CN1243015C - Separating and purifying technology for micro algae phycoerythrin - Google Patents
Separating and purifying technology for micro algae phycoerythrin Download PDFInfo
- Publication number
- CN1243015C CN1243015C CN 200410063071 CN200410063071A CN1243015C CN 1243015 C CN1243015 C CN 1243015C CN 200410063071 CN200410063071 CN 200410063071 CN 200410063071 A CN200410063071 A CN 200410063071A CN 1243015 C CN1243015 C CN 1243015C
- Authority
- CN
- China
- Prior art keywords
- phycoerythrin
- ammonium sulfate
- algae
- dialysis
- precipitation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108010004729 Phycoerythrin Proteins 0.000 title claims abstract description 38
- 241000195493 Cryptophyta Species 0.000 title claims abstract description 22
- 238000005516 engineering process Methods 0.000 title abstract description 3
- 238000000502 dialysis Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 238000001556 precipitation Methods 0.000 claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 14
- 239000012153 distilled water Substances 0.000 claims abstract description 13
- 229920002271 DEAE-Sepharose Polymers 0.000 claims abstract description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 239000007787 solid Substances 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 6
- 238000005342 ion exchange Methods 0.000 claims abstract description 5
- 238000005119 centrifugation Methods 0.000 claims abstract 3
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 15
- 238000007710 freezing Methods 0.000 claims description 13
- 230000008014 freezing Effects 0.000 claims description 13
- 239000000287 crude extract Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 238000010521 absorption reaction Methods 0.000 claims description 10
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 9
- 238000004255 ion exchange chromatography Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 238000005194 fractionation Methods 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 abstract description 9
- 239000012530 fluid Substances 0.000 abstract description 6
- 238000004108 freeze drying Methods 0.000 abstract description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000001488 sodium phosphate Substances 0.000 abstract description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 238000005070 sampling Methods 0.000 abstract 1
- 229910000406 trisodium phosphate Inorganic materials 0.000 abstract 1
- 235000019801 trisodium phosphate Nutrition 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 22
- 241001494715 Porphyridium purpureum Species 0.000 description 9
- 238000004440 column chromatography Methods 0.000 description 6
- 108060006184 phycobiliprotein Proteins 0.000 description 6
- 238000013016 damping Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- -1 dialysis Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000192700 Cyanobacteria Species 0.000 description 1
- 241000199914 Dinophyceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000206643 Rhodella Species 0.000 description 1
- 241000293712 Rhodella reticulata Species 0.000 description 1
- 241000206572 Rhodophyta Species 0.000 description 1
- 210000002659 acromion Anatomy 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001997 free-flow electrophoresis Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000886 photobiology Effects 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to technology by which micro algae phycoerythrin is leached by distilled water, is fractionally precipitated by ammonium sulfate, and is separated and purified by ion-exchange post chromatography. Freeze-drying algae powder, algae mud and distilled water are leached overnight at 4 DEG C, and adopts high-speed refrigerated centrifugation; solid ammonium sulfate is added and is fractionally precipitated when definite saturation is achieved, and a trisodium phosphate buffer solution balances dialysis; after centrifugation is carried out for 20 min, supernatant fluid is purified with one step by ion-exchange post chromatography; thereby, phycoerythrin is obtained. The present invention obtains the phycoerythrin by the fractional precipitation of ammonium sulfate and one-step purification of DEAE-Sepharose FF ion-exchange post chromatography, has the characteristics of favorable purification effect, large sampling quantity and high sample yield, and can be used for amplifying production, etc.; the purity of the phycoerythrin obtained by separation reaches 4.85, total yield is 52%, and a strip is displayed in polyacrylamide gel electrophoresis.
Description
Technical field
The present invention relates to a kind of method that adopts distilled water lixiviate, ammonium sulfate precipitation and ion-exchange chromatography separation and purification micro algae phycoerythrin.
