A sweet potato peptide that competitively inhibits α-glucosidase activity

Abstract

The present invention relates to the field of biotechnology, and in particular to a sweet potato peptide that competitively inhibits alpha-glucosidase activity. Its amino acid sequence is shown in SEQ ID NO.1. The present invention provides a sweet potato peptide that competitively inhibits alpha-glucosidase activity, comprising a sweet potato protein peptide that has good inhibition of alpha-glucosidase activity and can delay starch digestion in vivo, and inhibition kinetics show that it is a competitive inhibitor.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a sweet potato peptide capable of competitively inhibiting alpha-glucosidase activity. Background Art

[0002] Glucose is the most important energy source for mammals and maintains normal cell life activities. Breaking the body's blood sugar balance can lead to hypoglycemia or hyperglycemia. When the body's fasting blood sugar is higher than 6.1 mmol / L or the blood sugar is higher than 7.8 mmol / L two hours after a meal, it is considered hyperglycemia. Continuous hyperglycemia causes abnormal glucose metabolism in the body, inducing metabolic diseases such as insulin resistance, hyperinsulinemia, and diabetes. α-glucosidase (α-glucosidase) is one of the important enzymes for intestinal digestion of dietary carbohydrates and is a biological target for delaying blood sugar elevation. It can hydrolyze disaccharides such as sucrose, maltose and other oligosaccharides into monosaccharides that can be partially absorbed by the human small intestine. At present, α-glucosidase inhibitors such as acarbose, voglibose and miglitol are clinically used for the prevention and treatment of diabetes. However, long-term use of these drugs can cause obvious side effects, including stomach pain, diarrhea, flatulence and allergic reactions. Therefore, it is very necessary to find natural α-glucosidase inhibitors with high efficiency and few side effects from natural products to effectively reduce postprandial hyperglycemia.

[0003] Sweet potato (Dioscorea esculenta (Lour.) Burkill) is one of the important food crops in my country and an important raw material for the food industry, light industry and feed industry. Sweet potato is not only nutritious but also nutritionally balanced. Sweet potato is widely distributed in my country and is mainly used for starch production, but its nutrients such as protein are not fully utilized. my country produces more than 1 million tons of sweet potato starch annually, and it is estimated that the amount of protein-rich wastewater generated by the production of sweet potato starch reaches 20 million to 40 million tons each year. Research on the comprehensive utilization of sweet potato protein will provide a scientific basis for avoiding resource waste and increasing the added value of sweet potatoes, and has broad application prospects and good economic value.

[0004] At present, there are literature reports on the antioxidant and hypotensive activities of sweet potato protein peptides, but there are no reports on the preparation of glucose-lowering peptides from sweet potato protein. From the perspective of enzyme inhibition kinetics, enzyme inhibitors are divided into competitive inhibitors, anti-competitive inhibitors, non-competitive inhibitors, mixed inhibitors, etc. The mode of action and application of sweet potato peptides as inhibitors of α-glucosidase have not been reported. Summary of the invention

[0005] The technical problem to be solved by the present invention is that currently there are literature reports on the antioxidant and blood pressure lowering activities of sweet potato protein polypeptides, but there is no report on the preparation of peptides that inhibit α-glucosidase activity from sweet potato protein.

[0006] To solve the above problems, the present invention provides a sweet potato peptide that competitively inhibits α-glucosidase activity, comprising a sweet potato protein peptide that has good inhibition of α-glucosidase activity and can delay starch digestion in vivo. Inhibition kinetics show that it is a competitive inhibitor.

[0007] To achieve the above object, the present invention is achieved by the following technical means: a sweet potato peptide that competitively inhibits the activity of α-glucosidase, and its amino acid sequence is shown in SEQ ID NO.1.

[0008] SEQ ID NO.1:

[0009] The amino acid sequence is Gln-Ala-Gly-Pro-Asp-Pro-Trp, abbreviated as QAGPDPW.

