CN101899425B - Method for separating and purifying scallop phenol oxidase - Google Patents
Method for separating and purifying scallop phenol oxidase Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying scallop phenol oxidase. The method is characterized by comprising the following steps of: (1) obtaining haemolymph from adductor muscles of scallops, centrifuging, suspending and crushing the haemolymph, then centrifuging the crushed haemolymph, and taking the supernate, namely obtaining blood cell crushed supernate; (2) performing continuous gradient non-denaturing electrophoresis on the blood cell crushed supernate serving as a sample; (3) positioning a phenol oxidase protein band of non-denaturing electrophoresis by using a specific substrate coloring method; and (4) performing glue cutting on a destination band, reclaiming the phenol oxidase protein band by an electro-elution method, and obtaining purified scallop phenol oxidase protein after dialysis and freeze-drying. The method has the advantages that: (1) the method has short purification time; (2) the method has simple and convenient operation, simple equipment requirement and low cost, and greatly reduces the treatment steps of the sample; and (3) the purified sample has high purity, only presents one band by non-denaturing and denaturing electrophoresis detection, keeps the bioactivity of enzyme well and improves the purifying efficiency.
Description
Technical field
The present invention relates to the improvement of the phenol oxidase separating and purifying technology of bivalve shellfish, be specifically related to a kind of separation purification method of scallop phenol oxidase, belong to shellfish immunological technique field.
Background technology
Scallop is the low invertebrates of waiting, and resists the invasion of exotic disease substance through non-specific immunity.Phenol oxidase (phenoloxidase, be called for short PO) is one of important factor of non-specific immunity, induces at microbial polysaccharide etc. to be activated down, and the relevant substrate generation of catalysis quinone finally produces melanochrome.A series of products that the PO catalyzed reaction generates can limit and isolate exotic; Suppress pathogenic bacteria exoprotein and chitinous activity; Pathogen kill; Healing wound; Play opsonization; Promote hemocyte to engulf and packing; The mediation aggegation, solidify the generation with other bacteriocidal substances.At present in invertebrates, the saltout method of column chromatography (anion-exchange chromatography, hydrophobic chromatography, sephadex chromatography, sepharose chromatography, affinity chromatography and isoelectrofocusing chromatography) of neutral salt (mainly referring to ammonium sulfate) is adopted in the separation and purification of PO mostly.The content of PO is very low in the scallop body; And its activity is the very responsive and inactivation very easily of environment to external world; Saltout and column chromatography bonded method length consuming time (at least need 72 hours), treatment step is various can't in the process of purifying, keep the PO activity, and this method target protein recovery is low.Therefore the method that needs the separation purification scallop PO of a kind of simple and effective of exploitation.
Non-sex change electrophoresis (native-PAGE); It is a kind of quick, easy analysis of protein authenticate technology; The characteristic that it keeps albumen to be had in vivo to greatest extent; Normal combining with substrate color development technology, and the non-sex change electrophoresis of continuous gradient separate targets albumen more efficiently and accurately in order to the molecular weight and the isozyme of definite enzyme.Electroelution technology (electroelution) is a kind of proteic electrophoretic technique of gel that is used for reclaiming.At present, use the comprehensive of technology such as the non-sex change electrophoresis of continuous gradient-specific substrate color development-electroelution, also do not see relevant report in the research aspect the separation and purification invertebrates PO.
Summary of the invention
The purpose of this invention is to provide a kind of quick, easy, separation purification method of scallop phenol oxidase efficiently.
The present invention confirms the target protein position with the specific substrate chromophoric method again through the phenol oxidase of the non-sex change electrophoretic separation of continuous gradient scallop, and reclaims the phenol oxidase that target protein obtains purifying through electroelution method; The phenol oxidase BA and the enzymatic property of the definite purification of Physiology and biochemistry method are formed and used to the phenol oxidase purity and the subunit thereof of purifying out through the electrophoresis checking at last.
