Polypeptide as well as preparation and application thereof

Abstract

The invention discloses a polypeptide as well as preparation and application thereof, and belongs to the technical field of active peptides. According to the present invention, three active peptides are prepared from walnut meal protein, the amino acid sequences of the three polypeptides are respectively Leuu-Pro-Gln-Phe (LPQF), Leuu-Pro-Ser-Phe (LPSF) or Vla-Pro-Phe-Pro (VPFP), and the detection results show that the three polypeptides have inhibition activity on dipeptidyl peptidase-IV (DPP-IV), such that the three polypeptides can be used for preparing the blood sugar reducing drug.

Description

Technical Field

[0001] This invention relates to a polypeptide, its preparation and application, and belongs to the field of bioactive peptide technology. Background Technology

[0002] Diabetes is one of the most prevalent and deadliest chronic diseases worldwide. It was reported that 537 million adults had diabetes in 2021, and this number is projected to rise to 643 million by 2030 and 783 million by 2045, with type 2 diabetes accounting for approximately 90% of all cases. Currently used synthetic hypoglycemic drugs (such as saxagliptin and sitagliptin) have been shown to have many negative effects, such as weight gain, cardiovascular problems, hypoglycemia, and other adverse reactions. Dipeptidyl peptidase-IV (DPP-IV) inhibitors represent a newer and more advanced treatment option for type 2 diabetes.

[0003] DPP-Ⅳ is a metabolic enzyme distributed in human tissues, responsible for cleaving and inactivating the intestinal insulin-stimulating hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). This leads to a decrease in endogenous GLP-1 and GIP levels, inhibiting insulin release from pancreatic β-cells while promoting glucagon secretion from pancreatic α-cells, thus suppressing insulin levels and increasing blood glucose levels. Therefore, inhibiting DPP-Ⅳ activity can, to some extent, prevent the degradation of GLP-1 and GIP, thereby better regulating blood glucose. Against this backdrop, some food-derived protein bioactive peptides with strong DPP-Ⅳ inhibitory activity are considered a safe means of preventing and controlling the occurrence and development of diabetes. Therefore, the development of food-derived protein bioactive peptides that inhibit DPP-Ⅳ to prevent and control the occurrence and development of diabetes has become a global research hotspot.

[0004] Walnuts, one of the world's four major nuts, possess extremely high nutritional value and have long been considered an excellent food for warming and nourishing the lungs and kidneys, promoting heart and brain health, and strengthening the body. Walnut kernels are rich in oil, with a high content of unsaturated fatty acids, which is a crucial material basis for the economic benefits of walnut industrialization. Walnut oil production generates a large amount of walnut meal, which is high in protein and a rich source of bioactive peptides. A small portion is developed into walnut protein powder, but the majority is still used as animal feed. Whether walnut meal can be effectively developed is a new economic potential area for the walnut industry.

[0005] Peptides are defined as fragments of proteins, typically composed of 2-20 amino acid residues, and possess a variety of biological activities. The potential of bioactive peptides in preventing chronic diseases, particularly diabetes, is particularly recognized. Bioactive peptides in food sources generally refer to two categories: those that effectively promote bodily functions or states to improve human health, and those that impart umami and rich flavor to food. Therefore, walnut meal, a byproduct of walnut oil production, can be used to develop walnut meal protein peptides with stable and simple preparation technology and good blood sugar lowering effects. This would not only provide a new raw material for the prevention and auxiliary control of blood sugar levels in diabetic patients but also find a new source of profit growth for the walnut industry. Summary of the Invention

[0006] One objective of this invention is to provide a polypeptide whose sequence is Leu-Pro-Gln-Phe (LPQF), Leu-Pro-Ser-Phe (LPSF), or Vla-Pro-Phe-Pro (VPFP).

[0007] Preferably, the polypeptides described in this invention can be obtained not only from defatted walnut meal, but also through artificial synthesis.

[0008] A second objective of this invention is to provide a method for preparing the polypeptide, wherein the polypeptide is prepared from defatted walnut meal, and the specific steps are as follows: (1) Defatted walnut meal was modified by enzymatic hydrolysis using food industry protease to obtain protease hydrolysis products.

