CN103387965B - The method of separation and purification edge pipe Enteromorpha iron superoxide dismutase - Google Patents
The method of separation and purification edge pipe Enteromorpha iron superoxide dismutase Download PDFInfo
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Abstract
The present invention is a kind of method of separation and purification edge pipe Enteromorpha (Enteromorpha linza) iron superoxide dismutase (Fe-SOD).This purification process comprises 50 mM phosphoric acid buffer extracts, 50%-70% ammonium sulfate precipitation, ion exchange chromatography and gel permeation chromatography four steps.Application the method 103.6 times by edge pipe Enteromorpha iron superoxide dismutase purifying, the rate of recovery is 19.1%, and the Rate activity of final enzyme reaches 1750 U/mg albumen.The invention also discloses the some properties of edge pipe Enteromorpha iron superoxide dismutase.
Description
Technical field
The present invention relates to a kind of method of separation and purification edge pipe Enteromorpha iron superoxide dismutase.
Background technology
Superoxide-dismutase (EC 1.15.1.1) is the enzyme that the reaction of catalyzes superoxide free radical anion forms hydrogen peroxide and molecular oxygen, be antioxidase important in organism, superoxide-dismutase is distributed widely in various organism, as animal, plant, microorganism etc.It has special physiologically active, is the primary material of scavenging free radicals in organism.Superoxide-dismutase level height in vivo means index directly perceived that is old and feeble and death; Now confirm, the disease caused by oxyradical reaches kind more than 60.It can resist and the infringement blocked because oxyradical causes cell, and repairs damaged cell in time, restore because of free radical cause to cell damage.Superoxide-dismutase is widely used in food, cosmetic and field of medicaments, and the superoxide-dismutase of marine source has the features such as catalytic temperature is low, antifatigue.From marine plant, obtaining superoxide-dismutase is the key of researching and developing this fermentoid from now on.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, provides a kind of method being separated simple, cost-saving separation and purification edge pipe Enteromorpha iron superoxide dismutase.
Edge pipe Enteromorpha involved in the present invention collects in the seawater in marine site, Lianyun Harbour, Jiangsu Province, China, and the collecting sample time is in mid-June, 2012.
Technical problem to be solved by this invention is realized by following technical scheme, the present invention is a kind of method of separation and purification edge pipe Enteromorpha iron superoxide dismutase, be characterized in, its step is as follows: (1) sample preparation: the edge pipe Enteromorpha gathered first is removed silt and other impurity through seawater cleaning, clean three times respectively with tap water and deionization again, extract water; Then shred and put into refiner, and add 3 milliliters of 50mM phosphoric acid buffers, pH 7.4, ratio containing 1 mM EDTA in 1 gram of Enteromorpha, the 50mM phosphoric acid buffer of 4 DEG C is added in refiner, mixing also refrigerates 2 h under homogenate being placed in after abundant broken Enteromorpha cell 4 DEG C of temperature, then filtration is taken out, get filtrate centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor;
(2) ammonium sulfate precipitation: first add ammonium sulfate to 50% saturation ratio in above-mentioned supernatant liquor, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor to continue to add ammonium sulfate to 70% saturation ratio, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, dissolve throw out with the phosphoric acid buffer of 10 mM, pH7.4 and enzyme liquid of dialysing to obtain;
(3) ion exchange chromatography: above-mentioned enzyme liquid is loaded in chromatography column, chromatography column selects the Q-sepharose FF anion-exchange chromatography filler of GE healthcare, be installed on Bio-Rad double fluid phase FPLC chromatograph, 10 mM first used by post bed, the molten balance of phosphoric acid buffer of pH7.4, setting program is sample introduction respectively, the linear elution of sample and column regeneration, the concentration of linear elution solution is 800 mM sodium-chlor, flow velocity 0.8 mL/min, collect the detection that elutriant carries out superoxide-dismutase and protein concentration, merge there being the elutriant of the elution peak of superoxide-dismutase and ultrafiltration and concentration, carry out gel permeation chromatography,
(4) gel permeation chromatography: the Superdex 200 10/30 GL chromatography column above-mentioned elutriant follow procedure being entered GE healthcare, elutriant is 10 mM, pH7.4, containing the phosphoric acid buffer of 150 mM sodium-chlor, it is 0.3 mL/min that elution flow rate controls, collect elutriant and carry out the detection of superoxide-dismutase and protein concentration, by have the elutriant of the elution peak of superoxide-dismutase carry out dialysis also lyophilize obtain edge pipe Enteromorpha iron superoxide dismutase.
The inventive method advantages of simple, have good operability, and cost is low, and application the method 103.6 times by edge pipe Enteromorpha iron superoxide dismutase purifying, the rate of recovery is 19.1%, and the Rate activity of final enzyme reaches 1750 U/mg albumen.
