CN1654629A - Process for preparing intact phycobilisome - Google Patents
Process for preparing intact phycobilisome Download PDFInfo
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- CN1654629A CN1654629A CN 200510042029 CN200510042029A CN1654629A CN 1654629 A CN1654629 A CN 1654629A CN 200510042029 CN200510042029 CN 200510042029 CN 200510042029 A CN200510042029 A CN 200510042029A CN 1654629 A CN1654629 A CN 1654629A
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Abstract
The method of preparing intact phycobilisome belongs to the field of marine biotechnology. The cyanophyta and red algae material has the cells ultrasonic wave crushed, the phycobilisome dissociated from the thylakid membrane with detergent, and detergent, chlorophyll and cell fragment eliminated through conventional centrifugation. Through further polyglycol precipitation and Sepharose CL-4B column chromatography, intact phycobilisome is prepared. The said process omits the complicated traditional ultracentrifugal sucrose density gradient process and uses conventional apparatus in mass preparation of phycobilisome in greatly lowered cost. Therefore, the present invention lays the foundation for applying phycobilisome in super-sensitive biomedicine detection.
Description
(1) technical field
The present invention relates to a kind of preparation method of intact phycobilisome, relate to particularly from red algae and blue-green algae and separate the method for preparing intact phycobilisome, belong to the marine biotechnology field.
(2) background technology
Phycobiliprotein is that a class photosynthesis is caught photopigment-protein complex, mainly is present in blue-green algae, red algae, latent algae and the minority dinoflagellate.In photosynthesis, play a part to catch and transmit luminous energy, have intensive fluorescence.In blue-green algae and red algae, form the supramolecular structure phycobilisome by 2-3 kind phycobiliprotein, form transmission ofenergy sequence efficiently.At the mid-80, the scholar of American Studies algae photosynthesis proposition as fluorescent marker, is used for diagnostic reagent with phycobiliprotein.Because its unique advantage, the diagnostic reagent of phycobiliprotein and phycobiliprotein mark enters the world market in the early 1990s.
Compare with fluorescent marker commonly used, phycobiliprotein has following advantage: production process safety, nontoxic, and luminous energy absorbs strong, the fluorescent yield height surpasses 90%, and bias light interference and false positive rate are low, stable in the scope of pH4-11, can make double-colored, three looks and four color markers.So the range of application of this class reagent constantly enlarges.But,, be not applied to popular reagent for clinical diagnosis as yet owing to cost an arm and a leg.The whole imports of China also only limit to the diagnosis of carrying out with cell streaming instrument.
Phycobiliprotein has its special advantages as fluorescent probe, but one phycobiliprotein molecule, because its molecule is less, the pigment group that contains in the molecule is less, therefore, the brightness that produces fluorescence is not enough, makes in high-resolution detection medium sensitivity not high.U.S. Mo Senman (J.P.Morseman) proposes, and applying immobilized intact phycobilisome can be used for multiple biomedical the detection as fluorescent probe.Because contain more pigment group in the phycobilisome, therefore, its brightness is higher, and excitation wavelength is the absorbing wavelength of phycoerythrin (PE) or Phycocyanins, C-(PC), and fluorescent emission is from APC, therefore, excitation wavelength and emission wavelength are avoided the phase mutual interference at a distance of farther.Wherein, the quick technology of preparing of intact phycobilisome is the key that can this technology of restriction be applied cheaply.At present; the technology of preparing of phycobilisome all adopts the sucrose density centrifugation method, because the sucrose density gradient legal system is equipped with the phycobilisome complex operation, and essential ultra-high speed whizzer; thereby, seek easy, rapid, scale preparation phycobilisome alternative method and seem particularly important.
(3) summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of preparation method of intact phycobilisome is provided, technology is easy, rapid, and intact phycobilisome is produced on a large scale.
The preparation method of intact phycobilisome of the present invention, step is as follows:
(1) fragmentation of raw material: with fresh blue-green algae and red algae is raw material, is dissolved in the Sodium phosphate dibasic-potassium phosphate buffer of pH=6.8～7.0, adopts the ultrasonic disruption cell.
Above-mentioned blue-green algae is selected from spirulina plalensis or spirulina maxim, and red algae is selected from unicellular red algae Porphyridium cruentum, large-scale red algae multitube algae or Ceramium kondoi.
(2) phycobilisome dissociates: add stain remover tween X-100 (TritonX-100) in the frond of fragmentation, phycobilisome is disintegrated down from thylakoid membrane.Centrifugal, remove stain remover and broken cell, get the crude extract of phycobilisome.
Add-on the present invention of stain remover is not particularly limited, and is by state of the art, suitable so that phycobilisome is dissociated into from thylakoid membrane.
