Bifunctional peptide from cyclina sinensis as well as preparation method and application of bifunctional peptide

Abstract

The invention relates to a bifunctional peptide from cyclina sinensis, and belongs to the technical field of polypeptides. The amino acid sequence of the bifunctional peptide disclosed by the invention is His-Tyr-Pro-Gly-Gln-Pro-Tyr, and the amino acid sequence of the bifunctional peptide disclosed by the invention is as shown in the specification. The invention relates to an oligopeptide with alpha-glucosidase inhibitory activity and ACE (angiotensin converting enzyme) inhibitory activity. The invention also discloses a preparation method of the bifunctional peptide. The bifunctional peptide can also be synthesized by adopting a peptide synthesis method or a synthesis and modification method. The bifunctional peptide provided by the invention takes protein from marine cyclina sinensis as a starting point, is cut into active peptide with a specific sequence structure through control of cutting conditions of protease, has remarkable alpha-glucosidase inhibitory activity and angiotensin converting enzyme (ACE) inhibitory activity, has ICvalues of 0.4662 mg / mL and 0.09976 mg / mL respectively, and can be used for preparing the marine cyclina sinensis bifunctional peptide. Therefore, the tea has double physiological functions of reducing blood sugar and blood pressure. The bifunctional peptide provided by the invention can be used for preparing medicines or health-care foods for reducing blood sugar and / or blood pressure, has a clear sequence structure and dual biological activity, and provides a new way for developing multifunctional ocean-derived peptide products.

Description

TECHNICAL FIELD

[0001] The application provides a preparation method of a bifunctional peptide, in particular, an alpha-glucosidase inhibiting peptide and / or an angiotensin converting enzyme (ACE) inhibiting peptide, and a preparation method and use thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Bioactive peptides are a class of short-chain amino acid sequences with positive effects on body functions and specific physiological activities, usually consisting of 2-20 amino acid residues. Compared with traditional proteins and chemical drugs, bioactive peptides have many advantages such as small molecular weight, easy absorption by the human body, strong targeting, diverse activities, and small side effects. In recent years, bioactive peptides have become a research hotspot in the fields of functional foods, medicines and cosmetics. Their physiological functions are wide-ranging, including but not limited to: antioxidant, antibacterial, antihypertensive, immunomodulatory, antifatigue, and antitumor. These functional properties make bioactive peptides show great application potential in the development of new health foods, dietary supplements, drug lead compounds and functional cosmetic raw materials. At present, the sources of bioactive peptides are increasingly diverse, among which marine organisms are considered a valuable resource for discovering bioactive peptides with novel structures and unique functions due to their unique living environment and rich biodiversity.

[0003] Cyclina sinensis is a common economic marine bivalve in China, widely distributed in intertidal zones along the coast and rich in resources. Cyclina sinensis has delicious meat and is rich in nutrients, including high-quality protein, various amino acids, trace elements and taurine, which lays a material foundation for its use as a high-quality raw material for developing bioactive peptides. At present, many studies have been devoted to exploring the biological activity potential of Cyclina sinensis. For example, the published patent document CN108277249A discloses a preparation method of a Cyclina sinensis-derived peptide, which is confirmed to be effective in relieving non-alcoholic fatty liver; the published patent document CN108396046A discloses another preparation method of a Cyclina sinensis-derived peptide with immunomodulatory function; meanwhile, studies such as the published patent document CN201611021694.2 have developed Cyclina sinensis-derived peptides with antibacterial activity. It is particularly worth noting that the published patent document CN105821103A has identified a Cyclina sinensis-derived active peptide that can efficiently inhibit angiotensin, and its final amino acid sequence determined by high-performance liquid chromatography purification is Trp-Pro-Met-Gly-Phe. However, through comprehensive retrieval, it is found that the functions of the Cyclina sinensis-derived active peptides disclosed in the prior art are all directed to a single biological activity, and there is currently a lack of Cyclina sinensis-derived bifunctional peptides that can simultaneously have angiotensin inhibiting activity and other important physiological functions. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a new bifunctional active peptide with significant angiotensin converting enzyme (ACE) inhibitory activity and alpha-glucosidase inhibitory activity, and good safety, in view of the single function of the active peptide from Cyclina sinensis in the prior art.

