A polypeptide with high biological activity and application thereof

Abstract

The application provides a kind of high bioactivity polypeptide and application, it is related to active peptide preparation technical field.The application obtains high bioactivity polypeptide by enzyme hydrolysis to abalone.The polypeptide obtained is analyzed and screened, and the high bioactivity polypeptide with amino acid sequence as shown in SEQ ID NO.1-9 is obtained.The polypeptide of the application has higher bioactivity, the polypeptide with amino acid sequence as shown in SEQ ID NO.1-9 has higher angiotensin converting enzyme inhibitory, and the IC 50 Between 0.15-0.37mg / mL, so it can better inhibit the activity of angiotensin converting enzyme;In addition, the polypeptide of the application also has antioxidant property.The polypeptide of the application effectively improves the condition that the bioactivity of polypeptide is low in industrial production, and simultaneously provides technical support and supplement for the preparation and screening of marine bioactive peptide.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide preparation technology, and in particular to a highly bioactive polypeptide and its applications. Background Technology

[0002] With economic development, people's demands for quality of life are gradually increasing, and their awareness of health is gradually improving, leading to a rise in the consumption of health products. Bioactive peptides, as a type of functional food, not only possess rich nutritional value but also exhibit various physiological functions such as antioxidation, anti-cancer, antibacterial, blood pressure lowering, and immune regulation, playing an indispensable role in people's daily lives. Bioactive peptides are diverse and widely sourced, obtainable from animal and plant proteins. Plant protein sources include legumes and grains, while animal sources include milk, meat products, and eggs.

[0003] Abalone is a marine gastropod, a single-shelled mollusc, and is widely loved by consumers. As an important marine product, its market price is lower than that of high-end, expensive fish, but its nutritional value is no less, possessing high edible and medicinal value. Abalone is rich in protein, polysaccharides, taurine, and trace elements, and its peptide products contain 30%-50% collagen, far exceeding other fish and shellfish. Furthermore, the content of taurine and selenium in abalone is much higher than in common marine shellfish such as mussels, oysters, and clams, making it an excellent resource for developing marine bioactive peptides. Currently, the most common method for preparing bioactive peptides is enzymatic hydrolysis, where the main active enzymes are mostly non-specific enzymes such as alkaline proteases, neutral proteases, trypsin, and papain. The resulting products have complex compositions, leading to lower biological activity. Therefore, research on how to obtain highly bioactive peptides is still lacking. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a highly bioactive polypeptide in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a highly bioactive polypeptide and its applications. This invention utilizes enzymatic hydrolysis of abalone to screen for highly bioactive polypeptides in the hydrolysate. The amino acid sequences of the polypeptides of this invention are shown in SEQ ID NO. 1-9, and the polypeptides of this invention exhibit high bioactivity. Firstly, the polypeptides of this invention possess high angiotensin-converting enzyme (ACE) inhibitory activity, with an IC50 of 1 / 3 oz. ... 50 The concentration is between 0.15-0.37 mg / mL, thus effectively inhibiting angiotensin-converting enzyme activity; in addition, the polypeptide of this invention also has antioxidant properties, inhibiting ABTS· + EC 50The concentration ranges from 0.12 to 1.08 mg / mL. The peptides provided by this invention effectively improve the situation of low peptide bioactivity in industrial production, and at the same time provide technical support and supplementation for the preparation and screening of marine bioactive peptides.

[0006] The first object of the present invention is to provide a highly bioactive polypeptide having an amino acid sequence as shown in any one of SEQ ID NO. 1-9.

[0007] Furthermore, SEQ ID NO.1: SYPPLGRF;

[0008] SEQ ID NO.2: WGDHGW;

[0009] SEQ ID NO.3: SSYPPLGRF;

[0010] SEQ ID NO.4: WPNRPP;

[0011] SEQ ID NO.5: WSDRIPF;

[0012] SEQ ID NO.6: GWDKFWPE;

[0013] SEQ ID NO.7: PDRPW;

[0014] SEQ ID NO.8: FYDHIF;

[0015] SEQ ID NO.9: ADWDFLPAK.

