CN103275215A - Method for extracting metallothionein from fresh-water mussel - Google Patents
Method for extracting metallothionein from fresh-water mussel Download PDFInfo
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Abstract
The invention discloses a method for extracting metallothionein from a fresh-water mussel, which comprises the following four process steps of metal ion induced culture, homogenate centrifugation, denaturing and impurity removal, and secondary centrifugal separation and purification. The process provides a reliable and efficient extraction method for application of fresh-water mussels MT in industrial production of enterprises; and the extraction method is simple to operate, and the process is easy to control, thus being of far reaching importance in scientific research as well as production and deep processing of production enterprises.
Description
Technical field:
Patent of the present invention relates to a kind of method of extracting metallothionein(MT) from the mollusk freshwater mussel, belongs to biotechnology and protein engineering field.
Background technology:
Metallothionein(MT) (Metallothionein, be called for short MT) is that a class extensively is present in lower molecular weight in the organism, is rich in metal binding protein halfcystine, that can be produced by metal inducement; Extensively be present in animals and plants, the microorganism, because it relates to many physiology and pathological process, have important biological function.Trace does not have plain storage, transhipment and metabolism in the body comprising participating in, and the antagonism ionizing rays is removed free radical and to the detoxification of heavy metal, also participated in the stress reaction of body, fetal development and cellular metabolism regulation and control, the adjusting of endocrine system.Its research and development relates to every field such as agricultural, medicines and health protection, biotechnology, environment protection.For the research of metallothionein(MT), mainly concentrate on both at home and abroad in laboratory extraction, purifying and detection and the application.In actual production, main by synthetic with metal inducement animal liverss such as cadmium metal or zinc at present, prepare metallothionein(MT) through extraction, separation and purification then; Owing to be subjected to inducing the restriction with extracting method, also contain other foreign protein in the metallothionein(MT) after the separation and purification, productive rate is low, and can't realize producing in enormous quantities and industrialization, has influenced the Application and Development of metallothionein(MT) greatly.
China is culture fishery big country, abundant aquatic product resource is arranged, thereby the metallothionein(MT) in the development and use aquatic mollusk body has broad prospects, and induces in batches for mollusk both at home and abroad and extracts metallothionein(MT) to carry out the relevant research of suitability for industrialized production less relatively.Inducing not ideal enough with extraction process is the low major cause of preparation metallothionein(MT) productive rate, at present to the inducing of metallothionein(MT), mainly take the method for animal injection, because dosage and the temporal differences of injection, metallothionein(MT) in the animal body is synthetic unstable, causes the wasting of resources.
The software animal of indication of the present invention has angle back of the body anodon, cristaria plicata, hydriopsis cumingii.Freshwater mussel is precious from head to foot, and particularly manually bringing up the used freshwater mussel of pearl is the cradle of pearl, not only can form natural peral, also can manually bring up pearl; Except growing cultured pearls, clam shell can be obtained through refining pearl powder and pearl nucleus, 15 seed amino acids that pearl powder has needed by human body to want, roughly the same with the composition of pearl and effect, have clearing heat and detoxicating, the benefit that makes eye bright the moon, tranquilizing mind, dephlogistication and promoting nuscle growth, relieving cough and reducing sputum, the dysentery function such as long-pending that disappears only; From the freshwater mussel in-vivo tissue, extract metallothionein(MT), can be used as medicine, health care of food and cosmetics additive, because having special heavy metal detoxification function, for providing safe and reliable raw material, development protective foods, makeup, medicine guarantee, remedied the weak point of eliminating because of the Health hazard of heavy metals exceeding standard product, processed for the application of healthy products and brought Gospel.And remaining clam shell processing respectively, retort sterilization and mechanical disintegration namely can be made into cheap animality high protein feed, have improved the comprehensive utilization ratio of freshwater mussel industry resource.The comprehensive traditional animal metallothionein(MT) extracting method of the present invention, induce the synthetic of freshwater mussel in-vivo metal sulfoprotein with the metal ion solution aquaculture model, solve the limitation of the extraction of laboratory little metal sulfoprotein, helped large-scale industrialization to extract the interior metallothionein(MT) of freshwater mussel body.
