CN102241754A - Method for separating and purifying phycoerythrin from bangia fusco-purpurea - Google Patents
Method for separating and purifying phycoerythrin from bangia fusco-purpurea Download PDFInfo
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- CN102241754A CN102241754A CN2010101709638A CN201010170963A CN102241754A CN 102241754 A CN102241754 A CN 102241754A CN 2010101709638 A CN2010101709638 A CN 2010101709638A CN 201010170963 A CN201010170963 A CN 201010170963A CN 102241754 A CN102241754 A CN 102241754A
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- acid buffer
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Abstract
The invention discloses a method for separating and purifying phycoerythrin from bangia fusco-purpurea. According to the method, foreign proteins with small molecular weights are removed at the same time of salt removal by a membrane technology, and the phycoerythrin is effectively separated from the foreign proteins based on the principles that the bonding strength between anion exchange resin and the phycoerythrin is obviously changed in solutions of different salt contents and different pH values, and the like, thereby obtaining high-purity and high-yield phycoerythrin. In different components, the purity of the phycoerythrin is generally higher than 3.0 (commonly accepted standard) and is 5.5 at most, and the yield is about 0.8g/100g bangia fusco-purpurea. The method has a simple process and good separating effect, and realizes quick and effective separation and purification of the phycoerythrin.
Description
Technical field
The invention discloses a kind of extracting method of phycoerythrin, particularly relate to the separation purification method of phycoerythrin in a kind of red hair algae.
Background technology
Phycoerythrin is the important photopigment albumen of catching of many marine algas, and phycobiliprotein such as it and Phycocyanins, C-, Allophyxoxyanin are formed bar-shaped phycobilisome, and luminous energy can be caught and transmit to phycobilisome.Phycoerythrin mainly is distributed in the red algae of marine alga, because the existence of pigment molecular, it has stronger absorption at visible region 480-570nm wave band, and can produce intensive fluorescence.Phycoerythrin can be used as natural pigment and is applied to industries such as food, makeup, dyestuff, also can be made into fluorescent reagent, is used for clinical diagnose and research fields such as immunochemistry and biotechnology.Phycoerythrin still is a kind of important physical active substance, can be made into photosensitizers and is applied to photodynamic tumor treatment.At present, ammonium sulfate precipitation is mainly adopted in the separation and purification of phycoerythrin, ion-exchange chromatography, and the method that hydroxyapatite column chromatography, gel permeation chromatography combine is carried out, and the separation and purification process is loaded down with trivial details, complicated operation, length consuming time, and cost height.
Summary of the invention
The object of the present invention is to provide a kind of technology simple, the separation purification method of phycoerythrin in the red hair algae of good separating effect.
Process design principle of the present invention is: in ion exchange chromatography, phycoerythrin depends on the electrostatic attraction of opposite charges group to each other to the bonding force of anionite, and this magnetism is relevant with the pH value and the salt concn of solution, because pH value decision anionite and proteinic degree of ionization, when the pH value near the iso-electric point of phycoerythrin when (pI is about 3.8), anionite and protein interactions weaken, and protein is eluted easily; The subtle change of salt concn also can directly influence the loading capacity of anionite to the protein electric charge in the solution simultaneously.So the type of elution that the present invention adopts salt concn linear gradient elution and pH value linear gradient elution to combine is carried out purifying to phycoerythrin.
Concrete steps of the present invention:
1, extract: with red hair algae with 1: 20-25 (m/v) ratio is dissolved in the distilled water, through freezing-melt, stir, organize to smash to pieces, behind the ultrasonic disruption, filtration, centrifugal, supernatant liquor is a crude extract.
2, saltout: in crude extract, add a certain amount of ammonium sulfate, make solution reach 35% ammonium sulfate saturation ratio, left standstill 2 hours, abandon precipitation after centrifugal.In supernatant liquor, add ammonium sulfate to 50% saturation ratio, left standstill 2 hours, abandon supernatant liquor after centrifugal, with 0.02mol/L phosphoric acid buffer (pH=7.0) dissolution precipitation, the liquid of must saltouing.The ultra-filtration membrane concentrating and desalinating of holding back with the 30kDa molecular weight is also removed the foreign protein of molecular weight less than 30kDa.An amount of phosphoric acid buffer that adds guarantees that desalination is complete in the concentration process.
