CN1916168A - Method for extracting superoxide dismutase from blood of cattle, sheep - Google Patents
Method for extracting superoxide dismutase from blood of cattle, sheep Download PDFInfo
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- CN1916168A CN1916168A CN 200610051207 CN200610051207A CN1916168A CN 1916168 A CN1916168 A CN 1916168A CN 200610051207 CN200610051207 CN 200610051207 CN 200610051207 A CN200610051207 A CN 200610051207A CN 1916168 A CN1916168 A CN 1916168A
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Abstract
This invention discloses a method for extracting superoxide dismutase from cow and sheep blood. The method comprises: subjecting fresh cow and sheep blood to anti-coagulation, precipitation, 3-6 stages of temperature gradient treatment, salting out, ultrafiltration, concentration and freeze-drying to obtain two groups of superoxide dismutase products with slightly different specific activity and purity. Compared with present techniques, the method has such advantages as simple process, short production period, high yield, and high product specific activity, and stable product quality, low cost and high profit.
Description
Technical field:
The present invention relates to the extracting method of a kind of superoxide-dismutase (SOD), particularly a kind of method of from ox, sheep blood, extracting superoxide-dismutase.
Background technology:
Superoxide-dismutase (Superoxide dismutase, be abbreviated as SOD) be a kind of metalloenzyme that can catalysis ultra-oxygen anion free radical disproportionation reaction, have delaying senility, strengthen hypoxia tolerance and antifatigue, effect such as radioprotective, promotion infant growth, be widely used in industries such as medicine, food, makeup.At present from ox, what the method for extraction superoxide-dismutase adopted usually in the sheep blood is to get fresh ox, the centrifugal collection erythrocyte of sheep blood, vigorous stirring makes its haemolysis, adding ammonium sulfate behind the haemolysis saltouts, the precipitation of collection 50% to 90%, utilize the supercentrifuge high speed centrifugation to remove most of foreign protein, utilize trichloromethane again, ethanol, acetone and other organic solvent precipitates, further remove foreigh protein removing, through oversalting, remove residual ammonium sulfate, pass through column chromatography system (generally all through twice column chromatography) at last again, could separate obtaining the SOD alive than height ratio.
But the technology more complicated of this method, the production cycle is long; Owing to used expensive column chromatography system, column packing and supercentrifuge, cost is higher, and through behind the column chromatography, the yield of product is reduced greatly.
Summary of the invention:
The objective of the invention is to: provide that a kind of technology is simple, with short production cycle, cost is low, product yield and higher than living, the stay-in-grade method of from ox, sheep blood, extracting superoxide-dismutase, thus overcome the deficiencies in the prior art.
The present invention is achieved in that it may further comprise the steps;
(1), in fresh bovine blood or sheep blood, add the antithrombotics account for blood weight 0.2-0.5%, be to place 30-100 minute under 4-8 ℃ the low temperature in temperature, treat the solution layering, obtain precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 40-50 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 10-30 minute, remove precipitation after centrifugal 10-30 minute, obtain supernatant liquor for 4000-6000 rev/min;
(3), above-mentioned supernatant liquor is carried out obtain supernatant liquor a after 3-6 level thermograde is handled;
(4), supernatant liquor a being carried out two-stage saltouts: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 45-70%, fully stirs ammonium sulfate is dissolved fully, and placement is after 12-36 hour under temperature is 4-8 ℃, remove precipitation after centrifugal 10-30 minute for 4000-6000 rev/min, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 85-95%, fully stir ammonium sulfate dissolved fully, temperature be 4-8 ℃ place 12-36 hour down after, 4000-6000 rev/min centrifugal 10-30 minute, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 4-8 ℃ in temperature to be placed 12-36 hour down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5), with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.05-0.2M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration fully, after concentrated and lyophilize, can obtain respectively than two groups of superoxide-dismutase finished products slightly different of living with purity.
Used antithrombotics is a Trisodium Citrate in the above-mentioned steps (1).