(3) concentrating of phycobilisome: use the polyethylene glycol precipitation phycobilisome, collecting precipitation.To precipitate and be dissolved in again in pH=6.8～7.0 Sodium phosphate dibasics-potassium dihydrogen phosphate.
(4) purifying of intact phycobilisome: above-mentioned phycobilisome solution is crossed Sepharose CL-4B column chromatography, collect the blue-violet solution of wash-out front end, obtain complete phycobilisome solution.
The fragmentation of above-mentioned steps (1) raw material is specially: fresh frond 0.75～1.0g, be dissolved in Sodium phosphate dibasic-potassium phosphate buffer (pH=6.8～7.0) of 15～20mL 1molL-1, ultrasonic disruption, broken power is 12～15W, broken 5～6 times, each 1.5～2min, 2～3min at interval.
Above-mentioned steps (2) phycobilisome dissociates and is specially: adding concentration of volume percent in the frond solution of fragmentation is 1.9～2.1%TritonX-100 (V/V), under the room temperature, slowly stirred 1 hour～1.5 hours, centrifugal 3-4 time of 13000rpm～15000rpm, remove the cell debris of TritonX-100, chlorophyll and the bottom on top layer, collect out the solution at centrifuge tube middle part, be the phycobilisome crude extract, wherein be mixed with a spot of broken phycobilisome.
Used polyoxyethylene glycol is a polyethylene glycol 6000 in the concentrating of above-mentioned steps (3) phycobilisome, used Sodium phosphate dibasic-potassium phosphate buffer pH=6.8～7.0, concentration 1molL
-1, the consumption of Sodium phosphate dibasic-potassium phosphate buffer is not particularly limited being advisable by dissolution precipitation.
Above-mentioned steps (4) Sepharose CL-4B post is 2.0 * 30cm, with pH=6.98 concentration 0.8molL
-1～0.9molL
-1Sodium phosphate dibasic-potassium phosphate buffer wash-out, flow velocity are 0.4～0.5mLmin
-1
The present invention is a material with red algae and blue-green algae, using ultrasound ripple smudge cells, stain remover TritonX-100 from thylakoid membrane dissociate phycobilisome, conventional centrifugal removal TritonX-100, chlorophyll and cell debris, polyethylene glycol precipitation, after Sepharose CL-4B column chromatography obtains pure intact phycobilisome.Present method has been saved traditional sucrose density tonsure ultracentrifugation method to equipment requirements height, complicated operation and the shortcoming that is difficult to prepare in a large number, excellent results is to use conventional equipment, simple to equipment requirements, prepare and greatly reduce the preparation cost of phycobilisome easily in a large number, thereby lay a good foundation for phycobilisome being applied to overdelicate biomedical the detection, have good application prospects and economic benefit.
(4) description of drawings
Fig. 1 is the absorption and the room temperature fluorescence spectrum of the phycobilisome of embodiment 1 chromatography preparation.Curve 1: fluorescence excitation spectrum, Em=680nm; Curve 2: absorption spectrum; Curve 3: fluorescence emission, Ex=580nm curve 4: fluorescence emission spectrum, Ex=436nm.
(5) embodiment
Embodiment 1: the preparation method of intact phycobilisome comprises the steps:
(1) fragmentation of raw material: fresh blue-green algae spirulina plalensis 0.5g, unicellular red algae Porphyridium cruentum 0.4g are raw material, are dissolved in the 20mL 1molL of pH=6.9
-1In Sodium phosphate dibasic-potassium phosphate buffer, adopt the ultrasonic disruption cell, broken power is 15W, broken 5 times, and each 2min, 2min at interval.
(2) phycobilisome dissociates: add stain remover TritonX-100 concentration 2.0% (volume) in the frond of fragmentation, frond and stain remover volume ratio are 1: 0.2, under the room temperature, slowly stir 1.5 hours, and phycobilisome is disintegrated down from thylakoid membrane.Centrifugal 4 times of 14000rpm removes the cell debris of TritonX-100, chlorophyll and the bottom on top layer, collects out the solution at centrifuge tube middle part, is the phycobilisome crude extract, wherein is mixed with a spot of broken phycobilisome.
(3) concentrating of phycobilisome: precipitate phycobilisome with polyethylene glycol 6000, collecting precipitation.To precipitate and be dissolved in pH=6.8～7.0 concentration 1molL again
-1Sodium phosphate dibasic-potassium dihydrogen phosphate in.
(4) purifying of intact phycobilisome: above-mentioned phycobilisome solution is crossed the Sepharose CL-4B column chromatography of 2.0 * 30cm, with pH=6.98 concentration 0.9molL
-1Sodium phosphate dibasic-potassium phosphate buffer wash-out, flow velocity are 0.45mLmin
-1Collect the blue-violet solution of wash-out front end, the phycobilisome solution that obtains finishing.