3, fractionation by adsorption: the phycoerythrin mother liquor after the desalination pumped into be filled with in the anionite-exchange resin adsorption column that functional group is triethylamine or tetramethyl-amine, the control flow velocity is 1mL/min, after absorption finishes, fully wash pillar to the absorbancy under the 280nm with 0.02mol/L phosphoric acid buffer (pH=5.6 contains 0.05mol/L NaCl) and arrive baseline.
4, desorb: the resin after absorption is finished is via the 0.02mol/L phosphoric acid buffer (pH=5.6 contains 0.05mol/L NaCl) and the 0.2mol/L NaH of equal volume
2PO
4Solution (containing 0.2mol/L NaCl) mixes the linear gradient elution liquid drip washing desorb of forming, and collects elutriant, phycoerythrin purity (A in most eluant components
565/ A
280) being higher than 3.2, the phycoerythrin yield is about the red hair of 0.8g/100g algae.
5, resin regeneration: after the desorb, resin with 2mol/L NaCl solution drip washing 4 hours, is pulled down resin earlier again, soaks 0.5 hour in 0.1mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
The present invention obtains the ammonium sulfate precipitation saturation ratio (35%-50%) of phycoerythrin in the red hair of the most suitable separation and purification algae by carrying out concrete ammonium sulfate precipitation experiment; During desorb anionite-exchange resin, by use mixing the gradient elution mode, i.e. the mode that combines of salt concn linear gradient elution and pH value linear gradient elution, obtain purity greater than 3.2 and yield be the phycoerythrin of 0.8% (with respect to red mao of algae dry weight).
The present invention utilizes the characteristics of phycoerythrin molecular weight big (240kDa) behind ammonium sulfate precipitation, the ultra-filtration membrane concentrating and desalinating that adopts the 30kDa molecular weight to hold back.Compare with long dialysis desalting consuming time, membrane concentration has reduced sample volume when realizing quick desalination, saved the last sample time of ion exchange column; Removed the foreign protein of molecular weight, significantly improved purification efficiency less than 30kDa.
Because the present invention utilizes the phycoerythrin in the red hair of the fast effective separation and purification of the anionite-exchange resin algae, thereby it is simple to have technology, the advantage of good separating effect has been simplified the purification step that original needs carry out repeatedly column chromatography greatly.
To sum up, the present invention mainly adopts ammonium sulfate precipitation, and membrane technique is handled desalination and removed foreign protein, further utilizes the anion exchange chromatography method, the phycoerythrin in the red hair of the separation and purification fast and effeciently algae.The present invention has adopted the mode of mixing gradient elution that the phycoerythrin that is adsorbed in anion-exchange column carried out wash-out, efficiently purifying the phycoerythrin in the red hair algae.The present invention adopts membrane technique to accelerate desalination speed and has effectively removed the lower molecular weight foreign protein.On the anion-exchange column elution process, obviously be different from the past single salt concn gradient elution or single pH gradient elution mode, (It is generally accepted standard [12,13]) and yield are the phycoerythrin of 0.8% (with respect to red hair algae dry weight) greater than 3.2 to have obtained purity.
Description of drawings
Fig. 1 is the spectrogram of the phycoerythrin behind the purifying;
Fig. 2 is the SDS-PAGE electrophorogram of purifying phycoerythrin.
M, standard protein; Swimming lane 1, the phycoerythrin crude extract; Swimming lane 2,35-50% ammonium sulfate precipitation component; Swimming lane 3, DEAE-Sepharose column purification phycoerythrin, running gel are silver dyeing.
Embodiment
1, extract: get the red hair of 100mL distilled water swelling 5g algae powder, after freeze thawing, add 200mL distilled water stir, organize smash to pieces, ultrasonic disruption, filter then, centrifugal, collect the about 300mL of supernatant liquor, be the phycoerythrin crude extract.
2, saltout: in the 300mL crude extract, add the 58.20g solid ammonium sulfate, make solution reach 35% ammonium sulfate saturation ratio, left standstill the centrifugal precipitation of abandoning 2 hours.Add 29.14g ammonium sulfate to 50% saturation ratio in supernatant liquor, left standstill 2 hours, abandon supernatant liquor after centrifugal, with 0.02mol/L phosphoric acid buffer (pH=7.0) dissolution precipitation, volume is about 100mL.Further utilize the ultra-filtration membrane concentrating and desalinating that the 30kDa molecular weight holds back and remove the foreign protein of molecular weight less than 30kDa.An amount of phosphoric acid buffer that adds guarantees that desalination is complete in the concentration process.
