Polypeptide with angiotensin converting enzyme inhibition and preparation method and application thereof

Abstract

The invention provides a polypeptide with angiotensin converting enzyme inhibition as well as a preparation method and application thereof, and relates to the technical field of active peptide preparation. According to the method, the abalone is crushed, the compound enzyme is added for primary enzymolysis, then the specific protease mixture containing prolyl endopeptidase, chymotrypsin and protease K is added for secondary enzymolysis, and the obtained polypeptide liquid has high ACE (angiotensin converting enzyme) inhibition. The obtained polypeptide is analyzed and screened to obtain the polypeptide with the amino acid sequence as shown in SEQ ID NO.1-5. The polypeptide disclosed by the invention has high ACE (angiotensin converting enzyme) inhibition, and IC50 (50% of IC50) of the polypeptide to ACE is 0.05 mg / mL-0. 40mg / mL, so that the polypeptide can be used as an angiotensin converting enzyme inhibitor to play a role in reducing blood pressure.

Description

Technical Field

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

[0002] Bioactive peptides (BAPs) are peptide compounds that are beneficial to the life activities of biological organisms or have physiological effects. They are a class of polypeptides with a relative molecular mass of less than 6000Da and multiple biological functions. The complexity of their molecular structures varies. They are molecular polymers between amino acids and proteins, ranging from two amino acids to dozens of amino acids connected by peptide bonds. Moreover, 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 because of their effects on lowering blood pressure. Angiotensin converting enzyme (ACE) inhibitory peptides bind to the active pocket of ACE, thereby inhibiting the activity of ACE, preventing it from converting angiotensin I (Ang I) to Ang II, and then preventing the inactivation of vasodilator hormones, thereby achieving the purpose of reducing hypertension.

[0003] Abalone is a marine gastropod, a single-shell mollusk with tender meat and rich taste, which is deeply loved by consumers at home and abroad. my country is a major country in abalone farming. Abalone is not only rich in nutritional value, but a large number of studies have also shown that abalone contains active ingredients such as peptides, polysaccharides, free amino acids, fatty acids, etc. At present, the most commonly used method for preparing bioactive peptides is enzymatic hydrolysis, in which the main acting enzymes are mostly complex enzymes such as alkaline protease, neutral protease, trypsin, papain, etc. The obtained product composition is relatively complex, resulting in its low biological activity. At present, there is still a lack of research on how to extract high angiotensin-converting enzyme inhibitory peptides from abalone. Summary of the invention

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

[0005] In order to solve the above technical problems, the present invention provides a polypeptide with angiotensin converting enzyme inhibitory activity and a preparation method and application thereof. The present invention crushes abalone, adds a composite enzyme for preliminary enzymolysis, and then adds a specific protease mixture containing prolyl endopeptidase, chymotrypsin and proteinase K for secondary enzymolysis, and the obtained polypeptide has high ACE inhibitory activity. The obtained polypeptide is analyzed and screened to obtain a polypeptide with an amino acid sequence as shown in SEQ ID NO1-5. The polypeptide of the present invention has high ACE inhibitory activity and has an IC of 1.1777-1.180 of angiotensin converting enzyme. 50 Between 0.05mg / mL-0.40mg / mL, it can therefore act as an angiotensin-converting enzyme inhibitor to lower blood pressure.

[0006] The first object of the present invention is to provide a polypeptide having ACE inhibitory activity, wherein the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs. 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 invention is to provide a gene encoding the above polypeptide.

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

[0014] Furthermore, the blood pressure lowering product exerts its effect by inhibiting the activity of angiotensin converting enzyme.

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

[0016] S1, abalone is heated and crushed to obtain abalone crude product, and a composite enzyme is added to the abalone crude product for preliminary enzymolysis to obtain a preliminary enzymolysis product, wherein the composite enzyme includes papain, neutral protease, alkaline protease, trypsin and lipase;

[0017] S2, adding a specific protease mixture to the initial enzymatic hydrolysis product for secondary enzymatic hydrolysis to obtain the polypeptide;

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

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

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

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

[0022] Furthermore, the pH of the preliminary enzymatic hydrolysis in step S1 is 6.5-7.5.

[0023] Furthermore, the temperature of the preliminary enzymatic hydrolysis in step S1 is 50-60°C.

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

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

[0026] Furthermore, the temperature of the secondary enzymatic hydrolysis in step S2 is 30-40°C.