In the above-mentioned steps (3), when carrying out the processing of 3-6 level thermograde, elementary temperature is 40-50 ℃, the level temperature is 70-80 ℃ eventually, per two inter-stage thermograde differences are 5-10 ℃, it all is to be warming up to this grade temperature rapidly by room temperature earlier to keep reducing to room temperature more rapidly after 10-30 minute that every grade of gradient is handled, and carries out after every grade of cooling removing precipitation behind 4000-6000 rev/min centrifugally operated 10-30 minute.
Compared with prior art, production technique of the present invention is simple, and is with short production cycle, and cost is low, the yield of finished product enzyme and ratio high, steady quality alive.The ratio of enzyme is lived and is improved 10 times nearly in the whole heat-processed, and the yield of finished product enzyme can reach more than 96%, and dehematize red eggs white effect is good; Owing to adopted thermograde facture and two-stage to saltout, only use 4000-6000 rev/min low speed centrifuge can reach the purpose that the removal precipitation obtains high yield product, cost is lower; And can produce two kinds simultaneously than the product different of living with purity, the pharmaceutical industries that supply and demand wants high purity, height ratio to live, and do not need very height ratio makeup, food and health products trade alive.Through check, adopt the staging life of the high purity SOD enzyme freeze-drying prods that method of the present invention produces to reach more than 3 years; The ultraviolet light absorption spectrum of SOD enzyme: adopt the UV scanning of 730 type spectrophotometers to the SOD enzyme, the obtained the maximum absorption that records the Cu.Zn-SOD of ox blood is 261nm, A
261/ A
280=1.34, comply with the national standard requirements fully.
Embodiment:
Embodiments of the invention 1: the method for from ox blood, extracting superoxide-dismutase
(1), in the fresh ox blood of 10kg, add the sodium citrate anticoagulant of 20g, be to place 80 minutes under 6 ℃ the low temperature in temperature, treat the solution layering, obtain the 8500ml precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 50 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 15 minutes, remove precipitation after centrifugal 15 minutes, obtain the 5000ml supernatant liquor for 4000 rev/mins;
(3), the supernatant liquor that step (2) is obtained carries out 3 grades of thermogrades processing: the 5000ml supernatant liquor that step (2) is obtained is warming up to 40 ℃ rapidly by room temperature, keep reducing to rapidly again after 20 minutes room temperature, remove precipitation after centrifugal 10 minutes for 4000 rev/mins, obtain the supernatant liquor one of 4200ml; Supernatant liquor one is warming up to 50 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 15 minutes for 4000 rev/mins, obtain the supernatant liquor two of 3000ml; Supernatant liquor two is warming up to 60 ℃ rapidly by room temperature, keeps reducing to rapidly again after 10 minutes room temperature, remove precipitation after centrifugal 10 minutes, obtain 1500ml supernatant liquor a for 4500 rev/mins;
(4), supernatant liquor a carried out two-stage saltout: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 60%, fully stir ammonium sulfate is dissolved fully, temperature be 6 ℃ place 24 hours down after, remove precipitation after centrifugal 15 minutes for 4500 rev/mins, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 90%, fully stirring is dissolved ammonium sulfate fully, and placement is after 24 hours down to be 6 ℃ in temperature, and 5000 rev/mins centrifugal 10 minutes, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 6 ℃ in temperature to be placed 24 hours down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5), with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.10M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, after concentrated and lyophilize, can obtain purity and ratio two groups of slightly different superoxide-dismutase finished products alive respectively, the superoxide-dismutase finished product is the light green lyophilized powder, and its gross weight is about 7.5-10g.