[0010] Furthermore, the above sweet potato peptide is artificially synthesized in vitro.

[0011] The application of the above sweet potato peptide in the preparation of products that inhibit the activity of α-glucosidase. Sweet potato peptide can compete with the substrate for the corresponding α-glucosidase catalytic active site. Since the number of enzyme catalytic sites is certain, when QAGPDPW preferentially binds to the active site of the enzyme, the corresponding substrate cannot bind to the active site on the enzyme, which reduces the binding rate between the enzyme and the oligosaccharide, so that the glycosidic bond of the oligosaccharide cannot be cleaved, and a large amount of oligosaccharides cannot be catalyzed by the enzyme into monosaccharides that can be absorbed by the human body, thereby avoiding postprandial hyperglycemia.

[0012] The beneficial effects of the present invention are:

[0013] (1) A new peptide segment was obtained from sweet potato peptides. The inhibition kinetics showed that it was a competitive inhibitor of α-glucosidase, non-toxic and non-sensitizing. It not only had high α-glucosidase inhibitory activity in vitro, but also showed α-glucosidase inhibitory activity in vivo, providing a new raw material for products that inhibit α-glucosidase activity.

[0014] (2) These new peptides are all derived from sweet potato protein, opening up a new direction for the research of sweet potato protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the chemical structural formula of the polypeptide shown in SEQ ID NO.1.

[0016] Figure 2 It is the secondary mass spectrum of the polypeptide shown in SEQ ID NO.1.

[0017] Figure 3 This is the docking result of QAGPDPW and α-glucosidase.

[0018] Figure 4 The inhibitory activity of QAGPDPW on α-glucosidase.

[0019] Figure 5 It is the blood glucose level of mice within 3 hours after meal treated with QAGPDPW (Figure A represents the blood glucose level of normal mice treated with QAGPDPW after intragastric administration of starch; Figure B represents the blood glucose level of normal mice treated with QAGPDPW after intragastric administration of maltose; CD is the corresponding area under the curve).

[0020] Figure 6 It is the enzymatic reaction rate diagram of QAGPDPW to α-glucosidase.

[0021] Figure 7 It is a Lineweaver-Burk diagram. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0024] Embodiment 1:

[0025] A sweet potato peptide for competitively inhibiting alpha-glucosidase activity, including an amino acid sequence as shown in SEQ ID NO.1.

[0026] SEQ ID NO.1:

[0027] QAGPDPW.

[0028] Its chemical structure and secondary mass spectrum are as follows Figure 1-2 As shown, sweet potato peptides can compete with substrates for the corresponding α-glucosidase catalytic active sites. Since the number of enzyme catalytic sites is fixed, when QAGPDPW preferentially binds to the active site of the enzyme, the corresponding substrate cannot bind to the active site on the enzyme, which reduces the binding rate between the enzyme and the oligosaccharides, making it impossible for the glycosidic bonds of the oligosaccharides to be cleaved, and a large number of oligosaccharides cannot be catalyzed by the enzyme into monosaccharides that can be absorbed by the human body, thereby avoiding postprandial hyperglycemia.

[0029] Embodiment 2:

[0030] The use of the sweet potato peptide described in Example 1 in the preparation of a product for inhibiting α-glucosidase activity.

[0031] Embodiment 3:

[0032] The method for preparing the sweet potato peptide comprises the following steps:

[0033] (1) The sweet potatoes are washed and peeled, and 4 to 5 times the volume of water is added to crush and extract. The extract is allowed to stand for 6 to 8 hours and then filtered through gauze to remove the sweet potato fiber and large granular starch to obtain a supernatant. The supernatant is adjusted to a pH value of 4, allowed to stand and precipitate, and then centrifuged to obtain a precipitate. The precipitate is freeze-dried to obtain sweet potato crude protein.

