CN105821103B - Preparation method of clam angiotensin converting enzyme inhibitory peptide - Google Patents
Preparation method of clam angiotensin converting enzyme inhibitory peptide Download PDFInfo
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- CN105821103B CN105821103B CN201511011197.XA CN201511011197A CN105821103B CN 105821103 B CN105821103 B CN 105821103B CN 201511011197 A CN201511011197 A CN 201511011197A CN 105821103 B CN105821103 B CN 105821103B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Abstract
The invention discloses a method for preparing angiotensin converting enzyme inhibitory peptide of clam, which comprises the following steps: (1) pre-treating the clams; (2) carrying out enzymolysis; (3) performing ultrafiltration separation; (4) separating and purifying by a rapid protein purification system; (5) and (5) separating and purifying by using a high performance liquid chromatography system. The method takes the clam as a raw material, the clam is subjected to enzymolysis through trypsin, and the enzymolysis solution is separated and purified by using an ultrafiltration technology, a rapid protein purification system and a high performance liquid chromatography system to obtain the new angiotensin converting enzyme inhibitory peptide (ACE inhibitory peptide), the extraction and separation process is reasonable, the operability is strong, and an experimental basis is provided for preparing the high-activity ACE inhibitory peptide from the clam.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a preparation method of a clam angiotensin converting enzyme inhibitory peptide.
Background
Hypertension is a cardiovascular disease which afflicts today and can cause serious complications, with great harm to human health. Angiotensin-converting enzyme (ACE) plays an important role in the regulation of blood pressure of a body, on one hand, the ACE converts inactive Angiotensin I into Angiotensin II which can strongly contract systemic arterioles and increase blood pressure; on the other hand, ACE can promote the decomposition of bradykinin with the function of reducing blood pressure, and causes the increase of blood pressure.
Nowadays, synthetic ACE inhibitors such as captopril, enalapril and lisinopril have strong blood pressure lowering effect, but have side effects such as dizziness, nausea, severe cough, abnormal taste and renal function damage after long-term administration. Many studies show that the short peptide derived from food has the efficacy of reducing blood pressure, and the skin of Pacific cod such as Dai-Hung Ngo and the like is taken as a raw material, is subjected to enzymolysis by papain, and is separated and purified to obtain the antihypertensive peptide: Thr-Cys-Ser-Pro; hasan F and the like carry out enzymolysis on bonito flesh by using pepsin, and high-activity antihypertensive peptide (IKYGD) is obtained after separation and purification; the bioactive peptide derived from food has the characteristics of safety, no toxic or side effect, easy absorption by organisms and the like. Therefore, the preparation and isolation of highly active ACE inhibiting peptides from food sources is still a focus of research.
The Cyclina sinensis (Cyclinasinensis) is a common shellfish in China coastal region, and belongs to a high-quality protein source. The Cyclina sinensis extract has effects of resisting oxidation, resisting tumor and improving immunity. However, no research report related to the clam antihypertensive peptide is found at present.
Disclosure of Invention
The invention aims to provide a method for preparing the angiotensin converting enzyme inhibitory peptide of the clam, which has reasonable extraction and separation process, strong operability and good product stability.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing angiotensin converting enzyme inhibitory peptide of clam comprises the following steps:
(1) pre-treating clams: removing shells and stripping meat of the clams, cleaning the clams with clear water, homogenizing by a high-speed tissue mincing machine, soaking for 3-5 h with 0.1mol/L NaOH, filtering, soaking and washing filter residues with purified water until the pH value is neutral, and dehydrating to obtain clam meat residues for later use. The pre-treatment of the clam mainly comprises protein removal and lipid removal.
(2) Enzymolysis: adding trypsin into the clam meat residue obtained in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 2-3, adding 1000-1200U/g of enzyme, adjusting the pH value to 7.5-8.5, adjusting the enzymolysis temperature to 45-50 ℃, adjusting the enzymolysis time to 9-12 h, inactivating with a boiling water bath after the enzymolysis is finished, pre-cooling, and centrifuging to obtain a supernatant.