Background technology
Phycobiliprotein is a kind of water colo(u)r albumen, is present in red algae, blue-green algae, latent algae and some the dinoflagellate body, and be that the distinctive photosynthetical system of these algae is caught photopigment, in photosynthesis, work to capture luminous energy and transmit energy.According to structure and spectral response curve, phycobiliprotein is divided into: phycoerythrin (PE), Phycocyanins, C-(PC) and other structure Phycocyanins, C-(A-PC).Phycoerythrin passes to Phycocyanins, C-with the luminous energy of catching, and passes to allophycocyanin again, passes to the center pigment at last, realizes photosynthesis.Thereby phycoerythrin has important researching value at photosynthetic theoretical side originally.On the other hand, phycoerythrin is of many uses, both can be used as natural pigment and has been widely used in industry such as food, makeup, dyestuff, can be made into fluorescent reagent again, be used for fields such as clinical diagnose, immunochemistry and biotechnology, be used for optical dynamic treatment of tumor as photosensitizers.
A chromatoplast that is star is greatly arranged in the Porphyridium cruentum cell, include abundant phycoerythrin and Phycocyanins, C-, phycoerythrin accounts for phycobiliprotein 84%, and is wherein maximum with B-phycoerythrin content.Kost-Reyes (EurJ Biochem.1979,102 (1): 83-91) utilize carrier-free electrophoresis from the albumen of crossing post, to isolate B-phycoerythrin, b-phycoerythrin and R Phycocyanins, C-, at last the B-phycoerythrin OD of Huo Deing
545/ OD
280Ratio 〉=5.(J Photochemistry Photobiology B.1997 for Stadnichuk, 39:19-23) obtain Porphyridium cruentum B-phycoerythrin, the B-phycoerythrin OD that finally obtains by ToypearlDEAE-650M ion-exchange, hydroxyapatite and Sephadex G-200 gel separation
542/ OD
500=2.00, OD
542/ OD
280＞6.0.Bermejo (JChromatogr A.2001,917 (1-2): 135-145, J Biotech.2002,93:73-85; JChromatogr B.2003,709:317-325) wait the method separation and purification Porphyridium cruentum B-phycoerythrin that utilizes two step chromatographies, adopt reverse HPLC (high performance liquid chromatography) gradient half preparation method, with α, β and the γ subunit of C4 wide aperture post with the second cyanogen solution separating phycoerythrin of the aqueous solution that contains 0.05% trifluoroacetic acid (TFA) and 0.05%TFA, produce three approaching bands through polyacrylamide gel electrophoresis, this three band and its three subunits are corresponding.They also prepare the B-phycoerythrin and the R-Phycocyanins, C-of natural mode.Set up a kind of method of new and scalable separation and purification Porphyridium cruentum B-phycoerythrin, this experiment is in conjunction with expanded bed and two kinds of chromatography methods of DEAE-Mierocrystalline cellulose packed bed.Ma (Plant Sci.2003,164:253-257) grade utilizes the SephadexG-200 gel chromatography to separate Porphyridium cruentum B-phycoerythrin and R-Phycocyanins, C-, and utilize reacting to each other between these two kinds of albumen amino groups to form artificial B-phycoerythrin-R-Phycocyanins, C-covalency association, experimental result shows that the stability of this artificial covalency association is higher than the B-phycoerythrin, is expected to be used for the preparation of phycobiliprotein probe.Wen Shaohong (ocean circular .2000,19 (3): 90-93; China marine drug .2001,3:33-35) grade is with the water-soluble crude extract process ammonium sulfate precipitation and the hydroxyapatite column chromatography of Porphyridium cruentum, separation and purification obtains B-phycoerythrin (B-PE), and B-PE respectively has an absorption peak at 545nm and 563nm, has one to absorb acromion at 498nm.They also with the phycobiliprotein crude extract through ammonium sulfate precipitation, dialysis, hydroxyapatite and SephadexG-100 column chromatography, separation and purification obtains the B-phycoerythrin, purity (OD
545/ OD
280) being respectively 4.92 and 3.78, the polyacrylamide gel gradient electrophoresis obtains a band.
Summary of the invention
Purpose of the present invention just provides a kind of with the lixiviate repeatedly of algae powder, and ammonium sulfate branch precipitation obtains thick phycobiliprotein, obtains the method for highly purified phycoerythrin through the ion-exchange chromatography purifying.
