CN105203671A - HPLC (high performance liquid chromatography) and GPC (gel permeation chromatography) based separation and purification method for marine organism sourced polypeptide - Google Patents
HPLC (high performance liquid chromatography) and GPC (gel permeation chromatography) based separation and purification method for marine organism sourced polypeptide Download PDFInfo
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Abstract
The invention relates to an HPLC (high performance liquid chromatography) and GPC (gel permeation chromatography) based separation and purification method for marine organism sourced polypeptide. According to the method, urechis unicinctus viscera are selected and crushed, and urechis unicinctus visceral meal is prepared; the urechis unicinctus visceral meal is subjected to enzymatic hydrolysis after concentration; a product of the urechis unicinctus visceral meal subjected to enzymatic hydrolysis is concentrated, frozen and dried and then is subjected to Sephadex G-15 gel chromatography and HPLC separation and purification, and Gln-Pro-Met-Thr-Phe is obtained. The method has the advantages that an extraction and purification technology is more advanced, extraction and purification are fast and high in accuracy, and ACE (angiotensin I-converting enzyme) inhibition activity of the prepared polypeptide is remarkable.
Description
Technical field
The present invention relates to a kind of isolation and purification method of marine bioactivity polypeptide, particularly relate to high performance liquid chromatography and the gel permeation chromatography isolation and purification method of sea life source polypeptide.
Background technology
Urechis uniconctus (Urechisunicinctus), popular name sea intestines, extra large intestines, belong to Echiuroidea, Yi guiding principle, without pipe Yi order, sting Yi section.Body is thick, is about 100-300mm, wide about 25-27mm, long 200mm-250mm, and body surface is abound with the granular projection differed in size, and kiss is conical; Neuroseta 1 is right, thick; Perianal has a circle 9-13 bar brown caudal seta.Being distributed in Russia, Japan, Korea and China's coastal zone along the Huanghai Sea and the Bohai Sea, is the benthic Common Species of northern China coastal silt bank mesolittoral zone inferior segment and subtidal zone shoal water zone.It is individual loose, and meat flavour is delicious, and body wall flesh is rich in protein and multiple essential amino acid.Since ancient times, coastal all as famous and precious seafood in China, Japan and Korea, there is higher economic worth.Traditional method only its body wall edible of people, and discarded internal organ, dish is commonly called as extra large intestines.
The polypeptide that Urechis uniconctus extracts has certain antitumor and improve the effect of immune function of mice, tumour be the result of many reasons effect, but finally all to relate to the expression regulation of oncogene.The regulatory factors such as enzyme required when different tumours produces are different, select the little peptide of specificity to do to be less than regulatory factor etc. required when tumour occurs, close its avtive spot, can prevent tumour from occurring.Find now that a lot of tumor-related gene and tumour produce regulatory factor, the polypeptide of screening and these target spot specific bond, become the new focus finding cancer therapy drug, it is worth mentioning that, traditional antineoplastic, owing to lacking the selectivity to cancer cell, often produces bad reaction while treatment.Anti-tumour active polypeptide unlike this, shields to immune system while killing tumor cell.Existing experiment proves that Urechis uniconctus polypeptide suppresses hepatoma cell growth in vitro, and can strengthen mouse immunity, suppresses the growth of mouse S 180 sarcoma.But due in recent years because catching intensity is excessive, wild resource is inadequate.Now, China has also carried out the productivity research of Urechis uniconctus artificial culture seed successively.Patent of the present invention relates to carries out enzymolysis acquisition antihypertensive activity polypeptide, for the exploitation of Urechis uniconctus polypeptide active substance provides important references to Urechis uniconctus internal organ.
Summary of the invention
Technical matters to be solved by this invention is the preparation method of the active peptides providing a kind of employing high performance liquid chromatography that have good ACE inhibitory activity, that be derived from Urechis uniconctus to prepare for the above-mentioned state of the art.