Embodiments of the invention 2: the method for from ox blood, extracting superoxide-dismutase
(1), in the fresh ox blood of 10kg, add the sodium citrate anticoagulant of 30g, be to place 30 minutes under 4 ℃ the low temperature in temperature, treat the solution layering, obtain the 8500ml precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 40 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 10 minutes, remove precipitation after centrifugal 30 minutes, obtain the 5100ml supernatant liquor for 4000 rev/mins;
(3) supernatant liquor that step (2) is obtained carries out 4 grades of thermogrades processing: the 5100ml supernatant liquor that step (2) is obtained is warming up to 40 ℃ rapidly by room temperature, keep reducing to rapidly again after 10 minutes room temperature, remove precipitation after centrifugal 10 minutes, obtain 3600ml supernatant liquor one for 4000 rev/mins; Supernatant liquor one is warming up to 45 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 15 minutes, obtain 2500ml supernatant liquor two for 4000 rev/mins; Supernatant liquor two is warming up to 50 ℃ rapidly by room temperature, keeps reducing to rapidly again after 20 minutes room temperature, remove precipitation after centrifugal 15 minutes, obtain 1600ml supernatant liquor three for 4000 rev/mins; Supernatant liquor three is warming up to 55 ℃ rapidly by room temperature, keeps reducing to rapidly again after 30 minutes room temperature, remove precipitation after centrifugal 20 minutes, obtain 1000ml supernatant liquor a for 4500 rev/mins;
(4), supernatant liquor a being carried out two-stage saltouts: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 45-70%, fully stirs ammonium sulfate is dissolved fully, and placement is after 12 hours under temperature is 4 ℃, remove precipitation after centrifugal 10 minutes for 4000 rev/mins, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 85%, fully stirring is dissolved ammonium sulfate fully, and placement is after 12 hours down to be 4 ℃ in temperature, and 5000 rev/mins centrifugal 15 minutes, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 4 ℃ in temperature to be placed 12 hours down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5) with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.05M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, after concentrated and lyophilize, can obtain purity and ratio two groups of slightly different superoxide-dismutase finished products alive respectively, the superoxide-dismutase finished product is the light green lyophilized powder, and its gross weight is about 7.5-10g.
Embodiments of the invention 3: the method for from ox blood, extracting superoxide-dismutase
(1), in the fresh ox blood of 10kg, add the sodium citrate anticoagulant of 50g, be to place 100 minutes under 8 ℃ the low temperature in temperature, treat the solution layering, obtain the 8500ml precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 50 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 30 minutes, remove precipitation after centrifugal 10 minutes, obtain the 5300ml supernatant liquor for 6000 rev/mins;
(3) supernatant liquor that step (2) is obtained carries out 6 grades of thermogrades processing: the 5300ml supernatant liquor that step (2) is obtained is warming up to 50 ℃ rapidly by room temperature, keep reducing to rapidly again after 10 minutes room temperature, remove precipitation after 4000/ minute centrifugal 30 minutes, obtain 3900ml supernatant liquor one; Supernatant liquor one is warming up to 55 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 25 minutes, obtain 2800ml supernatant liquor two for 4500 rev/mins; Supernatant liquor two is warming up to 60 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 30 minutes, obtain 1800ml supernatant liquor three for 4000 rev/mins; Supernatant liquor three is warming up to 65 ℃ rapidly by room temperature, keeps reducing to rapidly again after 20 minutes room temperature, remove precipitation after centrifugal 15 minutes, obtain 1400ml supernatant liquor four for 5000 rev/mins; Supernatant liquor four is warming up to 70 ℃ rapidly by room temperature, keeps reducing to rapidly again after 25 minutes room temperature, remove precipitation after centrifugal 25 minutes, obtain 1000ml supernatant liquor five for 4500 rev/mins; Supernatant liquor five is warming up to 80 ℃ rapidly by room temperature, keeps reducing to rapidly again after 30 minutes room temperature, remove precipitation after centrifugal 10 minutes, obtain 800ml supernatant liquor a for 6000 rev/mins;
(4), supernatant liquor a carried out two-stage saltout: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 70%, fully stir ammonium sulfate is dissolved fully, temperature be 8 ℃ place 36 hours down after, remove precipitation after centrifugal 15 minutes for 5000 rev/mins, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 95%, fully stirring is dissolved ammonium sulfate fully, and placement is after 36 hours down to be 8 ℃ in temperature, and 6000 rev/mins centrifugal 10 minutes, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 8 ℃ in temperature to be placed 36 hours down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5) with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.2M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, after concentrated and lyophilize, can obtain purity and ratio two groups of slightly different superoxide-dismutase finished products alive respectively, the superoxide-dismutase finished product is the light green lyophilized powder, and its gross weight is about 7.5-10g.
