CN101747427A - Method for separating functional polypeptide from fish collagen - Google Patents
Method for separating functional polypeptide from fish collagen Download PDFInfo
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- CN101747427A CN101747427A CN 201010117306 CN201010117306A CN101747427A CN 101747427 A CN101747427 A CN 101747427A CN 201010117306 CN201010117306 CN 201010117306 CN 201010117306 A CN201010117306 A CN 201010117306A CN 101747427 A CN101747427 A CN 101747427A
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- isin glue
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Abstract
The invention relates to a method for separating functional polypeptide from fish collagen. The method comprises the following steps: (1) dissolving fish collagen in water to prepare collagen solution with a pH of 6.0 to 9.0; (2) placing the collagen solution on an affinity chromatography column and circulating an upper sample to the affinity chromatography column in order to adsorb the sample to reach saturation; (3) using eluent to leach out the polypeptide which is not combined with chromatography medium ligand; (4) using eluant to elute the polypeptide which is combined with affinity chromatography medium ligand and controlling elution temperature between 15 and 37 DEG C; (5) utilizing a gel filtration chromatography or a membrane ultrafiltration method to concentrate the obtained eluent and obtaining concentrated solution; and (6) freeze-drying the concentrated solution and obtaining white powder, namely the functional polypeptide in the fish collagen. The method utilizes fibronectin as the ligand, and adopts the affinity chromatography to separate part of polypeptide in the fish collagen, thereby having the advantages of reasonable steps, simple method, high resolution, strong characteristics and the like.
Description
Technical field
The present invention relates to technical field of bioengineering, relating in particular to a kind of is the method for raw material separating functional polypeptide with the Isin glue collagen.
Background technology
We know, Isin glue collagen is meant that with fish scale or fish-skin be the mixture that raw material adopts thermal treatment, acid/alkaline purification, enzymic degradation or degrades and obtain in conjunction with multiple mode, and its major ingredient is the inhomogenous polypeptides matter of molecular weight that forms behind the collagen degradation in fish scale or the fish-skin.Isin glue collagen can be used as functional food or healthcare products, have the absorption that improves body immunity, promote calcium, improve functions such as blood microcirculation and additional amino acid nutrient composition, effectively identification and the polypeptide component that separates difference in functionality are blind spots in the Isin glue collagen product development in recent years from Isin glue collagen.
The emphasis of traditional Isin glue collagen product development mainly concentrates on Isin glue collagen preparation technology and to products molecule weight range and Effect on Performance.Korea S Hee-GukByun utilize the method for ion-exchange chromatography and gel filtration chromatography from collagen protein, to isolate to have the polypeptide that suppresses hypertensin conversion enzyme activity (ProcessBiochemistry, 2001,36:1155-1162); Japan MaruyamaS isolates the polypeptide (Biochimi.Biophysi.Acta that can suppress biologically active pdgf from collagen protein; 1993,1164,215-223); Korea S KimSK adopts the method for exchange of gel filtration chromatography coupled ion and reverse-phase chromatography to isolate component (JAgricFoodChem, 2001,49 (4): 1984-9) with anti-oxidant function from fishskin gelatin.In traditional Isin glue collagen not the separation method of homopolypeptide mainly separate based on character such as molecular weight ranges, electrically charged states, have shortcomings such as the low and characteristic of resolving power is not strong.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned the deficiencies in the prior art, provide a kind of step rationally, have a strong method of separating functional polypeptide from Isin glue collagen of resolving power height and characteristic based on affinity chromatography.
The technical scheme that the present invention solves the problems of the technologies described above employing is: a kind of from Isin glue collagen the method for separating functional polypeptide, the steps include:
(1) Isin glue collagen is water-soluble, make collagen solution, and the pH value of this solution is transferred to 6.0-9.0;
(2) with affinity column on the collagen solution, sample to affine chromatography column adsorption sample reaches capacity in the circulation;
(3) use eluent not go out with the drip washing of chromatography media aglucon bonded polypeptide;
(4) use eluent to go out with affinity chromatography medium aglucon bonded polypeptide wash-out, the Controllable Temperature of elution process is built in 15-37 ℃;
(5) utilize the method for gel-filtration chromatography or membrane ultrafiltration that the elutriant that obtains is concentrated, obtain concentrated solution;
(6) with the concentrated solution lyophilize, the gained white powder is the functional polypeptide in the Isin glue collagen.