Technical scheme of the present invention may further comprise the steps: (1) obtains hemolymph from the closed shell flesh of scallop, and the centrifuging and taking supernatant through centrifugal, resuspended, broken back obtains hemocyte fragmentation liquid supernatant; (2) be that sample carries out the non-sex change electrophoresis of continuous gradient with the broken liquid supernatant of hemocyte; (3) with the pyrocatechol be the specific substrate color development, location phenol oxidase protein band; (4) the purpose band is cut glue, reclaim the phenol oxidase protein band, the scallop phenol oxidase that after the dialysis freeze-drying, obtains purifying through electroelution method.
Separation purification method separation and purification bay scallop (Argopecten irradians) PO and chlamys farreri (Chlamys farreri) PO by described scallop phenol oxidase; It is following to obtain the result: bay scallop PO behind the purifying and chlamys farreri PO all only present a band through the non-sex change electrophoresis detection of continuous gradient, and molecular weight is respectively about 477kDa and 408kDa; Two kinds of PO all have only a band through the sex change electrophoresis detection, and bay scallop PO and chlamys farreri PO molecular weight are respectively about 54kDa and 80kDa, show that these two kinds of PO only are made up of a kind of subunit; Survey PO BA and enzymatic property through the enzyme plate method; The result shows: in bay scallop; PO total activity (U) behind the purifying be before the purifying sample total activity (U) 73.14%, PO specificity vigor (U/mg) is 413.08 times of sample specificity vigor (U/mg) before the purifying behind the purifying; In chlamys farreri, the PO total activity (U) behind the purifying be before the purifying sample total activity (U) 72.16%, PO specificity vigor (U/mg) is 533.33 times of sample specificity vigor (U/mg) before the purifying behind the purifying; The vigor of two kinds of enzymes all can receive the influence of temperature, pH, divalent-metal ion and PO inhibition, and different substrates are also shown different avidity, has certain Substratspezifitaet.
The invention has the advantages that: be sample 1. with the broken liquid supernatant of hemocyte; Combine with specific substrate color development and electroelution technology through the non-sex change electrophoresis of continuous gradient; Realized the purifying of scallop PO, whole process weak point consuming time (approximately need 48 hours) helps keeping preferably the activity of enzyme; 2. only need technology such as electrophoresis/wash-out and specific substrate color development can reach the purifying of PO; Equipment requirements is simple, and cost is low, and is easy and simple to handle; And significantly reduced treatment step, when keeping, also helped keeping the activity of enzyme proteic higher separating power to sample; The sample purity of 3. purifying is high, only presents a band through non-sex change and sex change electrophoresis detection, and the target protein recovery is high, has well kept the biological activity of enzyme, has improved purification efficiency.
Description of drawings
Bay scallop PO and chlamys farreri PO electrophorogram that Fig. 1 purifies for the present invention.
Wherein, (A) be the non-sex change electrophoresis result of continuous gradient; (B) be the sex change electrophoresis result.
Fig. 2 bay scallop PO that to be temperature purify to the present invention and chlamys farreri PO vigor influence figure.
Fig. 3 bay scallop PO that to be pH purify to the present invention and chlamys farreri PO vigor influence figure.
Fig. 4 bay scallop PO that to be divalent-metal ion purify to the present invention and chlamys farreri PO vigor influence figure.
Wherein, (A) be the bay scallop result; (B) be the chlamys farreri result.
Fig. 5 for the present invention purify bay scallop PO and chlamys farreri PO kinetic parameter figure.
Wherein, (A) be the bay scallop result; (B) be the chlamys farreri result.