Embodiments of the invention 4: the method for from sheep blood, extracting superoxide-dismutase
(1), in the fresh sheep blood of 10kg, add the sodium citrate anticoagulant of 20g, be to place 80 minutes under 6 ℃ the low temperature in temperature, treat the solution layering, obtain the 8600ml precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 50 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 15 minutes, remove precipitation after centrifugal 15 minutes, obtain the 5200ml supernatant liquor for 4000 rev/mins;
(3), the supernatant liquor that step (2) is obtained carries out 3 grades of thermogrades processing: the 5200ml supernatant liquor that step (2) is obtained is warming up to 40 ℃ rapidly by room temperature, keep reducing to rapidly again after 20 minutes room temperature, remove precipitation after centrifugal 10 minutes for 4000 rev/mins, obtain the supernatant liquor one of 4000ml; Supernatant liquor one is warming up to 50 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 15 minutes for 4000 rev/mins, obtain the supernatant liquor two of 3100ml; Supernatant liquor two is warming up to 60 ℃ rapidly by room temperature, keeps reducing to rapidly again after 10 minutes room temperature, remove precipitation after centrifugal 10 minutes, obtain 1600ml supernatant liquor a for 4500 rev/mins;
(4), supernatant liquor a carried out two-stage saltout: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 50%, fully stir ammonium sulfate is dissolved fully, temperature be 6 ℃ place 24 hours down after, remove precipitation after centrifugal 15 minutes for 4500 rev/mins, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 90%, fully stirring is dissolved ammonium sulfate fully, and placement is after 24 hours down to be 6 ℃ in temperature, and 5000 rev/mins centrifugal 10 minutes, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 6 ℃ in temperature to be placed 24 hours down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5), with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.10M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, after concentrated and lyophilize, can obtain purity and ratio two groups of slightly different superoxide-dismutase finished products alive respectively, the superoxide-dismutase finished product is the light green lyophilized powder, and its gross weight is about 7.5-10g.
Embodiments of the invention 5: the method for from sheep blood, extracting superoxide-dismutase
(1), in the fresh ox blood of 10kg, add the sodium citrate anticoagulant of 30g, be to place 30 minutes under 4 ℃ the low temperature in temperature, treat the solution layering, obtain the 8600ml precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 40 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 10 minutes, remove precipitation after centrifugal 30 minutes, obtain the 5300ml supernatant liquor for 4000 rev/mins;
(3) supernatant liquor that step (2) is obtained carries out 4 grades of thermogrades processing: the 5300ml supernatant liquor that step (2) is obtained is warming up to 40 ℃ rapidly by room temperature, keep reducing to rapidly again after 10 minutes room temperature, remove precipitation after centrifugal 10 minutes, obtain 3400ml supernatant liquor one for 4000 rev/mins; Supernatant liquor one is warming up to 45 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 15 minutes, obtain 2600ml supernatant liquor two for 4000 rev/mins; Supernatant liquor two is warming up to 50 ℃ rapidly by room temperature, keeps reducing to rapidly again after 20 minutes room temperature, remove precipitation after centrifugal 15 minutes, obtain 1700ml supernatant liquor three for 4000 rev/mins; Supernatant liquor three is warming up to 55 ℃ rapidly by room temperature, keeps reducing to rapidly again after 30 minutes room temperature, remove precipitation after centrifugal 20 minutes, obtain 1100ml supernatant liquor a for 4500 rev/mins;
(4), supernatant liquor a carried out two-stage saltout: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 60%, fully stir ammonium sulfate is dissolved fully, temperature be 4 ℃ place 12 hours down after, remove precipitation after centrifugal 10 minutes for 4000 rev/mins, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 85%, fully stirring is dissolved ammonium sulfate fully, and placement is after 12 hours down to be 4 ℃ in temperature, and 5000 rev/mins centrifugal 15 minutes, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 4 ℃ in temperature to be placed 12 hours down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5) with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.05M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, after concentrated and lyophilize, can obtain purity and ratio two groups of slightly different superoxide-dismutase finished products alive respectively, the superoxide-dismutase finished product is the light green lyophilized powder, and its gross weight is about 7.5-10g.
