CN101148658A - Double aqueous phase method for extracting superoxide dismutase from animal blood - Google Patents
Double aqueous phase method for extracting superoxide dismutase from animal blood Download PDFInfo
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- CN101148658A CN101148658A CNA2007100499560A CN200710049956A CN101148658A CN 101148658 A CN101148658 A CN 101148658A CN A2007100499560 A CNA2007100499560 A CN A2007100499560A CN 200710049956 A CN200710049956 A CN 200710049956A CN 101148658 A CN101148658 A CN 101148658A
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Abstract
The present invention is new process of extracting SOD from animal blood, and belongs to the field of biochemical technology. The available extraction process has the demerits of overuse of organic solvent causing environmental pollution and low safety, high product cost, long production period, etc. The new process of the present invention includes the following steps: adding anticoagulant to blood and adding protectant comprising lactose in 1-2 %, beta-cyclodextrin in 0.2-1 % and polyglycol-6000 in 0.1-1 %; adding the treated blood to solution comprising PEG400 in 15-22 % and (NH4)2SO4 in 15-22 % through stirring; letting standing for blood to delaminate, taking the supernatant, adding K2HPO4 in 20-50 % through stirring and standing for 30 min; taking the supernatant, chromatographically separating, freeze drying to obtain SOD and distilling the lower layer liquid to recover PEG.
Description
One, technical field:
Patent of the present invention: the method that animal blood double water-phase extracts superoxide-dismutase belongs to biochemical field
Two, technical background:
Superoxide-dismutase is called for short SOD, and English is Super Oxide Dismutase, extensively be present in the various organisms, and be a kind of acidic protein, on the enzyme molecule, the metal prothetic group is arranged by covalent bonds.Can be divided into three kinds according to metal prothetic group composition difference, i.e. CuZn-SOD, Mn-SOD and Fe-SOD.SOD is the enzyme of free radical in unique energy scavenger cell, free radical is to have unpaired electron, atom or ion, its chemical property is active, high oxidation susceptibility is arranged, to capture the electronics of biomolecules such as nucleic acid, amino acid, make these physical properties be evolved into the stronger hydroxy radical qiao of toxicity, can cause the multiple disease of body.Studies show that the aging of body, pathology and radiation injury are all relevant with the form of free radical, so that SOD has is anti-ageing, the function of radioprotective, anti-inflammatory, inhibition tumour and cancer.Research also shows, SOD has unique curative effect to stomach trouble, trachitis, tetter, burn, beriberi etc., to sober up, excited spirit, antifatigue, regaining one's strength, losing weight also has good effect, also brought into use SOD in industries such as makeup, food, healthcare products, medicine, drinks, beverages at present, its development prospect is very wide.China also makes encouraging progress having done big quantity research aspect the extraction SOD, but present extraction process exists a large amount of organic solvents of use, and be difficult to recovery, contaminate environment and safety, the cost of product is higher, and the production cycle is longer, the not high weak point of productive rate.At above weak point, we have done a large amount of tests with reference to relevant document, find that emerging aqueous two phase extraction technique carries out system to traditional technology and improve good effect is arranged.Double water-phase be meant between some superpolymer or superpolymer and inorganic salt between in water, mix and form immiscible two-phase with certain concentration.Owing to carrying out method of extraction in the difference of two alternate partition ratios, solute is aqueous two-phase extraction.Utilize the technical process of SOD in the aqueous two-phase extraction animal blood to be: animal blood → protection → fragmentation → aqueous two-phase extraction → reextraction → column chromatography → freeze-drying → elaboration SOD.
Three, summary of the invention:
(1), superoxide-dismutase double water-phase extraction process flow process is:
Fresh pig blood---→ check and accept---→ metering---→ cooling---→ anti-freezing---→ protection---→ molten film---→ broken hemocyte-→ cooling---→ aqueous two-phase extraction---→ reextraction---→ chromatography---→ lyophilize-→ quality inspection---→ finished product
(2), major equipment:
Water treatment machine, crusher, separating centrifuge, jacketed kettle, extracter, distillation withdrawing can, temperature control retort, chromatography column, clarifixator, vacuum freeze-drier etc.
