CN101629167A - Method for extracting superoxide dismutase - Google Patents
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Abstract
The invention relates to a method for extracting superoxide dismutase, in particular to a method for extracting superoxide dismutase from barbadosnut. The method for extracting superoxide dismutase is achieved by using seeds or leaves of barbadosnut as raw materials, and comprises the steps of the preparing crude extract, removing foreign protein, sectional salting-out ammonium sulfate, precipitating acetone, carrying out Sephadex G2100 molecular sieve column chromatography, and the like. The invention provides a new source for extracting superoxide dismutase, and optimizes technical parameters for extracting superoxide dismutase. The method for extracting superoxide dismutase can give consideration to both the specific activity and the recovery ratio of enzyme, and is beneficial to the large-scale production of enterprises.
Description
Technical field
The present invention relates to a kind of method of extracting superoxide-dismutase, relate in particular to a kind of method of from Cortex jatrophae, extracting superoxide-dismutase.
Background technology
Superoxide-dismutase (Superoxide dismutase is called for short SOD) is that a class is prevalent in a kind of metalloenzyme in animal, plant and the microorganism.According to the difference of avtive spot bind metal ion, superoxide-dismutase can be divided into four kinds of CuZn-SOD, Mn-SOD, Fe-SOD and Ni-SOD.Superoxide-dismutase is the antioxidase of special removing ultra-oxygen anion free radical in the organism, can be disproportionated into molecular oxygen and hydrogen peroxide by the catalysis ultra-oxygen anion free radical, thereby alleviate the harm of oxyradical to body.
Oxyradical is relevant with the aging of most of disease and body.A large amount of accumulation of free radical can cause protein denaturation and crosslinked, make intravital many enzymes and hormone lose biological activity, system's vigor such as the immunological competence of body, neural reflex ability, motor capacity reduce, can also destroy simultaneously nucleic acid construct and cause the whole machine body metabolism not normal etc., finally make body generation pathology.Superoxide-dismutase is a topmost antioxidase in the biological cell, and it can resist and block the infringement that causes because of the oxyradical pair cell, and in time repairs damaged cell, restores the pair cell injury that causes because of free radical.The also extremely strong ultra-oxygen anion free radical of pair cell destructive force unnecessary in the organism is generated hydrogen peroxide and oxygen by disproportionation reaction, and hydrogen peroxide is decomposed into H by intravital xitix and catalase (CAT) subsequently
2O and O
2Thereby, remove O
2 -The oxidative stress that is caused.Play an important role in the generation of this ultra-oxygen anion free radical in keeping organism and the running balance of elimination.Therefore, superoxide-dismutase all is widely used in fields such as medicine, makeup and food.Clinically, superoxide-dismutase can be used for delaying senility, anti-inflammatory, radioprotective, anti-curing oncoma, cancer etc.; In cosmetic applications, superoxide-dismutase has removing beverage, crease-resistant, anti-inflammatory, prevents the function of skin aging; In foodstuffs industry, superoxide-dismutase can be used as efficacy factor or food enrichment is used, and multiple functional food such as existing at present superoxide-dismutase milk, beverage, can, beer appear on the market.
The separation and purification of superoxide-dismutase has two kinds of approach, and a kind of is directly to separate to obtain pure product from biological starting material, and another kind is the albumen that obtains gene recombination by the method for genetically engineered and fermentation engineering.Superoxide-dismutase product major part in the market is directly to separate to obtain from biological starting material, particularly extracts from animal blood.Pernicious transmissible diseases such as the epidemic disease of crowing along with mad cow disease all over the world, bird flu, mouth report that frequently the risk of production animal source blood products strengthens, and cause the extraction of superoxide-dismutase because starting material are limited, have the problem that output is few, be difficult to satisfy market demand.And the method processing step complexity of existing extraction superoxide-dismutase, the ratio of enzyme is lived and purity is difficult to reach overall equilbrium.
Summary of the invention
The object of the present invention is to provide a kind of method of extracting superoxide-dismutase, it is raw material with the jatropha curcas seed, can take into account the ratio of enzyme and live and the rate of recovery, helps enterprise's scale operation.