[0027] Beneficial effects of the present invention:

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

[0029] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0030] Figure 1 is a flow chart of the method for preparing and screening the polypeptides of the present invention;

[0031] Figure 2 is a graph showing the results of measuring the ACEi activity of the enzymatic hydrolysate obtained in Example 2 of the present invention;

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

[0033] Figure 4 This is a graph showing the results of measuring ACEi activity after gel filtration sequence fractions of the present invention. DETAILED DESCRIPTION

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

[0035] Compound enzymes: papain (>200000u / g) was provided by Shenzhen Xiameng Biotechnology Co., Ltd., China; neutral protease (>2.4AU-A / g) and alkaline protease (>6.5AU-N / g) were from Novozymes (China) Biotechnology Co., Ltd.; trypsin (>250000u / g) was from Shanghai MacLean Biochemical Technology Co., Ltd., China; lipase (>30000u / 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] ACE inhibition assay:

[0037] With FAPGG as substrate (FAPGG: N-[3-(2-furyl) acryloyl]-L-phenylalanyl-glycyl-glycine, FAPGG is a common substrate of angiotensin converting enzyme (ACE), and is often used for in vitro quantitative determination of angiotensin converting enzyme activity), reactants are added to the ELISA plate according to the table, and the absorbance A1 before the reaction is measured at 340nm by means of an ELISA reader, and then the absorbance value A2 is measured after incubation at 37°C for 30min, and ΔA is calculated, ΔA=A1-A2, and the change in absorbance value per unit time represents the ACE enzyme activity. The calculation of ACE inhibition rate is as follows:

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

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

[0040] Example 1

[0041] (1) Abalone is steamed at 110° C. for 4 hours, water is added and mixed evenly, and abalone meat slurry is obtained after crushing; the abalone meat slurry is frozen for more than 12 hours, and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat dry powder.

[0042] (2) Take 10 g of the dried abalone meat powder in (1), add 300 mL of water and mix evenly, place it in a constant temperature water bath at 55° C. and a stirrer speed of 800 rpm, add a composite enzyme with a mass concentration of 3% (based on the mass of the dried abalone meat powder) for enzymolysis for 4 h, and use sodium hydroxide to adjust the pH value to 6.5-7.5 during the enzymolysis process. After the enzymolysis is completed, inactivate the enzyme at 90-95° C. for 10-15 min; then centrifuge the system at a speed of 10000 rpm for 10 min, take the supernatant, freeze the supernatant for more than 12 h, and then place it in a low-temperature freeze dryer for more than 4 d to obtain abalone crude enzymolysis product dry powder.

[0043] (3) 10 g of the abalone crude enzymatic hydrolysate powder in (2) was dissolved in ultrapure water and fixed to 200 mL, placed in a constant temperature water bath at 37° C. and a stirrer speed of 800 rpm, mixed and added with a specific protease having a mass concentration of 0.5% (based on the mass of the abalone crude enzymatic hydrolysate) for enzymatic hydrolysis for 4 h to obtain an abalone hydrolysate; among the specific proteases, the mass concentration of prolyl endopeptidase was 0.125%, the mass concentration of chymotrypsin was 0.25%, and the mass concentration of proteinase K was 0.125%.

[0044] (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 10000 rpm for 10 min, and the supernatant is collected to obtain the abalone polypeptide liquid of the present invention.

[0045] Example 2

[0046] The preparation method of the abalone polypeptide liquid of this embodiment is the same as that of embodiment 1, except that the mass concentration of the specific protease added in this embodiment is 1%, wherein 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) Abalone is steamed at 110° C. for 4 hours, water is added and mixed evenly, and abalone meat slurry is obtained after crushing; the abalone meat slurry is frozen for more than 12 hours, and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat dry powder.

[0048] (2) Take 10 g of the dried abalone meat powder in (1), add 300 mL of water and mix evenly, place it in a constant temperature water bath at 55° C. and a stirrer speed of 800 rpm, add a composite enzyme with a mass concentration of 3% (based on the mass of the dried abalone meat powder) for enzymolysis for 4 h, and use sodium hydroxide to adjust the pH value to 6.5-7.5 during the enzymolysis process. After the enzymolysis is completed, inactivate the enzyme at 90-95° C. for 10-15 min; then centrifuge the system at a speed of 10000 rpm for 10 min, take the supernatant, freeze the supernatant for more than 12 h, and then place it in a low-temperature freeze dryer for more than 4 d to obtain abalone crude enzymolysis product dry powder.