[0034] (2) Weigh an appropriate amount of sweet potato crude protein, add distilled water at a ratio of 1:20 (w / v), heat at 90°C for 10 min, and then high-pressure treat at 200 MPa for 6 min. Then adjust the pH to 10 with 1 mol / L NaOH, add 4% (w / w) alkaline protease, and hydrolyze in a constant temperature water bath at 55°C for 3 h. After the enzymatic hydrolysis, centrifuge at 4°C, 10,000 r / min for 15 min to obtain the supernatant to obtain the sweet potato enzymatic hydrolysate (SPP).

[0035] (3) The enzymatic hydrolyzate obtained in Example 1 was filtered through a microporous filter membrane (pore size 0.22 μm). Then, it was treated in an ultrafiltration system with an ultrafiltration membrane having a molecular weight Mw cutoff value of 3 to obtain sweet potato peptide SPP-1 with Mw < 3 kDa.

[0036] (4) The sweet potato peptide is identified through separation, purification and sequence identification.

[0037] LC-MS / MS identification of the sequence of the inhibitory peptide of sweet potato protein α-glucosidase. After desalting, the peptide sample was centrifuged and dried, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and loaded into a bottle for online LCMS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100Å) in an appropriate volume, and then washed and desalted with a volume of 20 μl. After desalting and retention on the pre-column, the sample was separated on a C18 reverse phase column (Acclaim PepMap RSLC, 75 μm×25 cm C18-2μm 100Å). The gradient used in the experiment was increased from 5% to 38% of mobile phase B (80% acetonitrile, 0.1% formic acid) within 60 min. The mass spectrometer was operated with a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a nanospray NanoFlex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 320 °C.

[0038] Effect verification:

[0039] 1. Molecular docking of peptide with α-glucosidase (PDB ID: 2QMJ). The best docking conformation was selected based on the binding energy. The docking result file pdbqt was converted into a pdb file and then imported into Pymol software and the online website PLIP (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) for result visualization analysis. The chemical structure was drawn using ChemDraw, and then QAGPDPW was molecularly docked with α-glucosidase (e.g. Figure 3As shown in Table 1). The binding energy required for the docking of the peptide with α-glucosidase is shown in Table 1. The binding energy of the ligand and the receptor is used to evaluate the degree of binding between the two. The smaller the binding energy, the easier it is for the two to bind and the stronger the interaction. The binding energy of QAGPDPW with α-glucosidase is less than -9 kcal / mol. Therefore, it is speculated that QAGPDPW has a strong α-glucosidase inhibitory activity. The interaction force with α-glucosidase is shown in Table 2. QAGPDPW forms hydrogen bonds with the amino acid residues Tyr299, Asp443, Arg526, Asp542, Gln603, and Tyr605 of α-glucosidase, and forms hydrophobic interactions with Tyr299, Phe450, Phe575, and Ala576. The binding of active peptides to macromolecules mainly depends on various intermolecular forces such as hydrogen bond interaction force, van der Waals force, hydrophobic interaction force, and electrostatic interaction force, and hydrogen bond interaction force plays an important role in stabilizing the docking complex. Therefore, QAGPDPW matches well with α-glucosidase and has strong binding ability, which is consistent with the binding energy results of virtual screening. In addition, Asp542 and Arg526 are considered to be essential amino acid residues for the active pocket of α-glucosidase to bind to the substrate. Therefore, QAGPDPW can occupy the key binding site of α-glucosidase through hydrogen bonding, and bind to α-glucosidase under the influence of hydrophobic interaction, thereby inhibiting α-glucosidase in vivo to delay the rise of blood sugar.

[0040] Table 1: Binding energy required for peptide docking with α-glucosidase:

[0041] .

[0042] Table 2: Interaction forces with α-glucosidase:

[0043] .