[0005] Another technical problem to be solved by the present application is to provide a preparation method and use of the bifunctional active peptide.

[0006] The technical problem to be solved by the present application is solved by the following technical scheme. The present application is a bifunctional peptide from Cyclina sinensis 'Jianghai No.1', which has the following characteristics: the amino acid sequence of the bifunctional peptide is His-Tyr-Pro-Gly-Gln-Pro-Tyr, abbreviated as HYPGQPY.

[0007] The present application also discloses a preparation method of the bifunctional peptide, which has the following characteristics: proteins are extracted from the soft body of Cyclina sinensis 'Jianghai No.1', and then subjected to enzymolysis by neutral protease, followed by separation and purification, and freeze-drying to obtain the bifunctional peptide; the enzymolysis conditions are as follows: pH is 6.5-7.5, temperature is 40-55℃, enzymolysis time is 3-4h, and enzyme-substrate ratio is 1500-2000 U / g.

[0008] The further preferred technical scheme of the preparation method of the bifunctional peptide is that the enzymolysis conditions are as follows: pH is 7.0, temperature is 50℃, enzymolysis time is 3.5h, and enzyme-substrate ratio is 1700 U / g.

[0009] The further preferred technical scheme of the preparation method of the bifunctional peptide is that the separation and purification means comprises ultrafiltration and Sephadex G-15 gel chromatography.

[0010] The further preferred technical scheme of the preparation method of the bifunctional peptide is that the specific steps of separation and purification are as follows: (1) The enzymolysis product is first subjected to ultrafiltration separation by using an ultrafiltration membrane with a molecular weight cut-off range of 3 KDa and 10 KDa, so as to divide it into three components with different molecular weights, i.e., a component with a molecular weight greater than 10 KDa, a component with a molecular weight between 3 KDa and 10 KDa, and a component with a molecular weight less than 3 KDa; (2) Collecting the component with the best alpha-glucosidase inhibitory activity and ACE inhibitory activity, and then separating the component by Sephadex G-15 gel chromatography, eluting the component with pure water at a flow rate of 0.8 ml / min-1.2 ml / min, detecting the absorption peak at a wavelength of 280 nm, and determining the alpha-glucosidase inhibitory activity and ACE inhibitory activity of the elution component corresponding to each absorption peak; and obtaining the active component containing the bifunctional peptide.

[0011] The preparation method of the bifunctional peptide has the further preferred technical solutions as follows: (1) Preparation of the green clam protein: washing fresh green clams, removing shells, and taking soft bodies, homogenizing, and freeze-drying to obtain green clam freeze-dried powder; (2) Enzymatic hydrolysis of the green clam protein: resuspending the green clam freeze-dried powder at a substrate concentration of 5%, adjusting the pH to 7.0, and preheating to 50 DEG C; adding neutral protease at an enzyme amount of 1700 U / g, and hydrolyzing in a 50 DEG C constant-temperature water bath for 3.5 h, during which the pH is controlled to be stable; after the enzymatic hydrolysis is completed, boiling water is used for enzyme inactivation for 15 min, and then the supernatant is collected by centrifugation, which is the green clam polypeptide crude extract; (3) Separation, purification and identification of the enzymatic hydrolysis product: sequentially passing the crude extract through ultrafiltration membranes with molecular weight cut-off of 10 KDa and 3 KDa, and collecting the permeate component with a molecular weight of less than 3 KDa; further purifying the component by a Sephadex G-15 gel chromatography column, using pure water as the eluent, at a flow rate of 1 mL / min, detecting at 280 nm, and collecting the active component; finally, performing sequence identification by LC-MS / MS to obtain the bifunctional peptide with an amino acid sequence of His-Tyr-Pro-Gly-Gln-Pro-Tyr.