In order to realize the foregoing invention purpose, the inventor provides following technical scheme through repetition test:
A kind of extracting method of superoxide-dismutase, it is that the raw material extraction obtains with jatropha curcas seed or blade.
The concrete steps of the extracting method of described superoxide-dismutase are:
(1), the preparation of crude enzyme liquid:
Jatropha curcas seed or blade are cleaned, add the pH value and be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer of 1mmol/L EDTA, the weightmeasurement ratio of described jatropha curcas seed and phosphate buffered saline buffer is 100: 70～80 (g/ml), homogenate, filter, collect filtrate, the centrifugal supernatant liquor that gets is crude enzyme liquid;
(2), remove foreign protein:
Crude enzyme liquid thermally denature in 55 ℃ of water-baths is handled 25min,, go precipitation, in supernatant liquor, add 0.1mol/L hydrochloric acid conditioning solution pH to 5.0,, collect supernatant liquor at 4 ℃ of centrifugal 20min of following 6000r/min in 4 ℃ of centrifugal 20min of following 6000r/min;
(3), the ammonium sulfate segmentation is saltoutd:
In step (2) gained supernatant liquor, add the ammonium sulfate powder, to the solution saturation ratio be 45%, stir 30min, leave standstill 2h, at 4 ℃ of centrifugal 20min of following 6000r/min, remove precipitation, adding ammonium sulfate powder to solution saturation ratio in supernatant liquor is 90%, stirs 30min, in 4 ℃ of following standing over night, take out next day, at 4 ℃ of centrifugal 40min of following 6000r/min, abandons supernatant, is 7.8 with resolution of precipitate in the pH value, concentration is 0.05mol/L, include in the phosphate buffered saline buffer of 1mmol/L EDTA, to dissolve gained enzyme liquid and pack in the dialysis tubing, in 4 ℃ of dialysed overnight, the sulfate radical-free ion detects in enzyme liquid;
(4), acetone precipitation: in step (3) gained enzyme liquid, add isopyknic 4 ℃ of cold acetones, in 4 ℃ of natural sedimentation 60min, at 4 ℃ of centrifugal 40min of following 6000r/min, abandon supernatant, precipitate with distilled water diluting, pack in the dialysis tubing, with the pH value be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer dialysis equilibrium of 1mmol/L EDTA, collect the enzymic activity part and promptly get superoxide-dismutase liquid.
Gained superoxide-dismutase liquid promptly gets superoxide-dismutase dry powder after lyophilize, can be used for various commercial uses.
The centrifugal concrete grammar that gets supernatant liquor is in the described step (1):
Filtrate with collecting at 4 ℃ of centrifugal 40min of following 6000r/min, precipitates suspended substance fully, gets supernatant and preserves stand-by, precipitation adds phosphate buffered saline buffer again, and fully dissolving is once more at 4 ℃ of centrifugal 40min of following 6000r/min, discard precipitation, merge supernatant liquor twice, be crude enzyme liquid.
In the extracting method of above-mentioned superoxide-dismutase, can also may further comprise the steps after the acetone precipitation:
Sephadex G2100 molecular sieve column chromatography (60cm * 2cm): step (4) gained enzyme liquid is added in the SephadexG2100 molecular sieve chromatography, with the pH value be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer wash-out of 1mmol/L EDTA, elution speed is controlled at 4mL/10min, collect active peak, after 4 ℃ of dialysis, promptly get superoxide-dismutase liquid.
Superoxide-dismutase is being removed O
2 -Play an important role in the process, be widely used in multiple fields such as agricultural, medical science, industry, pharmacy, daily necessities and food.Whether the superoxide-dismutase that is applied to medicine, food, cosmetic industry is toxic, is the problem that people extremely pay close attention to.The superoxide-dismutase that extracts from plant, microorganism, prepares, usage safety performance is higher.The plant that can be used as the extraction superoxide-dismutase of at present existing bibliographical information or the kind of microorganism are less, mainly contain corn, garlic, marine alga, spirulina etc., there is the problem that output is few, be difficult to satisfy market demand in the extraction that causes superoxide-dismutase because starting material are limited.And in the preparation technology of existing superoxide-dismutase, what people mainly paid close attention to is the superoxide-dismutase that how to obtain higher purifying multiple, and seldom consider the rate of recovery problem of superoxide-dismutase, similarly seldom consider the relation between the rate of recovery of the purifying multiple of enzyme and enzyme especially.