The preparation method of active peptides prepared by this employing high performance liquid chromatography is as follows:
1) choose Urechis uniconctus internal organ, after break process, spend the night with acetone degreasing, centrifugal supernatant discarded, obtains Urechis uniconctus visceral meal after crushed after being dried at being deposited in 35 DEG C to 40 DEG C;
2) the Urechis uniconctus visceral meal got described in step 1) adds distilled water in proportion, and heat 4h in the thermostat water bath of 80 DEG C after, centrifuging and taking supernatant is sample after the supernatant concentration obtained;
3) get step 2) described in sample add distilled water in proportion, then pH6.8 is regulated, add papain, heat in 60 DEG C of water-baths, to go out enzyme with 100 DEG C of heating water bath 10min to 15min afterwards, get supernatant after the centrifugal 20min of 4500rpm in centrifuges, this supernatant is adopted trypsin hydrolysis 2-4h, controlling Extracting temperature is 60 DEG C, pH:10;
4) step 3) has been extracted rear tune pH to neutral rear 100 DEG C of heating water bath 10 to 15min and have been gone out enzyme, gets supernatant after centrifugal with 4500 ~ 6000rpm;
5) after supernatant concentration step 4) process obtained, use 95% alcohol settling, make ethanol final concentration be about 70% ~ 80%, in 4 DEG C of standing 5h to 20h, then supernatant concentration freeze drying is obtained Urechis uniconctus protein hydrolysate;
6) separation and purification of biologically active peptide: described Urechis uniconctus protein hydrolysate is adopted SephadexG-15 gel chromatography column chromatography, high performance liquid chromatography separation and purification obtains
gln-Pro-Met-Thr-Phe.
SephadexG-15 gel chromatography column chromatography is containing, for example lower step:
Get described Urechis uniconctus protein hydrolysate, be dissolved into 100mg/ml solution with distilled water, SephadexG-15 gel chromatographic columns 2.0cm × 100cm carries out separation and purification, and mobile phase is distilled water, and elution speed is 0.5ml/min, collects each peak.
The separation and purification of high performance liquid chromatography has following steps:
With the further separation and purification of high performance liquid chromatography.Chromatographic condition is as follows: chromatographic column adopts specification to be 250mm × 4.6mm, the anti-phase C18 bonded silica gel post of 5 μm; Mobile phase: A water, B acetonitrile; Collect main chromatographic peak, freeze-drying is for subsequent use to be obtained
gln-Pro-Met-Thr-Phe.
Step 2) described in ratio be mass ratio 1:10 to 1:50 or mass volume ratio 50g:500mL to 50g:2500mL.
Ratio described in step 3) is mass ratio 1:1 to 1:5 or mass volume ratio 50g:50mL to 50g:2500mL.
Papain described in step 3) accounts for 2% of sample solution massfraction.
Compared with prior art, the invention has the advantages that: extract, purifying process is more advanced, quick, accuracy is high, obtained polypeptide A CE inhibit activities is remarkable.
Accompanying drawing explanation
Fig. 1 is the present invention
gln-Pro-Met-Thr-Phemass spectrum (ESI-MS) figure;
Fig. 2 is the present invention
gln-Pro-Met-Thr-Phestructural formula;
Fig. 3 is inventive gel purifying figure;
Fig. 4 is liquid phase purifying figure of the present invention.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Embodiment: the biologically active peptide in Urechis uniconctus internal organ source, is specially the small peptide with following sequence:
gln-Pro-Met-Thr-Phe.
Its preparation process is as follows: (1) internal organ shred rear with organizing pulper to stir into homogenate.Acetone stirs degreasing and spends the night, centrifugal supernatant discarded, dries after spending the night and pulverize in the air dry oven being deposited in 40 DEG C.Get Urechis uniconctus visceral meal by 1:20(mass volume ratio, 50g:1000mL) add distilled water, heat 4h in the thermostat water bath of 80 DEG C after, centrifuging and taking supernatant, in triplicate above-mentioned steps, the supernatant finally obtained is laboratory sample, concentrated.Sample is pressed 1:5(mass volume ratio, 10g:50mL) adding distilled water, then regulate pH6.8, add 2%(massfraction) papain heats 4h in 60 DEG C of water-baths, to go out enzyme with 100 DEG C of heating water bath 15min afterwards, get supernatant after the centrifugal 20min of 4500rpm in centrifuges.Then trypsin hydrolysis 4h is adopted, Extracting temperature 60 DEG C, pH:10, enzyme concentration 0.5%(massfraction).Adjust pH to neutral rear 100 DEG C of heating water bath 15min to go out enzyme after extraction completes, get supernatant after the centrifugal 20min of 4500rpm in centrifuges.After being evaporated to 1/20 of original volume, use 95% alcohol settling, make ethanol final concentration be about 80%, place in 4 DEG C of refrigerators and spend the night.Supernatant concentrated frozen is dry.