[0012] The application further discloses another preparation method of the bifunctional peptide, which is characterized by synthesizing the bifunctional peptide by a peptide synthesis method or a synthesis and modification method; the synthesis or modification method includes but is not limited to biological synthesis, chemical synthesis, enzyme catalytic synthesis, connection, addition, coupling or derivation.

[0013] The bifunctional peptide can be used as an effective component in the preparation of alpha-glucosidase inhibitors and / or angiotensin converting enzyme (ACE) inhibitors.

[0014] Compared with the prior art, the bifunctional peptide has the following beneficial effects: 1.The present application provides a new type of Cyclina sinensis-derived polypeptide with clear structure and dual physiological activity, which breaks through the limitation of single function of existing Cyclina sinensis peptides. The peptide can efficiently inhibit ACE enzyme and alpha-glucosidase, and the IC values thereof are 0.09976 mg / mL and 0.4662 mg / mL, respectively, which provides a new core raw material for developing the next generation of comprehensive health care food with both blood pressure-lowering and blood sugar-lowering functions. 50

[0015] 2.The preparation method of the present application uses Cyclina sinensis which is rich in resources as raw material, and successfully mines the dual functional peptide through multi-target active-oriented screening strategy combined with specific enzymolysis and purification process, which not only improves the high-value utilization rate of Cyclina sinensis, but also provides a new technical path for the development of marine bioactive peptides.

[0016] 3.The product of the present application is a natural food-derived peptide, which is expected to have high safety and small side effects, can effectively avoid the adverse reactions that may be caused by chemical synthesis drugs, and has a broad application prospect in the fields of functional food, dietary supplements and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 ACE enzyme and alpha-glucosidase inhibitory activities of each component of Cyclina sinensis protease hydrolysate after ultrafiltration separation; Figure 2 Sephadex G-15 gel separation chromatogram of Cyclina sinensis protease hydrolysate; Figure 3 ACE enzyme and alpha-glucosidase inhibitory activities of each component of Sephadex G-25 gel separation; Figure 4 Total ion flow chart of subcomponent 3; Figure 5 Secondary mass spectrum chart of HYPGQPY; Figure 6 Molecular docking conformation and interaction of HYPGQPY and ACE enzyme (1O86); Figure 7 Molecular docking conformation and interaction of HYPGQPY and alpha-glucosidase (3WY1); DETAILED DESCRIPTION

[0018] The present application will be further described below in conjunction with examples, so that those skilled in the art can further understand the present application, but the present application is not limited in the scope of the described examples.

[0019] Example 1: Preparation of dual functional peptide (1) Raw material pretreatment: ​The green clam "Jianghai No.1" used in this experiment is a new variety cultivated by our school research group. After washing the fresh green clam, the soft body part is taken out and homogenized to break the tissue. The green clam homogenate is freeze-dried to obtain green clam freeze-dried powder, which is stored at -20°C.

[0020] (2) Preparation of polypeptide by enzymatic hydrolysis: The enzyme is a commercial enzyme provided by Suzhou Caben Life Science and Technology Co., Ltd. Neutral protease is used for enzymatic hydrolysis. The optimal conditions for the enzyme are pH 6.5-7.5 and temperature 40-55°C. The green clam freeze-dried powder is reconstituted at a substrate concentration of 5%, adjusted to pH 7 with 1 M NaOH, and preheated to 50°C. Neutral protease is added at an enzyme dosage of 1700 U / g, and the enzymatic hydrolysis is carried out in a 50°C constant temperature water bath for 3.5 h. During this period, the pH is controlled within the optimal range of the enzyme. After the enzymatic hydrolysis is completed, the enzyme is inactivated in a boiling water bath for 15 min, quickly cooled, and centrifuged at 8000 x g for 20 min. The supernatant is collected, which is the crude polypeptide extract of green clam.