Cortex jatrophae is happiness light sun plant, suit on arid, barren, the soil of degenerating, to grow, in the dry-hot valley area plantation of the torrid zone, subtropics and rainfall rareness, inclement condition, general 1 year one ripe, branch, the extremely strong fertility fecundity of dried tool, growth rapidly, the surviving rate height, vitality is strong, and it is simple to plant, extensive management can be to form forest community in flakes partially.Not only artificial afforestration is easy for it, and the natural regeneration ability is strong, and plantation just has harvest the first year, and output increases year by year, and picking fruit can reach 50 years.In recent years, because the promotion of conceding the land to forestry, plant in a large number various places.Only the Cortex jatrophae stock number on one ground, Sichuan is promptly more than 260,000 mu at present, and following national Cortex jatrophae cultivated area can reach more than 3,000 ten thousand mu at least, has shown good resources development and utilization prospect.
In the preparation technology of superoxide-dismutase, the ratio of enzyme is lived and the rate of recovery of enzyme is subjected to the influence of each processing condition easily, and is difficult to obtain the rate of recovery preferably in the superoxide-dismutase that obtains higher purifying multiple.The contriver is through repetition test, proposed with the thermally denature method remove behind the foreigh protein removing again with the ammonium sulfate segmentation saltout, the method for acetone precipitation is further purified superoxide-dismutase, and selected by experiment each processing condition, the ratio of the jatropha curcas seed superoxide-dismutase of extraction is lived and the rate of recovery can access and takes into account.The contriver finds in the process of extracting the jatropha curcas seed superoxide-dismutase, the ammonium sulfate segmentation is saltoutd and is concentrated in the superoxide-dismutase process, the saturation ratio of ammoniumsulphate soln has bigger influence to the activity of total protein content in the supernatant liquor and total superoxide-dismutase, thereby the contriver has designed a plurality of concentration gradients, detect with the protein content of biuret method the superoxide-dismutase of each concentration gradient extraction, and carry out active coloring, determine the saturation ratio of required ammonium sulfate according to active detected result.
The making method that biuret method is measured superoxide-dismutase protein content typical curve is: be standard with the bovine serum albumin, with Coomassie brilliant blue G250 production standard curve under the 595nm wavelength, get 6 clean tube, by 0～5 numbering, in each test tube, add reagent according to table 1 data, fully mixing is at room temperature placed 0.5h, is blank with No. 0 pipe, under 595nm, measure light absorption value (OD), with each tubulin matter content (mg) is X-coordinate, and the OD value is an ordinate zou, makes typical curve.Carry out protein content determination in the sample liquid again, sample thief 0.9mL, the making method of secundum legem curve, according to the A595 value of being measured, the reference standard curve is in the hope of the unknown solution protein concn.Repeat 3 times and average, obtain protein content in the sample (mg), and then calculate the ratio vigor of each purification phase SOD by calculating.
Table 1 typical curve data
| Reagent | ??0 | ??1 | ??2 | ??3 | ??4 | ??5 |
| Protein reference liquid/mL | ??0 | ??0.2 | ??0.4 | ??0.6 | ??0.8 | ??1.0 |
| Distilled water/mL | ??1.0 | ??0.8 | ??0.6 | ??0.4 | ??0.2 | ??0 |
| Biuret reagent/mL | ??4.0 | ??4.0 | ??4.0 | ??4.0 | ??4.0 | ??4.0 |
Under the condition that aerobic exists, riboflavin generation photoreduction produces O
2 -, O
2 -Restoring NBT and generate blue first hairpin, therefore be blue at the gel position that does not have superoxide-dismutase, is the water white transparency bright band at the gel position that superoxide dismutase activity is arranged.According to this active coloring principle, with the superoxide-dismutase behind the concentrate drying, being dissolved in the pH value is 7.8, concentration is 0.05mol/L, include in the phosphate buffered saline buffer of 1mmol/L EDTA, carry out discontinuous polyacrylamide gel electrophoresis (PAGE) 6h, concentrated gum concentration is 3%, resolving gel concentration is 10%, dye with superoxide dismutase activity after the electrophoresis, gel immerses in the 0.2%NBT-0.01% riboflavin solution, lucifuge is placed 50min, use the distilled water cleaning and dipping then, put an amount of time of illumination under the 20W fluorescent lamp, until Clear ﹠ Transparent superoxide dismutase activity bands of a spectrum on the bluish voilet background, occurring.
