CN102337252A - Method for extracting superoxide dismutase (SOD) from pig blood - Google Patents
Method for extracting superoxide dismutase (SOD) from pig blood Download PDFInfo
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- CN102337252A CN102337252A CN2010102300604A CN201010230060A CN102337252A CN 102337252 A CN102337252 A CN 102337252A CN 2010102300604 A CN2010102300604 A CN 2010102300604A CN 201010230060 A CN201010230060 A CN 201010230060A CN 102337252 A CN102337252 A CN 102337252A
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Abstract
The invention discloses a method for extracting superoxide dismutase (SOD) from pig blood. The method comprises the following steps of separating blood cells from blood plasma by a separation-cleaning-disruption integrated one-step separation technology and preparing a blood solution, treating the blood solution by an ethanol-chloroform dissolution and precipitation technology to obtain a crude enzyme solution, treating the crude enzyme solution by an acetone precipitation method to obtain a SOD crude protein, heating supernate obtained by the previous step for degeneration, collecting supernate, removing precipitates, carrying out further separation and purification of the supernate by a spiral gradient elution technology to improve enzyme unit activity and obtain a purified SOD enzyme solution, filtering the purified SOD enzyme solution by a membrane for desalination, and carrying out freeze drying to obtain a purified SOD product. The method simplifies production technology and production equipment, and satisfies requirements of large-scale industrialized production.
Description
Technical field:
The invention belongs to technical field of bioengineering, particularly a kind of method of from pig blood, extracting superoxide-dismutase SOD.
Background technology:
Superoxide-dismutase (SOD) is a kind of metalloenzyme of ability catalysis ultra-oxygen anion free radical disproportionation reaction; It mainly acts on is to remove the ultra-oxygen anion free radical that produces in the biological oxidation process single-mindedly; It is the key defence line of organism defence toxicity of biological active oxygen; Can prevent or treat the multiple disease that causes by ultra-oxygen anion free radical; It is a kind of medicinal enzyme on way of great use; Human thrombus's disease, arteriosclerosis, hyperglycemia there are certain prevention and assisting therapy effect, also have anti-ageing, radioprotective and prevent that effect such as cell mutation has and to remove in the body the too much characteristic of oxyradical, thereby be widely used in fields such as medical science, food, makeup.
From 1969 isolated SOD first from ORBC since, people separated from animal erythrocyte, muscle, liver organization and plants such as ox, pig, sheep, horse in succession and are purified into SOD.China's major part all is to adopt biochemical method to extract SOD now;, microbial fermentation synthetic and chemical extraction like gene; In the SOD purifying, use methods such as sepharose method regulating YIN and YANG ion-exchange resin simultaneously mostly, shortcomings such as these method ubiquity complex manufacturing, cost height, cycle length.
China has abundant pig blood resource as pork industry big country, but most of pig blood except simply process do feed with, then directly abandoned, not only greatly wasted the pig blood resource, the environment of having gone back severe contamination.In the pig blood abundant nutrients, except that containing abundant albumen, also contain with superoxide-dismutase (SOD) isoreactivity material, effective extraction that pig blood is carried out SOO will greatly improve the added value of pig blood, produce good economic benefit.
Summary of the invention:
The object of the invention just is to overcome the deficiency that exists in the above-mentioned ordinary method, and a kind of method of from pig blood, extracting superoxide-dismutase SOD is provided, simple, with short production cycle, the suitable suitability for industrialized production of this method technology.
