Polypeptides having angiotensin converting enzyme inhibitory activity, methods of making and use thereof

Abstract

The application provides a polypeptide with angiotensin converting enzyme inhibitory activity and a preparation method and application thereof, and relates to the technical field of active peptide preparation. The abalone is pulverized, a complex enzyme is added for primary enzymolysis, and then a specific protease mixture containing prolyl endopeptidase, chymotrypsin and protease K is added for two-step enzymolysis, so that the obtained polypeptide solution has high ACE inhibitory activity. The obtained polypeptide is analyzed and screened to obtain polypeptides with amino acid sequences as shown in SEQ ID NO. 1-5. The polypeptide has high ACE inhibitory activity, and the IC 50 0.05mg / mL-0.40mg / mL, and therefore can play a blood pressure lowering effect as an angiotensin converting enzyme inhibitor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active peptide preparation, in particular to a polypeptide with angiotensin converting enzyme inhibitory activity and a preparation method and application thereof. BACKGROUND

[0002] Bioactive peptides (BAP) are peptide compounds that are beneficial to the life activities of organisms or have physiological effects. They are a class of polypeptides with a relative molecular mass of less than 6000 Da and a variety of biological functions. Their molecular structures vary in complexity, and they are molecular polymers between amino acids and proteins, ranging from two amino acids to tens of amino acids connected by peptide bonds. These polypeptides can be modified by phosphorylation, glycosylation or acylation. In recent years, marine bioactive peptides have become a research hotspot in the food industry due to their diverse functions, wide sources, strong specificity, and low toxicity and side effects. Among them, angiotensin converting enzyme (ACE) inhibitory peptides have been widely studied due to their blood pressure lowering effects. ACE inhibitory peptides bind to the active pocket of ACE, thereby inhibiting the activity of ACE, preventing the conversion of angiotensin I (Ang I) to Ang II, and preventing the inactivation of vasodilator bradykinin, thereby achieving the purpose of reducing high blood pressure.

[0003] Abalone is a marine gastropod, a single-shell soft-bodied animal, with tender and rich meat, which is deeply loved by consumers at home and abroad. China is a major abalone farming country. Abalone is not only rich in nutritional value, but also has active ingredients such as polypeptides, polysaccharides, free amino acids, and fatty acids. The most commonly used method for preparing bioactive peptides is enzymatic hydrolysis, in which the main enzymes are usually complex enzymes such as alkaline protease, neutral protease, trypsin, and papain. The obtained product components are relatively complex, resulting in low biological activity. There is still a lack of research on how to extract polypeptides with high angiotensin converting enzyme inhibitory activity from abalone. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the lack of a polypeptide with high angiotensin converting enzyme inhibitory activity in the prior art.

[0005] To solve the above technical problems, the present application provides a polypeptide with angiotensin converting enzyme inhibitory activity, and a preparation method and application thereof. The polypeptide is obtained by crushing abalones, adding a compound enzyme for primary enzymolysis, and then adding a specific protease mixture containing prolyl endopeptidase, chymotrypsin and protease K for secondary enzymolysis. The polypeptide obtained has high ACE inhibitory activity. The polypeptide with the amino acid sequence shown in any one of SEQ ID NO. 1-5 is obtained by analyzing and screening the polypeptide. The polypeptide of the present application has high ACE inhibitory activity, and the IC 50 between 0.05 mg / mL and 0.40 mg / mL, and thus can play a blood pressure lowering effect as an angiotensin converting enzyme inhibitor.

[0006] The first object of the present application is to provide a polypeptide with ACE inhibitory activity, wherein the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 1-5.

[0007] Further, SEQ ID NO. 1: FDRLF;

[0008] SEQ ID NO. 2: SPPFFDGMTR;

[0009] SEQ ID NO. 3: FDFRQF;

[0010] SEQ ID NO. 4: GFDFRQF;

[0011] SEQ ID NO. 5: PEHFPF.

[0012] The second object of the present application is to provide a gene encoding the above polypeptide.

[0013] The third object of the present application is to provide an application of the above polypeptide in the preparation of a blood pressure lowering product.

[0014] Further, the blood pressure lowering product plays a role by inhibiting the activity of angiotensin converting enzyme.

[0015] The fourth object of the present application is to provide a preparation method of the above polypeptide, comprising the following steps:

[0016] S1, heating and crushing abalones to obtain abalone crude products, adding a compound enzyme to the abalone crude products for primary enzymolysis to obtain a primary enzymolysis product, wherein the compound enzyme comprises papain, neutral protease, alkaline protease, trypsin and lipase;

[0017] S2, adding a specific protease mixture to the primary enzymolysis product for secondary enzymolysis to obtain the polypeptide;

[0018] The specific protease mixture comprises prolyl endopeptidase, chymotrypsin and protease K.