Referring to Fig. 1-Fig. 5:
The non-sex change electrophorogram of Fig. 1 (A) expression continuous gradient.Wherein: the 1st, the proteic coomassie brilliant blue staining result of standard molecular weight; The broken liquid supernatant of 2 expression bay scallop hemocytes coomassie brilliant blue staining result; The broken liquid supernatant of 3 expression chlamys ferreri blood cells coomassie brilliant blue staining result; Bay scallop PO coomassie brilliant blue staining result behind the 4 expression purifying, molecular weight is about 477kDa; Chlamys farreri PO coomassie brilliant blue staining result behind the 5 expression purifying, molecular weight is about 408kDa; Be the result of substrate color development with the pyrocatechol behind the broken liquid supernatant of the 6 expression bay scallop hemocytes electrophoresis, molecular weight is about 477kDa; Be the result of substrate color development with the pyrocatechol behind the broken liquid supernatant of the 7 expression chlamys ferreri blood cells electrophoresis, molecular weight is about 408kDa.Fig. 1 (B) expression sex change electrophorogram.Wherein: the 1st, the proteic coomassie brilliant blue staining result of standard molecular weight; Bay scallop PO coomassie brilliant blue staining result behind the 2 expression purifying, molecular weight is about 54kDa; Chlamys farreri PO coomassie brilliant blue staining result behind the 3 expression purifying, molecular weight is about 80kDa.
Fig. 2 representes the bay scallop PO that temperature is purified to the present invention and the influence of chlamys farreri PO vigor, and bay scallop PO vigor optimum temperuture is about about 40 ℃, and when temperature was higher or lower than 40 ℃, the vigor of enzyme descended along with the rising of temperature or reduction; Chlamys farreri PO vigor optimum temperuture is about about 30 ℃, and when temperature was higher or lower than 30 ℃, the vigor of enzyme was along with the rising of temperature or reduction and descend.
Fig. 3 representes the bay scallop PO that pH purifies to the present invention and the influence of chlamys farreri PO vigor, and bay scallop PO and chlamys farreri PO vigor ph optimum are about about 8.0, and when pH was higher or lower than 8.0, the vigor of enzyme descended along with the rising of pH or reduction.
Fig. 4 (A) expression divalent-metal ion is to the influence of the bay scallop PO vigor of the present invention's purification, Ca
2+, Mg
2+Enzyme activity there are obvious promoter action, Fe
2+, Mn
2+Enzyme activity there are obvious restraining effect, Cu
2+, Zn
2+Different and different to the enzyme activity influence with concentration of metal ions; Fig. 4 (B) expression divalent-metal ion is to the influence of the chlamys farreri PO vigor of the present invention's purification, Ca
2+, Mg
2+Enzyme activity there are obvious promoter action, Fe
2+Enzyme activity there are obvious restraining effect, Cu
2+, Zn
2+, Mn
2+Influence to enzyme activity is different and different with concentration of metal ions.
The bay scallop PO kinetic parameter that Fig. 5 (A) expression the present invention purifies, this PO is respectively 0.51mM, 0.23mM, 0.87mM to the Michaelis-Menton constant Km of three kinds of substrate L-DOPA, pyrocatechol, Resorcinol; The chlamys farreri PO kinetic parameter that Fig. 5 (B) expression the present invention purifies, this PO is respectively 0.61mM, 0.2mM, 0.84mM to the Michaelis-Menton constant Km of three kinds of substrate L-DOPA, pyrocatechol, Resorcinol.
Embodiment
Further specify the present invention below in conjunction with accompanying drawing and through specific embodiment.
Instance 1: the non-sex change electrophoresis of continuous gradient-specific substrate color development-electroelution method separation and purification bay scallop (Argopectenirradians) PO
(1) the broken liquid supernatant of preparation hemocyte
1. get blood with sterilizing syringe from the closed shell flesh of bay scallop; Centrifugal (4 ℃, 3000rpm, 15min); Collection hemocyte deposition, approximately the centrifugal hemocyte deposition that obtains of 100ml hemolymph is resuspended with the 20mM Tris-HCl damping fluid (pH 7.0) of 1.5ml precooling to 4 ℃;
2. blood cell suspension is broken under 4 ℃ of conditions with the ultrasonic disruption appearance, centrifugal (4 ℃, 14000rpm 30min), gets supernatant, is the broken liquid supernatant of hemocyte.