At first selecting saturation ratio is that 30%～55% ammoniumsulphate soln is removed foreign protein, the centrifugal 30min of 6000r/min, discard precipitation, measuring total protein content and total superoxide dismutase activity in the supernatant liquor, the results are shown in Table 2, is 60%～90% ammonium sulfate saturated solution again with concentration, make superoxide-dismutase be the state of saltouing, be precipitated out, measure its total protein content and total enzyme and live, the results are shown in Table 3.
The 1st step of table 2 ammonium sulfate precipitation result
| Concentration (%) | Total protein content/mg | Total SOD enzyme work/U | Than work/Umg -1 |
| ??30 | ??9.9735 | ??109.8262 | ??11.0118 |
| ??35 | ??7.9812 | ??115.5350 | ??14.4759 |
| ??40 | ??6.7401 | ??169.3929 | ??25.1321 |
| ??45 | ??4.1165 | ??138.9064 | ??33.7438 |
| ??50 | ??5.8840 | ??159.8606 | ??27.1687 |
| ??55 | ??8.1026 | ??156.0002 | ??19.2531 |
The 2nd step of table 3 ammonium sulfate precipitation result
| Concentration (%) | Total protein content/mg | Total SOD enzyme work/U | Than work/Umg -1 |
| ??60 | ??0.5812 | ??13.3505 | ??22.9706 |
| ??65 | ??0.7437 | ??20.0809 | ??27.0013 |
| ??70 | ??0.8180 | ??25.5380 | ??31.2200 |
| ??75 | ??1.2011 | ??44.2522 | ??36.8431 |
| ??80 | ??1.6569 | ??69.4857 | ??41.9372 |
| ??85 | ??1.8433 | ??89.7881 | ??48.7105 |
| ??90 | ??1.9317 | ??100.8025 | ??52.1833 |
Table 2～3 results show, handle crude enzyme liquid with the ammonium sulfate precipitation method, in saturation ratio is 45% and during saturation ratio 90%, SOD is higher than living, be respectively 33.7438Umg-1 and 52.1833Umg-1, illustrate that separating effect is the most obvious, therefore if purify with this method, its purifying multiple is respectively 4.1756 and 6.4574.
The inventive method should be accepted or rejected purification step according to application target in practice, if be used for large-scale industrial production, then preferably require the ratio of enzyme to live high, the rate of recovery is also high, this the two overall equilbrium can bring good economic benefit to enterprise, can produce the purified product that height ratio is lived again.Take a broad view of in the table 4 comprehensive purification result to Cortex jatrophae SOD, the ratio of taking into account enzyme live and the rate of recovery the two, even it is maximum that the two product reaches, just can only arrive this step of acetone purifying, though the final SOD specific activity of enzyme of the column chromatography in one step of back can reach 90.0172Umg-1, recovery rate but has only 21.4562%, and this can't but be a loss greatly concerning the large-scale production of enterprise, and what optimum enterprise produced can only be to this step of acetone purifying.After through the SephadexG2100 chromatography, the ratio of Cortex jatrophae SOD enzyme is lived and is 90.0172Umg-1, and the rate of recovery is 21.4562%, and the purification multiple is 11.14 times, and the used time of technology is about 48h; Comprehensive Experiment data relatively, clearly, refining effect is than a certain method of single use or the comprehensive use of all methods is all good before this.In sum, if want realistic production to put on market, then can suitably reduce purification step, purifying technique is only till the acetone precipitation.If will obtain the highly finished product that height ratio is lived, then this technology can satisfy the demands fully.