(3) And (3) ultrafiltration separation: filtering the supernatant with 0.45 μm filter membrane, and ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: and (4) separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system to obtain five active components, respectively sampling and determining the angiotensin converting enzyme inhibitory activity of the five active components, and taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A. The angiotensin converting enzyme inhibitory activity is measured by High Performance Liquid Chromatography (HPLC), which is a prior art and therefore not described herein.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain seven active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the seven active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: an angiotensin converting enzyme inhibitory peptide of Trp-Pro-Met-Gly-Phe (WPMGF).
Preferably, in step (2), stirring is continued during the enzymatic hydrolysis.
Preferably, in the step (2), the temperature is precooled to 4-6 ℃. Precooling to 4-6 ℃ is helpful for improving the centrifugal effect.
Preferably, in the step (2), the centrifugal process conditions are as follows: the rotating speed is 5000-10000 rpm/min, and the centrifugation time is 10-30 min.
Preferably, in the step (4), the specific method for performing separation and purification by the rapid protein purification system is as follows: using Superose 1210/300 GL sepharose column (filler particle size 10 μm, 10X 300mm), diluting ultrafiltrate to 0.1g/mL, filtering through 0.22 μm filter membrane, injecting sample amount: 500 mu L of the solution; ultrapure water elution, flow rate: 0.5 mL/min; detection wavelength: 280 nm; automatic collection: 3.5 mL/tube, collect each peak.
Preferably, in the step (5), the specific method for separating and purifying by the high performance liquid chromatography system is as follows: adopting ZORBAXSB-C18 preparative chromatographic column (filler particle size 5 μm, 4.6 × 250 mm); mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.5 mL/min; detection wavelength: 214 and 280 nm; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 μ L, and each peak was collected.
Preferably, in the step (5), the purity detection method of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatically injecting samples, wherein the sample injection amount is 500 mu L; detection wavelength: 214 and 280 nm.
Therefore, the invention has the following beneficial effects: the method takes the green clams as raw materials, the green clams are subjected to enzymolysis through trypsin, and the enzymolysis liquid is separated and purified by using an ultrafiltration technology, a rapid protein purification system and a high performance liquid chromatography system to obtain a new angiotensin converting enzyme inhibitory peptide (ACE inhibitory peptide).
Detailed Description
The invention is further described below by means of specific embodiments.
Example 1
(1) Pre-treating clams: removing shell and meat of Cyclina sinensis, cleaning Carnis Mactrae with clear water, homogenizing with high-speed tissue mincing machine, soaking in 0.1mol/L NaOH for 3 hr, filtering, soaking the filter residue with purified water until pH is neutral, and dewatering to obtain Cyclina sinensis meat residue.
(2) Enzymolysis: adding trypsin into the clam meat residue obtained in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 2, adding 1000U/g of enzyme, adjusting the pH value to 7.5, carrying out enzymolysis at 45 ℃ for 9h, continuously stirring during enzymolysis, inactivating the mixture by using boiling water bath after the enzymolysis is finished, and centrifuging the mixture for 10min at the rotating speed of 5000rpm/min after precooling the mixture to 4 ℃ to obtain supernatant;
(3) and (3) ultrafiltration separation: filtering the supernatant with 0.45 μm filter membrane, and ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system (Apurifier UPC-100 type rapid protein purifier, GE Life sciences company) to obtain five active components, respectively sampling and measuring angiotensin converting enzyme inhibitory activity of the five active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A, and carrying out separation and purification by the rapid protein purification system according to the specific method: using Superose 1210/300 GL sepharose column (filler particle size 10 μm, 10X 300mm), diluting ultrafiltrate to 0.1g/mL, filtering through 0.22 μm filter membrane, injecting sample amount: 500 mu L of the solution; ultrapure water elution, flow rate: 0.5 mL/min; detection wavelength: 280 nm; automatic collection: 3.5 mL/tube, collect each peak.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain seven active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the seven active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: the specific method for separating and purifying the angiotensin converting enzyme inhibitory peptide of Trp-Pro-Met-Gly-Phe (WPMGF) by a high performance liquid chromatography system comprises the following steps: a ZORBAX SB-C18 preparative chromatographic column (the filler particle size is 5 mu m, and the particle size is 4.6 multiplied by 250mm) is adopted; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.5 mL/min; detection wavelength: 214 and 280 nm; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 mu L, collecting each peak, and the purity detection method of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 mu L of the solution; detection wavelength: 214 and 280 nm.