(2) be that sample carries out the non-sex change electrophoresis of continuous gradient with the broken liquid supernatant of hemocyte
1. prepare the non-sex change electrophoretic buffer of pH 8.8Tris-Glycin (6.06g Tris, 28.82g Glycin, 2L DW), precooling to 4 ℃.
2. use the continuous gradient separation gel of constant flow pump preparation acrylamide concentration scope as 6%-20%, concentrated gum concentration is 5%, does not contain sodium lauryl sulphate (SDS) in the glue.
3. the broken liquid supernatant of hemocyte is mixed every hole application of sample 110 μ l, permanent power 3W electrophoresis 12h under 4 ℃ of conditions with the sample buffer that equal-volume does not contain SDS and denaturing agent.
(3) be the specific substrate color development with the pyrocatechol, locate the electrophoretic PO protein band of non-sex change
Swimming lane in the gel behind the electrophoresis is downcut, is dipped in the 1% pyrocatechol solution, color development about 5 minutes to the red-brown band occurring.
(4) electroelution method reclaims PO albumen, the bay scallop PO albumen that after the dialysis freeze-drying, obtains purifying
1. with reference to pyrocatechol color development result, all the other bands that do not contain PO in the color development gel are downcut, in the non-sex change electrophoretic buffer of pH 8.8Tris-Glycin, (Model 422, Bio-Rad) for continuous current 20mA electroelution 12h under 4 ℃ of conditions.
2. the solution that obtains behind the wash-out 12h that under 4 ℃ of conditions, in zero(ppm) water, dialyses, every 3h changes first water.
3. the solution that obtains after the dialysis is carried out vacuum lyophilization, the albumen that obtains is bay scallop PO.
(5) non-sex change electrophoresis of continuous gradient and sex change electrophoretic method are identified the bay scallop PO purity of protein and the molecular weight subunit of purifying
1. the non-sex change electrophoresis of continuous gradient is identified the bay scallop PO purity of protein of purifying
A prepares the non-sex change electrophoretic buffer of pH 8.8Tris-Glycin (6.06g Tris, 28.82g Glycin, 2L DW), precooling to 4 ℃.
B uses the continuous gradient separation gel of constant flow pump preparation acrylamide concentration scope as 6%-20%, and concentrated gum concentration is 5%, does not contain sodium lauryl sulphate (SDS) in the glue.
C mixes the bay scallop PO aqueous solution of purifying with the sample buffer that equal-volume does not contain SDS and denaturing agent, every hole application of sample 110 μ l, and permanent power 3W electrophoresis 12h under 4 ℃ of conditions takes out gel, uses coomassie brilliant blue staining.
2. sex change electrophoresis (SDS-PAGE) is confirmed bay scallop PO protein protomer composition
A prepares pH 8.8Tris-Glycin sex change electrophoretic buffer (3.03g Tris, 14.41g Glycin, 1g SDS, 1L DW)
B preparation resolving gel concentration is 12%, concentrate gum concentration is 5% polyacrylamide gel, contains 0.15%SDS in the glue
C mixes the bay scallop PO aqueous solution of purifying with the sample buffer that equal-volume contains SDS and beta-mercaptoethanol, in boiling water, boil 3-5min, the sample of handling is added go up in the appearance hole, appearance 20 μ l on every hole;
D with low current (30-40mA), treats that sample is after concentrated glue partially concd becomes a line when initial under constant current conditions; Strengthen electric current (50-70mA); Electrophoresis to tetrabromophenol sulfonphthalein indicator can stop electrophoresis when reaching bottom margin, takes out gel, uses coomassie brilliant blue staining.