For realizing that the technical scheme that purpose of the present invention adopts is: at first little algae powder is pressed 1: 10～15 adding distil waters in the refrigerator overnight lixiviate, 4 ℃ of high speed freezing centrifuges, the centrifugal 20min of 6000r/min get supernatant liquor after the lixiviate.Sedimentary algae mud continues 2 lixiviates of spending the night by 1: 8.0～12.5 adding distil waters in refrigerator, in 4 ℃, the centrifugal 20min of 6000r/min, respectively get supernatant liquor, merge 3 lixiviate gained supernatant liquors (being the phycoerythrin crude extract), grouping, add solid ammonium sulfate 20,40,60,80% saturation ratio respectively, standing over night behind the mixing, 4 ℃, the centrifugal 20min of 6000r/min, get precipitation, precipitation is dissolved in a small amount of distilled water packs in the dialysis tubing, 4 ℃ of distill water dialysis 24h use the 0.01mol/L sodium phosphate buffer equilibrium dialysis 24h of pH7.0 again.Dialyzed sample in 4 ℃, the centrifugal 20min of 6000r/min, is got supernatant liquor, be all product.With the DEAE-Sepharose FF post that installs 0.01mol/L sodium phosphate buffer balance, be 7.0 until effluent liquid pH value.Sample is splined on the good capital of balance, washes post with 0.01～0.02mol/L sodium phosphate buffer and get back to baseline, make gradient elution with 0.5～1.0mol/LNaCl then, flow velocity 1.5mL/min until 280nm place absorption peak.Each pipe of collecting is surveyed the absorbance value at its 280nm and 545nm place, and make 280nm chromatography curve.According to absorption peak and OD
545/ OD
280Ratio is collected sample, and dialysis, concentrated, centrifugal, freeze-drying get sample.The concrete technical scheme of the present invention comprises following concrete step:
1, the extraction of phycoerythrin: take by weighing a certain amount of freeze-dried algae powder, press 1: 10～15 adding distil waters in the refrigerator overnight lixiviate as Rhodella reticulate, Porphyridium cruentum (Porphydidium cruentum), in 4 ℃ of high speed freezing centrifuges, the centrifugal 20min of 6000r/min, get supernatant liquor after the lixiviate.Sedimentary algae mud continues 2 lixiviates of spending the night by 1: 8.0～12.5 adding distil waters, in 4 ℃ of high speed freezing centrifuges, the centrifugal 20min of 6000r/min, gets supernatant liquor.Merge above-mentioned 3 lixiviate gained supernatant liquors and be the phycoerythrin crude extract.
2, ammonium sulfate precipitation: crude extract grouping, add solid ammonium sulfate respectively, saturation ratio is 20%, 40%, 60% and 80%, carries out the fractionation precipitation of heteroproteins and target protein matter, after the standing over night, high speed freezing centrifuge, 4 ℃, the centrifugal 20min of 6000r/min get precipitation, precipitation is dissolved in a small amount of distilled water packs in the dialysis tubing, 4 ℃ of distill water dialysis 15～24h use pH7.0 damping fluid equilibrium dialysis 15～24h then.In high speed freezing centrifuge, 4 ℃ of centrifugal 20min of 6000r/min get supernatant liquor, are all product with dialyzed sample.Measure phycoerythrin sample purity respectively, with the saturation ratio of determining that ammonium sulfate precipitation is adopted through ammonium sulfate precipitation.
3, DEAE-Sepharose Fast Flow column chromatography:,, be pH7.0 until effluent liquid with pH7.0 damping fluid balance with the DEAE-Sepharose FF post that installs.Sample is splined on the good capital of balance, wash post with sample-loading buffer and get back to baseline until 280nm place absorption peak, make gradient elution with 0.5～1.0mol/L NaCl solution then, each pipe of collecting is surveyed the absorbance value at its 280nm and 545nm place, and make 280nm chromatography curve.According to absorption peak and OD
545/ OD
280Ratio is collected purer sample, and dialysis, concentrated, centrifugal, freeze-drying get the phycoerythrin sample.