[0016] Furthermore, the polypeptide is prepared by the following steps:

[0017] S1. Add a non-specific enzyme to the abalone extract to react and obtain a crude extract;

[0018] S2. Add a mixed enzyme containing prolyl endopeptidase, chymotrypsin and proteinase K to the crude extract and react to obtain the polypeptide by screening.

[0019] Furthermore, the non-specific enzymes include papain, neutral protease, alkaline protease, trypsin, and lipase, with a mass ratio of papain:neutral protease:alkaline protease:trypsin:lipase of 1:2:2:2:1.

[0020] Furthermore, the abalone extract in step S1 is obtained by heating and pulverizing abalone, wherein the heating temperature is 110-130℃ and the heating time is 4-6 hours.

[0021] Furthermore, the reaction temperature in step S1 is 40℃-60℃, and the pH of the reaction is 6.5-7.5.

[0022] Further, in step S1, a non-specific enzyme is added to the abalone extract for reaction. After the reaction is completed, enzyme inactivation treatment is performed, and the supernatant is collected after centrifugation to obtain the crude extract. The enzyme inactivation treatment is performed by heating the abalone extract after the reaction to 90-95°C for 10-15 minutes.

[0023] Furthermore, the mass ratio of prolyl endopeptidase: chymotrypsin: proteinase K is (1-2): (1-2): (1-2).

[0024] Furthermore, the mass ratio of the mixed enzyme to the crude extract is (1-3):100.

[0025] A second objective of this invention is to provide a gene encoding the aforementioned polypeptide.

[0026] A third objective of this invention is to provide an application of the above-mentioned polypeptide in the preparation of antihypertensive products.

[0027] The fourth objective of this invention is to provide an application of the above-mentioned polypeptide in the preparation of antioxidant products.

[0028] The beneficial effects of this invention are:

[0029] This invention provides a highly bioactive polypeptide and its applications. The invention utilizes multiple enzymes to perform stepwise enzymatic hydrolysis of abalone, yielding a polypeptide solution with high bioactivity. Polypeptides with amino acid sequences as shown in SEQ ID NO. 1-9 were screened and identified from this polypeptide solution. The polypeptide of this invention exhibits high bioactivity; firstly, it possesses high angiotensin-converting enzyme (ACE) inhibitory activity, with an IC50 of 1 / 3 oz. ... 50 The concentration is between 0.15-0.37 mg / mL, thus effectively inhibiting angiotensin-converting enzyme activity; in addition, the polypeptide of this invention also has antioxidant properties, inhibiting ABTS· + EC 50 The concentration ranges from 0.12 to 1.08 mg / mL. The peptides provided by this invention effectively improve the situation of low peptide bioactivity in industrial production, and at the same time provide technical support and supplementation for the preparation and screening of marine bioactive peptides. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0031] Figure 1This is a flowchart of the polypeptide preparation technology route of the present invention;

[0032] Figure 2 This is a schematic diagram of the Sephadex G-15 gel filtration chromatography chromatogram and fraction collection of the present invention;

[0033] Figure 3 This is a graph showing the ACE activity determination results of the gel filtration sequence fraction of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] Non-specific enzymes: Papain (>200,000 u / g) was provided by Shenzhen Xiameng Biotechnology Co., Ltd., China; neutral protease (>2.4 AU-A / g) and alkaline protease (>6.5 AU-N / g) were from Novozymes (China) Biotechnology Co., Ltd.; trypsin (>250,000 u / g) was from Shanghai Maclean Biochemical Technology Co., Ltd., China, catalog number T992772-100mg; lipase (>30,000 u / g) was from Shanghai Yuanye Biotechnology Co., Ltd., China. The mass ratio of papain:neutral protease:alkaline protease:trypsin:lipase was 1:2:2:2:1.

[0036] Methods for detecting ACE inhibition (ACEi):

[0037] Using FAPGG as a substrate (FAPGG: N-[3-(2-furanyl)acryloyl]-L-phenylalanyl-glycyl-glycine, FAPGG is a commonly used substrate for angiotensin-converting enzyme (ACE) and is often used for the in vitro quantitative determination of ACE activity), the reactants were added to the microplate according to the table. The absorbance A1 before the reaction was measured at 340 nm using a microplate reader. After incubation at 37°C for 30 min, the absorbance A2 was measured. ΔA was calculated as ΔA = A1 - A2. The change in absorbance per unit time represents the ACE enzyme activity. The ACE inhibition rate was calculated using the following formula:

[0038] ACE inhibition rate (%) = 1 - ΔA sample / ΔA blank × 100%

[0039] In the formula: ΔA blank represents the change in absorbance within 30 minutes when buffer solution is added; ΔA sample represents the change in absorbance within 30 minutes when inhibitor is added.