[0009] (2) Use an ultrafiltration membrane to separate the protease hydrolysis product obtained in step (1) to obtain the separated product.

[0010] (3) Determine the inhibitory activity of the separation product obtained in step (2) against DPP-Ⅳ, and screen out the component with the highest activity.

[0011] (4) The most active component was purified using a reversed-phase high-performance liquid chromatography system to obtain the purified product.

[0012] (5) Determine the inhibitory activity of the purified product against DPP-Ⅳ, and screen out the component with the highest activity to obtain 3 polypeptides.

[0013] Preferably, the food industrial protease in step (1) is bromelain.

[0014] Preferably, the ultrafiltration membrane used in step (2) is 10kDa and 3kDa.

[0015] Preferably, the separation product obtained in step (2) has three parts: molecular weight less than 3kDa, molecular weight between 3-10kDa, and molecular weight greater than 10kDa.

[0016] Preferably, the walnut meal is a byproduct of walnut oil extraction.

[0017] A third objective of this invention is to provide an application of the aforementioned polypeptide, the application of which has the following two aspects: (1) Application of the polypeptide in the preparation of hypoglycemic drugs.

[0018] (2) Application of the polypeptide in the preparation of health products that help maintain healthy blood sugar levels.

[0019] Beneficial effects of the present invention (1) The half-maximal inhibitory concentration (IC50) of the polypeptides LPQF, LPSF, and VPFP of the present invention against DPP-IV. 50 The concentrations were 50.03, 53.06, and 52.09 μg / mL (i.e., 99.34, 114.70, and 113.58 μmol / L), respectively. It is also highly water-soluble, can be absorbed by the human intestine, and has few side effects on the human body.

[0020] (2) The polypeptide described in this invention can be obtained not only through artificial synthesis, but also from defatted walnut meal, providing a new way for the resource utilization of defatted walnut meal. Attached Figure Description

[0021] Figure 1 To investigate the inhibitory activity of hydrolyzed walnut meal protein hydrolysates from different food industry proteases against DPP-Ⅳ.

[0022] Figure 2 The DPP-Ⅳ inhibitory activity of different components after ultrafiltration separation of bromelain hydrolysate was measured.

[0023] Figure 3 To investigate the DPP-Ⅳ inhibitory activity of different components after purification using a liquid chromatography system.

[0024] Figure 4 The half-maximal inhibitory concentrations (IC50) of LPQF, LPSF, and VPFP against DPP-Ⅳ are given. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0026] Unless otherwise specified, all methods are standard and all materials and reagents used are obtained commercially.

[0027] Example 1 Preparation of polypeptides from defatted walnut meal The defatted walnut meal used in this embodiment is a byproduct of water-based ecological walnut oil extraction. The specific steps are as follows. (1) The defatted walnut meal was dried by conventional air drying at 60°C, crushed, passed through a 40-mesh silk sieve, and the sieve-passed material was used for enzymatic hydrolysis.

[0028] (2) The sieves obtained in step (1) were enzymatically hydrolyzed with bromelain, alkaline protease, papain, complex protease, flavor protease, neutral protease, pancreatin, trypsin, animal hydrolytic protease and pepsin under the optimal working conditions of each enzyme. After the enzymatic hydrolysis was completed, the enzyme activity was inactivated by boiling water bath for 20 minutes to obtain the enzymatic hydrolysate.

[0029] (3) After cooling the enzymatic hydrolysate obtained in step (2) to room temperature, centrifuge at 5000 rpm for 20 min and take the supernatant to obtain the enzymatic hydrolysate. Take an appropriate amount of the enzymatic hydrolysate and freeze dry it under vacuum to obtain the dried product. The remaining enzymatic hydrolysate is reserved for later use.

[0030] (4) The inhibitory activity of the dried product obtained in step (3) against DPP-IV was determined, and the results are as follows: Figure 1 As shown, according to Figure 1 The test results showed that the product of bromelain hydrolyzing walnut meal protein had the strongest inhibitory activity against DPP-IV. Therefore, the enzymatic hydrolysate obtained by bromelain hydrolysis was selected for the next experiment.