(2) separation and purification of biologically active peptide; Urechis uniconctus protein hydrolysate is adopted SephadexG-15 gel chromatography column chromatography, high performance liquid chromatography separation and purification obtains a new pentapeptide, and external activity shows it and has good ACE inhibitory activity.
The separation and purification of biologically active peptide comprises the steps:
(1) SephadexG-15 gel chromatography column chromatography;
Get Urechis uniconctus visceral protein enzymolysis product distilled water and be dissolved into 100mg/ml solution, SephadexG-15 gel chromatographic columns 2.0cm × 100cm carries out separation and purification, mobile phase is distilled water, elution speed is 0.5ml/min, automatic fraction collector is collected, and often pipe is collected 3mL, 280nm and measured absorbance, collect each peak, freeze-drying is for subsequent use.
(2) separation and purification of high performance liquid chromatography;
With the further separation and purification of high performance liquid chromatography.Chromatographic condition is as follows: chromatographic column is anti-phase C18 bonded silica gel post (ZorbaxC18,250mm × 4.6mm, 5 μm); Mobile phase: A water, B acetonitrile, condition of gradient elution sees the following form 1; Flow velocity: 1.0ml/min, column temperature 30 DEG C, determined wavelength is 254nm and 280nm, collects main chromatographic peak, and freeze-drying is for subsequent use obtains small peptide sample.Broken line in liquid phase purifying figure is flow velocity signal, for reference.
Table 1 high performance liquid chromatography separation condition
| Time (min) | A (water, %) | B (acetonitrile, %) |
| 0 | 100 | 0 |
| 4 | 100 | 0 |
| 16 | 70 | 30 |
| 17 | 0 | 100 |
| 35 | 0 | 100 |
4. sequencing
Utilize amino acid sequence analysis instrument and mass spectrometer, amino acid sequence analysis is carried out to the component of collecting gained.
The mensuration of 5.ACE inhibit activities
By Cushman-Cheung (1971) method slightly modified.Principle is 37 DEG C, under pH8.3 condition, ACE catalyzing hydrolysis angiotensinⅠ (Ang I) analogies hippuroyl histidyl-leucine (HHL) produces hippuric acid (Hip), it has characteristic absorption peak at ultraviolet 228nm place, by the change calculations ACE inhibiting rate of product Hip.Concrete operations are as follows: use 1.5ml centrifuge tube, and blank group adds 100 μ l5mmol/lHHL, complement to 120 μ l with borate buffer (pH8.3), and 37 DEG C of water bath with thermostatic control insulation 5min, add 5 μ l0.1U/mlACE and start reaction; After sample sets adds 100 μ l5mmol/lHHL and 20 μ l hydrolyzates, 37 DEG C of water bath with thermostatic control insulation 5min, add 5 μ l0.1U/mlACE and start reaction.After two groups of equal 37 DEG C of constant temperature keep 30min afterwards, add 1mol/LHCl200 μ l stopped reaction, add 175 μ l borate buffers.With high effective liquid chromatography for measuring Hip content.Corresponding peak area is respectively S
contrastand S
sample.
ACE inhibiting rate (%)=(S
contrast-S
sample)/S
contrast× 100
The present invention is separated the new biologically active peptide of 1 of obtaining, and has good ACE inhibitory activity, its IC
50be 8.80 ± 0.52 μ g/ml.The biologically active peptide of gained of the present invention is white powder, soluble in water.
The invention has the beneficial effects as follows: have employed enzymolysis-column chromatography connection technology and deep level development has been carried out to jellyfish, has been embodied in: 1. using Urechis uniconctus internal organ as enzymolysis raw material; 2. adopt exclusion chromatography and high performance liquid chromatography separation and purification biologically active peptide from Urechis uniconctus enzymolysis product.3. this peptide has good external ACE inhibitory activity, can be used for preparing anti-hypertension health food.