Example 2
(1) Pre-treating clams: removing shell and meat of Cyclina sinensis, cleaning Carnis Mactrae with clear water, homogenizing with high-speed tissue mincing machine, soaking in 0.1mol/L NaOH for 4 hr, filtering, soaking the filter residue with purified water until pH is neutral, and dewatering to obtain Cyclina sinensis meat residue.
(2) Enzymolysis: adding trypsin into the clam meat residue obtained in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 2.5, adding 1100U/g of enzyme, adjusting the pH value to 8, adjusting the enzymolysis temperature to 48 ℃, adjusting the enzymolysis time to 10h, continuously stirring during the enzymolysis, inactivating with boiling water bath after the enzymolysis is finished, pre-cooling to 5 ℃, and centrifuging at 8000rpm/min for 20min to obtain supernatant.
(3) And (3) ultrafiltration separation: filtering the supernatant with 0.45 μm filter membrane, and ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system to obtain five active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the five active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A, and carrying out separation and purification by the rapid protein purification system by the specific method comprising the following steps of: using Superose 1210/300 GL sepharose column (filler particle size 10 μm, 10X 300mm), diluting ultrafiltrate to 0.1g/mL, filtering through 0.22 μm filter membrane, injecting sample amount: 500 mu L of the solution; ultrapure water elution, flow rate: 0.5 mL/min; detection wavelength: 280 nm; automatic collection: 3.5 mL/tube, collect each peak.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain seven active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the seven active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: the specific method for separating and purifying the angiotensin converting enzyme inhibitory peptide of Trp-Pro-Met-Gly-Phe (WPMGF) by a high performance liquid chromatography system comprises the following steps: a ZORBAX SB-C18 preparative chromatographic column (the filler particle size is 5 mu m, and the particle size is 4.6 multiplied by 250mm) is adopted; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.5 mL/min; detection wavelength: 214 and 280 nm; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 mu L, collecting each peak, and the purity detection method of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 mu L of the solution; detection wavelength: 214 and 280 nm.
Example 3
(1) Pre-treating clams: removing shell and meat of Cyclina sinensis, cleaning Carnis Mactrae with clear water, homogenizing with high-speed tissue mincing machine, soaking in 0.1mol/L NaOH for 5 hr, filtering, soaking the filter residue with purified water until pH is neutral, and dewatering to obtain Cyclina sinensis meat residue.
(2) Enzymolysis: adding trypsin into the clam meat residue obtained in the step (1) for enzymolysis, wherein the enzymolysis conditions are as follows: the material-liquid ratio is 1: 3, adding 1200U/g of enzyme, adjusting the pH value to 8.5, adjusting the enzymolysis temperature to 50 ℃, adjusting the enzymolysis time to 12h, continuously stirring during the enzymolysis, inactivating with boiling water bath after the enzymolysis is finished, pre-cooling to 6 ℃, and centrifuging at 10000rpm/min for 30min to obtain supernatant.
(3) And (3) ultrafiltration separation: filtering the supernatant with 0.45 μm filter membrane, and ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 3ku to obtain ultrafiltrate.