[0040] ABTS· + Methods for detecting clearance rate:

[0041] ABTS·+ The working solution was prepared by mixing ABTS solution (7.0 mM) with an equal volume of potassium persulfate solution (2.45 mM), incubating at room temperature in the dark for 12-16 h, and then diluting with 50% ethanol solution to an absorbance of 0.75-0.80 at 734 nm. 200 μL of ABTS… + Add the working solution to 10 μL of the sample and incubate at room temperature in the dark for 30 min. Measure the absorbance at 734 nm. Use PBS buffer instead of the sample as a control. ABTS· + The clearance rate (%) is calculated using the following formula:

[0042] ABTS· + Clearance rate (%) = (A blank - A sample) / A blank × 100%

[0043] Example 1

[0044] (1) After heating and steaming the abalone at 115℃ for 4.5h, water was added and mixed evenly. The mixture was then crushed using a pulverizer to obtain abalone extract. The abalone extract was frozen and dried at low temperature to remove moisture, resulting in dried meat powder.

[0045] (2) Take 10g of meat powder from step (1) and add 300mL of water to mix evenly. Under the conditions of constant temperature water bath at 55℃ and stirring speed at 800rpm, add non-specific enzyme with a mass concentration of 3% (based on the mass of meat powder) for enzymatic hydrolysis for 4h. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme in the system at 90-95℃ for 10-15min. Centrifuge the system after enzyme inactivation at 10000rpm for 10min. Take the supernatant as the crude extract. Freeze the crude extract and dry it at low temperature to remove moisture to obtain crude extract powder.

[0046] (3) Dissolve 10g of the crude extract powder from (2) in ultrapure water and bring the volume to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. At the same time, add a mixed enzyme solution with a mass concentration of 1% (based on the mass of the crude extract powder) for a second enzymatic hydrolysis treatment for 3h to obtain a second enzymatic hydrolysate. In the mixed enzyme solution, the mass concentration of prolyl endopeptidase is 0.25%, the mass concentration of chymotrypsin is 0.5%, and the mass concentration of proteinase K is 0.25% (based on the mass of the crude extract powder).

[0047] (4) Inactivate the enzyme in the secondary enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the polypeptide solution.

[0048] Example 2: Screening of highly bioactive peptides

[0049] (1) The polypeptide solution obtained in Example 1 was filtered using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and the components with a molecular weight below 3 kDa were collected.

[0050] (2) The components collected in step 1 were further separated using a Sephadex G-15 gel column (2.6 × 40 cm), eluted with ultrapure water at a flow rate of 0.6 mL / min, and fractions with different absorption peaks at 280 nm were collected. The ACE inhibitory activity was then determined. Chromatograms and fraction collection details are shown below. Figure 2 The ACE inhibitory activities of different fractions are shown in the figure. Figure 3 Fraction C showed the highest ACE inhibitory activity, while fraction D had the highest peptide abundance.

[0051] (3) The dominant fractions (i.e., fractions C and D) in step (2) were analyzed using peptidomics technology to obtain the amino acid sequences of all contained peptides. The potential biological activity of the obtained peptide sequences was scored and ranked using PeptideRanker, and peptides with a score >0.9 were screened (as shown in Table 1). A total of 9 peptides with amino acid sequences as shown in SEQ ID NO.1-9 were obtained.

[0052] (4) Use the PeptideRanker tool to screen peptides with an amino acid sequence score > 0.9 in step (3) for molecular docking prediction. The specific steps of molecular docking are as follows: Molecular docking is performed using AutoDock Vina 1.1.2 software. The crystal structure of the human ACE-lisinopril complex (1O86) is obtained from the RCSB protein database (https: / / www.rcsb.org / ). The peptide molecule is constructed using PyMol 2.6 and energy minimization is performed. The protein is processed using PyMol 2.6 to remove water molecules and small molecule ligands, and hydrogen is added, etc. The receptor protein and peptide are converted to PDBQT format using AutoDock Tools. The center of the docking box is defined according to the position of the crystal ligand, and the side length of the box is . The highest score from the Vina docking was selected as the final result, and visualization analysis was performed in PyMol 2.6. After molecular docking, it was found that all nine peptides could stably exist in the active pocket of angiotensin-converting enzyme, with binding energies ranging from -5.8 to -8.9 kcal / mol.