[0031] (5) The enzymatic hydrolysis products obtained from the bromelain digestion in step (3) were separated using ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa to obtain three components: one with a molecular weight less than 3 kDa, one with a molecular weight between 3 and 10 kDa, and one with a molecular weight greater than 10 kDa. The inhibitory activity of the three components against DPP-Ⅳ was measured, and the results are as follows: Figure 2 As shown, according to Figure 2 The results showed that the component with a molecular weight of less than 3 kDa exhibited the strongest inhibitory activity against DPP-IV, so the component with a molecular weight of less than 3 kDa was selected for the subsequent experiments.

[0032] (6) Fractions with molecular weight less than 3 kDa were purified using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The purification conditions were as follows: 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, the elution gradient was 8-60% B, and the duration was 35 minutes. The detection wavelength was 220 nm. Purified component P1 was obtained at 4.0-6.4 minutes, purified component P2 at 15.0-16.4 minutes, purified component P3 at 17.7-18.6 minutes, purified component P4 at 19.7-20.6 minutes, and purified component P5 at 23.9-25.4 minutes. The inhibitory activity of the five components against DPP-Ⅳ was determined, and the results are as follows: Figure 3 As shown. According to Figure 3 The results showed that component 4 (P4) had the highest inhibitory activity after targeted screening. P4 was then freeze-dried under vacuum to obtain walnut meal protein peptides.

[0033] (7) Using ultra-high performance liquid chromatography-quadrupole-orbitrap-MS (UPLC-Q-Orbitrap-MS) 2 Mass spectrometry information of peptides in P4 obtained in step (6) of high-throughput analysis. Chromatographic conditions: Infinity-lab Poroshell 120 EC-C18 column (1.9μm, 2.1×100mm), column temperature 30℃, mobile phase A is acetonitrile containing 0.1% formic acid, mobile phase B is water containing 0.1% formic acid, flow rate 0.20mL / min. Elution gradient is 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, desiccator temperature 350 °C. Data acquisition range: 200–2000 m / z; scanning modes: full mass spectrometry and dd-MS². Full mass spectrometry resolution: 70,000, while dd-MS² resolution: 17,500.

[0034] Mass spectrometry data were analyzed using Peaks Studio 8.0 software; and the components of P4 were analyzed by de novo sequencing. Fifty-seven peptides with an average confidence level (ALC%) greater than 85% were identified. The peptide components in P4 are shown in Table 1.

[0035] Table 1 Information on peptides identified in component P4 (8) The bioactivity of the 57 peptides in Table 4 was evaluated using the Peptide Ranker program (http: / / disilldeep.ucd.ie / PeptideRankert). Based on the activity scores, 7 peptides with potential biological activity were screened, as shown in Table 2. The inhibitory activity potential of the 7 peptides against DPP-IV was evaluated using the BIOPEP-UWM platform (https: / / biochemia.uwm.edu.pl / biopep-uwm / ). Three peptides with inhibitory activity were screened, namely LPQF, LPSF, and VPFP. The amino acid sites of the three peptides that inhibit DPP-IV were predicted, as shown in Table 3. According to the prediction in Table 3, the three peptides have abundant amino acid sites that inhibit DPP-IV.

[0036] Table 2 shows peptides with potential biological activity in component P4. Table 3 shows the three peptides from walnut meal protein peptides that exhibit DPP-IV inhibitory activity, screened using computer bioinformatics tools, and their corresponding amino acid sites for DPP-IV inhibition. Example 2 Predictive properties of absorption, distribution, metabolism, excretion, and toxicity of the three peptides obtained in Example 1 (ADMET) First, the amino acid sequences of the peptides shown in Table 3 were converted into the Simplified Molecular Input Line Canonical System (SMILES). AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1) was used to predict the ADMET properties of the three peptides screened above. Peptides with good solubility, easy absorption, good metabolic performance, and low (or non-toxic) toxicity were further analyzed through molecular docking studies. The screening results are shown in Table 4. According to Table 4, the three peptides screened above all exhibit good solubility, can be absorbed by the human intestine, have good metabolic performance, and are non-toxic.