[0019] Further, the mass ratio of papain: neutral protease: alkaline protease: trypsin: lipase is 1:2:2:2:1.

[0020] Further, the temperature of the heating in step S1 is 100-150 DEG C, and the heating time is 3-5 hours.

[0021] Further, the mass ratio of the protease to the crude product of abalone in step S1 is (2-4):100.

[0022] Further, the pH of the primary enzymolysis in step S1 is 6.5-7.5.

[0023] Further, the temperature of the primary enzymolysis in step S1 is 50-60 DEG C.

[0024] Further, the mass ratio of the specific protease mixture to the crude product of enzymolysis in step S2 is (0.5-2):100.

[0025] Further, the mass ratio of the prolyl endopeptidase: chymotrypsin: protease K is (1-2):(1-2):(1-2).

[0026] Further, the temperature of the secondary enzymolysis in step S2 is 30-40 DEG C.

[0027] Advantages of the present application:

[0028] The present application provides a polypeptide with angiotensin converting enzyme inhibitory activity, and a preparation method and application thereof. The polypeptide obtained by the present application has an amino acid sequence as shown in SEQ ID NO. 1-5, and has high ACE inhibitory activity, with an IC 50 between 0.05 mg / mL and 0.40 mg / mL, and thus can be used as an angiotensin converting enzyme inhibitor to lower blood pressure. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0030] Figure 1 is a flow chart of the preparation and screening method of the polypeptide of the present application;

[0031] Figure 2 is a graph of the ACEi activity determination results of the enzymolysis product obtained in Example 2 of the present application;

[0032] Figure 3 is a Sephadex G-15 gel filtration chromatogram and fraction collection schematic of the present application;

[0033] Figure 4 is a graph of ACEi activity determination results after gel filtration chromatography fractionation of the present application. DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with the drawings and specific examples so that those skilled in the art can better understand the present application and implement it, but the examples are not intended to limit the present application.

[0035] The complex enzymes: papain (>200000u / g) was provided by Shenzhen Sumeng Biotechnology Co., Ltd., China; neutral protease (>2.4AU-A / g), alkaline protease (>6.5AU-N / g) were from Novozymes (China) Biotechnology Co., Ltd.; trypsin (>250000u / g) was from Shanghai Maikelin Biochemical Technology Co., Ltd., China; lipase (>30000u / g) was provided by Shanghai Yuan Ye Biotechnology Co., Ltd., China. The mass ratio of papain: neutral protease: alkaline protease: trypsin: lipase was 1:2:2:2:1.

[0036] Detection method of ACE inhibition:

[0037] FAPGG was used as a substrate (FAPGG: N-[3-(2-furyl) acryloyl]-L-phenylalanyl-glycyl-glycine, FAPGG is a commonly used substrate for angiotensin-converting enzyme (ACE) and is commonly used for in vitro quantitative determination of angiotensin-converting enzyme activity), and the reagents were added in the enzyme-labeled plate according to the table. The absorbance A1 before the reaction was determined by means of an enzyme-labeled instrument at 340nm, and then the absorbance A2 was determined after incubation at 37℃ for 30min. ΔA was calculated, ΔA = A1-A2. The change in absorbance value per unit time represented the ACE enzyme activity, and the calculation of ACE inhibition rate was as follows:

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

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

[0040] Example 1

[0041] (1) After cooking abalone at 110℃ for 4h, water was added and mixed uniformly, and then the abalone was crushed to obtain abalone meat slurry; the abalone meat slurry was frozen for more than 12h, and then placed in a low-temperature freeze-drying machine for more than 4d to obtain abalone meat dry powder.

[0042] (2) Take 10 g of the dried abalone meat powder in (1) and mix with 300 mL of water. Place the mixture in a 55°C constant temperature water bath and stir at a speed of 800 rpm. Add 3% (based on the mass of the dried abalone meat powder) of a compound enzyme and hydrolyze for 4 hours. During the hydrolysis, use sodium hydroxide to control the pH value to be 6.5-7.5. After the hydrolysis, inactivate the enzyme at 90-95°C for 10-15 minutes. Then centrifuge the system at a speed of 10,000 rpm for 10 minutes. Take the supernatant, freeze it for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain dried abalone crude hydrolyzate.

[0043] (3) Take 10 g of the dried abalone crude hydrolyzate in (2) and dissolve it in ultrapure water to make up to 200 mL. Place the mixture in a 37°C constant temperature water bath and stir at a speed of 800 rpm. Mix and add 0.5% (based on the mass of the dried abalone crude hydrolyzate) of a specific protease to hydrolyze for 4 hours to obtain an abalone hydrolysate. In the specific protease, 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%.

