CN1243015C - Separating and purifying technology for micro algae phycoerythrin - Google Patents
Separating and purifying technology for micro algae phycoerythrin Download PDFInfo
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- 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
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- phycoerythrin
- ammonium sulfate
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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.
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| 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 | 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 |
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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 |
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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 |