CN1587275A - Separating and purifying technology for micro algae phycoerythrin - Google Patents

Abstract

The present invention relates to the technological process of separating and purifying micro algae phycoerythrin including distilled water leaching, staged precipitation with ammonium sulfate and ionic exchange column chromatography. Freeze dried algae powder and algae mud is leached with distilled water at 4 deg.c over night, high speed freeze centrifuged, mixed with solid ammonium sulfate to certain saturation degree for staged precipitation, balance dialyzed in buffering sodium phosphate solution and centrifuged for 20 min, and the supernatant is chromatographic separated with ion exchange column to obtain phycoerythrin. The present invention is of high purification effect, high yield and other advantages and may be used in enlarged production, and the obtained phycoerythrin has purity up to 4.85 and total yield of 52 %, and shows one strip in polyacrylamide gel electrophoresis.

Description

A technology for separation and purification of microalgae phycoerythrin

The invention relates to a technique for separating and purifying microalgae phycoerythrin by using distilled water leaching, ammonium sulfate fractional precipitation and ion exchange column chromatography.

Phycobiliprotein is a water-soluble pigment protein that exists in red algae, cyanobacteria, cryptophyta and certain dinoflagellates. It is a light-harvesting pigment in the photosynthetic system unique to these algae, and it plays a role in capturing light energy and transferring energy in photosynthesis role. According to the structure and spectral properties, phycobiliproteins are divided into: phycoerythrin (PE), phycocyanin (PC) and allosteric phycocyanin (A-PC). Phycoerythrin transfers the captured light energy to phycocyanin, then to allophycocyanin, and finally to the central pigment to achieve photosynthesis. Therefore, phycoerythrin has important research value in the original theory of photosynthesis. On the other hand, phycoerythrin has a wide range of uses. It can be widely used in food, cosmetics, dyes and other industries as a natural pigment, and can also be made into fluorescent reagents for clinical medical diagnosis, immunochemistry and bioengineering. For photodynamic therapy of tumors.

There is a large star-shaped pigment body in the Porphylococcus cell, which is rich in phycoerythrin and phycocyanin. Phycoerythrin accounts for 84% of phycobiliproteins, of which B-phycoerythrin contains the most. Kost-Reyes (EurJBiochem.1979, 102 (1): 83-91) uses carrier-free electrophoresis to separate B-phycoerythrin, b-phycoerythrin and R phycoerythrin from the protein passing through the column, and the finally obtained B - Phycoerythrin OD ₅₄₅ /OD ₂₈₀ ratio > 5. Stadnichuk (J Photochemistry Photobiology B.1997, 39: 19-23) separated Porphyrococcus B-phycoerythrin through ToypearlDEAE-650M ion exchange, hydroxyapatite and Sephadex G-200 gel, and finally obtained B-phycoerythrin Red protein OD ₅₄₂ /OD ₅₀₀ =2.00, OD ₅₄₂ /OD ₂₈₀ >6.0. Bermejo (JChromatogr A.2001, 917 (1-2): 135-145, J Botech.2002, 93: 73-85; JChromatogr B.2003, 709: 317-325) etc. use two-step chromatography to separate and purify Phycoerythrin B-phycoerythrin from Porphyridum sp., Separation of Phycoerythrin by Reverse High Performance Liquid Chromatography Gradient Semi-Preparative Method Using C4 Wide Pore Column and Containing 0.05% Trifluoroacetic Acid (TFA) in Water and 0.05% TFA in Acetocyanide The α, β and γ subunits of polyacrylamide gel electrophoresis produce three close bands, which correspond to its three subunits. They also produced natural forms of B-phycoerythrin and R-phycocyanin. A new and scalable method for the separation and purification of B-phycoerythrin from Porphyridium sp. was established. This experiment combined two chromatography methods of expanded bed and DEAE-cellulose packed bed. Ma (Plant Sci.2003, 164: 253-257) etc. used SephadexG-200 gel chromatography to separate Porphyrococcus B-phycoerythrin and R-phycocyanin, and utilized the amino group between these two proteins The mutual reaction forms artificial B-phycoerythrin-R-phycocyanin covalent association. The experimental results show that the stability of this artificial covalent association is higher than that of B-phycoerythrin, and it is expected to be used for the detection of phycobiliprotein probes. preparation. Wen Shaohong (Ocean Bulletin. 2000, 19(3): 90-93; China Ocean Pharmaceuticals. 2001, 3: 33-35) etc. made the water-soluble crude extract of Porphyridium algae through ammonium sulfate precipitation and hydroxyapatite column layer Analysis, separation and purification to obtain B-phycoerythrin (B-PE), B-PE has an absorption peak at 545nm and 563nm, and an absorption shoulder at 498nm. They also separated and purified the crude phycobiliprotein extract by ammonium sulfate precipitation, dialysis, hydroxyapatite and SephadexG-100 column chromatography to obtain B-phycoerythrin with a purity (OD ₅₄₅ /OD ₂₈₀ ) of 4.92 and 3.78, respectively. , a band was obtained by polyacrylamide gel gradient electrophoresis.