[0044] (4) Inactivate the enzyme in the abalone hydrolysate in (3) at 90-95°C for 10-15 minutes. Then centrifuge the system at a speed of 10,000 rpm for 10 minutes. Take the supernatant to obtain the abalone polypeptide solution of the present application.

[0045] Example 2

[0046] The preparation method of the abalone polypeptide solution of the present example is the same as that of Example 1, except that the mass concentration of the specific protease added in the present example is 1%, in which 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%. The specific steps are as follows:

[0047] (1) Cook abalone at 110°C for 4 hours, then add water and mix evenly. Crush the mixture to obtain abalone meat slurry. Freeze the abalone meat slurry for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain dried abalone meat.

[0048] (2) Take 10 g of the dried abalone meat powder in (1) and mix with 300 mL of water. Place the mixture in a 55°C constant temperature water bath and stir at a speed of 800 rpm. Add 3% (based on the mass of the dried abalone meat powder) of a compound enzyme and hydrolyze for 4 hours. During the hydrolysis, use sodium hydroxide to control the pH value to be 6.5-7.5. After the hydrolysis, inactivate the enzyme at 90-95°C for 10-15 minutes. Then centrifuge the system at a speed of 10,000 rpm for 10 minutes. Take the supernatant, freeze it for more than 12 hours, and then place it in a low-temperature freeze dryer for more than 4 days to obtain dried abalone crude hydrolyzate.

[0049] (3) Take 10 g of the abalone crude enzymolysis product dry powder in (2) and dissolve it in ultrapure water and make up to 200 mL. Place it in a 37℃ constant temperature water bath and stir at a speed of 800 rpm. Mix and add specific protease with a mass concentration of 1% (based on the mass of the abalone crude enzymolysis product) to carry out enzymolysis treatment for 4 h. An abalone enzymolysis liquid is obtained. In the specific protease, 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%.

[0050] (4) The abalone enzymolysis liquid in (3) is inactivated at 90-95℃ for 10-15 min. Then the system is centrifuged at a speed of 10,000 rpm for 10 min. The supernatant is taken to obtain the abalone polypeptide liquid of the present application.

[0051] Example 3

[0052] The preparation method of the abalone polypeptide liquid of the present embodiment is the same as that of Example 1, except that the mass concentration of the specific protease added in the present embodiment is 2%, wherein 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%. The specific steps are as follows:

[0053] (1) After the abalone is cooked at 110℃ for 4 h, water is added and mixed uniformly. After crushing, abalone meat slurry is obtained. The abalone meat slurry is frozen for more than 12 h, and then placed in a low-temperature freeze dryer for more than 4 d to obtain abalone meat dry powder.

[0054] (2) Take 10 g of the abalone meat dry powder in (1) and add 300 mL of water to mix uniformly. Place it in a 55℃ constant temperature water bath and stir at a speed of 800 rpm. Add complex enzyme with a mass concentration of 3% (based on the mass of the abalone meat dry powder) to carry out enzymolysis for 4 h. The pH value is controlled to be 6.5-7.5 by using sodium hydroxide during the enzymolysis process. After the enzymolysis is completed, the enzyme is inactivated at 90-95℃ for 10-15 min. Then the system is centrifuged at a speed of 10,000 rpm for 10 min. The supernatant is taken. The supernatant is frozen for more than 12 h, and then placed in a low-temperature freeze dryer for more than 4 d to obtain abalone crude enzymolysis product dry powder.

[0055] (3) Take 10 g of the abalone crude enzymolysis product dry powder in (2) and dissolve it in ultrapure water and make up to 200 mL. Place it in a 37℃ constant temperature water bath and stir at a speed of 800 rpm. Mix and add specific protease with a mass concentration of 3% (based on the mass of the abalone crude enzymolysis product) to carry out enzymolysis treatment for 4 h. An abalone enzymolysis liquid is obtained. In the specific protease, 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%.

[0056] (4) The abalone enzymatic hydrolysate in (3) is inactivated at 90-95°C for 10-15 min, and then the system is centrifuged at a speed of 10,000 rpm for 10 min, and the supernatant is taken to obtain the abalone polypeptide solution of the present application.

[0057] Example 4

[0058] The preparation method of the abalone polypeptide solution of the present example is the same as that of Example 2, and the mass concentration of the specific protease added in the present example is also 1%, the difference lies in that 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%. The specific steps are as follows:

[0059] (1) After the abalone is cooked at 110°C for 4h, water is added and mixed uniformly, and then it is crushed to obtain abalone meat slurry; the abalone meat slurry is frozen for more than 12h, and then placed in a low-temperature freeze-drying machine for more than 4d to obtain abalone meat dry powder.