(4) And (3) separating and purifying a rapid protein purification system: separating and purifying the ultrafiltrate obtained in the step (3) by a rapid protein purification system to obtain five active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the five active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component A, and carrying out separation and purification by the rapid protein purification system by the specific method comprising the following steps of: using Superose 1210/300 GL sepharose column (filler particle size 10 μm, 10X 300mm), diluting ultrafiltrate to 0.1g/mL, filtering through 0.22 μm filter membrane, injecting sample amount: 500 mu L of the solution; ultrapure water elution, flow rate: 0.5 mL/min; detection wavelength: 280 nm; automatic collection: 3.5 mL/tube, collect each peak.
(5) Separating and purifying by a high performance liquid chromatography system: separating and purifying the component A by a High Performance Liquid Chromatography (HPLC) system to obtain seven active components, respectively sampling and determining angiotensin converting enzyme inhibitory activity of the seven active components, taking the component with the highest angiotensin converting enzyme inhibitory activity as a component B, and detecting the purity of the component B to obtain an amino acid sequence as follows: the specific method for separating and purifying the angiotensin converting enzyme inhibitory peptide of Trp-Pro-Met-Gly-Phe (WPMGF) by a high performance liquid chromatography system comprises the following steps: a ZORBAX SB-C18 preparative chromatographic column (the filler particle size is 5 mu m, and the particle size is 4.6 multiplied by 250mm) is adopted; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.5 mL/min; detection wavelength: 214 and 280 nm; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 mu L, collecting each peak, and the purity detection method of the component B comprises the following steps: an analytical column (packing particle size: 5 μm, 4.6X 150mm) of ZORBAX SB-C18 was used; mobile phase a was ultrapure water (with 0.05% TFA) and mobile phase B was acetonitrile (with 0.05% TFA), using gradient elution: 5min 20% B, 30min 100% B; flow rate: 1.0 mL/min; column temperature: 25 ℃; automatic sample introduction, sample introduction amount: 500 mu L of the solution; detection wavelength: 214 and 280 nm.
The extraction and separation process is reasonable and strong in operability, the clam is taken as a raw material, the enzymolysis liquid is separated and purified by using an ultrafiltration technology, a rapid protein purification system and a high performance liquid chromatography system after being subjected to enzymolysis by trypsin to obtain a new angiotensin converting enzyme inhibitory peptide (ACE inhibitory peptide), N-terminal degradation sequencing is carried out by an amino acid sequencer to obtain pentapeptide, and the amino acid sequence of the pentapeptide is as follows: Trp-Pro-Met-Gly-Phe (WPMGF) with relative molecular mass of 636.78Da and IC of WPMGF500.5025mg/ml, has higher ACE inhibitory activity (the ACE inhibitory rate is more than 87.58 percent), and provides experimental basis for preparing high-activity ACE inhibitory peptide from clam.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (1)
1. The clam angiotensin converting enzyme inhibitory peptide is characterized in that the amino acid sequence of the clam angiotensin converting enzyme inhibitory peptide is as follows: Trp-Pro-Met-Gly-Phe.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153055A (en) * | 2007-08-17 | 2008-04-02 | 华东理工大学 | Novel peptide with angiotonin transferase restraining liveness and method of producing the same |
| CN101845080A (en) * | 2010-01-08 | 2010-09-29 | 宁波大学 | Angiotensin converting enzyme inhibitory peptide and preparation method thereof |
| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN103242430A (en) * | 2013-05-31 | 2013-08-14 | 南京中医药大学 | Angiotensin-converting enzyme inhibitory peptide, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153055A (en) * | 2007-08-17 | 2008-04-02 | 华东理工大学 | Novel peptide with angiotonin transferase restraining liveness and method of producing the same |
| CN101845080A (en) * | 2010-01-08 | 2010-09-29 | 宁波大学 | Angiotensin converting enzyme inhibitory peptide and preparation method thereof |
| CN102586374A (en) * | 2012-01-17 | 2012-07-18 | 广西大学 | Angiotensin-converting enzyme inhibitory peptide and preparation method thereof |
| CN103242430A (en) * | 2013-05-31 | 2013-08-14 | 南京中医药大学 | Angiotensin-converting enzyme inhibitory peptide, and preparation method and application thereof |
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