Description of drawings
Fig. 1 is a jatropha curcas seed Sephadex G2100 molecular sieve chromatography elution curve among the embodiment.
Fig. 2 is a superoxide-dismutase sample purity qualification result among the embodiment.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment, allly all belong to scope of the present invention based on the technology that content of the present invention realized.
Embodiment
The used fresh manioca seed of present embodiment picks up from the Sichuan University school of life and health sciences Botanical gardens; Agents useful for same comprises: Sephadex G2100 (Pharmacia company product), protein Marker, bovine serum albumin, acetone, chloroform, ammonium sulfate etc. are homemade analytical pure.Chlorination nitro blue tetrazolium (NBT), methionine(Met), DEAE-cellulose (DEAE), Tetramethyl Ethylene Diamine (TEMED), Tutofusin tris (Tris), acrylamide (Chemical Reagent Co., Ltd., Sinopharm Group), methylene diacrylamide chemical reagent such as (Shanghai chemical reagents corporations of Chinese Medicine group) are analytical pure, the bovine serum albumin that standard protein adopts Shanghai biological chemical reagent company to produce, Xylene Brilliant Cyanine G G-250 (Shanghai chemical reagent packing factory); Used instrument comprises: UV29100 ultraviolet-visible pectrophotometer (Beijing Rayleigh analytical instrument factory); high speed freezing centrifuge (Made in Germany); freeze drier (Japan makes); electric high-speed tissue mashing machine (Jintan City high honour instrument Manufacturing Co., Ltd); DYY-III2 voltage stabilization and current stabilization electrophoresis apparatus (Beijing Liuyi Instrument Factory); electric-heated thermostatic water bath JK-S26 type (Hua Lian, Shanghai medicine equipment company limited); HD-3 Ultraviolet Detector (Shanghai Hu Xi analytical instrument factory); DHL-A computer constant flow pump (Shanghai Hu Xi analytical instrument factory); the full-automatic Fraction Collector of DBS-100 computer (Shanghai Hu Xi analytical instrument factory).
The preparation method of present embodiment comprises the steps:
(1), the preparation of crude enzyme liquid:
Take by weighing manioca seed 100g, clean, add the pH value and be 7.8, concentration is 0.05mol/L, include phosphate buffer 1 50Ml, a little quartz sand of 1mmol/L EDTA, homogenate, double gauze filters, and collects filtrate, at 4 ℃ of centrifugal 40min of following 6000r/min, suspended substance is precipitated fully, get supernatant and preserve stand-by, precipitation adds phosphate buffered saline buffer again, fully dissolving at 4 ℃ of centrifugal 40min of following 6000r/min, discards precipitation once more, merge supernatant liquor twice, be crude enzyme liquid.
(2), remove foreign protein:
Crude enzyme liquid thermally denature in 55 ℃ of water-baths is handled 25min, and foreign protein sex change precipitation is in 4 ℃ of centrifugal 20min of following 6000r/min, go precipitation, in supernatant liquor, add 0.1mol/L hydrochloric acid conditioning solution pH to 5.0,, collect supernatant liquor at 4 ℃ of centrifugal 20min of following 6000r/min;
(3), the ammonium sulfate segmentation is saltoutd:
In step (2) gained supernatant liquor, add the ammonium sulfate powder, to the solution saturation ratio be 45%, stir 30min with glass stick, leave standstill 2h, at 4 ℃ of centrifugal 20min of following 6000r/min, remove precipitation, adding ammonium sulfate powder to solution saturation ratio in supernatant liquor is 90%, stirs 30min, in 4 ℃ of following standing over night, take out next day, at 4 ℃ of centrifugal 40min of following 6000r/min, abandons supernatant, is 7.8 with resolution of precipitate in the pH value, concentration is 0.05mol/L, include in the phosphate buffered saline buffer of 1mmol/L EDTA, to dissolve gained enzyme liquid and pack in the dialysis tubing, in 4 ℃ of dialysed overnight, the sulfate radical-free ion detects in enzyme liquid;
(4), acetone precipitation: in step (3) gained enzyme liquid, add isopyknic 4 ℃ of cold acetones, in 4 ℃ of natural sedimentation 60min, at 4 ℃ of centrifugal 40min of following 6000r/min, abandon supernatant, precipitate with distilled water diluting, pack in the dialysis tubing, with the pH value be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer dialysis equilibrium of 1mmol/L EDTA, collect the enzymic activity part and promptly get superoxide-dismutase liquid.