[0060] (2) Take 10g of abalone meat dry powder in (1), add 300mL of water and mix uniformly, and then place it in a 55°C constant temperature water bath and an 800rpm stirrer at a speed of 800rpm, add a complex enzyme with a mass concentration of 3% (based on the mass of abalone meat dry powder) for enzymolysis for 4h, and use sodium hydroxide to control the pH value to be 6.5-7.5 during the enzymolysis process, and then inactivate the enzyme at 90-95°C for 10-15min after the enzymolysis is completed; then the system is centrifuged at a speed of 10,000 rpm for 10 min, and the supernatant is taken, and the supernatant is frozen for more than 12h, and then placed in a low-temperature freeze-drying machine for more than 4d to obtain abalone crude enzymolysis product dry powder.

[0061] (3) Take 10g of abalone crude enzymolysis product dry powder in (2), dissolve it in ultrapure water and make up to 200mL, and then place it in a 37°C constant temperature water bath and an 800rpm stirrer at a speed of 800rpm, mix and add specific proteases with a mass concentration of 1% (based on the mass of abalone crude enzymolysis product) for enzymolysis treatment for 4h to obtain an abalone enzymatic hydrolysate; among the specific proteases, 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%.

[0062] (4) The abalone enzymatic hydrolysate in (3) is inactivated at 90-95°C for 10-15 min, and then the system is centrifuged at a speed of 10,000 rpm for 10 min, and the supernatant is taken to obtain the abalone polypeptide solution of the present application.

[0063] The ACEi activity of the abalone polypeptide solution obtained in Examples 1-4 and Comparative Examples 1-2 was determined at 1mg / mL, and the results are shown in Table 1.

[0064] Table 1 ACEi activity determination of abalone polypeptide solution of Examples 1-4 and Comparative Examples 1-2

[0065]

[0066] As can be seen from Table 1, the technical scheme of the present application significantly improves the ACEi activity of the abalone polypeptide solution. Specifically, compared with Comparative Example 1, the ACEi activity of the abalone polypeptide solution obtained in Example 2 is increased by as much as 228%, fully proving the feasibility of the preparation method of the present application.

[0067] Example 5: Screening of abalone ACE inhibitory peptides

[0068] (1) The abalone polypeptide solution obtained in Example 2 was filtered using an ultrafiltration tube with a molecular weight cut-off of 3 kDa, and the component with a molecular weight of 0-3 kDa was collected;

[0069] (2) The component collected in step 1 was further separated using a Sephadex G-15 gel column (2.6 x 40 cm), eluted with ultrapure water at a flow rate of 0.6 mL / min, and the different absorption peak fractions at 280 nm were collected and the ACE inhibitory activity was determined. The chromatogram and fraction collection are shown in Figure 3 , and the ACE inhibitory activities of different fractions are shown in Figure 4 . The ACE inhibitory activity of fraction C is the highest, and the polypeptide abundance of fraction D is the highest.

[0070] (3) The fractions with advantages in step (2) (i.e., fraction C and fraction D) were analyzed using peptidomics technology to obtain the amino acid sequences of all polypeptides contained therein;

[0071] (4) The polypeptides with a score > 0.9 in the amino acid sequences in step (3) were screened using the PeptideRanker tool, and after molecular docking prediction, the polypeptides were chemically synthesized and the biological activity was verified

[0072] Specifically, the peptidomics analysis results showed that components C and D contained a total of 1937 different peptide sequences, and the PeptideRanker was used to score and rank the potential biological activity of the 1937 peptide sequences. The amino acid sequences of 5 polypeptides with a score > 0.9 are shown in Table 2 as SEQ ID NO. 1-5. After molecular docking, the 5 polypeptides can stably exist in the active pocket of the ACE enzyme, and the binding energy values are -6.2 to -9.7 kcal / mol. After the 5 polypeptides were chemically synthesized, the ACEi activity was determined, and the IC 50 values were between 0.05 and 0.40 mg / mL. Among them, the ACEi activity of the peptide FDRLF (0.05 mg / mL) was the best. The 5 peptide sequences did not match in BIOPEP-UWM and EROP-Moscow, proving that the 5 polypeptides are new sequences that have not been reported.