Summary of the invention:
The present invention adopts following technical scheme to realize its goal of the invention, a kind of method of from freshwater mussel, extracting metallothionein(MT), its adopt metal ion inducing culture, homogenate centrifugal, hold the sex change impurity elimination, secondary centrifuging is separated four processing steps of purifying and is finished;
Metal ion inducing culture: select for use the freshwater mussel of clean live body to put into and contain Cd
2+(concentration of metal ions of 10mg/L) carries out batch induces, and induction time is 2d, during feeding not, dissect then and take out fresh freshwater mussel muscle and liver organization, and carry out-20 ℃ freezing, the freshwater mussel muscle tissue after freezing is for further processing through the rubbing of thawing;
Homogenate is centrifugal: temperature 20-30 ℃ down place 8-10h after, with 5000～10000 rev/mins, the centrifugal 30min of whizzer, again with the negative pressure essence filter the active ingredient of slurry in the freshwater mussel muscle tissue; Adopt the Tirs-HCl damping fluid to carry out homogenate; preparation contains 0.15mol/L NaCl to promote protein dissolution; and proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF; 0.1,0.2mmol/L), reducing agent dithiothreitol (DTT; 1mmol/L) or B2 mercaptoethanol (10mmol/L) (SH) structure-activity stablizer (SH) is not degraded or oxidation with the sulfydryl of protection MT as sulfydryl.The freshwater mussel muscle that is extracted and liver organization and be generally 1/2～1/5 with the mass volume ratio (g/mL) of damping fluid;
Hold the sex change impurity elimination: namely, sex change is except foreign protein, and 10000r/min is in 4 ℃ of centrifugal 30min, abandon precipitation, get supernatant liquor, 85 ℃ of water-bath thermally denature 5min, the frozen water cooling with 8 layers of filtered through gauze, is got clear liquid, leave standstill about 2h, 8000r/min in 4 ℃ of centrifugal 10min, abandon precipitation, get supernatant liquor, obtain desired homogenate, write down volume V, be accurate to 1mL, every group establish 5 parallel;
Secondary centrifuging is separated purification: the purpose of secondary centrifuging is to realize that thermally denature flocculation foreign protein obtains the high purity concentrated solution with separating of extraction agent.Be 0.01mol/L with the Tris-HCl buffer concentration, PH8.6 puts 85 ℃ of water-bath thermally denature 5min, carries out centrifugal 8000r/min second time, 10min.The mass volume ratio of freshwater mussel slurries and damping fluid is 1/2.Centrifugal 10min just can be with material sedimentations such as cell debris, plastosomes when 10000r/min.Be improving velocity of separation, is under the situation of 8000r/min keeping centrifugal, and centrifugation time is optimized.The time of secondary centrifuging is little to the measured value influence of absorbancy, and is basicly stable in 10～60min; For saving extraction time, the time of secondary centrifuging is advisable with 10min, and the concentrated slurry of obtaining makes the MT lyophilized powder through conventional steps such as lyophilizes.
Owing to adopt technique scheme, in advance the live body freshwater mussel be exposed to Cd
2+(10mg/L) induce the synthetic of cadmium-metallothionein(MT) in the mass concentration solution; The centrifugal employing of homogenate Tirs-HCl damping fluid, stablizer be proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.1,0.2mmol/L), reducing agent dithiothreitol (DTT, 1mmol/L) or B2 mercaptoethanol (10mmol/L).At homogenized phase, optimum process condition is: the concentration 0.01mol of Tris-HCl damping fluid/L, and pH8.6, the mass volume ratio of mussel meat tissue and damping fluid are 1/2, the stripping quantity of MT in damping fluid reaches maximum with this understanding.The optimum process condition in removal of impurities stage is: homogenate is abandoned precipitation at 10000r/min, 4 ℃, centrifugal 30min, gets supernatant liquor, put 85 ℃ of water-bath thermally denature 5min, except foreign protein, cool off with frozen water again, with 8 layers of filtered through gauze, get clear liquid, leave standstill about 2h, 8000r/min in 4 ℃ of centrifugal 10min, abandon precipitation, get supernatant liquor, obtain desired homogenate, write down volume V, be accurate to 1mL.Above-mentioned process secondary centrifuging finally obtains high density MT concentrated solution, makes the MT lyophilized powder through conventional steps such as lyophilizes.