Accompanying drawing explanation
Fig. 1 ion exchange chromatography color atlas.
Fig. 2 gel permeation chromatography color atlas.
The gel electrophoresis of Fig. 3 SDS polypropylene milling amine, swimming lane 1 is edge pipe Enteromorpha superoxide-dismutase, and swimming lane 2 is Marker.
Fig. 4 gel permeation chromatography measures the molecular mass of enzyme.
Fig. 5 inhibitor method detects the type of superoxide-dismutase.Swimming lane 1: enzyme through deionized water process, swimming lane 2: enzyme through 5 mM potassium cyanide process, swimming lane 3: enzyme through 5 mM hydrogen peroxide treatment, swimming lane 4: enzyme is through 5 mM SDS process.
The catalytic temperature curve of Fig. 6 enzyme and thermostability.■ represents catalytic temperature, ● represent thermostability.
The pH stability diagram of Fig. 7 enzyme.
Embodiment
Below further describe concrete technical scheme of the present invention, so that those skilled in the art understands the present invention further, and do not form the restriction to its right.
Embodiment 1, a kind of method of separation and purification edge pipe Enteromorpha iron superoxide dismutase, its step is as follows: (1) sample preparation: the edge pipe Enteromorpha gathered first is removed silt and other impurity through seawater cleaning, then cleans three times respectively with tap water and deionization, extract water; Then shred and put into refiner, and add 3 milliliters of 50mM phosphoric acid buffers, pH 7.4, ratio containing 1 mM EDTA in 1 gram of Enteromorpha, the 50mM phosphoric acid buffer of 4 DEG C is added in refiner, mixing also refrigerates 2 h under homogenate being placed in after abundant broken Enteromorpha cell 4 DEG C of temperature, then filtration is taken out, get filtrate centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor;
(2) ammonium sulfate precipitation: first add ammonium sulfate to 50% saturation ratio in above-mentioned supernatant liquor, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor to continue to add ammonium sulfate to 70% saturation ratio, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, dissolve throw out with the phosphoric acid buffer of 10 mM, pH7.4 and enzyme liquid of dialysing to obtain;
(3) ion exchange chromatography: above-mentioned enzyme liquid is loaded in chromatography column, chromatography column selects the Q-sepharose FF anion-exchange chromatography filler of GE healthcare, be installed on Bio-Rad double fluid phase FPLC chromatograph, 10 mM first used by post bed, the molten balance of phosphoric acid buffer of pH7.4, setting program is sample introduction respectively, the linear elution of sample and column regeneration, the concentration of linear elution solution is 800 mM sodium-chlor, flow velocity 0.8 mL/min, collect the detection that elutriant carries out superoxide-dismutase and protein concentration, merge there being the elutriant of the elution peak of superoxide-dismutase and ultrafiltration and concentration, carry out gel permeation chromatography,
(4) gel permeation chromatography: the Superdex 200 10/30 GL chromatography column above-mentioned elutriant follow procedure being entered GE healthcare, elutriant is 10 mM, pH7.4, containing the phosphoric acid buffer of 150 mM sodium-chlor, it is 0.3 mL/min that elution flow rate controls, collect elutriant and carry out the detection of superoxide-dismutase and protein concentration, by have the elutriant of the elution peak of superoxide-dismutase carry out dialysis also lyophilize obtain edge pipe Enteromorpha iron superoxide dismutase.