[0073] The molecular docking method comprises: using AutoDock Vina 1.1.2 software for molecular docking. The human ACE-ralinopril complex (1O86) crystal structure is derived from the RCSB protein database (https: / / www.rcsb.org / ). The polypeptide molecule is constructed by PyMol 2.6 and energy minimized. The protein is treated by PyMol 2.6, and water molecules and small molecule ligands are removed, and hydrogen is added. The receptor protein and the peptide segment are converted into PDBQT format using AutoDock Tools. The docking box center is defined according to the crystal ligand position, and the box edge length is The highest score of Vina docking is selected as the final result, and visual analysis is performed in PyMol 2.6.

[0074] Table 2 Biological activity determination of polypeptides screened in Example 5

[0075]

[0076] Comparative Example 1

[0077] (1) The abalone is cooked at 110°C for 4h, then mixed with water, crushed to obtain abalone meat slurry; the abalone meat slurry is frozen for more than 12h, then placed in a low-temperature freeze-drying machine for more than 4d to obtain abalone meat dry powder.

[0078] (2) Take 10g of abalone meat dry powder in (1), mix the abalone meat and water mixture uniformly, and place it in a 55°C constant temperature water bath, 800rpm stirrer speed condition, add 3% (based on the mass of abalone meat dry powder) of complex enzyme with a mass concentration of 3% (based on the mass of abalone meat dry powder) enzyme hydrolysis for 4h, and adjust the pH value to 6.5-7.5 with sodium hydroxide during the enzyme hydrolysis process. Enzyme inactivation at 90-95°C for 10-15min; then centrifuge the system at 10000rpm for 10min, take the supernatant, and obtain the abalone meat crude enzyme hydrolysate.

[0079] Comparative Example 2

[0080] (1) The abalone is cooked at 110°C for 4h, then mixed with water, crushed to obtain abalone meat slurry; the abalone meat slurry is frozen for more than 12h, then placed in a low-temperature freeze-drying machine for more than 4d to obtain abalone meat dry powder;

[0081] (2) take 10 g of dried abalone meat powder in (1), add 300 mL of water to mix the mixture of abalone meat and water uniformly, place it in a 55℃ constant temperature water bath, and stir at a speed of 800 rpm; add 0.25% pralidomide endopeptidase, 0.5% chymotrypsin, and 0.25% protease K with a mass concentration (based on the mass of dried abalone meat powder) of 0.25% to carry out enzymolysis for 4 h; control the pH value to be 6.5-7.5 with sodium hydroxide during the enzymolysis process, and inactivate the enzyme at 90-95℃ for 10-15 min after the enzymolysis is completed; then centrifuge the system at a speed of 10000 rpm for 10 min, take the supernatant, and obtain the enzymolysis liquid.

[0082] Obviously, the above examples are only examples for clearly illustrating, and are not limitation to the embodiments. Other different forms of changes or variations can be made by those skilled in the art on the basis of the above description. All the embodiments do not need to be exhausted here, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A polypeptide having angiotensin converting enzyme inhibitory activity, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID NO.

2.

2. A gene encoding the polypeptide of claim 1.

3. Use of the polypeptide of claim 1 in the preparation of a blood pressure lowering product.

4. Use according to claim 3, characterized in that, The blood pressure lowering product functions by inhibiting the activity of angiotensin converting enzyme.

5. A method of producing the polypeptide of claim 1, comprising, The method comprises the following steps: S1, heating and crushing abalones to obtain an abalone crude product, adding a compound enzyme to the abalone crude product for preliminary enzymatic hydrolysis to obtain a preliminary enzymatic hydrolysis product, wherein the compound enzyme is composed of papain, neutral protease, alkaline protease, trypsin and lipase; S2, adding a specific protease mixture to the preliminary enzymatic hydrolysis product for secondary enzymatic hydrolysis to obtain the polypeptide; The specific protease mixture is composed of prolyl endopeptidase, chymotrypsin and protease K, and the mass ratio of the prolyl endopeptidase: chymotrypsin: protease K is (1-2):(1-2):(1-2), and the mass ratio of the papain: neutral protease: alkaline protease: trypsin: lipase is 1:2:2:2:

1.

6. The production method according to claim 5, wherein The heating temperature in step S1 is 100-150℃, and the heating time is 3-5 hours.

7. The preparation method according to claim 5, characterized in that The mass ratio of the protease to the abalone crude product in step S1 is (2-4):

100.

8. The preparation method according to claim 5, characterized in that The pH of the preliminary enzymatic hydrolysis in step S1 is 6.5-7.5, and the temperature of the preliminary enzymatic hydrolysis is 50-60℃.

9. The preparation method according to claim 5, characterized in that The mass ratio of the specific protease mixture to the enzymatic hydrolysis crude product in step S2 is (0.5-2):100.

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