CN105203671B - The high performance liquid chromatography and gel permeation chromatography isolation and purification method of marine organisms source polypeptide - Google Patents
The high performance liquid chromatography and gel permeation chromatography isolation and purification method of marine organisms source polypeptide Download PDFInfo
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- CN105203671B CN105203671B CN201510743823.8A CN201510743823A CN105203671B CN 105203671 B CN105203671 B CN 105203671B CN 201510743823 A CN201510743823 A CN 201510743823A CN 105203671 B CN105203671 B CN 105203671B
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Abstract
The present invention relates to a kind of high performance liquid chromatography of marine organisms source polypeptide and gel permeation chromatography isolation and purification method, by choosing Urechis uniconctus internal organ, after broken, prepare Urechis uniconctus visceral meal, digested after concentration, concentrated frozen is dried and uses the gel chromatography column chromatographies of Sephadex G 15, and high performance liquid chromatography separation purifies to obtain Gln Pro Met Thr Phe.It is an advantage of the invention that extraction, purifying process is more advanced, quick, the degree of accuracy is high, obtained polypeptide A CE inhibitory activity is notable.
Description
Technical field
The present invention relates to a kind of isolation and purification method of marine bioactivity polypeptide, more particularly to marine organisms source polypeptide
High performance liquid chromatography and gel permeation chromatography isolation and purification method.
Background technology
Urechis uniconctus (Urechis unicinctus), popular name sea intestines, extra large intestines, belong to Echiuroidea, Yi guiding principles, nothing
Pipe Yi mesh, thorn Yi sections.Body is thick, is about 100-300 mm, wide about 25-27 mm, long 200mm-250 mm, and body surface is abound with size
The granular projection not waited, kiss cone；Neuroseta 1 is right, thick；Perianal has a circle 9-13 bar brown caudal setas.It is distributed in
Russia, Japan, Korea and China's coastal zone along the Huanghai Sea and the Bohai Sea, are area and subtidal zone phytal zone under the coastal silt bank intertidal zone of northern China
Benthic Common Species.Its individual is loose, and meat flavour is delicious, and body wall flesh is rich in protein and a variety of essential amino acids.
Since ancient times, it is coastal as rare seafood in China, Japan and Korea, there is higher economic value.The biography of people
System way only eats its body wall, and discarded internal organ, dish are commonly called as extra large intestines.
The polypeptide of Urechis uniconctus extraction plays the role of certain antitumor and raising immune function of mice, the generation of tumour
It is the result of many reasons effect, but will be finally related to the expression regulation of oncogene.Different tumours is required when producing
The regulatory factor such as enzyme it is different, select specific small peptide to make to be less than regulatory factor required when tumour occurs etc., close its activity
Site, tumour can be prevented.It has now been found that many tumor-related genes and tumour produce regulatory factor, screening and these targets
Point specific bond polypeptide, it has also become find the new focus of cancer therapy drug, it is worth mentioning at this point that, traditional antineoplastic due to
Lack the selectivity to cancer cell, often produce adverse reaction while treatment.Anti-tumour active polypeptide unlike this, is killing
Immune system is shielded while hindering tumour cell.Having tested proves that Urechis uniconctus polypeptide suppresses human liver cancer in vitro
Cell growth, and mouse immunity can be strengthened, suppress the growth of mouse S 180 sarcoma.But due in recent years due to catching intensity
Excessive, wild resource is inadequate.Now, China has also carried out the production Journal of Sex Research of Urechis uniconctus artificial culture seed successively.
Patent of the present invention is related to carrying out Urechis uniconctus internal organ enzymolysis acquisition antihypertensive activity polypeptide, is Urechis uniconctus polypeptide active matter
The exploitation of matter provides important references.
The content of the invention
The technical problems to be solved by the invention are to provide one kind for the above-mentioned state of the art there is preferable ACE to suppress
The preparation method of the active peptides active, from Urechis uniconctus prepared using high performance liquid chromatography.