3, fractionation by adsorption: the phycoerythrin mother liquor after the desalination is pumped into the adsorption column (in 2.5 * 6cm) that is filled with DEAE-Sepharose Fast Flow anionite-exchange resin, flow velocity 1mL/min, after absorption finishes, washed pillar 6 hours with 0.02mol/L phosphoric acid buffer (pH=5.6 contains 0.05mol/L NaCl).
4, desorb: the resin after absorption is finished is via 120mL 0.02mol/L phosphoric acid buffer (pH=5.6, contain 0.05mol/LNaCl) and linear gradient elution liquid drip washing desorb that mix to form of 120mL 0.2mol/L NaH2P04 solution (containing 0.2mol/L NaCl), collect elutriant (2mL/ pipe), phycoerythrin purity (A565/A280) is higher than 3.2 in most eluant components, be up to 5.5, the phycoerythrin yield is the red hair of a 0.785g/100g algae.The visible light feature of phycoerythrin and bibliographical information basically identical are seen Fig. 1 behind the purifying.
It under the phycoerythrin normality in the known red algae form existence with (α β) 6 γ, after the present embodiment phycoerythrin is handled through 2 mercapto ethanol, carry out SDS-PAGE and silver-colored staining analysis, the result as shown in Figure 2, at molecular weight is that the protein band at 21.5kDa place is the α and the β subunit of phycoerythrin, because of both relative molecular masses quite approaching, so the phenomenon of overlapping occurred.The relative molecular mass of α subunit is about 19kDa, and the relative molecular mass of β subunit is about 21kDa, and the relative molecular mass of γ subunit is about 27kDa.Thereby electrophorogram explanation phycoerythrin of the present invention is consistent with report.
5, resin regeneration: after the desorb, resin is pulled down resin with 2mol/L NaCl solution drip washing 4 hours again, soaks 0.5 hour in 0.1mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
1, extract: get the red hair of 200mL distilled water swelling 10g algae powder, after freeze thawing, add 550mL distilled water stir, organize smash to pieces, ultrasonic disruption, filter then, centrifugal, collect the about 750mL of supernatant liquor, be the phycoerythrin crude extract.
2, saltout: in the 750mL crude extract, add the 145.50g solid ammonium sulfate, make solution reach 35% ammonium sulfate saturation ratio, left standstill 2 hours, abandon precipitation after centrifugal, in supernatant liquor, add 71.78g ammonium sulfate to 50% saturation ratio, left standstill 2 hours, abandon supernatant liquor after centrifugal, with 0.02mol/L phosphoric acid buffer (pH=7.0) dissolution precipitation, the liquid of must saltouing, volume is about 180mL.Further utilize the ultra-filtration membrane concentrating and desalinating that the 30kDa molecular weight holds back and remove the foreign protein of molecular weight less than 30kDa.An amount of phosphoric acid buffer that adds guarantees that desalination is complete in the concentration process.
3, fractionation by adsorption: the phycoerythrin mother liquor after the desalination is pumped into the adsorption column (in 2.5 * 9cm) that is filled with Q-Sepharose Fast Flow anionite-exchange resin, flow velocity 1ml/min, after absorption finishes, washed pillar 6 hours with 0.02mol/L phosphoric acid buffer (pH=5.6 contains 0.05mol/L NaCl).
4, desorb: the resin after absorption is finished is via 180mL 0.02mol/L phosphoric acid buffer (pH=5.6, contain 0.05mol/L NaCl) and linear gradient elution liquid drip washing desorb that mix to form of 180mL 0.2mol/L NaH2P04 solution (containing 0.2mol/L NaCl), collect elutriant (3ml/tube), phycoerythrin purity (A565/A280) is higher than 3.2 in most eluant components, be up to 5.2, the phycoerythrin yield is the red hair of a 0.814g/100g algae.
5, resin regeneration: after the desorb, resin is pulled down resin with 2mol/L NaCl solution drip washing 4 hours again, soaks 0.5 hour in 0.1mol/L NaOH solution, cleans resin near neutral with a large amount of distilled water then, and resin regeneration finishes, and can be recycled.
1, extract: get the red hair of 500mL distilled water swelling 20g algae powder, after freeze thawing, add 1000mL distilled water fully stir, organize smash to pieces, ultrasonic disruption, filter then, centrifugal, collect the about 1500mL of supernatant liquor, be the phycoerythrin crude extract.