Compared with prior art this method has following advantage: have single factor design optimization freshwater mussel mainly organize in the leaching process of MT; Because the conformation of MT molecule is comparatively firm, have stronger thermotolerance, can in 80 ℃ of water, preserve the 30min unchangeability, and the protein of most of kinds begins sex change when high 40～50 ℃ of temperature.Utilize this characteristic can remove the thermally labile high molecular weight protein, further eliminate the interference component in the extracting solution.Consider when temperature is lower than 80 ℃, the sex change of non-MT albumen is abundant inadequately, and temperature is when too high, among the MT-the easy inactivation of SH, select 85 ℃ of water-baths that above-mentioned supernatant liquor after centrifugal is handled, during heating in water bath 5～15min, absorbancy remains on the higher stable level, show in the freshwater mussel tissue MT at this moment between in can inactivation; When heating surpassed 15min, along with time lengthening, absorbancy significantly reduced (p＜0.05), shows that thermally denature has taken place part MT.The stability of MT molecule and effectively separate other protein two aspects and consider between collateral security heating period, choose in the suitableeest heating in water bath time removal of impurities process, the purpose of heating in water bath is to utilize the thermostability of MT to remove thermally labile high molecular weight protein in the homogenate, and centrifugal only for obtaining a kind of means of supernatant liquor.The characteristics of iso-electric point between pH3.5～6.0 according to hydrobiont MT, adopt the buffered soln of pH6～8 can effectively prevent the protein denaturation that exsomatizes, the buffered soln pH scope (7.4～9.0) of protein active is optimized impurity elimination, when pH of buffer is 8.6, the measured value maximum of freshwater mussel MT, illustrate that this pH is conducive to the stable of MT most, can avoid near the albumen precipitation that occurs of iso-electric point, can avoid the protein denaturation under the higher pH condition again, determine that thus the pH of Tris-HCl buffered soln the best is 8.6.
Above-mentioned technology provides reliably for freshwater mussel MT is applied to enterprise's suitability for industrialized production, extracting method efficiently; And this extracting method is simple to operate, and process is easy to control, will be significant to production and the deep processing of scientific research and manufacturing enterprise.
Embodiment:
Be described further below in conjunction with summary of the invention:
Embodiment 1:
Induce in the freshwater mussel body tissue that cadmium MT is synthetic to be selected for use the freshwater mussel of clean live body to put into the cadmium content measuring method to contain Cd
2+(concentration of metal ions of 10mg/L) carries out batch induces, and induces the synthetic of cadmium-metallothionein(MT), and induction time is 2d, and cadmium-metallothionein(MT) can be stablized and reaches maximum in the freshwater mussel body, during feeding not.Take out the fresh freshwater mussel muscle liver organization of dissecting then ,-20 ℃ freezing.The accurate freshwater mussel standard MT sample liquid of drawing quantitatively dilutes (making Cd concentration in the typical curve concentration range) with deionized water in advance, shakes up.Record absorbancy on the atomic absorption spectrophotometer, by the extracted amount (the wet freshwater mussel muscle of mg Cd/g and liver) of freshwater mussel muscle and liver re-computation cadmium.To wet freshwater mussel muscle and liver organization with wet method digestion after, cadmium content in freshwater mussel muscle and the liver organization is measured in dilution.Measure its cadmium concentration on the atomic absorption spectrophotometer, molecular mass by Cd-MT is that 6500u and per molecule Cd-MT contain 7 cadmium atoms calculating, the content that obtains Cd-MT is about the wet freshwater mussel muscle of 4.53mg/g and liver organization, sulfhydryl content to record again, survey ultraviolet absorption value outward by 412nm, the content that obtains Cd-MT in freshwater mussel muscle and the liver organization is about 4.48mg/g.