The advantage that the present invention has is phycoerythrin through DEAE-Sepharose FF ion-exchange chromatography single step purification, and purity reaches OD
545/ OD
280=4.85, the gained sample shows a band in polyacrylamide gel electrophoresis, illustrates that to reach electrophoresis pure, total recovery 52%.Adopt DEAE-Sepharose FF ion-exchange chromatography to have that purification effect is good, applied sample amount big, the sample yield is higher and can be used for amplifying characteristics such as production.Compare with the phycoerythrin separating purifying method of having reported, adopt DEAE-Sepharose FF ion-exchange chromatography to be better than adopting hydroxyapatite column chromatography, Sephadex G-100 gel filtration chromatography.
Embodiment
The present invention is described further below in conjunction with specific embodiment.
Embodiment 1.
1, the extraction of phycoerythrin: take by weighing 5g Porphyridium cruentum lyophilized powder and add 50mL distilled water in the refrigerator overnight lixiviate in 1: 10 ratio, fully after the lixiviate in 4 ℃ of centrifugal 20min of 6000r/min of high speed freezing centrifuge, get supernatant liquor.Sedimentary algae mud adds the lixiviate 2 times that continues to spend the night of 50mL distilled water.Merge 3 lixiviate gained supernatant liquors and be the phycoerythrin crude extract.
2, ammonium sulfate precipitation: crude extract grouping, add solid ammonium sulfate respectively, saturation ratio is 20%, 40%, 60% and 80%, carries out the fractionation precipitation of heteroproteins and target protein matter behind the mixing, standing over night, high speed freezing centrifuge, 4 ℃, the centrifugal 20min of 6000r/min get precipitation, precipitation is dissolved in a small amount of distilled water packs in the dialysis tubing, 4 ℃ of distill water dialysis 24h use 0.01mol/L sodium phosphate buffer (PBS) pH7.0 damping fluid equilibrium dialysis 24h then.In high speed freezing centrifuge, 4 ℃ of centrifugal 20min of 6000r/min get supernatant liquor, are all product with dialyzed sample.Measure phycoerythrin sample purity respectively, with the saturation ratio of determining that ammonium sulfate precipitation is adopted through ammonium sulfate precipitation.
3, phycoerythrin DEAE-Sepharose FF column chromatography purification:,, be pH7.0 until effluent liquid with 0.01mol/L sodium phosphate buffer balance with the DEAE-SepharoseFF post that installs.Sample is splined on the good capital of balance, washes post with sample-loading buffer and get back to baseline until 280nm place absorption peak, make gradient elution with 0.5mol/LNaCl then, the elutriant cumulative volume is 300mL, flow velocity 1.5mL/min, and every pipe is collected about 5mL.Each pipe of collecting is surveyed the absorbance value at its 280nm and 545nm place, and make 280nm chromatography curve.According to absorption peak and OD
545/ OD
280Ratio is collected purer sample, dialysis, concentrate, freeze-drying gets Porphyridium cruentum phycoerythrin sample.
Embodiment 2
1, the extraction of phycoerythrin: take by weighing 5gRhodella reticulate lyophilized powder and add 75mL distilled water in the refrigerator overnight lixiviate in 1: 15 ratio, fully after the lixiviate in 4 ℃ of centrifugal 20min of 6000r/min of high speed freezing centrifuge, get supernatant liquor.Sedimentary algae mud added 60mL distilled water by 1: 12.5 and continues 2 lixiviates of spending the night.Merge 3 lixiviate gained supernatant liquors and be the phycoerythrin crude extract.
2, ammonium sulfate precipitation, the crude extract grouping adds solid ammonium sulfate respectively, and saturation ratio is 20%, 40%, 60% and 80%, carry out the fractionation precipitation of heteroproteins and target protein matter behind the mixing, standing over night, high speed freezing centrifuge, 4 ℃, the centrifugal 20min of 6000r/min, get precipitation, precipitation is dissolved in a small amount of distilled water packs in the dialysis tubing, 4 ℃ of distill water dialysis 24h use 0.02mol/L sodium phosphate buffer (PBS) pH7.0 damping fluid equilibrium dialysis 24h then.In high speed freezing centrifuge, 4 ℃ of centrifugal 20min of 6000r/min get supernatant liquor, are all product with dialyzed sample.Measure phycoerythrin sample purity respectively, with the saturation ratio of determining that ammonium sulfate precipitation is adopted through ammonium sulfate precipitation.