(3), operating parameters and operation instructions
1, gets the raw materials ready: the blood of collecting that the slaughterhouse slaughtered the same day through the healthy animal of animal test quarantine;
2, anti-freezing is handled: in the pig blood of collecting, in the ratio adding trisodium citrate of 3.8g/kg pig blood, and stir;
3, protective treatment: the lactose of adding blood weight 1.5%, 0.5% beta-cyclodextrin, 0.25% polyethylene glycol 6000 are as protective material;
4, molten film: the triton x-100 that adds blood weight 0.3% stirs as molten film;
5, smudge cells: the blood of handling well is carried out cytoclasis by high pressure homogenizer;
6, cooling: the blood that fragmentation is good cools off by interchanger;
7, extraction: cooled blood is passed through by PEG-(NH
4)
2SO
4The aqueous two-phase system of forming left standstill 15 minutes, the blood AUTOMATIC ZONING, and it is standby to draw supernatant liquid;
8, reextraction: press 24kg K
2HPO
4/ 100L supernatant liquor adds K
2HPO
4Carry out reextraction, draw supernatant and be used for chromatography, the PEG of lower floor recycles;
9, chromatography: DEAE Sephadex A-50 chromatography column carries out chromatography on the extraction liquid that step 8 extraction is obtained.The DEAE Sephadex A-50 that will handle well the in advance chromatography column (26 * 150cm) of packing into, buffer (2.0mmol/L PBS+0.1mmol/L EDTA) equilibrate overnight with pH7.6, behind the sample upper prop, carry out gradient elution with pH7.62.0～200mmol PBS (all adding 0.1mmol/L EDTA), flow rate control is at 5L/h.
10, drying: elutriant is collected postlyophilization, gets finished product.
Four, embodiment:
Embodiments of the invention are as follows:
Example (one)
1, gets the fresh pig blood of 50kg, behind the water-soluble trisodium citrate of adding 0.19kg100ml, stir;
2, add 0.75kg lactose, 0.25kg beta-cyclodextrin, 0.125kg polyethylene glycol 6000 in the pig blood that in step 1, stirs, and stir;
3, in step 2, add the 0.15kg triton x-100 in the blood after the stirring, and stir;
4, the blood that step 3 is stirred carries out cytoclasis by high pressure homogenizer;
5, with the broken good interchanger that passes through in the step 4, be cooled to about 5 ℃;
6, in 50kg distilled water, add 11kg PEG 400, stir, stand-by;
7, in 50kg distilled water, add 8.5kg (NH
4)
2SO
4, stir, stand-by;
8, the solution that step 6,7 is obtained mixes, and leaves standstill 10 minutes;
9, the smudge cells that step 5 is obtained is poured in the solution that step 8 obtains, and leaves standstill 15 minutes, takes out supernatant liquid then;
10, add 3kg K in the supernatant liquid that obtains toward step 9
2HPO
43H
2The O solid stirs, and leaves standstill 20 minutes, takes out supernatant liquid then;
11, DEAE Sephadex A-50 chromatography column carries out chromatography on the extraction liquid that step 10 extraction is obtained, and gets light blue liquid;
12, the light blue liquid that step 11 chromatography is obtained carries out freeze-drying, gets light blue solid, i.e. finished product 5.8g.