Fig. 1 shows that the absorption peak of spirulina plalensis phycobilisome does not have the foliage green charateristic avsorption band (curve 2) of 436nm at the 620nm place.With the optical excitation of 580nm wavelength, the emission wavelength of phycobilisome is at 680nm (curve 3), and this emission wavelength with the phycobilisome of sucrose density gradient supercentrifugal process preparation is identical, illustrates that this kind phycobilisome is complete; And with the optical excitation of 436nm wavelength, its emission wavelength still is 680nm (curve 4), chlorophyllous characteristic fluorescence peak do not occur, and its 680nm excitation peak is at 621nm, do not occur chlorophyllous characteristic peaks (curve 1) yet, illustrate not contain chlorophyll in the phycobilisome solution.
Embodiment 2:
As described in embodiment 1, different is that step (1) raw material is fresh spirulina plalensis (blue-green algae) 0.4g and multitube algae (large-scale red algae) 0.5g, is dissolved in the 16mL 1molL of pH=7.0
-1In Sodium phosphate dibasic-potassium phosphate buffer, adopt the ultrasonic disruption cell, broken power is 14W, broken 6 times, and each 1.5min, 3min at interval.
Embodiment 3:
As described in embodiment 1, different is that step (1) raw material is spirulina plalensis 0.3g and multitube algae 0.7g.
Embodiment 4:
As described in embodiment 1, different is that step (1) raw material is spirulina plalensis 0.5g and Ceramium kondoi 0.5g.
Claims (7)
1, a kind of preparation method of intact phycobilisome, step is as follows:
(1) fragmentation of raw material: with fresh blue-green algae and red algae is raw material, is dissolved in the Sodium phosphate dibasic-potassium phosphate buffer of pH=6.8～7.0, adopts the ultrasonic disruption cell;
(2) phycobilisome dissociates: in the frond of fragmentation, add stain remover tween X-100, phycobilisome is disintegrated down from thylakoid membrane, and centrifugal, remove stain remover and broken cell, get the crude extract of phycobilisome;
(3) concentrating of phycobilisome: use the polyethylene glycol precipitation phycobilisome, collecting precipitation; To precipitate and be dissolved in again in pH=6.8～7.0 Sodium phosphate dibasics-potassium dihydrogen phosphate;
(4) purifying of intact phycobilisome: above-mentioned phycobilisome solution is crossed Sepharose CL-4B column chromatography, collect the blue-violet solution of wash-out front end, obtain complete phycobilisome solution.
2, the preparation method of intact phycobilisome as claimed in claim 1 is characterized in that, described blue-green algae is selected from spirulina plalensis or spirulina maxim.
3, the preparation method of intact phycobilisome as claimed in claim 1 is characterized in that, described red algae is selected from unicellular red algae Porphyridium cruentum, large-scale red algae multitube algae or Ceramium kondoi.
4, the preparation method of intact phycobilisome as claimed in claim 1 is characterized in that, described step (1) raw material 0.75g～1.0g is dissolved in Sodium phosphate dibasic-potassium phosphate buffer of 15～20mL 1molL-1.
5, the preparation method of intact phycobilisome as claimed in claim 1 is characterized in that, the ultrasonic disruption of described step (1), broken power are 12～15W, broken 5～6 times, and each 1.5～2min, 2～3min at interval.
6, the preparation method of intact phycobilisome as claimed in claim 1, it is characterized in that, described step (2) phycobilisome dissociates and is specially: the adding concentration of volume percent is 1.9～2.1% stain remover tween X-100, under the room temperature, slowly stirred 1 hour～1.5 hours centrifugal 3-4 time of 13000rpm～15000rpm.
7, the preparation method of intact phycobilisome as claimed in claim 1 is characterized in that, described step (4) Sepharose CL-4B post is 2.0 * 30cm, with pH=6.98 concentration 0.8molL
-1～0.9molL
-1Sodium phosphate dibasic-potassium phosphate buffer wash-out, flow velocity are 0.4～0.5mLmin
-1
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275967A (en) * | 2013-05-29 | 2013-09-04 | 中国海洋大学 | Method for obtaining botryococcus braunii single cells by combining Tween80 and ultrasonic waves |
| WO2023115326A1 (en) * | 2021-12-21 | 2023-06-29 | 佛山蓝强生物科技有限公司 | Composition for enhancing photothermal stability of phycobiliproteins, and preparation method therefor and use thereof |
-
2005
- 2005-01-17 CN CN 200510042029 patent/CN1654629A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275967A (en) * | 2013-05-29 | 2013-09-04 | 中国海洋大学 | Method for obtaining botryococcus braunii single cells by combining Tween80 and ultrasonic waves |
| WO2023115326A1 (en) * | 2021-12-21 | 2023-06-29 | 佛山蓝强生物科技有限公司 | Composition for enhancing photothermal stability of phycobiliproteins, and preparation method therefor and use thereof |
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