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Method for preparing high purity phycobiliprotein with primary column chromatography

A technology of phycobiliprotein and column chromatography, which is applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of high production costs, complicated preparation methods of high-purity phycobiliproteins, and inability to produce large quantities. Achieve low production cost, solve utilization and high-value problems, and moderate particle size

Inactive Publication Date: 2009-01-14
GUANGDONG OCEAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the defects of existing high-purity phycobiliprotein preparation methods such as complex preparation methods, high production costs, and inability to produce in large quantities, the purpose of the present invention is to provide a simple and effective method suitable for mass production of high-purity phycoerythrin, phycocyanin and allophycoerythrin. blue protein method
Contents of the invention

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. Preparation of phycobiliprotein crude extract

[0019] Take the dry powder of Spirulina platensis produced in Sanya, Hainan, use pH = 7, 0.02-0.5M phosphate buffer as the extractant, the solid-liquid ratio of algae powder and phosphate buffer is 1:10, stir well and freeze for a certain period of time , thawed at room temperature, repeated freezing and thawing several times in this way, and then centrifuged at 4800r / min for 10min, took its supernatant, added 20% to 60% ammonium sulfate and divided it into salt solution, salting out, and centrifugation four times, and a blue precipitate was obtained. It is the crude extract of algae blue, after dialysis and desalination, the purity is A 620 / A 280 = 2.06 phycobiliproteins.

[0020] 2. Preparation of hydroxyapatite

[0021] (1) Solution preparation: Prepare 0.01M, pH=7.0 phosphate buffer, 1MCaCl 2 and 1MNa 2 HPO 4 solution.

[0022] (2) Preparation of hydroxyapatite: 500mL 1MCaCl was added at a rate of 12mL / min ...

Embodiment 2

[0027] 1. Preparation of phycobiliprotein crude extract

[0028] Take the fresh Anabaena anabaena produced in Zhanjiang, Guangdong, add phosphate buffer as the extractant, the solid-to-liquid ratio is 1:6, and prepare the crude phycobiliprotein extract by implementing the same steps as in Example 1, and the crude extract has a purity of A 620 / A 280 = 2.19.

[0029] 2. Preparation of hydroxyapatite

[0030] (1) Solution preparation: Prepare 0.01M, pH=7.0 phosphate buffer, 1MCaCl 2 and 1MNa 2 HPO 4 solution.

[0031] (2) Preparation of hydroxyapatite: 500mL 1MCaCl was added at a rate of 12mL / min 2 solution and 500mL 1MNa 2 HPO 4 The solution was added to the beaker and stirred. After the dropwise addition was completed, the static calcium hydrogen phosphate precipitated. Pour off the supernatant, wash the precipitate with about 3L of tap water, add 25mL of 40% NaOH solution in a water bath at 80°C for 2h, and wash the precipitate with tap water at the same temperature ...

Embodiment 3

[0036] 1. Preparation of phycobiliprotein crude extract

[0037] Take fresh laver produced in Shantou, Guangdong, and grind it with a tissue grinder at 12000 rpm. After breaking the cell wall and adding the extractant, centrifuge at 4800r / min for 20min, take the supernatant, add 20-60% ammonium sulfate for several times of salting out, and centrifuge at 10000r / min for 1 min, and the precipitation is the crude extraction of phycobiliprotein. material, and then dialyzed for desalting to obtain a purity of A 620 / A 280 = 2.02 of phycobiliproteins.

[0038] 2. Preparation of hydroxyapatite

[0039] (1) Solution preparation: Prepare 0.01M, pH=7.0 phosphate buffer, 1MCaCl 2 and 1MNa 2 HPO 4 solution.

[0040] (2) Preparation of hydroxyapatite: 500mL 1MCaCl was added at a rate of 12mL / min 2 solution and 500mL 1MNa 2 HPO 4 The solution was added to the beaker and stirred. After the dropwise addition was completed, the static calcium hydrogen phosphate precipitated. Pour off...

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Abstract

The invention relates to a method for preparing high purity phycoerythrin, phycocyanin and allophycocyanin from the algae, belonging to the bioengineering extraction separation technology. The method is as follows: red algae or cyanobacteria is adopted as a raw material, and phosphate buffer is used as an extractant; after algae cells are broken, the raw material of the red algae or the cyanobacteria is salt dissolved, salted out and dialyzed in grade by ammonium sulfate, and crude extract of phycobiliprotein is obtained; the crude extract is in chromatography by a hydroxyapatite column for one time, is gradient eluted by the phosphate buffer, and is frozen and dried, and high purity phycoerythrin, phycocyanin and allophycocyanin are obtained. The purity of the crude extract of the phycobiliprotein prepared by the effective method in the earlier stage reaches the standard of pharmaceutical grade (A620 / A 280 is more than 2.0), thereby simplifying the follow-up purifying procedures, so the high purity phycoerythrin, phycocyanin and allophycocyanin can be respectively obtained by only one time column chromatography. When the method is used to extract high purity phycobiliprotein from the fresh algae and the dry algae, the process is simple, the production cost is low, and the yield of the products is high. Thus, the method is suitable for mass production.

Description

technical field [0001] The invention provides a simple and rapid method for simultaneously extracting and separating high-purity phycocyanin, phycoerythrin and allophycocyanin from cyanobacteria and red algae. The invention belongs to the technical field of bioengineering extraction and separation. Background technique [0002] Phycobiliproteins (Phyeobi lipmteins, PBP) are a class of pigment complex proteins present in the phycobilisomes (Phycobili-somes, PBS) of some algae (mainly red algae and cyanobacteria). Phycobilisomes are a supramolecular light-harvesting pigment complex unique to red algae and cyanobacteria, which are fixed on the outer surface of thylakoid membranes by anchoring proteins. Phycobiliprotein has strong fluorescence and emits orange-red fluorescence, but itself is red, purple or blue, so it is a colored polypeptide. It constitutes phycobilisomes together with connecting polypeptides, which account for 80% and 20% of phycobilisomes respectively. Phy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C07K1/14
Inventor 冯亚非李先文温燕梅部音利
Owner GUANGDONG OCEAN UNIVERSITY
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