Embodiment 2:
Freshwater mussel muscle tissue after the process of the homogenate separation and Extraction MT of freshwater mussel body tissue is freezing is through the rubbing of thawing, spare matter and separate, after placing 8-10h under temperature 20-30 ℃, with 5000～10000 rev/mins, the centrifugal 30min of whizzer, again with the negative pressure essence filter the active ingredient in the slurry tissue.Adopt the Tirs-HCl damping fluid to carry out homogenate; preparation contains 0.15mol/L NaCl to promote protein dissolution; and proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF; 0.1,0.2mmol/L), reducing agent dithiothreitol (DTT; 1mmol/L) or B2 mercaptoethanol (10mmol/L) (SH) structure-activity stablizer (SH) is not degraded or oxidation with the sulfydryl of protection MT as sulfydryl.The freshwater mussel muscle that is extracted and liver organization and be 1/2～1/5 with the mass volume ratio (g/mL) of damping fluid.After the preparation homogenate, get supernatant liquor and measure MT content with enzyme-linked immunosorbent assay (ELISA).With coating buffer by dilution in 1: 100 after, add 100 μ L in the shrinkage pool of enzyme plate, add a cover 4 ℃ and spend the night, with pH7.4 Tris-HCl (including the Tween-20 of massfraction 0.05%) damping fluid washing 3 times; Add confining liquid (bovine serum albumin of 1.5% massfraction) 200 μ L, 37 ℃, washing is 3 times behind 1 h; Add 37 ℃ of the anti-freshwater mussel MT of 100 μ L rabbits polyclonal antibodies (1:1000 doubly), 2 h are with damping fluid washing 3 times, 100 μ L add in the hand-hole with HRP-goat anti-rabbit igg antibody (dilution in 1: 1000), 37 ℃, 1.5h is with damping fluid washing 3 times, distilled water wash 3 times, add 100 μ LTMB in the hole and use liquid, room temperature adds 50 μ L2mol/L H2SO4 after placing 15min again, survey A450 behind the 5min, and compare its result with oxyphorase/cadmium saturation method.
Embodiment 3:
MT thermally denature impurity elimination process in the freshwater mussel body tissue: 10000r/min abandons precipitation in 4 ℃ of centrifugal 30min, gets supernatant liquor, 85 ℃ of water-bath thermally denature 5min, the frozen water cooling is with 8 layers of filtered through gauze, get clear liquid, leave standstill about 2h, 8000r/min in 4 ℃ of centrifugal 10min, abandon precipitation, get supernatant liquor, obtain desired homogenate, write down volume V, be accurate to 1mL, every group establish 5 parallel, be for further processing.
Embodiment 4:
The secondary centrifuging separating and purifying method of MT and cubage method in the freshwater mussel body tissue: be 0.01mol/L with the Tris-HCl buffer concentration, PH8.6 puts 85 ℃ of water-bath thermally denature 5min, carries out centrifugal 8000r/min second time, 10min.The mass volume ratio of freshwater mussel slurries and damping fluid is 1/2, obtains the MT concentrated solution.(SH) content makes the MT lyophilized powder up to 83% through conventional steps such as lyophilizes to dredge base.Final mensuration and the extraction yield that extracts the sulfhydryl content of concentrated solution calculates, and the mensuration of sulfhydryl content detects with Ellman reagent, takes by weighing a certain amount of freshwater mussel MT sample (dredging basic content 0.1～0.5 μ mol), is made into less than 50 μ L solution.Add 10 μ L 1.2mol/L HCl and 200 μ L 0.1mol/LEDTA reaction 10min and slough metal.Add 200 μ L Ellman ' s reagent and (contain 5mmol/L DTNB, 1mmol/L EDTA, 6mol/L Guanidinium hydrochloride, 0.1mol/L PBS, pH7.3), mixing 3min forms yellow complex with DTNB after making the MT sex change, with 0.1mol/L PBS, pH7.3 is diluted to 4mL and surveys ultraviolet absorptivity A at the 412nm place, replaces sample extracting solution with 50 μ L extraction agents simultaneously, carries out blank assay by same step and obtains absorbance A o.With Δ A(A-Ao) MT level in the characterizing tissues.Extraction yield calculates: because 1mol MT (molecular weight 9799) can be in conjunction with 7mol Cd2+(nucleidic mass 112.4), so the MT content in every gram tissue (μ gg-1) is in the freshwater mussel tissue
MT?=?［(?C
Cd－?C
KB)×V×9799×S)?］/?(?G×7×112.4×N)
In the formula: C
CdBe Cd in the supernatant liquor
2+Mass concentration, μ gmL-1; C
KBBe the final supernatant liquor reading of blank sample; The volume of the supernatant liquor of getting when S is mensuration, mL; G is the quality of sampling tissue, and gV is the tissue homogenate volume, mL; N is the sample volume after the homogenate, mL.Dredging basic concentration calculates: C=DA/ ε, ε in the formula=1.23 * 104 (mol/L)-1cm-1, A is light absorption value, D is extension rate.Data processing adopts Excel7.0 version and SPSS data analysis software to carry out statistical study.Experimental data is represented with mean number ± standard deviation, adopts T-test to carry out otherness and test of significance simultaneously.