[0049] (3) 10 g of the abalone crude enzymatic hydrolysate powder in (2) was dissolved in ultrapure water and fixed to 200 mL, placed in a constant temperature water bath at 37° C. and a stirrer speed of 800 rpm, mixed and added with a specific protease having a mass concentration of 1% (based on the mass of the abalone crude enzymatic hydrolysate) for enzymatic hydrolysis for 4 h to obtain an abalone hydrolysate; among the specific proteases, the mass concentration of prolyl endopeptidase was 0.25%, the mass concentration of chymotrypsin was 0.5%, and the mass concentration of proteinase K was 0.25%.

[0050] (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 10000 rpm for 10 min, and the supernatant is collected to obtain the abalone polypeptide liquid of the present invention.

[0051] Example 3

[0052] The preparation method of the abalone polypeptide liquid of this embodiment is the same as that of embodiment 1, except that the mass concentration of the specific protease added in this 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) Abalone is steamed at 110° C. for 4 hours, water is added and mixed evenly, and abalone meat slurry is obtained after crushing; the abalone meat slurry is frozen for more than 12 hours, and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat dry powder.

[0054] (2) Take 10 g of the dried abalone meat powder in (1), add 300 mL of water and mix evenly, place it in a constant temperature water bath at 55° C. and a stirrer speed of 800 rpm, add a composite enzyme with a mass concentration of 3% (based on the mass of the dried abalone meat powder) for enzymolysis for 4 h, and use sodium hydroxide to adjust the pH value to 6.5-7.5 during the enzymolysis process. After the enzymolysis is completed, inactivate the enzyme at 90-95° C. for 10-15 min; then centrifuge the system at a speed of 10000 rpm for 10 min, take the supernatant, freeze the supernatant for more than 12 h, and then place it in a low-temperature freeze dryer for more than 4 d to obtain abalone crude enzymolysis product dry powder.

[0055] (3) 10 g of the abalone crude enzymatic hydrolysate powder in (2) was dissolved in ultrapure water and fixed to 200 mL, placed in a constant temperature water bath at 37° C. and a stirrer speed of 800 rpm, mixed and added with a specific protease having a mass concentration of 3% (based on the mass of the abalone crude enzymatic hydrolysate) for enzymatic hydrolysis for 4 h to obtain an abalone hydrolysate; among the specific proteases, the mass concentration of prolyl endopeptidase was 0.5%, the mass concentration of chymotrypsin was 1%, and the mass concentration of proteinase K was 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 10000 rpm for 10 min, and the supernatant is collected to obtain the abalone polypeptide liquid of the present invention.

[0057] Example 4

[0058] The preparation method of the abalone polypeptide liquid of this embodiment is the same as that of Example 2, and the mass concentration of the specific protease added in this embodiment is also 1%, except 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) Abalone is steamed at 110° C. for 4 hours, water is added and mixed evenly, and abalone meat slurry is obtained after crushing; the abalone meat slurry is frozen for more than 12 hours, and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat dry powder.

[0060] (2) Take 10 g of the dried abalone meat powder in (1), add 300 mL of water and mix evenly, place it in a constant temperature water bath at 55° C. and a stirrer speed of 800 rpm, add a composite enzyme with a mass concentration of 3% (based on the mass of the dried abalone meat powder) for enzymolysis for 4 h, and use sodium hydroxide to adjust the pH value to 6.5-7.5 during the enzymolysis process. After the enzymolysis is completed, inactivate the enzyme at 90-95° C. for 10-15 min; then centrifuge the system at a speed of 10000 rpm for 10 min, take the supernatant, freeze the supernatant for more than 12 h, and then place it in a low-temperature freeze dryer for more than 4 d to obtain abalone crude enzymolysis product dry powder.

[0061] (3) 10 g of the abalone crude enzymatic hydrolysate powder in (2) was dissolved in ultrapure water and fixed to 200 mL, placed in a constant temperature water bath at 37° C. and a stirrer speed of 800 rpm, mixed and added with a specific protease having a mass concentration of 1% (based on the mass of the abalone crude enzymatic hydrolysate) for enzymatic hydrolysis for 4 h to obtain an abalone hydrolysate; among the specific proteases, the mass concentration of prolyl endopeptidase was 0.17%, the mass concentration of chymotrypsin was 0.33%, and the mass concentration of proteinase K was 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 10000 rpm for 10 min, and the supernatant is collected to obtain the abalone polypeptide liquid of the present invention.

[0063] The abalone polypeptide liquids obtained in Examples 1-4 and Comparative Examples 1-2 were subjected to ACEi activity assay at 1 mg / mL. The results are shown in Table 1.