Embodiments of the invention 6: the method for from sheep blood, extracting superoxide-dismutase
(1), in the fresh ox blood of 10kg, add the sodium citrate anticoagulant of 50g, be to place 100 minutes under 8 ℃ the low temperature in temperature, treat the solution layering, obtain the 8600ml precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 50 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 30 minutes, remove precipitation after centrifugal 10 minutes, obtain the 5500ml supernatant liquor for 6000 rev/mins;
(3) supernatant liquor that step (2) is obtained carries out 6 grades of thermogrades processing: the 5500ml supernatant liquor that step (2) is obtained is warming up to 50 ℃ rapidly by room temperature, keep reducing to rapidly again after 10 minutes room temperature, remove precipitation after 4000/ minute centrifugal 30 minutes, obtain 4000ml supernatant liquor one; Supernatant liquor one is warming up to 55 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 25 minutes, obtain 2900m1 supernatant liquor two for 4500 rev/mins; Supernatant liquor two is warming up to 60 ℃ rapidly by room temperature, keeps reducing to rapidly again after 15 minutes room temperature, remove precipitation after centrifugal 30 minutes, obtain 1900ml supernatant liquor three for 4000 rev/mins; Supernatant liquor three is warming up to 65 ℃ rapidly by room temperature, keeps reducing to rapidly again after 20 minutes room temperature, remove precipitation after centrifugal 15 minutes, obtain 1500ml supernatant liquor four for 5000 rev/mins; Supernatant liquor four is warming up to 70 ℃ rapidly by room temperature, keeps reducing to rapidly again after 25 minutes room temperature, remove precipitation after centrifugal 25 minutes, obtain 1200ml supernatant liquor five for 4500 rev/mins; Supernatant liquor five is warming up to 80 ℃ rapidly by room temperature, keeps reducing to rapidly again after 30 minutes room temperature, remove precipitation after centrifugal 10 minutes, obtain 900ml supernatant liquor a for 6000 rev/mins;
(4), supernatant liquor a carried out two-stage saltout: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 70%, fully stir ammonium sulfate is dissolved fully, temperature be 8 ℃ place 36 hours down after, remove precipitation after centrifugal 15 minutes for 5000 rev/mins, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 95%, fully stirring is dissolved ammonium sulfate fully, and placement is after 36 hours down to be 8 ℃ in temperature, and 6000 rev/mins centrifugal 10 minutes, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 8 ℃ in temperature to be placed 36 hours down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5) with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.2M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration (10000M) fully, after concentrated and lyophilize, can obtain purity and ratio two groups of slightly different superoxide-dismutase finished products alive respectively, the superoxide-dismutase finished product is the light green lyophilized powder, and its gross weight is about 7.5-10g.