As above design, technical scheme of the present invention is: a kind of method of from pig blood, extracting superoxide-dismutase SOD is characterized in that: carry out according to following steps:
(1) in pig blood, add antithrombotics, the centrifugal 10～30min of the centrifugal 3000rpm slurry of dehematizing is collected red corpuscle; The 0.9%NaCl solution that adds 3～5 times of erythrocyte volumes then cleans; Centrifugal 10～the 30min of centrifugal 4000rpm; After the abandoning supernatant, collect red corpuscle and add the deionized water of 1～3 times of volume of erythrocyte volume, obtain hemolysate after leaving standstill 10-15h under 0～4 ℃ of water-bath;
(2) in hemolysate, slowly add 95% ethanol of cold preparatory mistake below 4 ℃ after, slowly add again precooling chloroform, stir then, leave standstill, centrifugal back collects supernatant and obtain crude enzyme liquid;
(3) in crude enzyme liquid, add acetone, stirring, centrifugal then, taking precipitate;
(4) throw out is dissolved with the deionized water that adds 1-3 times of volume, and after heat-treating, below the ice bath to 4 ℃, then, add K rapidly
2HPO
4Damping fluid, stirring, centrifugal back obtain supernatant;
(5) adopt the spiral gradient elution technique that supernatant is carried out purification process, obtain purifying enzyme liquid;
(6) purifying enzyme liquid is removed salt ion through dialysis technology, and the exquisitest enzyme liquid carries out lyophilize, can obtain final pig blood SOD finished product.
Above-mentioned antithrombotics adopts Potassium Oxalate Solution, and its concentration is 2～5%, and its volume is 10% of a pig blood volume.
The pH=9 of above-mentioned deionized water.
95% alcoholic acid add-on is 0.2～0.5 times of hemolysate volume in the above-mentioned steps (2); The add-on of chloroform is 0.1～0.5 times of hemolysate volume; Hemolysate stirs 10-20min after adding 95% ethanol and chloroform, leaves standstill 0.5-1h, the centrifugal 10～30min of 4000rmp, collects supernatant and obtains crude enzyme liquid.
The add-on of the middle acetone of above-mentioned steps (3) is 1～3 times of crude enzyme liquid volume, and crude enzyme liquid stirs 10-15min, the centrifugal 10～30min of 4000rmp, taking precipitate after adding acetone.
Heat treated condition is 40～70 ℃ of temperature, time 10～30min in the above-mentioned steps (4); K
2HPO
4The add-on of damping fluid is 20～30 times of throw out volume, and throw out adds K
2HPO
4Stir 5-8min after the damping fluid, the centrifugal 10～30min of 4000rmp obtains supernatant then.
Spiral gradient elution technique employing concentration is that 2～50mmol/L, pH value are 7.2～9.0 K in the above-mentioned steps (5)
2HPO
4Damping fluid makes the sedimentary step by step method of impurity realize the purifying of SOD enzyme through the gradient principle of saltouing.
Advantage of the present invention is: comprehensive organism of the present invention and chemical process, adopt high efficiency separation, extraction and purification technique, and working method is simple, the cycle is short, pollution is little, is applicable to large-scale industrial production.After testing, the pig blood Cu/Zn-SOD that adopts this method to extract, its unit enzyme work can reach more than the 6000U/mg.
Embodiment:
Embodiment 1:
2～5% the Potassium Oxalate Solution that in pig blood, adds 10% volume, the centrifugal 20min of the 3000rpm slurry of dehematizing, collection red corpuscle.The 0.9%NaCl solution that adds 5 times then cleans, during constantly stir after, the centrifugal 20min of 4000rpm.After the abandoning supernatant, collect red corpuscle and add the deionized water (pH=9) of 2 times of volume ratios, obtain hemolysate after leaving standstill 10h under 4 ℃ of water-baths; Behind 95% ethanol of 0.25 volume of cold preparatory mistake, slowly add the chloroform of 0.2 volume of precooling again under in hemolysate, slowly adding 4 ℃, stir 15min.After leaving standstill 0.5h, the centrifugal 20min of 4000rmp collects supernatant and obtains crude enzyme liquid; The acetone that in crude enzyme liquid, adds 3 times of volumes, behind the stirring 10min, the centrifugal 20min of 4000rmp, taking precipitate; Throw out is dissolved with the deionized water that adds 2 times of volumes, and behind thermal treatment 15min under 60 ℃ of temperature, rapidly below the ice bath to 4 ℃.Then, the K that adds 20 times of volumes
2HPO
4Damping fluid, behind the stirring 5min, the centrifugal 20min of 4000rmp obtains supernatant.Employing concentration is that 8.5mmol/L, pH value are 8.2 K
2HP0
4Damping fluid precipitates impurity through the gradient principle of saltouing step by step, can obtain purifying enzyme liquid; Purifying enzyme liquid is removed salt ion through dialysis technology, and the exquisitest enzyme liquid carries out lyophilize, can obtain final pig blood SOD finished product.