3, phycoerythrin DEAE-Sepharose FF column chromatography purification is with the DEAE-SepharoseFF post that installs, with 0.02mol/L sodium phosphate (pH7.O) damping fluid balance, until effluent liquid pH7.0.Sample is splined on the good capital of balance, washes post with sample-loading buffer and get back to baseline until 280nm place absorption peak, make gradient elution with 1.Omol/LNaCl then, the elutriant cumulative volume is 500mL, flow velocity 2.OmL/min, and every pipe is collected about 5mL.Each pipe of collecting is surveyed the absorbance value at its 280nm and 545nm place, and make 280nm chromatography curve.According to absorption peak and OD
545/ OD
280Ratio is collected purer sample, dialysis, concentrate, freeze-drying gets the Rhodella reticulata phycoerythrin.
Claims (3)
1, a kind of method that adopts ammonium sulfate precipitation and ion exchange chromatography separation and purification micro algae phycoerythrin is characterized in that:
I, algae powder adding distil water are in the refrigerator overnight lixiviate, 4 ℃ of high speed freezing centrifuges, the centrifugal 20min of 6000r/min after the lixiviate, get supernatant liquor, sedimentary algae mud adding distil water in refrigerator after 2 lixiviates of spending the night in 4 ℃ of high speed freezing centrifuges, the centrifugal 20min of 6000r/min, get supernatant liquor, merge the gained supernatant liquor and be the phycoerythrin crude extract;
II, phycoerythrin crude extract grouping back adding saturation ratio are respectively 20%, 40%, 60% and 80% solid ammonium sulfate and spend the night, carry out centrifugation, get the precipitation be dissolved in distilled water and the dialysis tubing of packing in distill water dialysis, the sodium phosphate buffer equilibrium dialysis, dialyzed sample is centrifugal, get supernatant liquor and be all product;
III, sample is splined on the good DEAE-Sepharose FF ion exchange column capital of balance, wash post with 0.01～0.02mol/L sodium phosphate buffer and get back to baseline until 280nm place absorption peak, carry out gradient elution with 0.5～1.0mol/LNaCl, flow velocity 1.5mL/min.
2, the method for ammonium sulfate precipitation according to claim 1 and ion exchange chromatography separation and purification micro algae phycoerythrin is characterized in that algae powder and algae mud are respectively by 1: 10～15 and 1: 8.0～12.5 adding distil water lixiviates.
3, the method for ammonium sulfate precipitation according to claim 1 and ion exchange chromatography separation and purification micro algae phycoerythrin, it is characterized in that precipitation behind the solid ammonium sulfate fractionation precipitation is dissolved in a small amount of distilled water and packs in the dialysis tubing, 4 ℃ of distill water dialysis 24h, be 7.0 0.01mol/L sodium phosphate buffer equilibrium dialysis 24h again with pH, in 4 ℃, the centrifugal 20min of 6000r/min, get supernatant liquor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410063071 CN1243015C (en) | 2004-07-13 | 2004-07-13 | Separating and purifying technology for micro algae phycoerythrin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410063071 CN1243015C (en) | 2004-07-13 | 2004-07-13 | Separating and purifying technology for micro algae phycoerythrin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1587275A CN1587275A (en) | 2005-03-02 |
| CN1243015C true CN1243015C (en) | 2006-02-22 |
Family
ID=34603811
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410063071 Expired - Fee Related CN1243015C (en) | 2004-07-13 | 2004-07-13 | Separating and purifying technology for micro algae phycoerythrin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1243015C (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412730B (en) * | 2017-04-27 | 2020-08-14 | 浙江工商大学 | Dihuang protein nano-particles and preparation method thereof |
| CN107200774B (en) * | 2017-06-14 | 2020-04-10 | 湖南炎帝生物工程有限公司 | Extraction and purification method of nostoc sphaeroides biliprotein and purified phycoerythrin |
| CN107840870A (en) * | 2017-12-15 | 2018-03-27 | 大连海洋大学 | A kind of method that centrifugation chromatogram prepares high-purity phycoerythrin |