[0021] (3) Isolation and purification of polypeptide: Ultrafiltration separation: The crude extract is sequentially passed through ultrafiltration membranes with molecular weight cut-off of 10 kDa and 3 kDa, and separated into three components with different molecular weights, <3 kDa, 3-10 kDa, and >10 kDa. The α-glucosidase and ACE enzyme inhibitory activities of each component are determined, as shown in Figure 1 . The α-glucosidase and ACE enzyme inhibitory activities of the <3 kDa component are better, reaching 46.88±2.40% and 66.54±3.03%, respectively. The <3 kDa component is collected and freeze-dried for future use.

[0022] Gel chromatography purification: The <3 kDa component is dissolved in deionized water to prepare a 50 mg / mL solution. It is loaded onto a Sephadex G-15 chromatography column (1.6 cm x 70 cm) equilibrated with deionized water. Elution is carried out with deionized water at a flow rate of 1 mL / min, and the absorbance is detected at a wavelength of 280 nm. The elution components corresponding to each absorption peak are collected, and the inhibitory activities of each component are determined. Referring to Figure 2 , the results show that five sub-components are collected, among which sub-component 3 has significantly higher inhibitory activity on both enzymes than other components, with α-glucosidase inhibition rate of 58.28±1.85% and ACE inhibition rate of 77.74±02.44%, as shown in Figure 4 . Therefore, sub-component 3 is determined as the dual-functional active core component, and sub-component 3 is collected and freeze-dried for future use.

[0023] Peptide identification: Sub-fraction 3 was analyzed by LC-MS / MS, and the sequence was identified by Shanghai Sangon Biotech Co., Ltd. (https: / / www.sangon.com / ).

[0024] Example 2: Screening of bifunctional peptides (1) Virtual screening of polypeptide sequences: Online activity prediction tools: The identified peptide sequences were input into online prediction tools such as PeptideRanker for scoring. First, PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to assess the bioactivity potential of the peptides. PeptideRanker bioactivity probability values ​​> 0.5 were selected, resulting in 10 candidate peptides: ANDYLKGKW; ANDYLKGKWR; ANEFLISKWK; AQRPDLPVGY; CCCMTCSF; DGPSSKDWRG; GGGGGY; GPNDDRFGPR; HYPGQPY; SWPKEDYGK. See Table 1 for reference. Table 1. Activity prediction table for peptide sequences

[0025] Using BIOPEP( https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php ToxinPred was used to screen and compare peptides to identify potential novel bioactive peptides. https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html Predicting peptide toxicity; using AllerTOP v2.1 ( https: / / ddg-pharmfac.net / allertop_test / Predicting peptide sensitization; using SwissADME ( https: / / www.swissadme.ch / index.php Predicting the gastrointestinal absorption of peptides; using PlifePred ( https: / / webs.iiitd.edu.in / raghava / plifepred / batch.php Predict the half-life of peptides using NovoPro (). https: / / www.novopro.cn / tools / calc_peptide_property.html Predict the pI and solubility of peptides.

[0026] (2) Molecular docking verification: Using molecular docking software such as AutoDock Vina, the above 10 candidate peptides were molecularly docked with the crystal structures of α-glucosidase (PDB: 3WY1) and ACE (PDB: 1O86), respectively. First, the crystal structures of α-glucosidase and ACE were obtained from the PDB protein database (https: / / www.rcsb.org / ). The receptors were then dehydrated and the protoligands were removed using PyMOL 3.0.3 software. PepSMI (… https: / / www.novoprolabs.com / tools / convert-peptide-to-smiles-string) The polypeptide was converted into SMILES format, and then the three-dimensional structure of the polypeptide was drawn and energy minimization optimization was performed using Chem3D 23.1.1.