Although describe the present invention in conjunction with preferred embodiment; so itself and be not used to limit the present invention; any those skilled in the art; without departing from the spirit and scope of the present invention; can implement various change, the displacement of coordinator and amendment here to the theme listed, therefore protection scope of the present invention be as the criterion when the scope limited depending on proposed claim.
Claims (7)
1. the high performance liquid chromatography of sea life source polypeptide and gel permeation chromatography isolation and purification method, is characterized in that: comprise the steps:
1) choose Urechis uniconctus internal organ, after break process, spend the night with acetone degreasing, centrifugal supernatant discarded, obtains Urechis uniconctus visceral meal after crushed after being dried at being deposited in 35 DEG C to 40 DEG C;
2) the Urechis uniconctus visceral meal got described in step 1) adds distilled water in proportion, and heat 4h in the thermostat water bath of 80 DEG C after, centrifuging and taking supernatant is sample after the supernatant concentration obtained;
3) get step 2) described in sample add distilled water in proportion, then pH6.8 is regulated, add papain, heat in 60 DEG C of water-baths, to go out enzyme with 100 DEG C of heating water bath 10min to 15min afterwards, get supernatant after the centrifugal 20min of 4500rpm in centrifuges, this supernatant is adopted trypsin hydrolysis 2-4h, controlling Extracting temperature is 60 DEG C, pH:10;
4) step 3) has been extracted rear tune pH to neutral rear 100 DEG C of heating water bath 10 to 15min and have been gone out enzyme, gets supernatant after centrifugal with 4500 ~ 6000rpm;
5) after supernatant concentration step 4) process obtained, use 95% alcohol settling, make ethanol final concentration be about 70% ~ 80%, in 4 DEG C of standing 5h to 20h, then supernatant concentration freeze drying is obtained Urechis uniconctus protein hydrolysate;
6) separation and purification of biologically active peptide: described Urechis uniconctus protein hydrolysate is adopted SephadexG-15 gel chromatography column chromatography, high performance liquid chromatography separation and purification obtains
gln-Pro-Met-Thr-Phe.
2. the high performance liquid chromatography of sea life source according to claim 1 polypeptide and gel permeation chromatography isolation and purification method, is characterized in that: described SephadexG-15 gel chromatography column chromatography is containing, for example lower step:
Get described Urechis uniconctus protein hydrolysate, be dissolved into 100mg/ml solution with distilled water, SephadexG-15 gel chromatographic columns 2.0cm × 100cm carries out separation and purification, and mobile phase is distilled water, and elution speed is 0.5ml/min, collects each peak.
3. the high performance liquid chromatography of sea life source according to claim 1 polypeptide and gel permeation chromatography isolation and purification method, is characterized in that: the separation and purification of high performance liquid chromatography has following steps:
With the further separation and purification of high performance liquid chromatography.
4. chromatographic condition is as follows: chromatographic column adopts specification to be 250mm × 4.6mm, the anti-phase C18 bonded silica gel post of 5 μm; Mobile phase: A water, B acetonitrile; Collect main chromatographic peak, freeze-drying is for subsequent use to be obtained
gln-Pro-Met-Thr-Phe.
5. the high performance liquid chromatography of sea life source according to claim 1 polypeptide and gel permeation chromatography isolation and purification method, is characterized in that: step 2) described in ratio be mass ratio 1:10 to 1:50 or mass volume ratio 50g:500mL to 50g:2500mL.
6. the high performance liquid chromatography of sea life source according to claim 1 polypeptide and gel permeation chromatography isolation and purification method, is characterized in that: the ratio described in step 3) is mass ratio 1:1 to 1:5 or mass volume ratio 50g:50mL to 50g:2500mL.
