High-purity biliprotein separating process

The technology of a phycobiliprotein and a separation method is applied in the separation field of high-purity phycobiliprotein, can solve the problems of long time, less than 1% yield, low phycocyanin purity, etc., and achieves the effects of low cost and high yield

Inactive Publication Date: 2002-04-17
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patents CN1106414A and CN1130028A describe methods for isolating phycocyanin from spirulina. The methods used in the above two patents are crude methods for spirulina phycocyanin. Although simple, the purity of phycocyanin obtained is extremely low, only Can be used as a food additive, not as a biochemical reagent at all
[0004] 1. It takes time and effort to use sucrose density gradient ultracentrifugation, because the equipment of ultracentrifuge is expensive, many units do not have ultracentrifuge equipment, and the method of ultracentrifugation is not suitable for large-scale separation and purification of phycobiliprotein;
[0005] 2. The separation material Sephadex G-200 used in the gel filtration method is expensive;
[0006] 3. The time used by the gel filtration method is too long, and it takes about a week to equilibrate the chromatography column;
[0007] 4. The yield of separating and purifying phycobiliprotein by the above method is too low, generally less than 1%
It is precisely because of the defects of the above-mentioned separation technology that the price of phycobiliprotein in the international market is too expensive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Get 20 grams of Spirulina platensis dry powder purchased from Shengli Oilfield, Dongying City, Shandong Province. Add 200ml of distilled water, place it in the refrigerator at 4°C for 24 hours, centrifuge at low speed to remove the residue, the supernatant is the crude extract of spirulina, the total volume is 151ml, OD 620 =46.660 (containing crude phycobiliprotein amount is 1003.26 mg); Weigh 1 gram of hydroxyapatite, be placed in a centrifuge tube with a cover, balance with 0.001M phosphate buffer, then add 4ml of spirulina crude extract ( The amount of phycocyanin contained is about 26.576 mg), the centrifuge tube was inverted for 10 minutes to make it fully adsorbed, and the supernatant was removed by centrifugation at low speed for 2 minutes; the hydroxyapatite adsorbed with phycobiliprotein was washed twice with the above buffer solution; Then add 0.030M phosphate buffer to elute phycocyanin until the eluate is colorless, combine the eluate, the total volume is 3...

Embodiment 2

[0026] The previous treatment steps are the same as in Example 1, instead of 0.003M phosphate buffer, use 0.05M phosphate buffer for elution until the hydroxyapatite is colorless; the total volume of the eluent is 52ml, OD 620 =0.909, containing about 6.732 mg of phycocyanin; the yield of phycocyanin from the crude extract of spirulina is about 25.330%. If the hydroxyapatite adsorption and elution process is performed again, the purity of phycocyanin A 620 / A 280 It can also reach 4 or more.

Embodiment 3

[0028] Sanchaxiancai is collected from Huiquan Bay, Qingdao. After washing the algae twice with seawater and tap water, add an equal volume of tap water to soak for 24 hours, filter to remove the residue of the algae, and measure the OD of the crude extract filtrate 560 =3.808; Weigh 2 grams of hydroxyapatite, put it in a centrifuge tube with a cover, balance it with 0.001M phosphate buffer (pH6.8), then add the crude extract of 16ml trigeminal saline (containing the amount of phycoerythrin) about 7.4mg), the centrifuge tube was inverted for 10 minutes to make it fully adsorbed, and the supernatant was removed by centrifugation at low speed for 2 minutes; the hydroxyapatite adsorbed with phycobiliprotein was washed twice with the above buffer solution; and then added 0.030M Phycoerythrin was eluted with phosphate buffer until the eluate was colorless, and the combined eluate had a total volume of 72ml, OD 565 =0.504, containing about 4.4mg of phycoerythrin; the yield of phycoe...

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Abstract

The present invention includes the following steps: hypotonic process to prepare extraction of algae, mixing hydroxyl apatite and the extraction of algae and eliminating the other components of extraction of algae in the supernatant; washing hydroxyl apatite with combined biliprotein with low concentration phosphate buffering liquid to eliminate other components; eluting phycocyanin or phycoerythrin from hydroxyl apatite with medium concentration phosphate buffering liquid and collecting phycocyanin or phycoerythrin; and eluting hydroxyl apatite with higher concentration phosphate buffering liquid to obtain allophycocyanin component. The present invention has high yield and purify of biliprotein and is favorable to production in industrial scale.

Description

(1) Technical field [0001] The invention relates to a simpler, more economical and more efficient method for separating and purifying high-quality, high-value-added phycobiliprotein products, which belongs to a new method for separating and purifying economically valuable substances from marine organisms, specifically high-purity A method for the isolation of phycobiliproteins. (2) Background technology [0002] Phycobiliproteins can be widely used in medical diagnosis, clinical medicine, etc. There are many types of phycobiliproteins, strong fluorescence, and easy to combine with biotin, antibodies, etc., so phycobiliproteins can be used as a new generation of fluorescent probes; phycobiliproteins have some unique curative effects, such as photosensitizers for photodynamic therapy of tumors, improving The immune function of the body, etc.; instead of artificially synthesized pigments, it is used as an additive for food and cosmetics, not only can avoid the possible side ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K2/00
Inventor 王广策孙海宝马圣媛曾呈奎
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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