The preparation method of the active peptides prepared using high performance liquid chromatography is as follows：
1）Urechis uniconctus internal organ are chosen, after break process, are stayed overnight with acetone degreasing, supernatant discarding is centrifuged, is deposited in 35 DEG C
At to 40 DEG C Urechis uniconctus visceral meal is obtained after crushed after being dried；
2）Take step 1）Described Urechis uniconctus visceral meal is proportionally added into distilled water, in 80 DEG C of thermostat water bath plus
Centrifuging and taking supernatant, it is sample after obtained supernatant concentration after hot 4h；
3）Take step 2）Described sample is proportionally added into distilled water, then adjusts pH 6.8, adds papain,
Heated in 60 DEG C of water-baths, rear 100 DEG C of heating water bath 10min to 15 min enzyme deactivations, the 4500 rpm centrifugations 20 in centrifuge
Supernatant is taken after min, the supernatant is used into trypsin hydrolysis 2-4 h, it is 60 DEG C to control Extracting temperature, pH：10；
4）Step 3）PH is adjusted after the completion of extraction to 100 DEG C of min enzyme deactivations of heating water bath 10 to 15 after neutrality, with 4500 ~
Supernatant is taken after 6000rpm centrifugations；
5）By step 4）After handling obtained supernatant concentration, using 95% ethanol precipitation, make ethanol final concentration be about 70% ~
80%, 5h to 20h is stood in 4 DEG C, is then freeze-dried supernatant concentration to obtain Urechis uniconctus protein hydrolysate；
6）Biologically active peptide isolates and purifies：Described Urechis uniconctus protein hydrolysate is used into Sephadex G-15
Gel chromatography column chromatography, high performance liquid chromatography separation purify to obtain Gln-Pro-Met-Thr-Phe.
Sephadex G-15 gel chromatography column chromatographies contain following steps：
Described Urechis uniconctus protein hydrolysate is taken, 100 mg/ml solution, Sephadex G- are dissolved into distilled water
The cm of the cm of 15 gel chromatographic columnses 2.0 × 100 is isolated and purified, and mobile phase is distilled water, and elution speed is 0.5 ml/min, is received
Collect each peak.
High performance liquid chromatography is isolated and purified with following steps：
Further isolated and purified with high performance liquid chromatography.Chromatographic condition is as follows：Chromatographic column uses specification as 250 mm × 4.6
Mm, 5 μm of anti-phase C18 bonded silica gel columns；Mobile phase：A water, B acetonitriles；Main chromatographic peak is collected, it is lyophilized standby to obtain Gln-
Pro-Met-Thr-Phe。
Step 2）Described ratio is mass ratio 1：10 to 1：50 or mass volume ratio 50g：500mL to 50g：2500mL.
Step 3）Described ratio is mass ratio 1：1 to 1：5 or mass volume ratio 50g：50mL to 50g：2500mL.
Step 3）Described papain accounts for the 2% of sample solution mass fraction.
Compared with prior art, the advantage of the invention is that：Extraction, purifying process is more advanced, quick, the degree of accuracy is high, system
The polypeptide A CE inhibitory activity obtained is notable.
Brief description of the drawings
Fig. 1 is Gln-Pro-Met-Thr-Phe of the present invention mass spectrum（ESI-MS）Figure；
Fig. 2 is Gln-Pro-Met-Thr-Phe of the present invention structural formula；
Fig. 3 is inventive gel purifying figure；
Fig. 4 is liquid phase purifying figure of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
Embodiment：The biologically active peptide in Urechis uniconctus internal organ source, the specially small peptide with following sequence：Gln-Pro-
Met-Thr-Phe。
Its preparation process is as follows：（1）Internal organ with tissue pulper stir into homogenate after shredding.Acetone stirring degreasing is stayed overnight, from
Heart supernatant discarding, it is deposited in 40 DEG C of air dry oven and is crushed after drying overnight.Urechis uniconctus visceral meal is taken by 1:20（Quality
Volume ratio, 50 g：1000 mL）Distilled water is added, after 4h is heated in 80 DEG C of thermostat water bath, centrifuging and taking supernatant, is repeated
Above-mentioned steps three times, the supernatant finally given are laboratory sample, concentration.Sample is pressed 1：5（Mass volume ratio, 10 g：50
mL）Distilled water is added, then adjusts pH 6.8, adds 2%（Mass fraction）Papain heats 4 h in 60 DEG C of water-baths,
Afterwards with 100 DEG C of min enzyme deactivations of heating water bath 15,4500 rpm take supernatant after centrifuging 20 min in centrifuge.Then use
The h of trypsin hydrolysis 4,60 DEG C of Extracting temperature, pH：10, enzyme concentration 0.5%（Mass fraction）.PH is adjusted after the completion of extraction to neutrality
100 DEG C of min enzyme deactivations of heating water bath 15 afterwards, 4500 rpm take supernatant after centrifuging 20 min in centrifuge.It is concentrated under reduced pressure into original
Behind the 1/20 of volume, using 95% ethanol precipitation, it is about 80% to make ethanol final concentration, is stood overnight in 4 DEG C of refrigerators.Supernatant is dense
Contracting freeze-drying.