[0027] The polypeptide and the receptor protein were hydrogenated using AutoDock Tools 1.5.7, and the parameter setting and docking were completed using Vina (the center grid box of 1O86: size_x = 24.0; size_y = 37.5; size_z = 22.5; the center grid box of 3WY1: size_x = 47.25; size_y = 26.25; size_z = 39.75). Other parameters were set to default values. Finally, visualization was performed using PyMOL 3.0.3, and Discovery Studio 2021 was used to analyze the molecular docking results. The results showed that the polypeptide (His-Tyr-Pro-Gly-Gln-Pro-Tyr) exhibited the lowest binding energy (-10.6 kcal / mol and -8.8 kcal / mol, respectively) with the two target enzymes, and could be stabilized in the active pocket of the enzyme through multiple hydrogen bonds and hydrophobic forces (as shown in FIG. 1). Figure 3 Therefore, His-Tyr-Pro-Gly-Gln-Pro-Tyr (HYPGQPY) was finally determined as the bifunctional peptide target sequence to be protected by the present application. The properties of the polypeptide sequences and the binding energy of molecular docking are shown in Table 2.

[0028] Table 2 Properties of polypeptide sequences and binding energy of molecular docking

[0029] Table 3 Properties of screened bifunctional inhibitory peptides

[0030] Secondary mass spectrum of HYPGQPY Figure 5 .

[0031] Example 3: Activity determination of bifunctional peptides The chemically synthesized peptide segment HYPGQPY was subjected to in vitro activity verification.

[0032] The above peptide segment was synthesized by Shanghai Sangon Biological Engineering Co., Ltd. (https: / / www.sangon.com / ) and subjected to the next step of activity test.

[0033] (1) Alpha-glucosidase inhibitory activity: The polypeptide sample (1 mg / mL) was mixed with a-glucosidase (0.2 U / mL) at 50 μL each, incubated at 37 ℃ for 20 min, 50 μL of substrate solution PNPG (5 mmol / L) was added, and the reaction was continued at 37 ℃ for 20 min. Finally, 100 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction, and the absorbance was measured at 405 nm after deducting the background value of the blank group. Acarbose was used as a positive control. The calculation formula is as follows:

[0034] A sample: sample + a-glucosidase + PNPG A sample blank: sample + PBS + PNPG A control: PBS + a-glucosidase + PNPG A control blank: PBS + PNPG (2) ACE enzyme inhibition activity: The ACE enzyme solution and FAPGG substrate solution were prepared using 50 mmol / L pH 8.3 HEPES buffer containing 300 mmol / L NaCl. The polypeptide sample (1 mg / mL) 40 μL, FAPGG substrate (1 mmol / L) 50 μL, and ACE enzyme (0.1 U / mL) 10 μL were mixed uniformly, and the initial absorbance values a1 and b1 of the blank group and the sample group were measured at 340 nm. After continuous reaction at 37 ℃ for 30 min, the absorbance values a2 and b2 of the blank group and the sample group were measured at 340 nm. Captopril (1 mg / mL) was used as a positive control. The calculation formula is as follows:

[0035] ΔA blank: HEPES + ACE enzyme + FAPGG, a1-a2 ΔB sample: sample + ACEM enzyme + FAPGG, b1-b2 The IC 50 value of the a-glucosidase inhibition activity of the polypeptide was 0.4662 mg / mL, and the IC 50 value of the ACE inhibition activity was 0.09976 mg / mL, confirming that it indeed has significant dual inhibition function. The HYPGQPY-ACE enzyme (1O86) molecular docking conformation and interaction diagram are referred to in Figure 6 ; and the HYPGQPY-a-glucosidase (3WY1) molecular docking conformation and interaction are referred to in Figure 7 .

[0036] Based on this, the bifunctional peptide can be used to prepare oral liquids, tablets, capsules, powders, and other forms of blood glucose-lowering and / or blood pressure-lowering drugs or health foods.

[0037] In summary, the present application provides a technical path for preparing the bifunctional peptide from the Chinese mitten crab "Jianghai No.1", and obtains a new polypeptide with clear sequence and double physiological activities, which has good development prospects.

Claims

1. A bifunctional peptide derived from clam, characterized in that: The amino acid sequence of the bifunctional peptide is His-Tyr-Pro-Gly-Gln-Pro-Tyr.

