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A method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin

A technology of allophycocyanin and phycocyanin, applied in the field of simultaneous separation and purification of reagent-grade phycocyanin and allophycocyanin, to achieve the effect of improving efficiency

Active Publication Date: 2019-10-18
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin, which is used to solve the problem of incomplete and effective simultaneous separation and purification in the prior art The problem of producing high value-added reagent grade phycocyanin and allophycocyanin

Method used

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  • A method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin
  • A method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin
  • A method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin

Examples

Experimental program
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Effect test

Embodiment 1

[0039] (1) Thaw fresh blue-green algae (water content 99%) stored frozen at -20°C (generally about 10 hours) at 25°C (the thawing time can be selected as about 6h), and then put them under the condition of -20°C Freeze, repeat this step at least 2 times to achieve the broken wall of cyanobacteria cells; coarsely filter the solution after the broken wall of cyanobacteria with multi-layer gauze, the number of gauze is preferably 200 mesh, remove algae residue, and then carry out 8000r / Min high-speed centrifugation, the centrifugation time is 20min, and the phycobiliprotein crude extract can be obtained after removing the residue;

[0040] (2) One-step salting-out: at 4°C, add medium (NH 4 ) 2 SO 4 The solid made the crude extract (NH 4 ) 2 SO 4 The concentration of the solution is 1.0mol / L, stir and mix well, let it stand for 30min, then perform high-speed centrifugation at 8000r / min at 4°C for 20min, discard the precipitate, and keep the supernatant;

[0041] (3) Two-ste...

Embodiment 2

[0050] (1) Thaw fresh blue-green algae (water content 99%) stored at -20°C (generally about 10 hours) at 25°C, and then freeze at -20°C. Repeat this step at least twice to Realize the breaking of cyanobacteria cells; coarsely filter the solution after breaking the walls of cyanobacteria with multi-layer gauze, the number of gauze is preferably 200 mesh, remove the algae residue, and then perform high-speed centrifugation at 8000r / min at 4°C for 20 minutes to remove The phycobiliprotein crude extract can be obtained from the slag;

[0051] (2) One-step salting-out: at 4°C, add medium (NH 4 ) 2 SO 4 The solid made the crude extract (NH 4 ) 2 SO 4 The concentration of the solution is 1.2mol / L, stir and mix well, let it stand for 30min, then perform high-speed centrifugation at 8000r / min at 4°C for 20min, discard the precipitate, and keep the supernatant;

[0052] (3) Two-step salting-out: at 4°C, continue to add (NH 4 ) 2 SO 4 The solid was added to the supernatant (NH ...

Embodiment 3

[0059] (1) Thaw fresh cyanobacteria (water content 99%) stored at -20°C (generally stored for about 10 hours) at 30°C, and then freeze at -20°C. Repeat this step at least twice to Realize the breaking of cyanobacteria cells; coarsely filter the solution after breaking the walls of cyanobacteria with multi-layer gauze, the number of gauze is preferably 200 mesh, remove the algae residue, and then perform high-speed centrifugation at 8000r / min at 4°C for 20 minutes to remove The phycobiliprotein crude extract can be obtained from the slag;

[0060] (2) One-step salting-out: at 4°C, add medium (NH 4 ) 2 SO 4 The solid made the crude extract (NH 4 ) 2 SO 4 The concentration of the solution is 1.4mol / L, stir and mix well, let it stand for 30min, then perform high-speed centrifugation at 8000r / min at 4°C for 20min, discard the precipitate, and keep the supernatant;

[0061] (3) Two-step salting-out: at 4°C, continue to add (NH 4 ) 2 SO 4 The solid was added to the supernata...

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Abstract

The invention provides a method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin. The method comprises the following steps: breaking walls of blue-green algae and carrying out solid-liquid separation to obtain a phycobiliprotein crude extraction solution; carrying out two-step salting-out to obtain sediment; dissolving the sediment by utilizing a phosphate buffering solution; by taking the phosphate buffering solution as a dialysis solution, dialyzing a dissolved solution so as to obtain a dialyzed phycobiliprotein solution; finally, carrying out cellulose column chromatography and hydroxylapatite column chromatography to obtain the reagent-grade phycocyanin and the reagent-grade allophycocyanin. According to the method provided by the invention, water-bloom fresh blue-green algae is used as a raw material and the reagent-grade phycocyanin and the reagent-grade allophycocyanin are separated and purified by adopting salting-out combined with column chromatography; compared with a traditional chromatography process, high-purity phycocyanin and allophycocyanin can be simultaneously extracted and purified under the premise of considering the recovery rate of protein, and the efficiency of comprehensively extracting and purifying high-value protein in blue-green alga cells is improved and can be amplified.

Description

technical field [0001] The invention relates to the technical field of biochemical separation, in particular to a method for simultaneously separating and purifying reagent-grade phycocyanin and allophycocyanin. Background technique [0002] Bloom cyanobacteria are rich in phycobiliproteins, and the structures of different phycobiliproteins are basically similar. They all contain two polypeptide chains α and β subunits with similar structures. The molecular weight is about 30,000 Daltons. Each subunit is related to a Molecule of phycocyanin binding. According to the absorption spectrum properties, phycobiliproteins can be divided into phycocyanin, allophycocyanin, phycoerythrin and phycoerythrin. Among them, phycocyanin and allophycocyanin generally exist in the form of trimers and hexamers. The higher the purity of phycocyanin and allophycocyanin, the higher the price, and the purity is calculated by A 620 / A 280 with A 650 / A 280 According to its purity, it can be cl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/795C07K1/36C07K1/30C07K1/16
Inventor 汪家权张发宇余金卫苏雨张晓萌匡武
Owner HEFEI UNIV OF TECH
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