（2）Biologically active peptide isolates and purifies；Urechis uniconctus protein hydrolysate is used into Sephadex G-15 gel colors
Column chromatography is composed, high performance liquid chromatography separation purifies to obtain a new pentapeptide, and external activity shows that there is preferable ACE to suppress for it
Activity.
Isolating and purifying for biologically active peptide comprises the following steps：
（1）Sephadex G-15 gel chromatography column chromatographies；
Urechis uniconctus visceral protein enzymolysis product is taken to be dissolved into 100 mg/ml solution with distilled water, Sephadex G-15 coagulate
The cm of the cm of glue chromatographic column 2.0 × 100 is isolated and purified, and mobile phase is distilled water, and elution speed is 0.5 ml/min, automatic portion
Collector is divided to collect, often pipe collects 3mL, 280 nm measure absorbances, collects each peak, freezes standby.
（2）High performance liquid chromatography isolates and purifies；
Further isolated and purified with high performance liquid chromatography.Chromatographic condition is as follows：Chromatographic column is anti-phase C18 bonded silica gel columns
(Zorbax C18, 250 mm×4.6 mm, 5 μm)；Mobile phase：A water, B acetonitriles, condition of gradient elution see the table below 1；Flow velocity：
1.0 ml/min, 30 DEG C of column temperature, Detection wavelength are 254 nm and 280 nm, collect main chromatographic peak, lyophilized standby to obtain small peptide
Sample.Broken line in liquid phase purifying figure is illustrated for flow velocity, for reference.
The high performance liquid chromatography separation condition of table 1
| Time (min) | A (water, %) | B (acetonitrile, %) |
| 0 | 100 | 0 |
| 4 | 100 | 0 |
| 16 | 70 | 30 |
| 17 | 0 | 100 |
| 35 | 0 | 100 |
4. sequencing
Using amino acid sequence analysis instrument and mass spectrograph, amino acid sequence analysis is carried out to the component for collecting gained.
The measure of 5.ACE inhibitory activity
By Cushman-Cheung (1971) method slightly modified.Principle is 37 DEG C, under the conditions of pH 8.3, ACE catalysis
Hydrolyze angiotensinⅠ (Ang I) analogies hippuroyl histidyl- leucine (HHL) and produce hippuric acid (Hip), it is ultraviolet 228
There is characteristic absorption peak at nm, ACE inhibiting rates are calculated by product Hip change.Concrete operations are as follows：With 1.5 ml from
Heart pipe, blank control group add the mmol/l HHL of 100 μ l 5, and 120 μ l are complemented to borate buffer (pH 8.3), 37 DEG C
Water bath with thermostatic control is incubated 5 min, adds the U/ml ACE of 5 μ l 0.1 and starts reaction；Sample sets add the mmol/l HHL of 100 μ l 5
After 20 μ l hydrolyzates, 37 DEG C of waters bath with thermostatic control are incubated 5 min, add the U/mlACE of 5 μ l 0.1 and start reaction.Two groups afterwards
After equal 37 DEG C of constant temperature keeps 30 min, the μ l stopped reactions of 1 mol/L HCl 200 are added, add 175 μ l borate buffers.
With high effective liquid chromatography for measuring Hip contents.Corresponding peak area is respectively SControlAnd SSample。
ACE inhibiting rates (%)=(SControl- SSample) /SControl×100
1 isolated new biologically active peptide of the present invention, has preferable ACE inhibitory activity, its IC50For 8.80 ±
0.52 μg/ml.The biologically active peptide of the gained of the present invention is white powder, soluble in water.
The beneficial effects of the invention are as follows：Employ enzymolysis-column chromatography connection technology and deep level development has been carried out to jellyfish, specifically
It is embodied in：1. it is used as enzymolysis raw material using Urechis uniconctus internal organ；2. using exclusion chromatography and high performance liquid chromatography from monocyclic
Biologically active peptide is isolated and purified in thorn Yi enzymolysis products.3. the peptide has preferable external ACE inhibitory activity, available for preparing
Anti- hypertension health food.