The present invention is based in the method for separating functional polypeptide from Isin glue collagen of affinity chromatography,, then save (1) described collagen protein dissolution process, directly the pH value of collagen solution is modulated 6.0-9.0 if collagen protein raw material to be separated is the aqueous solution.
Isin glue collagen of the present invention can be fish scale collagen or collagen of fish skin.
The aglucon of chromatography media can be a fibronectin in the affinity column of the present invention.
Eluent of the present invention can be to contain 2-8mol/L urea, the pH value damping fluid for 3.0-8.0, also can be the aqueous solution that contains the 5-50% alcoholic acid aqueous solution or 5-50% acetonitrile.If the aqueous solution that adopts the 5-50% alcoholic acid aqueous solution or 5-50% acetonitrile is as eluent, (5) the described method of gel-filtration chromatography or membrane ultrafiltration of utilizing of then saving concentrates the elutriant that obtains, obtain concentration process, elutriant directly carries out lyophilize.
Fibronectin can combine with the many polypeptid specificities in the collagen protein degradation product among the present invention, utilize fibronectin etc. as aglucon, the method of employing affinity chromatography is separated the part of polypeptide in the Isin glue collagen, against existing technologies, step of the present invention is reasonable, method is simple, has advantages such as resolving power height and characteristic are strong.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1:
A kind of from Isin glue collagen the method for separating functional polypeptide, its fish scale collagen aqueous solution with 2mg/ml is raw material, the method steps of separating functional polypeptide is from this fish scale collagen:
(1) pH with the raw material fish scale collagen aqueous solution transfers to 7.0;
(2) with sample on the fish scale collagen aqueous solution to affine chromatography column, circulation in sample extremely affine chromatography column adsorption sample reach capacity, that is to say until detecting collagen protein through in the liquid.Affinity column aglucon: human plasma fibronectin, column packing volume 40ml, packing material size: 60-120 micron;
(3) use 20ml0.2mol/L bicarbonate of ammonia as eluent drip washing chromatography column, flow velocity 1ml/min will not go out with the drip washing of chromatography media aglucon bonded polypeptide;
(4) use 20% ethanolic soln to wash chromatography column down, will go out, flow velocity 1ml/min, 30min component before collecting with affinity chromatography medium aglucon bonded polypeptide wash-out at 30 ℃;
(5) with the direct lyophilize of the component of collecting, the white powder of acquisition is the functional polypeptide in the fish scale collagen.
Utilize fibronectin etc. as aglucon among the present invention, adopt the method for affinity chromatography to separate part of polypeptide in the Isin glue collagen, step is reasonable, method is simple, has advantages such as the high and characteristic of resolving power is strong.
Embodiment 2:
A kind of from Isin glue collagen the method for separating functional polypeptide, the steps include:
(1) it is a raw material with the collagen of fish skin aqueous solution, and the pH of the raw material collagen of fish skin aqueous solution is modulated to 7.0, and concentration is controlled at 3-10mg/mL;
(2) with sample on the fish scale collagen aqueous solution to affine chromatography column, detect collagen protein to seeing through in the liquid.Affinity column aglucon behaviour plasma fibronectin connects albumen, column packing volume 40ml, packing material size: 60-120 micron;
(3) use 20ml0.2mol/L bicarbonate of ammonia as eluent drip washing chromatography column, flow velocity 1ml/min will not go out with the drip washing of chromatography media aglucon bonded polypeptide;
(4) use 8M urea cellulose solution (to contain 0.1NH
4HCO
3) wash chromatography column down at 20 ℃, will go out flow velocity 1ml/min, 35min component before collecting with affinity chromatography medium aglucon bonded polypeptide wash-out;
(5) elutriant collected is used gel permeation chromatography post (SephadexG10) remove urea element in the solution, obtain elutriant and concentrate;
(6) the direct lyophilize of the polypeptide after the desalination, the white powder of acquisition is the functional polypeptide in the fish scale collagen.