Embodiment 2, a kind of method of the edge of separation and purification as described in Example 1 pipe Enteromorpha iron superoxide dismutase, one, purification process step is as follows: 1.1 sample preparation: the edge pipe Enteromorpha gathered first is removed silt and other impurity through seawater cleaning, clean three times respectively with tap water and deionization again, extract water stand-by in-40 DEG C of refrigerator freezings;
The Enteromorpha processed is shredded, put into refiner, and add the 50mM phosphoric acid buffer of 3 milliliters of precoolings, pH 7.4, ratio containing 1 mM EDTA in 1 gram of Enteromorpha, the 50mM phosphoric acid buffer of 4 DEG C is added in refiner, mixing also refrigerates 2 h under homogenate being placed in after abundant broken Enteromorpha cell 4 DEG C of temperature, then takes out homogenate, by eight layers of clean filtered through gauze, get filtrate centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor for subsequent use;
1.2 ammonium sulfate precipitations: first add ammonium sulfate to 50% saturation ratio in above-mentioned supernatant liquor, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor to continue to add ammonium sulfate to 70% saturation ratio, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, dissolve throw out with the phosphoric acid buffer of 10 mM, pH7.4 and enzyme liquid of dialysing to obtain;
1.3 ion exchange chromatographies, by ammonium sulfate precipitation and the enzyme liquid of dialysing carries out ion exchange chromatography is further purified superoxide-dismutase.Above-mentioned enzyme liquid is loaded in chromatography column, chromatography column selects the Q-sepharose FF anion-exchange chromatography filler of GE healthcare, be installed on Bio-Rad double fluid phase FPLC chromatograph, 10 mM first used by post bed, the molten balance of phosphoric acid buffer of pH7.4, setting program is sample introduction respectively, the linear elution of sample and column regeneration, the concentration of linear elution solution is 800 mM sodium-chlor, flow velocity 0.8 mL/min, detect superoxide-dismutase and the protein concentration of elutriant, result as shown in Figure 1, elution process has appearance 4 absorbing proteins peaks altogether, wherein the 3rd absorption peak has superoxide dismutase activity, by the elutriant Collection and conservation at this peak, carry out next step experiment,
1.4 gel permeation chromatographies: the Superdex 200 10/30 GL chromatography column above-mentioned elutriant follow procedure being entered GE healthcare, 10 mM first used by elutriant, the molten balance of phosphoric acid buffer of pH7.4, the linear elution of sample and column regeneration, the concentration of linear elution solution is 150mM sodium-chlor, flow rate control is 0.3 mL/min, wash-out result as shown in Figure 2, there are 4 absorbing proteins peaks, detect elutriant, wherein the 3rd blob detection obtains superoxide dismutase activity, collect the elutriant containing enzyme activity, dialysis also lyophilize obtains edge pipe Enteromorpha iron superoxide dismutase.
two,the some properties of the edge pipe Enteromorpha iron superoxide dismutase of purifying of the present invention
The edge pipe Enteromorpha iron superoxide dismutase of purifying of the present invention, is studied its character, substantially finds out the part of properties of this enzyme.
The detection method of 2.1 superoxide dismutase activities:
The detection of superoxide dismutase activity adopts assay NBT photoreduction.20 uL enzyme liquid and 1.5 mL deionized waters and 1.43 mL Tris-HCl damping fluid (50 mM, pH 8.2) mixing, incubation 10 min in 25 DEG C of water-baths, add 50 uL 6 mM pyrogallol solution and start timing immediately, measure light absorption value in 325 nm places after 30 s, in 4 min, measure a numerical value every 30 s.Blank assay substitutes enzyme liquid by deionized water.
The relative molecular weight of 2.2 enzymes:
The edge pipe Enteromorpha superoxide-dismutase of purifying carries out sodium laurylsulfonate-polyacrylamide gels (SDS-PAGE), result as shown in Figure 3, show a band in figure, illustrate that this enzyme has been purified to electrophoresis pure, corresponding Marker learns that the molecular weight of this enzyme is approximately 23 kDa.
The edge pipe Enteromorpha superoxide-dismutase of purifying is carried out the molecular weight that gel permeation chromatography measures enzyme simultaneously.Select the Superdex 200 10/30 GL chromatography column of GE healthcare, elutriant is the phosphoric acid buffer (containing 150 mM sodium-chlor) of 10 mM pH7.4, flow rate control is 0.3 mL/min, molecule Marker selects phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg white albumin (44.3 kDa) and cytochrome C (12.3 kDa).Result as shown in Figure 4, the molecular weight of edge pipe Enteromorpha superoxide-dismutase is approximately 46 kDa, because present method measures the molecular weight of enzyme under non denatured condition, and SDS-PAGE measures molecular weight under Denaturing, and SDS may make the subunit in superoxide-dismutase molecule disconnect.Result shows that edge pipe Enteromorpha superoxide-dismutase is an enzyme comprising two subunits, and molecular weight is 46 kDa, and the molecular weight of each subunit is 23 kDa.
The detection of 2.3 Isozyme Types:
The enzyme of purifying is carried out active gel electrophoresis, after electrophoresis terminates, gel is cut apart respectively, and process in different inhibitor, then with NBT dyeing, finally irradiate colour developing under fluorescent light.Result as shown in Figure 5, swimming lane 1,2,3 and 4 is respectively through deionized water, 5 mM potassium cyanide, 5 mM hydrogen peroxide and 5 mM SDS process 1 h, this enzyme to potassium cyanide and SDS insensitive and easily suppressed by hydrogen peroxide, therefore edge pipe Enteromorpha superoxide-dismutase is a Fe-SOD.