7. the high performance liquid chromatography of sea life source according to claim 1 polypeptide and gel permeation chromatography isolation and purification method, is characterized in that: the papain described in step 3) accounts for 2% of sample solution massfraction.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106086129A (en) * | 2016-04-18 | 2016-11-09 | 浙江省海洋水产研究所 | A kind of preparation method of monocycle thorn visceral protein source zinc chelating peptide |
| CN107475341A (en) * | 2017-10-09 | 2017-12-15 | 赵德润 | A kind of preparation method of extra large intestines active peptide |
| CN107880090A (en) * | 2017-11-13 | 2018-04-06 | 青岛农业大学 | A kind of method that the anti-enzymes of BACE 1 of isolate are extracted from Urechis uniconctus |
| CN111876458A (en) * | 2020-07-23 | 2020-11-03 | 大连海洋大学 | Urechis unicinctus antioxidant peptide and preparation and application thereof |
| CN112457373A (en) * | 2020-12-17 | 2021-03-09 | 浙江海洋大学 | Urechis unicinctus polypeptide with angiotensin converting enzyme inhibitory activity and application thereof |
| CN112501230A (en) * | 2020-12-17 | 2021-03-16 | 浙江海洋大学 | Preparation method and application of urechis unicinctus ACE inhibitory peptide |
| CN113186243A (en) * | 2021-06-23 | 2021-07-30 | 大洲新燕(厦门)生物科技有限公司 | Method for extracting sea cucumber polypeptide from sea cucumber viscera |
| CN113528605A (en) * | 2021-09-16 | 2021-10-22 | 中国农业大学 | Method for preparing urechis unicinctus viscera antioxidant peptide by ultrasonic-assisted enzymolysis |
| CN114354798A (en) * | 2021-12-30 | 2022-04-15 | 广州广电计量检测无锡有限公司 | Method for separating and determining free amino acid in peptide product based on two-dimensional chromatography and application thereof |
| CN114657228A (en) * | 2022-05-17 | 2022-06-24 | 中国科学院烟台海岸带研究所 | Method for extracting ACE inhibitory peptide from urechis unicinctus and application of ACE inhibitory peptide |
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2015
- 2015-11-05 CN CN201510743823.8A patent/CN105203671B/en not_active Expired - Fee Related
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106086129A (en) * | 2016-04-18 | 2016-11-09 | 浙江省海洋水产研究所 | A kind of preparation method of monocycle thorn visceral protein source zinc chelating peptide |
| CN107475341A (en) * | 2017-10-09 | 2017-12-15 | 赵德润 | A kind of preparation method of extra large intestines active peptide |
| CN107880090A (en) * | 2017-11-13 | 2018-04-06 | 青岛农业大学 | A kind of method that the anti-enzymes of BACE 1 of isolate are extracted from Urechis uniconctus |
| CN111876458A (en) * | 2020-07-23 | 2020-11-03 | 大连海洋大学 | Urechis unicinctus antioxidant peptide and preparation and application thereof |
| CN111876458B (en) * | 2020-07-23 | 2022-10-11 | 大连海洋大学 | Urechis unicinctus antioxidant peptide and preparation and application thereof |
| CN112457373A (en) * | 2020-12-17 | 2021-03-09 | 浙江海洋大学 | Urechis unicinctus polypeptide with angiotensin converting enzyme inhibitory activity and application thereof |
| CN112501230A (en) * | 2020-12-17 | 2021-03-16 | 浙江海洋大学 | Preparation method and application of urechis unicinctus ACE inhibitory peptide |
| CN113186243A (en) * | 2021-06-23 | 2021-07-30 | 大洲新燕(厦门)生物科技有限公司 | Method for extracting sea cucumber polypeptide from sea cucumber viscera |
| CN113528605A (en) * | 2021-09-16 | 2021-10-22 | 中国农业大学 | Method for preparing urechis unicinctus viscera antioxidant peptide by ultrasonic-assisted enzymolysis |
| CN114354798A (en) * | 2021-12-30 | 2022-04-15 | 广州广电计量检测无锡有限公司 | Method for separating and determining free amino acid in peptide product based on two-dimensional chromatography and application thereof |
| CN114657228A (en) * | 2022-05-17 | 2022-06-24 | 中国科学院烟台海岸带研究所 | Method for extracting ACE inhibitory peptide from urechis unicinctus and application of ACE inhibitory peptide |
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