Although describing the present invention with reference to preferred embodiment, so it is not limited to the present invention, any this area
Technical staff, without departing from the spirit and scope of the present invention, various change can be implemented to the theme listed herein
Become, the displacement and modification of coordinate, therefore protection scope of the present invention is worked as the scope limited depending on the claim proposed and is defined.
Claims (7)
1. the high performance liquid chromatography and gel permeation chromatography isolation and purification method of marine organisms source polypeptide, it is characterized in that：Including such as
Lower step：
1）Urechis uniconctus internal organ are chosen, after break process, are stayed overnight with acetone degreasing, supernatant discarding is centrifuged, is deposited in 35 DEG C to 40
Urechis uniconctus visceral meal is obtained at DEG C after crushed after being dried；
2）Take step 1）Described Urechis uniconctus visceral meal is proportionally added into distilled water, in heating 4h in 80 DEG C of thermostat water bath
Afterwards, centrifuging and taking supernatant, it is sample after obtained supernatant concentration；
3）Take step 2）Described sample is proportionally added into distilled water, then adjusts pH to 6.8, papain is added, 60
Heated in DEG C water-bath, use 100 DEG C of heating water bath 10min to 15 min enzyme deactivations afterwards, 4500 rpm centrifugations, 20 min in centrifuge
After take supernatant, the supernatant is used into trypsin hydrolysis 2-4 h, control Extracting temperature be 60 DEG C, pH 10；
4）Step 3）PH to 100 DEG C of heating water bath 10min after neutrality is adjusted after the completion of extraction to 15 min enzyme deactivations, with 4500 ~
Supernatant is taken after 6000rpm centrifugations；
5）By step 4）After handling obtained supernatant concentration, using 95% ethanol precipitation, make ethanol final concentration of 70% ~ 80%, in
Supernatant concentration, is then freeze-dried to obtain Urechis uniconctus protein hydrolysate by 4 DEG C of standing 5h to 20h；
6）Biologically active peptide isolates and purifies：Described Urechis uniconctus protein hydrolysate is used into Sephadex G-15 gels
Column chromatography, high performance liquid chromatography separation purify to obtain Gln-Pro-Met-Thr-Phe.
2. high performance liquid chromatography and the gel permeation chromatography side of isolating and purifying of marine organisms source according to claim 1 polypeptide
Method, it is characterized in that：Described Sephadex G-15 gel chromatography column chromatographies contain following steps：Take described Urechis uniconctus egg
White enzymolysis product, 100 mg/ml solution are dissolved into distilled water, the cm of the cm of Sephadex G-15 gel chromatographic columnses 2.0 × 100
Isolated and purified, mobile phase is distilled water, and elution speed is 0.5 ml/min, collects each peak.
3. high performance liquid chromatography and the gel permeation chromatography side of isolating and purifying of marine organisms source according to claim 1 polypeptide
Method, it is characterized in that：High performance liquid chromatography is isolated and purified with following steps：Further isolated and purified with high performance liquid chromatography.
4. high performance liquid chromatography and the gel permeation chromatography side of isolating and purifying of marine organisms source according to claim 1 polypeptide
Method, it is characterized in that：The chromatographic condition is as follows：Chromatographic column uses specification as the mm of 250 mm × 4.6,5 μm of anti-phase C18 keys
Close silicagel column；Mobile phase：A water, B acetonitriles；Main chromatographic peak is collected, it is lyophilized standby to obtain Gln-Pro-Met-Thr-Phe.
5. high performance liquid chromatography and the gel permeation chromatography side of isolating and purifying of marine organisms source according to claim 1 polypeptide
Method, it is characterized in that：Step 2）Described ratio is mass ratio 1：10 to 1：50 or mass volume ratio 50g：500mL to 50g：
2500mL。
6. high performance liquid chromatography and the gel permeation chromatography side of isolating and purifying of marine organisms source according to claim 1 polypeptide
Method, it is characterized in that：Step 3）Described ratio is mass ratio 1：1 to 1：5 or mass volume ratio 50g：50mL to 50g：2500mL.
7. high performance liquid chromatography and the gel permeation chromatography side of isolating and purifying of marine organisms source according to claim 1 polypeptide
Method, it is characterized in that：Step 3）Described papain accounts for the 2% of sample solution mass fraction.
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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 |
| CN111876458B (en) * | 2020-07-23 | 2022-10-11 | 大连海洋大学 | Urechis unicinctus antioxidant peptide and preparation 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 |
| 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 |
| 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 |
| CN113528605B (en) * | 2021-09-16 | 2021-12-10 | 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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