[0053] Nine peptides were chemically synthesized using a solid-phase peptide synthesis method. After synthesis, their ACEi activity and antioxidant capacity were determined, and the IC50 of ACEi was measured. 50 Values ​​ranged from 0.12 to 0.54 mg / mL, ABTS· + EC 50The concentration range was 0.12 mg / mL to 1.08 mg / mL. Among them, the peptide SSYPPLGRF showed the best ACEi activity (IC50). 50 Of the peptides, PDRPW (at 0.15 mg / mL) exhibited the best antioxidant activity (EC). 50 (0.12 mg / mL). These nine peptide sequences did not match in either BIOPEP-UWM or EROP-Moscow, indicating that all nine peptides are previously unreported novel sequences.

[0054] Table 1. Results of bioactivity assays of the peptides screened in Example 1.

[0055]

[0056] Example 3

[0057] (1) After heating and steaming the abalone at 115℃ for 4.5h, water was added and mixed evenly. The mixture was then crushed using a pulverizer to obtain abalone extract. The abalone extract was frozen and dried at low temperature to remove moisture, resulting in dried meat powder.

[0058] (2) Take 10g of meat powder from step (1) and add 300mL of water to mix evenly. Under the conditions of constant temperature water bath at 55℃ and stirring speed at 800rpm, add non-specific enzyme with a mass concentration of 3% (based on the mass of meat powder) for enzymatic hydrolysis for 4h. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme in the system at 90-95℃ for 10-15min. Centrifuge the system after enzyme inactivation at 10000rpm for 10min. Take the supernatant as the crude extract. Freeze the crude extract and dry it at low temperature to remove moisture to obtain crude extract powder.

[0059] (3) Dissolve 10g of the crude extract powder from (2) in ultrapure water and bring the volume to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. At the same time, add a mixed enzyme solution with a mass concentration of 0.5% (based on the mass of the crude extract powder) for a second enzymatic hydrolysis treatment for 3h to obtain a second enzymatic hydrolysate. In the mixed enzyme solution, the mass concentration of prolyl endopeptidase is 0.125%, the mass concentration of chymotrypsin is 0.25%, and the mass concentration of proteinase K is 0.125% (based on the mass of the crude extract powder).

[0060] (4) Inactivate the enzyme in the secondary enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain abalone polypeptide solution.

[0061] Example 4

[0062] (1) After heating and steaming the abalone at 115℃ for 4.5h, water was added and mixed evenly. The mixture was then crushed using a pulverizer to obtain abalone extract. The abalone extract was frozen and dried at low temperature to remove moisture, resulting in dried meat powder.

[0063] (2) Take 10g of meat powder from step (1) and add 300mL of water to mix evenly. Under the conditions of constant temperature water bath at 55℃ and stirring speed at 800rpm, add non-specific enzyme with a mass concentration of 3% (based on the mass of meat powder) for enzymatic hydrolysis for 4h. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme in the system at 90-95℃ for 10-15min. Centrifuge the system after enzyme inactivation at 10000rpm for 10min. Take the supernatant as the crude extract. Freeze the crude extract and dry it at low temperature to remove moisture to obtain crude extract powder.

[0064] (3) Dissolve 10g of the crude extract powder from (2) in ultrapure water and bring the volume to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. Add a specific protease with a mass concentration of 3% (based on the mass of the crude enzymatic hydrolysis product of abalone) for 3h to obtain a secondary enzymatic hydrolysate. In the mixed enzyme solution, the mass concentration of prolyl endopeptidase is 0.5%, the mass concentration of chymotrypsin is 1%, and the mass concentration of proteinase K is 0.5%.

[0065] (4) Inactivate the enzyme in the secondary enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the polypeptide solution.