The object of the present invention is to provide a technique of repeatedly extracting algae flour, and then precipitating with ammonium sulfate to obtain crude phycobiliprotein, which is purified by ion-exchange column chromatography to obtain high-purity phycoerythrin.

The technical scheme adopted in order to realize the object of the present invention is: at first the microalgae algae powder is added distilled water by 1: 10～15 and is extracted overnight in the refrigerator, after the extraction, a high-speed refrigerated centrifuge is centrifuged at 4°C and 6000r/min for 20min, Take the supernatant. Add distilled water to the precipitated algae mud at a ratio of 1:8.0 to 12.5, continue leaching overnight in the refrigerator for 2 times, centrifuge at 4°C and 6000r/min for 20min, take supernatants from each, and combine the supernatants obtained from 3 times of leaching (that is, Phycoerythrin crude extract), divided into groups, respectively added solid ammonium sulfate 20, 40, 60, 80% saturation, mixed and left to stand overnight, centrifuged at 4°C, 6000r/min for 20min, took the precipitate, and dissolved the precipitate in a small amount of Put distilled water into a dialysis bag, dialyze with distilled water at 4°C for 24 hours, and then dialyze with 0.01mol/L sodium phosphate buffer solution with pH 7.0 for 24 hours. The dialyzed sample was centrifuged at 4° C. and 6000 r/min for 20 min, and the supernatant was taken as the loading sample. Equilibrate the packed DEAE-Sepharose FF column with 0.01mol/L sodium phosphate buffer until the pH of the effluent is 7.0. Load the sample on the well-balanced column top, wash the column with 0.01-0.02mol/L sodium phosphate buffer until the absorption peak at 280nm returns to the baseline, and then use 0.5-1.0mol/L NaCl as gradient elution at a flow rate of 1.5mL/ min. Measure the light absorption values at 280nm and 545nm of the collected tubes, and make a 280nm chromatographic curve. According to the absorption peak and the OD ₅₄₅ /OD ₂₈₀ ratio, the samples were collected, dialyzed, concentrated, centrifuged, and freeze-dried to obtain the samples. Concrete technical scheme of the present invention comprises the following specific steps:

1. Extraction of phycoerythrin: Weigh a certain amount of freeze-dried algae powder, such as Rhodella reticulate and Porphydidium cruentum, add distilled water at a ratio of 1:10 to 15, extract in the refrigerator overnight, and then centrifuge in a high-speed freezer Centrifuge at 4°C, 6000r/min for 20min, and take the supernatant. Add distilled water to the precipitated algae mud at a ratio of 1:8.0 to 12.5 to continue leaching twice overnight, centrifuge in a high-speed refrigerated centrifuge at 4°C and 6000r/min for 20min, and take the supernatant. The supernatant obtained by combining the above three extractions is the crude phycoerythrin extract.

2. Fractional precipitation of ammonium sulfate: the crude extract is divided into groups, and solid ammonium sulfate is added respectively, and the saturation is 20%, 40%, 60% and 80%, and the fractional precipitation of miscellaneous proteins and target proteins is carried out. After standing overnight, high-speed freezing Centrifuge at 4°C, 6000r/min for 20min, take the precipitate, dissolve the precipitate in a small amount of distilled water and put it into a dialysis bag, dialyze with distilled water at 4°C for 15-24h, and then dialyze with pH7.0 buffer solution for 15-24h. Centrifuge the dialyzed sample in a high-speed refrigerated centrifuge at 6000 r/min at 4°C for 20 min, and take the supernatant, which is the loading sample. The purity of the phycoerythrin samples subjected to ammonium sulfate fractional precipitation was measured respectively to determine the degree of saturation used in the ammonium sulfate fractionation precipitation.

3. DEAE-Sepharose Fast Flow column chromatography: Equilibrate the installed DEAE-Sepharose FF column with pH7.0 buffer until the effluent is pH7.0. Load the sample on the well-balanced column top, wash the column with loading buffer until the absorption peak at 280nm returns to the baseline, then use 0.5-1.0mol/L NaCl solution as gradient elution, and measure the Absorption values at 280nm and 545nm, and make 280nm chromatography curve. According to the absorption peak and the OD ₅₄₅ /OD ₂₈₀ ratio, a relatively pure sample was collected, dialyzed, concentrated, centrifuged, and freeze-dried to obtain a phycoerythrin sample.

