Efficient separation and purification method of Gracilaria verrucosa reagent grade R-phycoerythrin

A technology for separation and purification of phycoerythrin, which is applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems of long operation period, high cost, and large output loss, and achieve reduced production costs and simple operation , High recovery effect

Active Publication Date: 2015-01-21
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Generally, the separation and purification of phycoerythrin is carried out through several times of hydroxyapatite column chromatography combined with ion exchange chromatography or gel filtration chromatography. The previous purification methods have complicated operation steps and power consumption in the actual industrial production process. High and long operating cycle; According to literature reports, the cost of separation and purification accounts for 50-90% of the product cost. In addition, due to too many steps, the yield loss is large, and the target protein will be reduced by 20% for each additional step of the chromatography process. %, which is undoubtedly very expensive for industrial production

Method used

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  • Efficient separation and purification method of Gracilaria verrucosa reagent grade R-phycoerythrin
  • Efficient separation and purification method of Gracilaria verrucosa reagent grade R-phycoerythrin

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Embodiment 1

[0020] (1) Wash the large economical marine red algae asparagus with deionized water;

[0021] (2) Rough extraction of R-phycoerythrin: Superfine asparagus to a particle size of 0.1-0.5 mm, crush and add 10 times its volume to PBS buffer solution with a pH of 7.0 and a concentration of 50 mM / L, 4 Centrifuge at 15000rpm / min for 30min at 4℃ for 48 hours, then filter the supernatant after centrifugation and concentrate the filtrate with a 50kd concentrator;

[0022] (3) Preliminary purification of R-type phycoerythrin: Add solid ammonium sulfate to the crude phycoerythrin extract obtained in step (2) to make the saturation reach 25%, and then let it stand for 12 hours. Centrifuge at 4°C, 15000rpm / min for 30min, collect the supernatant, then add ammonium sulfate to the supernatant to make the saturation reach 45%, let stand for 24-48 hours, centrifuge at 4°C, 15000rpm / min for 30min , collect the precipitate and dissolve it with PBS buffer solution with a concentration of 25mM / L a...

Embodiment 2

[0026] (1) Wash the large economical marine red algae asparagus with deionized water;

[0027] (2) Rough extraction of R-phycoerythrin: Superfine asparagus to a particle size of 0.1-0.5mm, crush and add 30 times its volume to PBS buffer solution with a pH of 6.0 and a concentration of 50mM / L, 4 Centrifuge at 9000rpm / min for 20min at 4℃ for 24 hours, then filter the supernatant after centrifugation, and concentrate the filtrate with a 50kd concentrator plate;

[0028](3) Preliminary purification of R-type phycoerythrin: Add solid ammonium sulfate to the crude phycoerythrin extract obtained in step (2) to make the saturation reach 25%, and then let it stand for 12 hours. Centrifuge at 4°C, 12000rpm / min for 20min, collect the supernatant, then add ammonium sulfate to the supernatant to make the saturation reach 45%, let it stand for 6 hours, centrifuge at 4°C, 12000rpm / min for 20min, collect Precipitate and dissolve with PBS buffer solution with a pH of 6.0 and a concentration o...

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Abstract

The invention belongs to a preparation technology of marine natural active substances, and concretely relates to an efficient separation and purification method of Gracilaria verrucosa reagent grade R-phycoerythrin. The method comprises the following steps: soaking a crushed raw material marine red alga economic Gracilaria verrucosa into a PBS buffer solution with the volume 10-30 times the crushed raw material, centrifuging under 9000-15000rpm for 20-30min, collecting the obtained supernatant, and concentrating the supernatant; carrying out grading salting-out on the obtained concentrate by ammonium sulfate, collecting the obtained precipitate, dissolving by using the PBS buffer solution, and dialyzing for 24-48h after the dissolution; and carrying out strong anion Source-15Q chromatography column purification on the obtained dialysate, eluting with a PBS buffer solution containing NaCl as an eluent under a flow rate of 0.5ml / min, and collecting 360-510th min eluate to obtain an R-phycoerythrin solution with the purity of above 5.0. The method has the has the characteristics of high recovery rate of the above obtained product, good purification effect, easy amplification, large sample amount, and efficient separation and purification of phycoerythrins.

Description

technical field [0001] The invention belongs to the preparation technology of marine natural active substances, in particular to a high-efficiency separation and purification method of asparagus reagent grade R-type phycoerythrin. Background technique [0002] Phycobiliprotein (PBP) is another kind of photosynthetic pigment protein besides chlorophyll a, which exists in cyanobacteria, red algae, cryptophyta and a few dinoflagellates. According to the characteristics of phycobiliprotein ultraviolet absorption spectrum, phycobiliprotein can be divided into three categories: phycocyanin (phycocyanin, PC, λ max 610-620nm), allophycocyanin (allophycocyanin, APC, λ max 650-655nm) and phycoerythrin (phycoerythrin, PE, λ max 540-570nm); Usually, phycoerythrin can be divided into B-phycoerythrin, R-phycoerythrin and C-phycoerythrin according to different sources and spectral characteristics. Phycoerythrin is composed of apoprotein and several chromophores with tetrapyrrole as the ...

Claims

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Application Information

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IPC IPC(8): C07K14/405C07K1/36C07K1/34C07K1/30C07K1/18
CPCC07K14/795
Inventor 刘冰杜虹谷洋洋秦松陈洁辉
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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