Octapeptide with ADH activation capability and preparation method thereof

Abstract

The invention relates to an octapeptide with ADH activation capability and a preparation method thereof, and belongs to the technical field of biology. The octapeptide is abbreviated as NDDDFIRI, the molecular weight of the octapeptide is 1007.20, and the sequence of the octapeptide is as follows: Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile; the method comprises the following steps: selecting macromolecular resin, according to the characteristics of an amino acid sequence Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile, connecting hydroxyl of Ile with the resin, then carrying out a shrinkage reaction and treatment on amino of Arg and carboxyl of Ile, then adding Ile, carrying out a reaction on amino of Ile and carboxyl of Arg, sequentially adding amino acids from right to left, adding the last Asn amino acid, and then cutting off the resin to obtain the target polypeptide. According to the invention, hemp seed protein is taken as a raw material, a biological enzymolysis technology is combined with bioinformatics and a molecular docking technology to screen octapeptide with ethanol dehydrogenase activation activity, and polypeptide synthesis and in-vitro activity determination are carried out through a solid-phase synthesis technology. The polypeptide with the hemp seed protein source capable of activating the activity of ethanol dehydrogenase is obtained and can be used for developing medicines with the functions of dispelling the effects of alcohol and protecting the liver.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an octapeptide with ADH activation ability and a preparation method thereof. Background Art

[0002] The history of human alcohol consumption is long. Since ancient times, as a special food, wine has played an important role in daily life and social interactions. However, excessive drinking can not only lead to alcohol dependence but also increase the risk of people getting sick. Such as alcoholic fatty liver, cirrhosis, alcoholic hepatitis, digestive system diseases, etc.

[0003] Alcohol dehydrogenase (ADH) is the rate-limiting enzyme for normal human metabolism of alcohol, catalyzing the dehydrogenation of alcohol to produce acetaldehyde. Acetaldehyde is further catalyzed by aldehyde dehydrogenase (ALDH) to produce acetic acid harmless to humans, and finally decomposed into water and carbon dioxide and excreted from the body. Activating the activity of ADH can not only accelerate the metabolism of alcohol, the excretion of alcohol and metabolic intermediates from the body, but also reduce its adverse effects on the liver.

[0004] Bioactive peptides exhibit various biological activities due to different amino acid compositions and sequences, and are considered natural efficacy factors for regulating body metabolism and promoting human health. Bioactive peptides have the advantages of small molecular weight, easy absorption, wide sources of food proteins, and good safety, and are widely favored. Therefore, finding a safe and effective liver-protecting active peptide to prevent or treat ALD has become a research and attention hotspot. Hemp seeds contain 20% - 25% protein and are high-quality raw materials for preparing bioactive peptides. Summary of the Invention

[0005] The object of the present invention is to provide a bioactive peptide that activates the activity of ADH, thereby accelerating the metabolism of alcohol, the excretion of alcohol and metabolic intermediates from the body, and reducing its adverse effects on the liver. The present invention uses hemp seed protein as a raw material and prepares an octapeptide with ethanol dehydrogenase activation ability by using biological enzymolysis technology, chromatography technology, bioinformatics, molecular docking, and polypeptide coupling technology. This octapeptide has the ability to activate ethanol dehydrogenase and can be used for the development of functional foods for relieving alcohol and protecting the liver, and can be applied to the fields of biopharmaceuticals and functional foods.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] An octapeptide with ADH activation ability, the octapeptide is abbreviated as NDDDFIRI, with a molecular weight of 1007.20, and the sequence is: Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile.

[0008] A method for preparing the above-mentioned octapeptide with ADH activation ability, the method is as follows: select a high molecular resin, according to the characteristics of the amino acid sequence Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile, the hydroxyl group of Ile is connected to the resin, and then the amino group of Arg and the carboxyl group of Ile undergo a condensation reaction. After treatment, Ile is added again, and the amino group of Ile reacts with the carboxyl group of Arg. Amino acids are added in turn from right to left. After adding the last Asn amino acid, the resin is removed to obtain the target polypeptide.

[0009] Further, high performance liquid chromatography is used for purification. The chromatographic column is NanoChromcore TM120C18 (4.6*250mm*5um). Mobile phase A: 0.1% trifluoroacetic acid in 100% water; Mobile phase B: 0.1% trifluoroacetic acid in 100% acetonitrile; B rises from 25% to 45% within 20 min, the flow rate is 1.0 mL / min, the detection wavelength is 214 nm, it is quickly frozen in liquid nitrogen and freeze-dried to obtain the final product, and the required purity is above 98%.