Embodiment 2:
2～5% the Potassium Oxalate Solution that in pig blood, adds 10% volume, the centrifugal 30min of the 3000rpm slurry of dehematizing, collection red corpuscle.The 0.9%NaCl solution that adds 3 times then cleans, during constantly stir after, the centrifugal 20min of 4000rpm.After the abandoning supernatant, collect red corpuscle and add the deionized water (pH=9) of 3 times of volume ratios, obtain hemolysate after leaving standstill 10h under 4 ℃ of water-baths; In hemolysate, slowly add 4 ℃ down behind 95% ethanol of 0.5 volume of cold preparatory mistake, slowly add the chloroform of 0.5 volume of precooling again, stir 15min, leave standstill 0.5h after, the centrifugal 30min of 4000rmp collects supernatant and obtains crude enzyme liquid; The acetone that in crude enzyme liquid, adds 2 times of volumes, behind the stirring 10min, the centrifugal 20min of 4000rmp, taking precipitate; Throw out is dissolved with the deionized water that adds 2 times of volumes, and behind thermal treatment 20min under 50 ℃ of temperature, rapidly below the ice bath to 4 ℃.Then, the K that adds 25 times of volumes
2HPO
4Damping fluid, behind the stirring 5min, the centrifugal 20min of 4000rmp obtains supernatant.Employing concentration is that 8.5mmol/L, pH value are 8.2 K
2HPO
4Damping fluid precipitates impurity through the gradient principle of saltouing step by step, can obtain purifying enzyme liquid; Purifying enzyme liquid is removed salt ion through dialysis technology, and the exquisitest enzyme liquid carries out lyophilize, can obtain final pig blood SOD finished product.
Claims (7)
1. method of from pig blood, extracting superoxide-dismutase SOD is characterized in that: carry out according to following steps:
(1) in pig blood, add antithrombotics, the centrifugal 10～30min of the centrifugal 3000rpm slurry of dehematizing is collected red corpuscle; The 0.9%NaCl solution that adds 3～5 times of erythrocyte volumes then cleans; Centrifugal 10～the 30min of centrifugal 4000rpm; After the abandoning supernatant, collect red corpuscle and add the deionized water of 1～3 times of volume of erythrocyte volume, obtain hemolysate after leaving standstill 10-15h under 0～4 ℃ of water-bath;
(2) in hemolysate, slowly add 95% ethanol of cold preparatory mistake below 4 ℃ after, slowly add again precooling chloroform, stir then, leave standstill, centrifugal back collects supernatant and obtain crude enzyme liquid;
(3) in crude enzyme liquid, add acetone, stirring, centrifugal then, taking precipitate;
(4) throw out is dissolved with the deionized water that adds 1-3 times of volume, and after heat-treating, below the ice bath to 4 ℃, then, add K rapidly
2HPO
4Damping fluid, stirring, centrifugal back obtain supernatant;
(5) adopt the spiral gradient elution technique that supernatant is carried out purification process, obtain purifying enzyme liquid;
(6) purifying enzyme liquid is removed salt ion through dialysis technology, and the exquisitest enzyme liquid carries out lyophilize, can obtain final pig blood SOD finished product.
2. a kind of method of from pig blood, extracting superoxide-dismutase SOD according to claim 1, it is characterized in that: above-mentioned antithrombotics adopts Potassium Oxalate Solution, and its concentration is 2～5%, and its volume is 10% of a pig blood volume.
3. a kind of method of from pig blood, extracting superoxide-dismutase SOD according to claim 1 is characterized in that: the pH=9 of above-mentioned deionized water.