Step (4) gained enzyme liquid is added in the Sephadex G2100 molecular sieve chromatography, with the pH value be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer wash-out of 1mmol/L EDTA, elution speed is controlled at 4mL/10min, automatic collector is collected active peak, every pipe is collected eluate 3mL, collects 70 pipes altogether.The active peak of collecting is merged,, freeze as for 70 ℃ of refrigerator and cooled again and spend the night, with freeze drier enzyme liquid is made dry powder and promptly get the superoxide-dismutase finished product in 4 ℃ of dialysis.2 protein peak (see figure 1)s appear on the Sephadex G2100 molecular sieve chromatography elution curve, through determination of activity, active peak overlaps with the 2nd protein peak substantially, illustrate that the 2nd peak is main protein peak, the 1st peak is the foreign protein peak, and manioca seed superoxide-dismutase has obtained separation and purification preferably in the chromatography process.
Collect and respectively go on foot purifying gained enzyme liquid, detect its total protein content, total activity, alive, the rate of recovery of ratio, assay sees Table 4
Table 4 embodiment 1 total protein content, total activity, ratio are lived, rate of recovery detected result
| Purification step | Total protein m/mg | Total activity m/U | Than C/Umg alive -1 | Rate of recovery % | The purifying multiple |
| Crude extract | ??25.0129 | ??202.1342 | ??8.0512 | ??100 | ??1.00 |
| Thermally denature | ??14.6581 | ??176.8895 | ??12.0677 | ??87.5109 | ??1.4933 |
| 45% saturated ammonium sulphate is saltoutd | ??4.1165 | ??138.9064 | ??33.7438 | ??68.7199 | ??4.1756 |
| 90% saturated ammonium sulphate is saltoutd | ??1.9317 | ??100.8025 | ??52.1833 | ??49.8691 | ??6.4574 |
| Acetone precipitation | ??1.0031 | ??77.3739 | ??77.1348 | ??38.2785 | ??9.550 |
| The Sephadex chromatography | ??0.4818 | ??43.3703 | ??90.0172 | ??21.4562 | ??11.14 |
Wherein refer to the ratio of total activity and total protein than living, to represent the proteic vigor size of every mg, the enzyme activity in promptly every mg albumen has several units, or is expressed as U; The rate of recovery refers to the rate of recovery of total enzyme activity, and along with the carrying out of purification step, Tot Prot reduces thereupon, and simultaneously total enzyme is lived and also can be reduced, and supposes that enzyme work total in the crude extract is 100, and as radix, the total enzyme work that reduces gradually in the subsequent technique is the rate of recovery with this; The purifying multiple is meant the purifying multiple of enzyme activity, supposes that the proteic vigor of every mg is 1 in the crude extract, and through after the thermally denature, the proteic vigor of every mg brings up to 1.4933, and the enzyme activity in the unit albumen increases, and enzyme has obtained purifying.
The superoxide-dismutase finished product that extraction obtains is through electrophoretic process, and electrophorogram presents transparent bands of a spectrum as shown in Figure 2, shows that sample has obtained separation and purification preferably, and it is pure to have reached electrophoresis.
Claims (4)
1. a method of extracting superoxide-dismutase is characterized in that it is that the raw material extraction obtains with jatropha curcas seed or blade.