The result is as shown in Figure 1: in the non-sex change electrophoresis of continuous gradient, the bay scallop PO of separation and purification of the present invention only presents a band, and molecular weight is about 477kDa; In the sex change electrophoresis; The bay scallop PO of separation and purification of the present invention also presents a band; Molecular weight is about 54kDa; Show the success of the non-sex change electrophoresis of continuous gradient of the present invention-specific substrate color development-electroelution method separation and purification from the broken liquid supernatant of bay scallop hemocyte PO, and have higher degree, the oligomer that the subunit of this PO about by 54kDa formed.
(6) biochemistry of the bay scallop PO that the present invention is purified and the analysis of enzymatic property
1. the mensuration of bay scallop PO vigor
Adopt the enzyme plate method; Add 10 μ l 15mM L-DOPA (being dissolved in pH 7.0,20mM Tris-HCl damping fluid) and 10 μ l testing samples (the PO aqueous solution after broken liquid supernatant of hemocyte or the purification) in every hole; Hatch 40min in 37 ℃ behind the mixing; Add 0 ℃ of zero(ppm) water of 200 μ l then, use ELIASA under the 492nm wavelength, to measure photoabsorption.The PO vigor is defined as: it is 1 unit of activity (U) that the absorbance value PM increases by 0.001.The Bradford method is surveyed protein concentration, uses bovine serum albumin (BSA) as standard protein.The PO specificity vigor of sample is defined as U/mg albumen.
2. temperature is to the influence of the bay scallop PO vigor after purifying
Choose 10 ℃ of 6 thermogrades, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, measuring the PO specificity vigor after purifying according to the enzyme plate method under these 6 temperature respectively, each thermograde is done three repetitions.
3. pH is to the influence of the bay scallop PO vigor after purifying
Choose 6 pH gradients, 5,6,7,8,9,10, measuring the PO specificity vigor after purifying according to the enzyme plate method under these 6 pH respectively, each pH gradient is done three repetitions.
4. divalent-metal ion is to the influence of the bay scallop PO vigor after purifying
The 10 μ lPO aqueous solution respectively with the MgSO of 10 μ l different concns
4, ZnSO
4, MnCl2, FeCl
2, CuSO
4, CaCl
2(being dissolved in pH 7.0,20mM Tris-HCl damping fluid) solution is hatched 20min in advance in enzyme plate under 4 ℃ of conditions; Every then hole adds 10 μ l 15mM L-DOPA (being dissolved in pH 7.0,20mM Tris-HCl damping fluid); Hatch 40min under 37 ℃ of conditions; Last every hole adds 190 μ l0 ℃ zero(ppm) water, measures PO specificity vigor with the enzyme plate method.Each concentration of every metal ion species is done three repetitions, and control group is with 20mM Tris-HCl damping fluid (pH 7.0) substituted metal solion.
5. the PO inhibition is to the influence of the bay scallop PO vigor after purifying
The 10 μ lPO aqueous solution respectively with the common PO inhibition (being dissolved in pH 7.0,20mM Tris-HCl damping fluid) of 10 μ l different concns; Halfcystine, ascorbic acid usp/bp, S-WAT, Hydrocerol A, thio urea, sodium azide, EDTA Disodium, Thiocarb; Under 4 ℃ of conditions, in enzyme plate, hatch 20min in advance; Every then hole adds 10 μ l15mM L-DOPA (being dissolved in pH 7.0,20mM Tris-HCl damping fluid); Hatch 40min under 37 ℃ of conditions, last every hole adds 190 μ l0 ℃ zero(ppm) water, measures PO specificity vigor with the enzyme plate method.Each concentration of every kind of PO inhibition is done three repetitions, and control group replaces PO inhibition solution with 20mM Tris-HCl damping fluid (pH 7.0).