[0066] Example 5

[0067] (1) After heating and steaming the abalone at 115℃ for 4.5h, water was added and mixed evenly. The mixture was then crushed using a pulverizer to obtain abalone extract. The abalone extract was frozen and dried at low temperature to remove moisture, resulting in dried meat powder.

[0068] (2) Take 10g of meat powder from step (1) and add 300mL of water to mix evenly. Under the conditions of constant temperature water bath at 55℃ and stirring speed at 800rpm, add non-specific enzyme with a mass concentration of 3% (based on the mass of meat powder) for enzymatic hydrolysis for 4h. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme in the system at 90-95℃ for 10-15min. Centrifuge the system after enzyme inactivation at 10000rpm for 10min. Take the supernatant as the crude extract. Freeze the crude extract and dry it at low temperature to remove moisture to obtain crude extract powder.

[0069] (3) Take 10g of the crude extract powder from (2) and dissolve it in ultrapure water and make up to 200mL. Place it in a constant temperature water bath at 37℃ and stir at 800rpm. At the same time, add a mixed enzyme solution with a mass concentration of 1% (based on the mass of the crude extract powder) for a second enzymatic hydrolysis treatment for 3h to obtain a second enzymatic hydrolysate. In the mixed enzyme solution, the mass concentration of prolyl endopeptidase is 0.17%, the mass concentration of chymotrypsin is 0.33%, and the mass concentration of proteinase K is 0.5% (based on the mass of the crude extract powder).

[0070] (4) Inactivate the enzyme in the secondary enzymatic hydrolysate in (3) at 90-95℃ for 10-15 min, then centrifuge the system at 10000 rpm for 10 min, and take the supernatant to obtain the polypeptide solution.

[0071] The ACEi activity of the polypeptide solutions obtained in Examples 1, 3-5 and Comparative Examples 1-2 were measured at a concentration of 1 mg / mL, and the results are shown in Table 2.

[0072] Table 2. ACEi activity determination of abalone polypeptide solutions in Examples 1-4 and Comparative Examples 1-2

[0073]

[0074] As can be seen from Table 2, the technical solution of the present invention significantly improves the ACEi activity of abalone polypeptide liquid, proving the feasibility of the preparation method of the present invention.

[0075] Comparative Example 1

[0076] (1) After heating and steaming the abalone at 115℃ for 4.5h, water was added and mixed evenly. The mixture was then crushed using a pulverizer to obtain abalone extract. The abalone extract was frozen and dried at low temperature to remove moisture, resulting in dried meat powder.

[0077] (2) Take 10g of meat powder from step (1) and add 300mL of water to mix evenly. Under the conditions of constant temperature water bath at 55℃ and stirring speed at 800rpm, add non-specific enzyme with a mass concentration of 3% (based on the mass of meat powder) for enzymatic hydrolysis for 4h. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme in the enzymatic hydrolysis system at 90-95℃ for 10-15min. Centrifuge the enzyme-inactivated system at 10000rpm for 10min and take the supernatant as the polypeptide solution.

[0078] Comparative Example 2

[0079] (1) After heating and steaming the abalone at 115℃ for 4.5h, water was added and mixed evenly. The mixture was then crushed using a pulverizer to obtain abalone extract. The abalone extract was frozen and dried at low temperature to remove moisture, resulting in dried meat powder.

[0080] (2) Take 10g of the dried meat powder from (1) and add 300mL of water to mix the abalone meat and water evenly. Place it in a constant temperature water bath at 55℃ and stir at 800rpm. At the same time, add 0.25% prolyl endopeptidase, 0.5% chymotrypsin and 0.25% proteinase K by mass concentration (based on the mass of dried meat powder) for 3h of enzymatic hydrolysis. During the enzymatic hydrolysis, adjust the pH value to 6.5-7.5 with sodium hydroxide. After the enzymatic hydrolysis is completed, inactivate the enzyme at 90-95℃ for 10-15min. Then centrifuge the system at 10000rpm for 10min and take the supernatant as the polypeptide solution.

[0081] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A highly bioactive polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO.1-9.

2. A gene encoding the polypeptide of claim 1.

3. The use of the polypeptide according to claim 1 in the preparation of antihypertensive products.

4. The use of the polypeptide according to claim 1 in the preparation of antioxidant products.

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