[0044] 2. The peptides in Example 1 were artificially synthesized, and the purity of the synthesized peptides was >95%. After synthesis, the ability of each peptide to inhibit α-glucosidase was measured. Take 200 μL of sample and add 200 μL of α-glucosidase (0.2 U / mL), mix the above solution and incubate at 37°C for 5 minutes. Then add 200 μL of 2.5mM p-nitrobenzene-α-glucoside solution (pNPG) and 0.1 M phosphate buffer (pH 6.8) to 2 mL and incubate at 37°C for 20 minutes. Finally, add 200 μL of 0.4 M sodium carbonate to stop the reaction, and measure the absorbance at 405nm. The calculation formula for α-glucosidase inhibition rate is as follows:

[0045] α-glucosidase inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100%;

[0046] Where: A1—absorbance value of sample group,

[0047] A2—absorbance value of sample background,

[0048] A3—absorbance value without sample,

[0049] A4—Absorbance value of blank group without sample.

[0050] like Figure 4 As shown, the IC of QAGPDPW 50 The values ​​were 1.581 ± 0.168 mg / mL, respectively, indicating that QAGPDPW showed strong α-glucosidase inhibitory activity in in vitro experiments.

[0051] 3. Mice were gavaged with QAGPDPW and starch or maltose, and blood glucose levels within 3 hours after the meal were analyzed. Figure 5 A and Figure 5 B The results showed that the postprandial blood glucose of mice increased first and reached the maximum value within 0.5 h. The blood glucose concentration decreased over time and returned to the initial level within 3 h. After gavage with starch, the three pure peptides could inhibit the increase of postprandial blood glucose content; compared with the control group, the acarbose and QAGPDPW groups significantly reduced the area under the blood glucose curve ( p <0.05), which is consistent with the results of in vitro inhibition of α-glucosidase activity. Compared with the results of the starch test, the effect of this peptide on the postprandial blood glucose level of mice induced by maltose showed the same trend. Therefore, QAGPDPW has a significant hypoglycemic effect in vivo, and it is speculated that this peptide may have an inhibitory effect on α-glucosidase in vivo.

[0052] 4. Plot the enzymatic reaction rate of QAGPDPW to α-glucosidase at different peptide concentrations, e.g. Figure 6 As shown, all straight lines pass through the origin and show a good linear relationship. Moreover, the slope of the straight line decreases with the continuous increase of peptide, indicating that the activity of α-glucosidase is not completely inhibited, but its catalytic rate gradually decreases. It is reported that irreversible inhibitors can form a stable enzyme-inhibitor complex with the enzyme through covalent interaction, which completely inactivates the enzyme. Therefore, the experimental results show that QAGPDPW is a reversible α-glucosidase inhibitor.

[0053] Lineweaver-Burk plots were used to study the inhibition type and inhibition kinetic parameters of the inhibitors. The maximum initial digestion rate (Vmax) calculated from the intercept of the Lineweaver-Burk equation (1 / Vmax) remained almost unchanged (Table 3). Figure 7 As shown, the intersection point of QAGPDPW is located on the y-axis. In addition, the maximum initial digestion rate (Vmax) calculated from the intercept (1 / Vmax) of the Lineweaver-Burk equation line was almost unchanged (Table 3). At the same time, the inhibition constant (Ki) calculated from the slope of the equation (Km / vmax) increased along the increase of QAGPDPW concentration (Table 3). These parameter characteristics indicate that the inhibition type of QAGPDPW is competitive inhibition.

[0054] Table 3:

[0055] .

[0056] Toxicity prediction was performed using ToxinPred ( https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php Allergenicity prediction was performed using AllerTOP v2.0 ( http: / / www.ddg-pharmfac.net / AllerTOP / ). It has been proven that the above sweet potato peptides are non-toxic and non-allergenic.

[0057] Finally, it should be noted that although the above embodiments describe the specific implementation of the present invention, they are not intended to limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. All modifications or equivalent replacements should be included in the protection scope of the present invention.

Claims

1. A sweet potato peptide that competitively inhibits α-glucosidase activity, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The sweet potato peptide according to claim 1, characterized in that: The sweet potato peptide is artificially synthesized.

3. Use of the sweet potato peptide according to claim 1 or 2 in the preparation of a product for inhibiting α-glucosidase activity.

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