2. the concrete steps of the method for extraction superoxide-dismutase according to claim 1 are:
(1), the preparation of crude enzyme liquid:
Jatropha curcas seed or blade are cleaned, add the pH value and be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer of 1mmol/L EDTA, the weightmeasurement ratio of described jatropha curcas seed and phosphate buffered saline buffer is 100: 70～80 (g/ml), homogenate, filter, collect filtrate, the centrifugal supernatant liquor that gets is crude enzyme liquid;
(2), remove foreign protein:
Crude enzyme liquid thermally denature in 55 ℃ of water-baths is handled 25min,, go precipitation, in supernatant liquor, add 0.1mol/L hydrochloric acid conditioning solution pH to 5.0,, collect supernatant liquor at 4 ℃ of centrifugal 20min of following 6000r/min in 4 ℃ of centrifugal 20min of following 6000r/min;
(3), the ammonium sulfate segmentation is saltoutd:
In step (2) gained supernatant liquor, add the ammonium sulfate powder, to the solution saturation ratio be 45%, stir 30min, leave standstill 2h, at 4 ℃ of centrifugal 20min of following 6000r/min, remove precipitation, adding ammonium sulfate powder to solution saturation ratio in supernatant liquor is 90%, stirs 30min, in 4 ℃ of following standing over night, take out next day, at 4 ℃ of centrifugal 40min of following 6000r/min, abandons supernatant, is 7.8 with resolution of precipitate in the pH value, concentration is 0.05mol/L, include in the phosphate buffered saline buffer of 1mmol/L EDTA, to dissolve gained enzyme liquid and pack in the dialysis tubing, in 4 ℃ of dialysed overnight, the sulfate radical-free ion detects in enzyme liquid;
(4), acetone precipitation: in step (3) gained enzyme liquid, add isopyknic 4 ℃ of cold acetones, in 4 ℃ of natural sedimentation 60min, at 4 ℃ of centrifugal 40min of following 6000r/min, abandon supernatant, precipitate with distilled water diluting, pack in the dialysis tubing, with the pH value be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer dialysis equilibrium of 1mmol/L EDTA, collect the enzymic activity part and promptly get superoxide-dismutase liquid.
3. the method for extraction superoxide-dismutase according to claim 2 is characterized in that the centrifugal concrete grammar that gets supernatant liquor is in the described step (1):
Filtrate with collecting at 4 ℃ of centrifugal 40min of following 6000r/min, precipitates suspended substance fully, gets supernatant and preserves stand-by, precipitation adds phosphate buffered saline buffer again, and fully dissolving is once more at 4 ℃ of centrifugal 40min of following 6000r/min, discard precipitation, merge supernatant liquor twice, be crude enzyme liquid.
4. according to the method for claim 2 or 3 described extraction superoxide-dismutases, it is characterized in that after the acetone precipitation step further comprising the steps of:
Sephadex G2100 molecular sieve column chromatography: step (4) gained enzyme liquid is added in the Sephadex G2100 molecular sieve chromatography, with the pH value be 7.8, concentration is 0.05mol/L, include the phosphate buffered saline buffer wash-out of 1mmol/L EDTA, elution speed is controlled at 4mL/10min, collect active peak, after 4 ℃ of dialysis, promptly get superoxide-dismutase liquid.
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| CN102154232A (en) * | 2010-11-23 | 2011-08-17 | 郭雨忠 | Method for extracting SOD (Super Oxide Dlsmutase) from celery cabbages |
| CN103509763A (en) * | 2013-10-11 | 2014-01-15 | 中国科学院烟台海岸带研究所 | Process method for extracting leaf protein and superoxide dismutase from plants |
| CN105838685A (en) * | 2016-04-26 | 2016-08-10 | 句容市东方紫酒业有限公司 | Method for extracting SOD (superoxide dismutase) from mulberry wine lees |
| CN106011091A (en) * | 2016-06-22 | 2016-10-12 | 绍兴文理学院 | Method for extracting and purifying SOD freeze-dried powder from meat of hyriopsis cumingii |
| CN106399271A (en) * | 2016-11-01 | 2017-02-15 | 湖北宝迪农业科技有限公司 | Method for extracting superoxide dismutase from pig blood and superoxide dismutase |
| CN106399271B (en) * | 2016-11-01 | 2020-02-18 | 湖北宝迪农业科技有限公司 | Method for extracting superoxide dismutase from pig blood and superoxide dismutase |
| CN110760487A (en) * | 2018-07-27 | 2020-02-07 | 中国科学院地理科学与资源研究所 | Method for preparing superoxide dismutase enriched dry powder |
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| CN101629167B (en) | 2011-04-06 |
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