Utilize fibronectin etc. as aglucon among the present invention, adopt the method for affinity chromatography to separate part of polypeptide in the Isin glue collagen, step is reasonable, method is simple, has the advantages such as the high and characteristic of resolution ratio is strong.
Claims (4)
1. the method for a separating functional polypeptide from Isin glue collagen is characterized in that the steps include:
(1) Isin glue collagen is water-soluble, make collagen solution, and the pH value of this solution is transferred to 6.0-9.0;
(2) with affinity column on the collagen solution, sample to affine chromatography column adsorption sample reaches capacity in the circulation;
(3) use eluent not go out with the drip washing of chromatography media aglucon bonded polypeptide;
(4) use eluent to go out with affinity chromatography medium aglucon bonded polypeptide wash-out, the Controllable Temperature of elution process is built in 15-37 ℃;
(5) utilize the method for gel-filtration chromatography or membrane ultrafiltration that the elutriant that obtains is concentrated, obtain concentrated solution;
(6) with the concentrated solution lyophilize, the gained white powder is the functional polypeptide in the Isin glue collagen.
2. according to claim 1 from Isin glue collagen the method for separating functional polypeptide, it is characterized in that: said Isin glue collagen is fish scale collagen or collagen of fish skin.
3. according to claim 1 from Isin glue collagen the method for separating functional polypeptide, it is characterized in that: the aglucon of chromatography media is a fibronectin in the said affinity column.
4. according to claim 1 from Isin glue collagen the method for separating functional polypeptide, it is characterized in that: said eluent is to contain 2-8mol/L urea, pH value for the damping fluid of 3.0-8.0 or contain the 5-50% alcoholic acid aqueous solution or the aqueous solution of 5-50% acetonitrile.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010117306 CN101747427A (en) | 2010-03-04 | 2010-03-04 | Method for separating functional polypeptide from fish collagen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010117306 CN101747427A (en) | 2010-03-04 | 2010-03-04 | Method for separating functional polypeptide from fish collagen |
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| CN101747427A true CN101747427A (en) | 2010-06-23 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199192A (en) * | 2011-03-29 | 2011-09-28 | 中国科学院过程工程研究所 | Method for separating functional polypeptide from tortoise-shell glue |
| CN102199193A (en) * | 2011-03-28 | 2011-09-28 | 中国科学院过程工程研究所 | Method for separating functional polypeptide from antler glue based on affinity chromatography |
| CN103468771A (en) * | 2013-05-27 | 2013-12-25 | 河北考力森生物科技有限公司 | Method for extracting collagens from bovine achilles tendon |
-
2010
- 2010-03-04 CN CN 201010117306 patent/CN101747427A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 《Collagen Rel.Res.》 19811231 EVA ENGVALL等 Affinity Chromatography of Collagen on Collagen-Binding Fragments of Fibronectin 摘要、第507页倒数第3－4段、第511页倒数第2段－第512页第1段 1-4 第1卷, * |
| 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 19790710 Erkki Ruoslahti等 Isolation of a Tryptic Fragment Containing the Collagen-binding Site of Plasma Fibronectin 6054-6059 1-4 第254卷, 第13期 * |
| 《化学通报》 20021231 王碧等 色谱法在胶原蛋白分离分析中的应用（I） W70 1-4 第65卷, * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199193A (en) * | 2011-03-28 | 2011-09-28 | 中国科学院过程工程研究所 | Method for separating functional polypeptide from antler glue based on affinity chromatography |
| CN102199192A (en) * | 2011-03-29 | 2011-09-28 | 中国科学院过程工程研究所 | Method for separating functional polypeptide from tortoise-shell glue |
| CN103468771A (en) * | 2013-05-27 | 2013-12-25 | 河北考力森生物科技有限公司 | Method for extracting collagens from bovine achilles tendon |
| CN103468771B (en) * | 2013-05-27 | 2015-07-15 | 河北考力森生物科技有限公司 | Method for extracting collagens from bovine achilles tendon |
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Application publication date: 20100623 |