[0037] Table 4 shows the ADMET characteristic prediction of the screened peptides using SMILES codes. Example 3 Molecular docking of the three polypeptides prepared in Example 1 with DPP-IV As a template for molecular docking studies, the crystal structure of DPP-IV was obtained from the RCSB protein database (http: / / www.rcsb.org), with PDB ID 4PNZ. The three-dimensional structures of each peptide were 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 optimal binding conformation of the peptide to DPP-IV was predicted based on the consistency score (C-score) and total score (T-score). The interaction between the peptide and the active site of DPP-IV was constructed, yielding peptides LPQF, LPSF, and VPFP that bind stably to DPP-IV (PDB ID: 4PNZ).

[0038] DPP-IV contains three active pockets: S1, S2, and the crucial S3. The S1 pocket consists of amino acid residues including Ser630, Asn710, His740, Tyr631, Val656, Trp629, Tyr666, and Val711; the S2 pocket consists of Arg125, Glu205, Glu206, and Ser209; and the S3 pocket consists of Tyr547, Arg358, and Phe357. The results, shown in Table 5, indicate that all three peptides interact with either the S1, S2, or crucial S3 pockets of DPP-IV. LPQF, VPFP, and LPSF established seven (Tyr666 (2.97 Å), Glu205 (2.79 Å), His126 (3.02 Å), Arg125 (2.98 Å, 3.21 Å), Tyr547 (2.66 Å), Pro550 (2.86 Å)), four (Tyr666 (2.82 Å), Arg669 (2.75 Å, 3.35 Å), Glu206 (3.06 Å)), and six (Tyr662 (2.88 Å), Glu206 (2.97 Å), Tyr547 (2.97 Å, 3.08 Å), Arg125 (2.78 Å, 3.08 Å)) hydrogen bond interactions with DPP-IV, respectively. These three peptides also formed 5, 9, and 7 hydrophobic interactions with DPP-IV, respectively. Interestingly, all three peptides contain two key amino acid residues, Phe357 and Ser209, suggesting that this may be one of the important mechanisms by which they exert their DPP-IV inhibitory activity. In summary, the three peptides can alter the conformation of DPP-IV to exert their DPP-IV inhibitory activity.

[0039] Table 5. Interactions of LPQF, LPSF, and VPFP with DPP-IV (PDB ID: 4PNZ) Example 4 The half-maximal inhibitory rate (ICP-4) of the three peptides prepared in Example 1 against DPP-IV activity The walnut meal protein bioactive peptides LPQF, LPSF, and VPFP prepared in Example 1 were synthesized at Shanghai Jietai Biotechnology Co., Ltd., yielding products with a purity greater than 98%. Bioactive peptide solutions with concentrations of 250, 125, 62.5, 31.25, and 15.62 μg / mL were prepared using DPP analysis buffer (No. 700211; Cayman Chemicals, USA). The inhibitory activity of the three peptides on DPP-IV was determined using a DPP-IV inhibitor activity assay kit (No. 700210; Cayman Chemicals, USA). The experimental procedures were performed according to the kit instructions, and the peptide concentration at which half-maximal inhibition (IC50) was calculated. 50 (Value). Result as follows Figure 4 As shown, the IC50 of peptide LPQF 50 The concentration was 99.34 μmol / L (50.03 μg / mL); the IC50 of the peptide LPSF was... 50 The concentration was 114.70 μmol / L (53.06 μg / mL); the IC50 of the peptide VPFP was... 50 It is 113.58μmol / L (52.09μg / mL).

Claims

1. The application of a polypeptide in the preparation of hypoglycemic drugs, wherein the amino acid sequence of the polypeptide is Vla-Pro-Phe-Pro.

2. The use of a polypeptide in the preparation of health products that help maintain healthy blood sugar levels, wherein the amino acid sequence of the polypeptide is Vla-Pro-Phe-Pro.

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