[0010] Further, the vacuum degree of the freeze-drying is 50 Pa, the temperature is -80 °C, and the time is 16 h.

[0011] The beneficial effects of the present invention compared with the prior art are as follows: The present invention uses hemp seed protein as a raw material, and a bioinformatics and molecular docking technology combined with a biological enzymatic hydrolysis technology is used to screen an octapeptide with ethanol dehydrogenase activation activity. And through solid-phase synthesis technology for polypeptide synthesis and in vitro activity determination, a polypeptide with ethanol dehydrogenase activation activity from hemp seed protein is obtained, which can be used for the development of anti-alcohol and liver protection drugs. Description of the Drawings

[0012] Figure 1 It is a graph showing the activation rate of each component of hemp seed peptide on ethanol dehydrogenase.

[0013] Figure 2 It is a molecular docking diagram of the synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile and ethanol dehydrogenase.

[0014] Figure 3 It is an HPLC diagram of the synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile.

[0015] Figure 4 It is an MS diagram of the synthetic polypeptide Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile.

[0016] Figure 5 Activation rate graph of ethanol dehydrogenase by synthetic polypeptide Asn - Asp - Asp - Asp - Phe - Ile - Arg - Ile at different concentrations. Detailed implementation method

[0017] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, shall be covered by the protection scope of the present invention.

[0018] Example 1:

[0019] The octapeptide that activates ethanol dehydrogenase synthesized by the present invention is abbreviated as NDDDFIRI, with a molecular weight of 1007.20, and the sequence is: Asn - Asp - Asp - Asp - Phe - Ile - Arg - Ile.

[0020] 1. Preparation of hemp seed liver - protecting peptide

[0021] Weigh 10 g of hemp seed protein powder, add 50 mL of deionized water, fully dissolve and stir evenly, adjust the pH of the enzymatic hydrolysis solution to 6.2, add 3.36% neutral protease, carry out enzymatic hydrolysis at an enzymatic hydrolysis temperature of 44 °C and an enzymatic hydrolysis time of 2.75 h. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95 °C for 15 min, centrifuge at 10000 r / min at 4 °C for 15 min, and take the supernatant for vacuum freeze - drying for standby.

[0022] 2. Separation and purification of hemp seed liver - protecting peptide

[0023] Put anhydrous ethanol in a 4 °C refrigerator overnight, and add it dropwise to the prepared hemp seed peptide solution at 40 mg / mL until the ethanol volume fraction reaches 20%. Then let the solution stand at 4 °C for 1 h, and then centrifuge at 7500 g for 10 min. The precipitate part is denoted as E20. Add the supernatant part to cold anhydrous ethanol until the ethanol volume fraction reaches 40%, let it stand at 4 °C for 1 h, and the precipitate part obtained by centrifugation is denoted as E40. Add the supernatant part to cold anhydrous ethanol until the ethanol volume fraction reaches 60%, let it stand at 4 °C for 1 h, and the precipitate part obtained by centrifugation is denoted as E60, and the supernatant is denoted as SPT. All components are concentrated under reduced pressure at 55 °C to remove ethanol, and then freeze - dried. Measure the activation rate of ethanol dehydrogenase of each component to screen the component with the strongest activity ( Figure 1 ).

[0024] The activation rate of ethanol dehydrogenase was measured by the Warburg - Christian method. First, 1.5 mL (0.1 M, pH 8.8) of sodium pyrophosphate buffer, 1.0 mL of 0.01 M NAD +, 0.5 mL (11.5%, v / v) ethanol and 0.1 mL of hydrolyzed hemp seed protein were mixed at 25 °C and reacted for 5 min. Then, 0.1 mL of 0.25 U / mL ADH was added to the reaction system. The absorbance was immediately measured at 340 nm for 10 min. The linear part at the beginning of the reaction was plotted, and the slope was denoted as Ka. For the control group, 0.2 mL of distilled water was used instead of the sample, and the slope was denoted as Kc, which was calculated according to formula (1).

[0025]

[0026] 3. Virtual Screening and Molecular Docking of Hemp Seed Hepatoprotective Peptide Sequences

[0027] For the most active component SPT obtained by separation and purification, LC-MS / MS mass spectrometry was used to analyze and identify the polypeptide sequence. Dock 6.9 was used for virtual screening of the obtained peptide sequence; Peptide ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to score the potential biological activities of the alternative polypeptides; GRAVYCALCULATOR (https: / / www.gravy-calculator.de / ) was used to calculate the hydrophobicity; PepDraw (https: / / www.pepdraw.com / ) was used to calculate the relative molecular mass, net charge, and isoelectric point; the toxicity was determined by ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html). According to the docking scores of the peptide segments, the peptide segment NDDDFIRI with better docking effect with alcohol dehydrogenase was screened out, with a docking score of -19.1 kcal / mol, a molecular weight of 1007.20 Da, a polypeptide score of 0.6767, a hydrophilicity of 076, a net charge of -2, an isoelectric point of 3.94, and no toxicity.