6. bay scallop PO kinetic parameter is measured
The 10 μ lPO aqueous solution respectively with four kinds of substrates (being dissolved in pH7.0,20mM Tris-HCl damping fluid) of 10 μ l different concns; L-DOPA, pyrocatechol, Resorcinol, tyrosine; Under 37 ℃ of conditions, in enzyme plate, hatch 40min; Every then hole adds 0 ℃ of zero(ppm) water of 200 μ l, adopts the enzyme plate method to measure the PO vigor, uses Lineweaver-Burk double-reciprocal plot method to try to achieve the Michaelis-Menton constant of PO to above-mentioned four kinds of substrates.Each concentration of every kind of substrate is done three repetitions.
The result:
Bay scallop PO purification efficiency is as shown in table 1:
Table 1 bay scallop PO purification efficiency
Through the bay scallop PO total activity (U) behind the purifying is 73.14% of the preceding sample total activity (U) of purifying, and the bay scallop PO specificity vigor (U/mg) behind the purifying is 413.08 times of the preceding sample specificity vigor (U/mg) of purifying.
The influence of the bay scallop PO vigor of PO inhibition after to purifying is as shown in table 2:
The influence of the bay scallop PO vigor of table 2PO inhibition after to purifying
In above-mentioned PO inhibition; S-WAT is the strongest to the bay scallop PO vigor restraining effect of purifying; Ascorbic acid usp/bp and Thiocarb have remarkable restraining effect; Halfcystine and Hydrocerol A have obvious restraining effect, the EDTA Disodium restraining effect relatively a little less than, thio urea and sodium azide unrestraint effect.
As shown in Figure 2, the optimum temperuture of the bay scallop PO vigor of purifying is 40 ℃; As shown in Figure 3, ph optimum is about 8.0; Shown in Fig. 4 (A), Ca
2+, Mg
2+Bay scallop PO vigor to purifying has obvious promoter action, Fe
2+, Mn
2+The PO vigor there are obvious restraining effect, Cu
2+, Zn
2+Influence to the PO vigor changes with its concentration is different.Shown in Fig. 5 (A), bay scallop PO is to three kinds of substrates, and the Michaelis-Menton constant Km of L-DOPA, pyrocatechol, Resorcinol is respectively 0.51mM, 0.23mM, 0.87mM, but tyrosine is not by this PO catalysis.
Instance 2: the non-sex change electrophoresis of continuous gradient-specific substrate color development-electroelution method separation and purification chlamys farreri (Chlamysfarreri) PO
(1) the broken liquid supernatant of preparation hemocyte
1. get blood with sterilizing syringe from the closed shell flesh of chlamys farreri; Centrifugal (4 ℃, 3000rpm, 15min); Collection hemocyte deposition, approximately the centrifugal hemocyte deposition that obtains of 100ml hemolymph is resuspended with the 20mM Tris-HCl damping fluid (pH 7.0) of 1.5ml precooling to 4 ℃;
2. blood cell suspension is broken under 4 ℃ of conditions with the ultrasonic disruption appearance, centrifugal (4 ℃, 14000rpm 30min), gets supernatant, is the broken liquid supernatant of hemocyte.
(2) be that sample carries out the non-sex change electrophoresis of continuous gradient with the broken liquid supernatant of hemocyte
With step (2) in the instance 1, sample is replaced with the broken liquid supernatant of chlamys ferreri blood cells.
(3) be the specific substrate color development with the pyrocatechol, locate the electrophoretic PO protein band of non-sex change
With step (3) in the instance 1.
(4) electroelution method reclaims PO albumen, the chlamys farreri PO albumen that after the dialysis freeze-drying, obtains purifying
With instance 1 step (4).
(5) non-sex change electrophoresis of continuous gradient and sex change electrophoretic method are identified the chlamys farreri PO purity of protein and the molecular weight subunit of purifying
With step (5) in the instance 1, sample is replaced with the chlamys farreri PO aqueous solution of purification.
The result is as shown in Figure 1: in the non-sex change electrophoresis of continuous gradient, the chlamys farreri PO of separation and purification of the present invention only presents a band, and molecular weight is about 408kDa; In the sex change electrophoresis; The chlamys farreri PO of separation and purification of the present invention also presents a band; Molecular weight is about 80kDa; Show the success of the non-sex change electrophoresis of continuous gradient of the present invention-specific substrate color development-electroelution method separation and purification from the broken liquid supernatant of chlamys ferreri blood cells PO, and have higher degree, the oligomer that the subunit of this PO about by 54kDa formed.