The suitable catalyst temperature of 2.4 enzymes and thermostability:
The superoxide-dismutase of purifying measures enzyme activity in 0 DEG C, 10 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C of water-baths, result as shown in Figure 6,35 DEG C time, enzyme reaches maximum catalytic efficiency, and along with the rising of temperature or reduction, enzyme activity declines gradually.Experimental result also finds that enzyme also has certain catalytic capability 0 DEG C time, and result shows that edge pipe Enteromorpha iron superoxide dismutase is a kind of cold adapted.Experiment detects the thermostability of enzyme simultaneously 0 DEG C, 10 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C, by the edge pipe Enteromorpha iron superoxide dismutase of purifying after the water-bath (0 DEG C in mixture of ice and water) of said temperature is incubated 1 h, cooling simultaneously also measures enzyme activity at 35 DEG C, and calculates enzyme activity.As shown in Figure 6, enzyme is incubated 1 h in lower than the water-bath of 35 DEG C and keeps 100% enzyme activity result, declines gradually more than the stability of 40 DEG C of enzymes.
The pH stability of 2.5 enzymes:
Detect the pH stability of this superoxide-dismutase, first enzyme mixed from different damping fluids, and in 25 DEG C of water-baths incubation 1 h, then measure enzyme activity, and be 100% calculating enzyme activity with maximum enzyme vigor.The damping fluid of the different pH of 50 mM: acetate buffer solution (pH 3.0-6.0), phosphoric acid buffer (pH 6.0-7.5), Tris-HCl damping fluid (7.5-9.0) and carbonic acid buffer (pH 9.0-11.0).As shown in Figure 7, the pH stability of enzyme liquid is better, and within the scope of pH 5.0-10.0, keep more than 80% enzyme activity, vertex is at pH 7.0 for result.
2.6 metal ions are on the impact of enzyme:
Detect metal ion to the impact of enzyme, first mixed with different metal ion salt by enzyme, final concentration is 5 mM, and measures enzyme activity in 35 DEG C, and is 100% calculating enzyme activity with maximum enzyme vigor.Result is as shown in table 1, Cu
2+, Mn
2+, Zn
2+and Ca
2+slight promoter action is had, Ag to enzyme activity
+, Al
3+and Co
2+have restraining effect in various degree to enzyme, the impact of other ions enzyme is little.
Table 1 metal ion is on the impact of edge pipe Enteromorpha iron superoxide dismutase vigor
The N terminal amino acid sequence of 2.7 enzymes:
Adopt Edman method to measure the N terminal amino acid sequence of the edge pipe Enteromorpha superoxide-dismutase of purifying, front 11 aminoacid sequences that result records its N end are ALELKAPPYEL.
Claims (1)
1. a method for separation and purification edge pipe Enteromorpha iron superoxide dismutase, is characterized in that, its step is as follows:
(1) sample preparation: the edge pipe Enteromorpha gathered first is removed silt and other impurity through seawater cleaning, then cleans three times respectively with tap water and deionization, extract water; Then shred and put into refiner, and add 3 milliliters of 50mM phosphoric acid buffers, pH 7.4, ratio containing 1 mM EDTA in 1 gram of Enteromorpha, the 50mM phosphoric acid buffer of 4 DEG C is added in refiner, mixing also refrigerates 2 h under homogenate being placed in after abundant broken Enteromorpha cell 4 DEG C of temperature, then filtration is taken out, get filtrate centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor;
(2) ammonium sulfate precipitation: first add ammonium sulfate to 50% saturation ratio in above-mentioned supernatant liquor, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, get supernatant liquor to continue to add ammonium sulfate to 70% saturation ratio, leave standstill 1 h at 4 DEG C of temperature after, centrifugal 20 min under 12000 g centrifugal actions, dissolve throw out with the phosphoric acid buffer of 10 mM, pH7.4 and enzyme liquid of dialysing to obtain;
(3) ion exchange chromatography: above-mentioned enzyme liquid is loaded in chromatography column, chromatography column selects the Q-sepharose FF anion-exchange chromatography filler of GE healthcare, be installed on Bio-Rad double fluid phase FPLC chromatograph, 10 mM first used by post bed, the molten balance of phosphoric acid buffer of pH7.4, setting program is sample introduction respectively, the linear elution of sample and column regeneration, the concentration of linear elution solution is 800 mM sodium-chlor, flow velocity 0.8 mL/min, collect the detection that elutriant carries out superoxide-dismutase and protein concentration, merge there being the elutriant of the elution peak of superoxide-dismutase and ultrafiltration and concentration, carry out gel permeation chromatography,
(4) gel permeation chromatography: the Superdex 200 10/30 GL chromatography column above-mentioned elutriant follow procedure being entered GE healthcare, elutriant is 10 mM, pH7.4, containing the phosphoric acid buffer of 150 mM sodium-chlor, it is 0.3 mL/min that elution flow rate controls, collect elutriant and carry out the detection of superoxide-dismutase and protein concentration, by have the elutriant of the elution peak of superoxide-dismutase carry out dialysis also lyophilize obtain edge pipe Enteromorpha iron superoxide dismutase.
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