2, saltout: in the 1500mL crude extract, add the 291g solid ammonium sulfate, make solution reach 35% saturation ratio, after leaving standstill 2 hours, the centrifugal precipitation of abandoning of 8000g adds 143.56g ammonium sulfate to 50% saturation ratio in supernatant liquor, left standstill 2 hours, abandon supernatant liquor after centrifugal, with 0.02mol/L phosphoric acid buffer (pH=7.0) dissolution precipitation, the phycoerythrin crude extract of saltouing, volume is about 260mL.Further utilize the ultra-filtration membrane concentrating and desalinating that the 30kDa molecular weight holds back and remove the foreign protein of molecular weight less than 30kDa.An amount of phosphoric acid buffer that adds guarantees that desalination is complete in the concentration process.
3, fractionation by adsorption: the phycoerythrin mother liquor after the desalination is pumped into the Hitrap Capto Q anionite-exchange resin prepacked column of U.S. GE company (in 2 * 5mL), flow velocity 1.5mL/min, after absorption finishes, washed pillar 4 hours with 0.02mol/L phosphoric acid buffer (pH=5.6 contains 0.05mol/L NaCl).
4, desorb: the resin after absorption is finished is via 100mL 0.02mol/L phosphoric acid buffer (pH=5.6, contain 0.05mol/L NaCl) and linear gradient elution liquid drip washing desorb that mix to form of 100mL 0.2mol/L NaH2P04 solution (containing 0.2mol/L NaCl), collect elutriant (2.5mL/tube), phycoerythrin purity (A565/A280) is higher than 3.2 in most eluant components, be up to 5.3, the phycoerythrin yield is the red hair of a 0.832g/100g algae.
5, resin regeneration: after the desorb, resin with 0.1mol/L NaOH solution drip washing 30mL, cleans resin near neutral with a large amount of distilled water with 2mol/L NaCl solution drip washing 1 hour then, and resin regeneration finishes, and can be recycled.
Above-mentioned only is specific embodiments of the invention, but design concept of the present invention is not limited thereto, and allly utilizes this design that the present invention is carried out the change of unsubstantiality, all should belong to the behavior of invading protection domain of the present invention.
Claims (4)
1. the separation purification method of phycoerythrin in the red hair algae comprises the steps:
(1) extract: with red hair algae with 1: the 20-25m/v ratio is dissolved in the distilled water, through freezing-melt, stir, organize to smash to pieces, behind the ultrasonic disruption, filtration, centrifugal, supernatant liquor is a crude extract;
(2) saltout: adding ammonium sulfate to its saturation ratio in the phycoerythrin crude extract of step (1) gained is that 35%-50% saltouts, centrifugal, and collecting precipitation is also used phosphoric acid buffer (K
2HPO
4-NaH
2PO
4) dissolving, the liquid of must saltouing, the ultra-filtration membrane concentrating and desalinating of holding back with the 30kDa molecular weight and remove the foreign protein of molecular weight then less than 30kDa;
(3) fractionation by adsorption desorb: the concentrated solution after the desalination is through the anionite-exchange resin fractionation by adsorption, and phycoerythrin is adsorbed on the resin, and the resin after the absorption obtains phycoerythrin through mixing the phosphoric acid buffer drip washing desorb phycoerythrin of gradient.
2. the separation purification method of phycoerythrin in the red hair algae according to claim 1, it is characterized in that: it also comprises step (4): resin regeneration: after the phosphoric acid buffer desorb, resin is used 2mol/L NaCl solution drip washing 4 hours, again resin is pulled down, in 0.1mol/L NaOH solution, soaked 0.5 hour, clean resin near neutral with a large amount of distilled water then, resin regeneration finishes, and can be recycled.
3. the separation purification method of phycoerythrin in the red hair algae according to claim 1, it is characterized in that: the anionite-exchange resin described in the step (3) is the anionite-exchange resin of triethylamine or tetramethyl-amine.