| CN109879943B (en) * | 2019-04-12 | 2021-07-20 | 集美大学 | A kind of extraction method of phycoerythrin |
| CN113968900B (en) * | 2020-07-23 | 2023-07-25 | 中元汇吉生物技术股份有限公司 | Method for purifying C1q protein |
| CN111978383A (en) * | 2020-09-03 | 2020-11-24 | 福建师范大学 | Method for synergistically extracting phycoerythrin and grease from wet algae |
| CN112778411A (en) * | 2021-02-26 | 2021-05-11 | 通用生物系统（安徽）有限公司 | Extraction method of natural beta 2-microglobulin |
| CN114702557A (en) * | 2021-07-09 | 2022-07-05 | 杭州职业技术学院 | Preparation method of nostoc sphaeroides hemoglobin |
| CN113476891A (en) * | 2021-08-06 | 2021-10-08 | 中国科学院南海海洋研究所 | Method for simultaneously extracting multiple active substances from porphyridium |
-
2004
- 2004-07-13 CN CN 200410063071 patent/CN1243015C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1587275A (en) | 2005-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103880947B (en) | A kind of chromatography method of separating and purifying high-purity recombination human serum albumin | |
| CN1243015C (en) | Separating and purifying technology for micro algae phycoerythrin | |
| CN102329381A (en) | Method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof | |
| CN104774182B (en) | The extraction of erythrothioneine and purification process | |
| CN106478810B (en) | A method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin | |
| CN102532254A (en) | Method for separating and purifying recombinant human serum albumin (rHSA) from rice seeds | |
| Zheng et al. | Phycocyanin fluorescent probe from Arthrospira platensis: preparation and application in LED-CCD fluorescence density strip qualitative detection system | |
| Zang et al. | Preparation of high-purity R-phycoerythrin and R-phycocyanin from Pyropia yezoensis in membrane chromatography | |
| CN107298710A (en) | Extraction method of spirulina phycocyanin | |
| CN108103130A (en) | The combination technique of extraction separation small active peptides from marine protein resource | |
| CN106146652A (en) | A kind of method for extraction and purification of middle phycocyanin of delivering vegetables | |
| CN109320588A (en) | A kind of ACE inhibitory activity peptide in stichopus japonicus source | |
| CN101021452A (en) | Efficient fast micro algae phycoerythrin separating purifying method | |
| CN102167725A (en) | Method for preparing Raja porosa angiogenesis inhibiting factor RCAIF-I | |
| CN109369783A (en) | A kind of polypeptide RDP1 and its purification method and application | |
| CN104292316A (en) | Efficient separation and purification method of Gracilaria verrucosa reagent grade R-phycoerythrin | |
| CN104059127A (en) | Natural high-activity antihypertensive peptide, and preparation method and application thereof | |
| CN101935665B (en) | Preparation method and application of recombinant protein A gene and expression product thereof | |
| CN101538561B (en) | A method for extracting CELⅠ nuclease in celery | |
| CN101073666B (en) | Method for producing kallidin proenzyme raw-material medicine | |
| CN102796190A (en) | Method for extracting riboflavin binding protein from egg white | |
| CN101555277B (en) | A rapid combined purification method of thyroglobulin and red kidney bean phytohemagglutinin | |
| CN111870998A (en) | Device for purifying sea cucumber polysaccharide by virtue of tandem lectin affinity column and use method of device | |
| CN107641154B (en) | Squid bioactive peptide and preparation method thereof | |
| CN101362792A (en) | Lactoferrin affinity separation material and lactoferrin affinity purification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Xiamen Kingdomway Vitamin Corp. Assignor: Fujian Normal University Contract fulfillment period: 2008.6.20 to 2014.6.20 contract change Contract record no.: 2008350000027 Denomination of invention: Separating and purifying technology for micro algae phycoerythrin Granted publication date: 20060222 License type: Exclusive license Record date: 2008.9.16 |
|
| LIC | Patent licence contract for exploitation submitted for record |
Free format text: EXCLUSIVE LICENCE; TIME LIMIT OF IMPLEMENTING CONTACT: 2008.6.20 TO 2014.6.20 Name of requester: XIAMEN JINDAWEI VITAMIN CO.LTD. Effective date: 20080916 |