[0064] Table 1 Determination of ACEi activity of abalone polypeptide liquid in Examples 1-4 and Comparative Examples 1-2

[0065]

[0066] As can be seen from Table 1, the technical solution of the present invention significantly improves the ACEi activity of the abalone polypeptide liquid. Specifically, compared with Comparative Example 1, the ACEi activity of the abalone polypeptide liquid obtained in 3h in Example 2 of the present invention can be increased by as much as 228%, which fully proves the feasibility of the preparation method of the present invention.

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

[0068] (1) using an ultrafiltration tube with a molecular weight cut-off of 3 kDa to filter the abalone polypeptide liquid obtained in Example 2, collecting components with a molecular weight of 0-3 kDa;

[0069] (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 and their ACE inhibitory activity was determined. The chromatogram and fraction collection are shown in Figure 3 , ACE inhibitory activity of different fractions is shown in Figure 4 Fraction C had the highest ACE inhibitory activity, and fraction D had the highest peptide abundance.

[0070] (3) using peptidomics technology to analyze the advantageous fractions in step (2) (i.e., fraction C and fraction D) to obtain the amino acid sequences of all the peptides contained therein;

[0071] (4) Use the PeptideRanker tool to screen the peptides with an amino acid sequence score > 0.9 in step (3). After molecular docking prediction, chemically synthesize the peptides and verify their biological activity

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

[0073] The specific method of molecular docking includes: using AutoDock Vina 1.1.2 software for molecular docking. The crystal structure of human ACE-lisinopril complex (1O86) 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 processed using PyMol 2.6 to remove water molecules and small molecule ligands, add hydrogen, etc. The receptor protein and peptide segment 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 Vina docking with the highest score was selected as the final result and visualized and analyzed in PyMol 2.6.

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

[0075]

[0076] Comparative Example 1

[0077] (1) Abalone is steamed at 110° C. for 4 hours, water is added and mixed evenly, and abalone meat slurry is obtained after crushing; the abalone meat slurry is frozen for more than 12 hours, and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat dry powder.

[0078] (2) 10 g of the dried abalone meat powder in (1) was taken, and 300 mL of water was added to obtain a uniform mixture of abalone meat and water, which was placed in a constant temperature water bath at 55° C. and a stirrer speed of 800 rpm, and a composite enzyme with a mass concentration of 3% (based on the mass of the dried abalone meat powder) was added for enzymolysis for 4 h. During the enzymolysis process, the pH value was regulated to 6.5-7.5 with sodium hydroxide, and the enzyme was inactivated at 90-95° C. for 10-15 min after the enzymolysis was completed; then the system was centrifuged at a speed of 10000 rpm for 10 min, and the supernatant was taken to obtain a crude enzymatic hydrolyzate of abalone meat.

[0079] Comparative Example 2

[0080] (1) abalone is steamed at 110° C. for 4 hours, water is added and mixed evenly, and abalone meat is obtained after crushing; the abalone meat is frozen for more than 12 hours, and then placed in a low-temperature freeze dryer for more than 4 days to obtain abalone meat powder;

[0081] (2) Take 10 g of the dried abalone meat powder in (1), add 300 mL of water to mix the mixture of abalone meat and water, place it in a constant temperature water bath at 55° C. and a stirrer speed of 800 rpm, add 0.25% prolyl endopeptidase, 0.5% chymotrypsin, and 0.25% proteinase K in a mass concentration (based on the mass of the dried abalone meat powder) to mix for 4 h, and adjust the pH value to 6.5-7.5 with sodium hydroxide during the enzymolysis process. After the enzymolysis is completed, inactivate the enzyme at 90-95° C. for 10-15 min; then centrifuge the system at a speed of 10000 rpm for 10 min, take the supernatant, and obtain an enzymolysis solution.

[0082] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A polypeptide having angiotensin converting enzyme inhibitory activity, characterized in that: The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO.1-5.

2. A gene encoding the polypeptide according to claim 1.

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

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

5. A method for preparing the polypeptide according to claim 1, characterized in that: The following steps are involved: S1, abalone is heated and crushed to obtain abalone crude product, and a composite enzyme is added to the abalone crude product for preliminary enzymolysis to obtain a preliminary enzymolysis product, wherein the composite enzyme includes 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 includes prolyl endopeptidase, chymotrypsin and proteinase K.

6. The preparation method according to claim 5, characterized in that: The heating temperature in step S1 is 100-150° C., 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°C.

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

100.

10. The preparation method according to claim 5, characterized in that: The mass ratio of prolyl endopeptidase:chymotrypsin:proteinase K is (1-2):(1-2):(1-2).

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