Example (two)
1, gets the fresh pig blood of 100kg, add, stir with behind the water-soluble 0.38kg trisodium citrate of 200ml;
2, add 1.5kg lactose, 0.5kg beta-cyclodextrin, 0.25kg polyethylene glycol 6000 in the pig blood that in step 1, stirs, and stir;
3, in step 2, add the 0.3kg triton x-100 in the blood after the stirring, and stir;
4, the blood that step 3 is stirred carries out cytoclasis by high pressure homogenizer;
5, with the broken good interchanger that passes through in the step 4, be cooled to about 5 ℃;
6, in 100kg distilled water, add 22kg PEG 400, stir, stand-by;
7, in 100kg distilled water, add 17kg (NH
4)
2SO
4, stir, stand-by;
8, the solution that step 6,7 is obtained mixes, and leaves standstill 10 minutes;
9, the smudge cells that step 5 is obtained is poured in the solution that step 8 obtains, and leaves standstill 15 minutes, keeps supernatant liquid then;
10, add 6kg K in the supernatant liquid that obtains toward step 9
2HPO
43H
2The O solid stirs, and leaves standstill 20 minutes, takes out supernatant liquid then;
11, DEAE Sephadex A-50 chromatography column carries out chromatography on the extraction liquid that step 10 extraction is obtained, and gets light blue liquid;
12, the light blue liquid that step 11 chromatography is obtained carries out freeze-drying, gets light blue solid, i.e. finished product 13.3g.
Claims (2)
1. in raw materials pretreatment, add composite protectant and preserve superoxide dismutase activity.It is characterized in that: add in the fresh blood with behind the water-soluble trisodium citrate, the lactose, 0.2-1% beta-cyclodextrin, 0.1-1% polyethylene glycol 6000 that add blood weight 1-2% again reduce the reduction of enzyme work in the post-treatment process as protective material.
2. in abstraction process, adopt the aqueous two-phase extraction method to carry out separation and Extraction, thereby promptly utilize the partition ratio of biochemical substances superoxide-dismutase in immiscible two-phase different and make target molecule be concentrated in a purpose that reaches purifying in mutually.It is characterized in that: with distilled water or pure water preparation 15-22%PEG400,15-22% (NH
4)
2SO
4Solution, the processing blood behind adding cooling, the rupture of membranes stirs, and leaves standstill 15-30 minute, and the blood AUTOMATIC ZONING is drawn supernatant liquid standby (is called: a time water extracts); The K that in supernatant liquor, adds the 20-50% weight of supernatant liquid measure
2HPO
4, stirring and dissolving is even, leaves standstill 20-40 minute, and liquid is divided into two-layer up and down automatically, draws supernatant liquid standby (be called: the secondary water extracts), and the subnatant body is used for distillation and reclaims PEG.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100499560A CN101148658A (en) | 2007-09-06 | 2007-09-06 | Double aqueous phase method for extracting superoxide dismutase from animal blood |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100499560A CN101148658A (en) | 2007-09-06 | 2007-09-06 | Double aqueous phase method for extracting superoxide dismutase from animal blood |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434580A (en) * | 2016-10-21 | 2017-02-22 | 南阳理工学院 | New method for extracting superoxide dismutase form plants |
| CN111372451A (en) * | 2017-10-19 | 2020-07-03 | 斯特雷克股份有限公司 | Compositions for hemolysis and coagulation regulation and stabilization of extracellular vesicles |
| US11299764B2 (en) | 2015-11-20 | 2022-04-12 | Streck, Inc. | Single spin process for blood plasma separation and plasma composition including preservative |
-
2007
- 2007-09-06 CN CNA2007100499560A patent/CN101148658A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11299764B2 (en) | 2015-11-20 | 2022-04-12 | Streck, Inc. | Single spin process for blood plasma separation and plasma composition including preservative |
| CN106434580A (en) * | 2016-10-21 | 2017-02-22 | 南阳理工学院 | New method for extracting superoxide dismutase form plants |
| CN111372451A (en) * | 2017-10-19 | 2020-07-03 | 斯特雷克股份有限公司 | Compositions for hemolysis and coagulation regulation and stabilization of extracellular vesicles |
| CN111372451B (en) * | 2017-10-19 | 2022-08-12 | 斯特雷克股份有限公司 | Compositions for hemolysis and coagulation regulation and stabilization of extracellular vesicles |
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Open date: 20080326 |