4. a kind of method of from pig blood, extracting superoxide-dismutase SOD according to claim 1; It is characterized in that: 95% alcoholic acid add-on is 0.2～0.5 times of hemolysate volume in the above-mentioned steps (2); The add-on of chloroform is 0.1～0.5 times of hemolysate volume; Hemolysate stirs 10-20min after adding 95% ethanol and chloroform, leaves standstill 0.5-1h, the centrifugal 10～30min of 4000rmp, collects supernatant and obtains crude enzyme liquid.
5. a kind of method of from pig blood, extracting superoxide-dismutase SOD according to claim 1; It is characterized in that: the add-on of the middle acetone of above-mentioned steps (3) is 1～3 times of crude enzyme liquid volume; Crude enzyme liquid stirs 10-15min, the centrifugal 10～30min of 4000rmp, taking precipitate after adding acetone.
6. a kind of method of from pig blood, extracting superoxide-dismutase SOD according to claim 1, it is characterized in that: heat treated condition is 40～70 ℃ of temperature, time 10～30min in the above-mentioned steps (4); K
2HPO
4The add-on of damping fluid is 20～30 times of throw out volume, and throw out adds K
2HPO
4Stir 5-8min after the damping fluid, the centrifugal 10～30min of 4000rmp obtains supernatant then.
7. a kind of method of from pig blood, extracting superoxide-dismutase SOD according to claim 1 is characterized in that: spiral gradient elution technique employing concentration is that 2～50mmol/L, pH value are 7.2～9.0 K in the above-mentioned steps (5)
2HPO
4Damping fluid makes the sedimentary step by step method of impurity realize the purifying of SOD enzyme through the gradient principle of saltouing.
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| CN2010102300604A CN102337252A (en) | 2010-07-19 | 2010-07-19 | Method for extracting superoxide dismutase (SOD) from pig blood |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374554A (en) * | 2012-04-18 | 2013-10-30 | 吉林博大农林生物科技有限公司 | Method for extracting freeze-dried powder of blood of wood frog |
| CN105039273A (en) * | 2015-07-13 | 2015-11-11 | 青岛康大食品有限公司 | Method for purifying and preparing superoxide dismutase in rabbit blood |
| CN106967695A (en) * | 2017-05-31 | 2017-07-21 | 南宁学院 | A kind of method that superoxide dismutase is extracted from ox blood |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034369A (en) * | 1987-01-08 | 1989-08-02 | 苏州医学院 | The method of purification of superoxide-dismutase |
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2010
- 2010-07-19 CN CN2010102300604A patent/CN102337252A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034369A (en) * | 1987-01-08 | 1989-08-02 | 苏州医学院 | The method of purification of superoxide-dismutase |
Non-Patent Citations (3)
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| 丁全福: "《药理实验教程》", 31 December 1996, article "《药理实验常用抗凝剂》" * |
| 张宏等: "牛血超氧化物岐化酶（bovine superoxide dismutase）生产工艺研究", 《内蒙古自然大学学报（自然科学版）》, vol. 33, no. 5, 30 September 2002 (2002-09-30), pages 567 - 571 * |
| 杨佳友等: "猪血中功能性物质的分离纯化及其应用", 《肉类工业》, no. 3, 31 March 2010 (2010-03-31), pages 40 - 42 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374554A (en) * | 2012-04-18 | 2013-10-30 | 吉林博大农林生物科技有限公司 | Method for extracting freeze-dried powder of blood of wood frog |
| CN103374554B (en) * | 2012-04-18 | 2016-05-18 | 吉林博大农林生物科技有限公司 | The extracting method of wood frog blood freeze-dried powder |
| CN105039273A (en) * | 2015-07-13 | 2015-11-11 | 青岛康大食品有限公司 | Method for purifying and preparing superoxide dismutase in rabbit blood |
| CN106967695A (en) * | 2017-05-31 | 2017-07-21 | 南宁学院 | A kind of method that superoxide dismutase is extracted from ox blood |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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Application publication date: 20120201 |