2. A method for preparing the bifunctional peptide as described in claim 1, characterized in that: Protein was extracted from the soft tissue of the clam, then enzymatically hydrolyzed with a neutral protease, and purified and freeze-dried to obtain a bifunctional peptide with α-glucosidase inhibitory activity and ACE inhibitory activity. The enzymatic hydrolysis conditions were: pH 6.5-7.5, temperature 40℃-55℃, hydrolysis time 3h-4h, and enzyme-substrate ratio 1500-2000 U / g.

3. The method for preparing the bifunctional peptide according to claim 2, characterized in that: The enzymatic hydrolysis conditions were: pH 7.0, temperature 50 ℃, hydrolysis time 3.5 h, and enzyme to substrate ratio 1700 U / g.

4. The method for preparing the bifunctional peptide according to claim 2, characterized in that: The separation and purification methods include ultrafiltration and Sephadex G-15 gel chromatography.

5. The method for preparing the bifunctional peptide according to claim 2, characterized in that: The specific steps for separation and purification are as follows: (1) The enzymatic hydrolysis products were first separated by ultrafiltration using ultrafiltration membranes with molecular weight cutoff ranges of 3 KDa and 10 KDa, and were divided into three components with different molecular weight ranges: the component with molecular weight greater than 10 KDa, the component with molecular weight between 3 KDa and 10 KDa, and the component with molecular weight less than 3 KDa. (2) Collect the fractions with the best α-glucosidase inhibitory activity and ACE inhibitory activity, and then separate them by Sephadex G-15 gel chromatography with pure water as the eluent at a flow rate of 1 ml / min. Detect the absorption peaks at a wavelength of 280 nm and determine the α-glucosidase inhibitory activity and ACE inhibitory activity of the eluted fractions corresponding to each absorption peak; obtain the active fraction containing bifunctional peptides.

6. The method for preparing the bifunctional peptide according to claim 2, characterized in that, The specific steps of the preparation method are as follows: (1) Preparation of clam protein: Fresh clams are cleaned, shelled and the soft part is taken, homogenized and then freeze-dried to obtain clam freeze-dried powder; (2) Enzymatic hydrolysis of clam protein: The freeze-dried clam powder was reconstituted at a substrate concentration of 5%, the pH was adjusted to 7.0, and the temperature was preheated to 50°C. Neutral protease was added at an enzyme dosage of 1700 U / g, and the mixture was enzymatically hydrolyzed in a constant temperature water bath at 50°C for 3.5 h, during which the pH was kept stable. After the enzymatic hydrolysis was completed, the enzyme was inactivated in a boiling water bath for 15 min, and after rapid cooling, the supernatant was collected by centrifugation, which is the crude extract of clam polypeptide. (3) Separation, purification and identification of enzymatic hydrolysis products: The crude extract was passed through ultrafiltration membranes with molecular weight cutoff of 10 KDa and 3 KDa in sequence, and the permeate fraction with molecular weight less than 3 KDa was collected. The fraction was further purified by Sephadex G-15 gel chromatography column with pure water as the eluent, flow rate of 1 mL / min, and active fraction was detected and collected at 280 nm. Finally, the sequence was identified by LC-MS / MS to obtain a bifunctional peptide with amino acid sequence His-Tyr-Pro-Gly-Gln-Pro-Tyr.

7. A method for preparing the bifunctional peptide as described in claim 1, characterized in that, The bifunctional peptide is synthesized using peptide synthesis methods or synthesis and modification methods; the synthesis or modification methods include, but are not limited to: biosynthesis, chemical synthesis, enzyme-catalyzed synthesis, linkage, addition, coupling or derivatization.

8. The use of the bifunctional peptide as described in claim 1, characterized in that: The application of the aforementioned bifunctional peptide as an active ingredient in the preparation of α-glucosidase inhibitors and / or angiotensin-converting enzyme (ACE) inhibitors.

9. The use of the bifunctional peptide according to claim 8, characterized in that: The inhibitors are used to prepare drugs or health foods for the prevention and / or adjunctive treatment of hyperglycemia and / or hypertension.

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