Claims (3)
1, a kind of method of extracting superoxide-dismutase from ox, sheep blood is characterized in that: may further comprise the steps:
(1), in fresh bovine blood or sheep blood, add the antithrombotics account for blood weight 0.2-0.5%, be to place 30-100 minute under 4-8 ℃ the low temperature in temperature, treat the solution layering, obtain precipitation after removing most of supernatant liquor;
(2), above-mentioned precipitation is heated rapidly to 40-50 ℃ by room temperature, be incubated and reduce to room temperature rapidly after 10-30 minute, remove precipitation after centrifugal 10-30 minute, obtain supernatant liquor for 4000-6000 rev/min;
(3), above-mentioned supernatant liquor is carried out obtain supernatant liquor a after 3-6 level thermograde is handled;
(4), supernatant liquor a being carried out two-stage saltouts: at first add ammonium sulfate, making the liquid saturation ratio of saltouing is 45-70%, fully stirs ammonium sulfate is dissolved fully, and placement is after 12-36 hour under temperature is 4-8 ℃, remove precipitation after centrifugal 10-30 minute for 4000-6000 rev/min, supernatant liquor b; Continuation adds ammonium sulfate in supernatant liquor b, making the liquid saturation ratio of saltouing is 85-95%, fully stir ammonium sulfate dissolved fully, temperature be 4-8 ℃ place 12-36 hour down after, 4000-6000 rev/min centrifugal 10-30 minute, collecting precipitation, obtain precipitating one, remaining supernatant liquor is 4-8 ℃ in temperature to be placed 12-36 hour down, carried out suction filtration with G4 frosted funnel, collecting precipitation obtains precipitating two;
(5), with concentration be the potassium phosphate buffer dissolution precipitation one and precipitate two respectively of 0.05-0.2M, pH=7.0, residual ammonium sulfate is removed in dissolving back ultrafiltration fully, after concentrated and lyophilize, can obtain respectively than two groups of superoxide-dismutase finished products slightly different of living with purity.
2, the method for extracting superoxide-dismutase from ox, sheep blood according to claim 1, it is characterized in that: used antithrombotics is a Trisodium Citrate in the step (1).
3, the method for from ox, sheep blood, extracting superoxide-dismutase according to claim 1, it is characterized in that: in the step (3), when carrying out the processing of 3-6 level thermograde, elementary temperature is 40-50 ℃, the level temperature is 70-80 ℃ eventually, per two inter-stage thermograde differences are 5-10 ℃, it all is to be warming up to this grade temperature rapidly by room temperature earlier to keep reducing to room temperature more rapidly after 10-30 minute that every grade of gradient is handled, and carries out after every grade of cooling removing precipitation behind 4000-6000 rev/min centrifugally operated 10-30 minute.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200610051207 CN1916168A (en) | 2006-09-04 | 2006-09-04 | Method for extracting superoxide dismutase from blood of cattle, sheep |
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| CN 200610051207 CN1916168A (en) | 2006-09-04 | 2006-09-04 | Method for extracting superoxide dismutase from blood of cattle, sheep |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921736A (en) * | 2010-07-30 | 2010-12-22 | 云南呈贡光明亚南经贸有限责任公司 | Method for extracting superoxide dismutase from cattle and sheep blood largly industrially and blade type agitator applied to same |
| CN107365752A (en) * | 2017-02-22 | 2017-11-21 | 吉林天肽生物科技有限公司 | From the method for bovine cardiac tissue preparation superoxide dismutase and prepared superoxide dismutase and application thereof |
-
2006
- 2006-09-04 CN CN 200610051207 patent/CN1916168A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921736A (en) * | 2010-07-30 | 2010-12-22 | 云南呈贡光明亚南经贸有限责任公司 | Method for extracting superoxide dismutase from cattle and sheep blood largly industrially and blade type agitator applied to same |
| CN101921736B (en) * | 2010-07-30 | 2011-07-06 | 云南呈贡光明亚南经贸有限责任公司 | Method for extracting superoxide dismutase from cattle and sheep blood largly industrially and blade type agitator applied to same |
| CN107365752A (en) * | 2017-02-22 | 2017-11-21 | 吉林天肽生物科技有限公司 | From the method for bovine cardiac tissue preparation superoxide dismutase and prepared superoxide dismutase and application thereof |
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