration that achieved the half-maximal inhibition rate (IC) was calculated. 50 The result is as follows Figure 4 As shown, the IC of peptide LPQF 50 The IC value of peptide LPSF is 99.34 μmol / L (50.03 μg / mL). 50 is 114.70 μmol / L (53.06 μg / mL); the IC of peptide VPFP 50 It is 113.58μmol / L (52.09μg / mL).

Claims

1. Use of a polypeptide in the preparation of a hypoglycemic medicament, the amino acid sequence of the polypeptide being Leu-Pro-Gln-Phe.

2. Use of a polypeptide in the preparation of a health product for helping to maintain a healthy level of blood glucose, the amino acid sequence of the polypeptide being Leu-Pro-Gln-Phe.

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