(6) biochemistry of the chlamys farreri PO that the present invention is purified and the analysis of enzymatic property
With step (6) in the instance 1, sample is replaced with the chlamys farreri PO aqueous solution of broken liquid supernatant of chlamys ferreri blood cells or purification.
The result:
Chlamys farreri PO purification efficiency is as shown in table 3:
Table 3 chlamys farreri PO purification efficiency
Through the PO total activity (U) behind the purifying is before the purifying sample total activity (U) 72.16%, PO specificity vigor (U/mg) is 533.33 times of sample specificity vigor (U/mg) before the purifying behind the purifying.
The influence of the chlamys farreri PO vigor of PO inhibition after to purifying is as shown in table 4:
The influence of the chlamys farreri PO vigor of table 4PO inhibition after to purifying
In above-mentioned PO inhibition; S-WAT and ascorbic acid usp/bp are the strongest to the bay scallop PO vigor restraining effect of purifying; Thiocarb has remarkable restraining effect; Halfcystine and Hydrocerol A have obvious restraining effect, the EDTA Disodium restraining effect relatively a little less than, thio urea and sodium azide unrestraint effect.
As shown in Figure 2, the optimum temperuture of the chlamys farreri PO vigor of purifying is 40 ℃; As shown in Figure 3, ph optimum is about 8.0; Shown in Fig. 4 (B), Ca
2+, Mg
2+Chlamys farreri PO vigor to purifying has obvious promoter action, Fe
2+Enzyme activity there are obvious restraining effect, Cu
2+, Zn
2+, Mn
2+Influence to enzyme activity is different and different with concentration of metal ions.Shown in Fig. 5 (B), the chlamys farreri PO of purifying is to three kinds of substrates, and the Michaelis-Menton constant Km of L-DOPA, pyrocatechol, Resorcinol is respectively 0.61mM, 0.2mM, 0.84mM, but tyrosine is not by this PO catalysis.
Those of ordinary skill in the art can understand, and in protection scope of the present invention, makes amendment for the foregoing description, and it all is possible adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (2)
1. the separation purification method of a scallop phenol oxidase, it is characterized in that it may further comprise the steps: blood is got from the closed shell flesh of scallop in (1), and is centrifugal; Collection hemocyte deposition; Resuspended with the Tris-HCl damping fluid, use the ultrasonic disruption appearance broken, centrifugal again; Get supernatant, obtain the broken liquid supernatant of hemocyte; (2) be that sample carries out the non-sex change electrophoresis of continuous gradient with the broken liquid supernatant of hemocyte; (3) be the specific substrate color development with the pyrocatechol, locate the electrophoretic phenol oxidase protein band of non-sex change; (4) the purpose band is cut glue, reclaim the phenol oxidase protein band, the scallop phenol oxidase albumen that after the dialysis freeze-drying, obtains purifying through electroelution method.
2. the separation purification method of scallop phenol oxidase according to claim 1 is characterized in that the described scallop of step (1) is bay scallop or chlamys farreri.
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| Sdijanski S等.Eletophoretic separation and identificationg of phenoloxidases in hemolymph and midgut of adult Anophelews stepyhensi mosquitoes.《Journal of Parasitology》.1997,第83卷(第4期),摘要. * |
| 任东涛等.百合花粉萌发过程中的肌动蛋白和肌球蛋白的研究.《科学通报》.1998,第43卷(第5期),515. * |
| 张蕾等.大鼠海马83kD蛋白诱导圣经干细胞项乙酰胆碱酯酶阳性神经元的定向分化.《中国组织工稃研究与临床康复》.2009,第13卷(第32期),摘要. * |
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