Claims (1)
1. method of from freshwater mussel, extracting metallothionein(MT), it is characterized in that its adopt metal ion inducing culture, homogenate centrifugal, hold the sex change impurity elimination, secondary centrifuging is separated four processing steps of purifying and is finished;
Metal ion inducing culture: select for use the freshwater mussel of clean live body to put into and contain Cd
2+(concentration of metal ions of 10mg/L) carries out batch induces, and induction time is 2d, during feeding not, dissect then and take out fresh freshwater mussel muscle and liver organization, and carry out-20 ℃ freezing, the freshwater mussel muscle tissue after freezing is for further processing through the rubbing of thawing;
Homogenate is centrifugal: temperature 20-30 ℃ down place 8-10h after, with 5000～10000 rev/mins, the centrifugal 30min of whizzer, again with the negative pressure essence filter the active ingredient of slurry in the freshwater mussel muscle tissue; Adopt the Tirs-HCl damping fluid to carry out homogenate, preparation contains 0.15mol/L NaCl to promote protein dissolution, and proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF, 0.1,0.2mmol/L), reducing agent dithiothreitol (DTT, 1mmol/L) or B2 mercaptoethanol (10mmol/L) (SH) structure-activity stablizer (SH) is not degraded or oxidation with the sulfydryl of protection MT as sulfydryl; The freshwater mussel muscle that is extracted and liver organization and be generally 1/2～1/5 with the mass volume ratio (g/mL) of damping fluid;
Hold the sex change impurity elimination: i.e. sex change is except foreign protein, and 10000r/min abandons precipitation in 4 ℃ of centrifugal 30min, get supernatant liquor, 85 ℃ of water-bath thermally denature 5min, frozen water cooling, with 8 layers of filtered through gauze, get clear liquid, leave standstill about 2h, 8000r/min in 4 ℃ of centrifugal 10min, abandon precipitation, get supernatant liquor, obtain desired homogenate, write down volume V, be accurate to 1mL, every group establish 5 parallel;
Secondary centrifuging is separated purification: the purpose of secondary centrifuging is to realize that thermally denature flocculation foreign protein obtains the high purity concentrated solution with separating of extraction agent; Be 0.01mol/L with the Tris-HCl buffer concentration, PH8.6 puts 85 ℃ of water-bath thermally denature 5min, carries out centrifugal 8000r/min second time, 10min; The mass volume ratio of freshwater mussel slurries and damping fluid is 1/2; Centrifugal 10min just can be with material sedimentations such as cell debris, plastosomes when 10000r/min; Be improving velocity of separation, is under the situation of 8000r/min keeping centrifugal, and centrifugation time is optimized; The time of secondary centrifuging is little to the measured value influence of absorbancy, and is basicly stable in 10～60min; For saving extraction time, the time of secondary centrifuging is advisable with 10min, and the concentrated slurry of obtaining makes the MT lyophilized powder through conventional steps such as lyophilizes.
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