4. the separation purification method of phycoerythrin in the red hair algae according to claim 1 is characterized in that: the phosphoric acid buffer described in the step (3) is pH=5.6, contain 0.05mol/L NaCl equal volume the 0.02mol/L phosphoric acid buffer and contain the 0.2mol/L NaH of 0.2mol/L NaCl
2PO
4The linear gradient eluent of solution phase combined group carries out wash-out to the anionite-exchange resin that adsorbs phycoerythrin, collects elutriant, obtains the phycoerythrin component of purifying.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103951738A (en) * | 2014-04-18 | 2014-07-30 | 哈尔滨工业大学 | Aqueous two-phase method for efficiently extracting phycoerythrin |
| CN104131055A (en) * | 2014-06-09 | 2014-11-05 | 集美大学 | Preparation method for phycoerythrin ACE inhibitory peptide |
| CN106117326A (en) * | 2015-12-09 | 2016-11-16 | 烟台大学 | A kind of centrifuging combines the method that anion-exchange chromatography medium prepares phycoerythrin |
| CN108865978A (en) * | 2018-07-25 | 2018-11-23 | 辽宁润基生物科技有限公司 | A method of separation and purifying excretion body |
| CN108976290A (en) * | 2018-08-23 | 2018-12-11 | 泉州师范学院 | A kind of preparation method of red hair algae anti-oxidation peptide |
| CN108977423A (en) * | 2018-08-17 | 2018-12-11 | 集美大学 | A method of the separating-purifying angiotensin converting enzyme from pig lung |
| CN109021072A (en) * | 2018-08-23 | 2018-12-18 | 泉州师范学院 | A kind of red hair algae anti-oxidation peptide and preparation method thereof |
| WO2018227664A1 (en) * | 2017-06-14 | 2018-12-20 | 湖南炎帝生物工程有限公司 | Methods for extracting and purifying nostoc sphaeroides kutzing phycobiliprotein, and purified phycoerythrin |
| CN110256542A (en) * | 2019-06-03 | 2019-09-20 | 中国海洋大学 | A kind of preparation method of red algae phycoerythrin |
| CN115181194A (en) * | 2022-06-21 | 2022-10-14 | 浙江大学 | Rapid purification degradation and structure analysis method of pectin |
| CN117510355A (en) * | 2023-11-09 | 2024-02-06 | 信联电子材料科技股份有限公司 | Method for extracting gamma-aminobutyric acid from plants |
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| CN103951738A (en) * | 2014-04-18 | 2014-07-30 | 哈尔滨工业大学 | Aqueous two-phase method for efficiently extracting phycoerythrin |
| CN104131055A (en) * | 2014-06-09 | 2014-11-05 | 集美大学 | Preparation method for phycoerythrin ACE inhibitory peptide |
| CN104131055B (en) * | 2014-06-09 | 2017-02-22 | 集美大学 | Preparation method for phycoerythrin ACE inhibitory peptide |
| CN106117326A (en) * | 2015-12-09 | 2016-11-16 | 烟台大学 | A kind of centrifuging combines the method that anion-exchange chromatography medium prepares phycoerythrin |
| WO2018227664A1 (en) * | 2017-06-14 | 2018-12-20 | 湖南炎帝生物工程有限公司 | Methods for extracting and purifying nostoc sphaeroides kutzing phycobiliprotein, and purified phycoerythrin |
| CN108865978A (en) * | 2018-07-25 | 2018-11-23 | 辽宁润基生物科技有限公司 | A method of separation and purifying excretion body |
| CN108977423A (en) * | 2018-08-17 | 2018-12-11 | 集美大学 | A method of the separating-purifying angiotensin converting enzyme from pig lung |
| CN109021072A (en) * | 2018-08-23 | 2018-12-18 | 泉州师范学院 | A kind of red hair algae anti-oxidation peptide and preparation method thereof |
| CN108976290A (en) * | 2018-08-23 | 2018-12-11 | 泉州师范学院 | A kind of preparation method of red hair algae anti-oxidation peptide |
| CN109021072B (en) * | 2018-08-23 | 2021-04-30 | 泉州师范学院 | Rhodophyta antioxidant peptide and preparation method thereof |
| CN108976290B (en) * | 2018-08-23 | 2021-06-25 | 泉州师范学院 | Preparation method of rambutan antioxidant peptide |
| CN110256542A (en) * | 2019-06-03 | 2019-09-20 | 中国海洋大学 | A kind of preparation method of red algae phycoerythrin |
| CN115181194A (en) * | 2022-06-21 | 2022-10-14 | 浙江大学 | Rapid purification degradation and structure analysis method of pectin |
| CN117510355A (en) * | 2023-11-09 | 2024-02-06 | 信联电子材料科技股份有限公司 | Method for extracting gamma-aminobutyric acid from plants |
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Application publication date: 20111116 |