[0028] The crystal structure of alcohol dehydrogenase (PDB ID: 1U3T) was obtained from the RCSB Protein Data Bank database (http: / / www.rcsb.org / ). The semi-flexible docking of NDDDFIRI with alcohol dehydrogenase was performed using Discovery studio 2019 software to determine the key amino acid residues and interaction forces between it and alcohol dehydrogenase (see Figure 2) The peptide NDDDFIRI forms 10 hydrogen bonds with ADH1A, involving ASN1, ASP4, ARG7, ASP2, ASP3 in the NDDDFIRI peptide and GLU107, GLY316, PRO295, ASN105, LYS315, TYR110, ASN109 in ADH1A. And the interactions include van der Waals forces, hydrogen bonds, hydrophobic interactions, salt bridge interactions, and π-alkyl interactions.

[0029] 4. Solid-phase synthesis, separation, purification and identification of hemp seed hepatoprotective peptide

[0030] A high molecular resin (Bio-Engineering (Shanghai) Co., Ltd.) was selected. According to the characteristics of the amino acid sequence Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile, the hydroxyl group of Ile was connected to the resin, and then the amino group of Arg underwent a condensation reaction with the carboxyl group of Ile. After treatment, Ile was added again, and the amino group of Ile reacted with the carboxyl group of Arg. Amino acids were added sequentially from right to left. After adding the last Asn amino acid, the resin was removed to obtain the target polypeptide.

[0031] Purification was carried out by high performance liquid chromatography. The chromatographic column was NanoChromcore TM120C18 (4.6*250mm*5um). Mobile phase A: 0.1% trifluoroacetic acid in 100% water; Mobile phase B: 0.1% trifluoroacetic acid in 100% acetonitrile; B increased from 25% to 45% within 20 min, the flow rate was 1.0 mL / min, and the detection wavelength was 214 nm. It was quickly frozen in liquid nitrogen and freeze-dried (vacuum degree: 50 Pa, temperature: -80 °C, time: 16 hours) to obtain the final product, with a required purity of over 98%, and the structure was identified by MS (as Figure 3 、 Figure 4 shown).

[0032] 5. Verification of the in vitro activity of solid-phase synthesized polypeptides

[0033] Study on the activation rate of ADH by Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile at different concentrations. Using the above-mentioned method for measuring the ADH activation rate, active peptide solutions with concentrations of 0, 1, 3, 5, 7, and 9 mM were respectively prepared, and the in vitro activation activity of ADH by active peptides at different concentrations was measured. The results are shown in Figure 5 .

Claims

1. An octapeptide with ADH activation ability, characterized in that: The octapeptide is abbreviated as NDDDFIRI, with a molecular weight of 1007.20 and the sequence: Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile.

2. A method for preparing an octapeptide with ADH activation ability according to claim 1, characterized in that: The method is as follows: A high molecular resin is selected. According to the characteristics of the amino acid sequence Asn-Asp-Asp-Asp-Phe-Ile-Arg-Ile, the hydroxyl group of Ile is connected to the resin, and then the amino group of Arg undergoes a condensation reaction with the carboxyl group of Ile. After treatment, Ile is added, and the amino group of Ile reacts with the carboxyl group of Arg. Amino acids are added sequentially from right to left. After adding the last Asn amino acid, the resin is removed to obtain the target polypeptide.

3. The preparation method of an octapeptide with ADH activation ability according to claim 2, characterized in that: Purification is carried out by high performance liquid chromatography. The chromatographic column is NanoChromcore TM120 C18 (4.6*250mm*5um). Mobile phase A: 0.1% trifluoroacetic acid in 100% water; Mobile phase B: 0.1% trifluoroacetic acid in 100% acetonitrile; B is increased from 25% to 45% within 20 min, the flow rate is 1.0 mL / min, the detection wavelength is 214 nm, quick-frozen with liquid nitrogen, and freeze-dried to obtain the final product, with the required purity reaching over 98%.

4. The preparation method of an octapeptide with ADH activation ability according to claim 3, characterized in that: The vacuum degree of the freeze-drying is 50 Pa, the temperature is -80 °C, and the time is 16 h.

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