The present invention has the advantage that phycoerythrin is purified in one step by DEAE-Sepharose FF ion exchange column chromatography, and the purity reaches OD ₅₄₅ /OD ₂₈₀ = 4.85, and the obtained sample shows a band in polyacrylamide gel electrophoresis, indicating that the electrophoresis has been achieved. Pure, the total yield is 52%. The DEAE-Sepharose FF ion-exchange column chromatography has the characteristics of good purification effect, large sample loading, high sample yield and can be used for scale-up production. Compared with the reported separation and purification methods of phycoerythrin, DEAE-Sepharose FF ion exchange column chromatography is superior to hydroxyapatite column chromatography and Sephadex G-100 gel column chromatography.

The present invention will be further described below in conjunction with specific embodiments.

Example 1

1. Extraction of phycoerythrin: Weigh 5g of Porphyridum lyophilized powder and add 50mL of distilled water in the refrigerator at a ratio of 1:10 to extract overnight. Take the supernatant. Add 50 mL of distilled water to the precipitated algae mud and continue to extract twice overnight. The supernatant obtained by combining the 3 extractions is the crude phycoerythrin extract.

2. Fractional precipitation of ammonium sulfate: the crude extract is divided into groups, and solid ammonium sulfate is added respectively, and the saturation is 20%, 40%, 60% and 80%. High-speed refrigerated centrifuge, 4°C, 6000r/min centrifuge for 20min, take the precipitate, dissolve the precipitate in a small amount of distilled water and put it into a dialysis bag, dialyze with 4°C distilled water for 24h, and then use 0.01mol/L sodium phosphate buffer (PBS) pH7. 0 buffer equilibrium dialysis for 24 hours. Centrifuge the dialyzed sample in a high-speed refrigerated centrifuge at 6000 r/min at 4°C for 20 min, and take the supernatant, which is the loading sample. The purity of the phycoerythrin samples subjected to ammonium sulfate fractional precipitation was measured respectively to determine the degree of saturation used in the ammonium sulfate fractionation precipitation.

3. Phycoerythrin DEAE-Sepharose FF column chromatography purification: equilibrate the packed DEAE-Sepharose FF column with 0.01mol/L sodium phosphate buffer until the effluent reaches pH 7.0. Load the sample on the top of the well-balanced column, wash the column with loading buffer until the absorption peak at 280nm returns to the baseline, then use 0.5mol/L NaCl as gradient elution, the total volume of the eluent is 300mL, and the flow rate is 1.5mL /min, each tube collected about 5mL. Measure the light absorption values at 280nm and 545nm of the collected tubes, and make a 280nm chromatographic curve. According to the absorption peak and the OD ₅₄₅ /OD ₂₈₀ ratio, a relatively pure sample was collected, dialyzed, concentrated, and freeze-dried to obtain a Porphyrophycoerythrin sample.

Example 2

1. Extraction of phycoerythrin: Weigh 5g of Rhodella reticulate freeze-dried powder and add 75mL of distilled water in the refrigerator for overnight extraction at a ratio of 1:15. Serum. Add 60 mL of distilled water to the precipitated algae mud at a ratio of 1:12.5 to continue leaching overnight twice. The supernatant obtained by combining the 3 extractions is the crude phycoerythrin extract.

2. Fractional precipitation of ammonium sulfate, the crude extract is divided into groups, and solid ammonium sulfate is added respectively, and the saturation is 20%, 40%, 60% and 80%. After mixing, carry out the fractional precipitation of miscellaneous proteins and target proteins, and let stand overnight. High-speed refrigerated centrifuge, 4°C, 6000r/min centrifuge for 20min, take the precipitate, dissolve the precipitate in a small amount of distilled water and put it into a dialysis bag, dialyze with 4°C distilled water for 24h, and then use 0.02mol/L sodium phosphate buffer (PBS) pH7. 0 buffer equilibrium dialysis for 24 hours. Centrifuge the dialyzed sample in a high-speed refrigerated centrifuge at 6000 r/min at 4°C for 20 min, and take the supernatant, which is the loading sample. The purity of the phycoerythrin samples subjected to ammonium sulfate fractional precipitation was measured respectively to determine the degree of saturation used in the ammonium sulfate fractionation precipitation.

3. Phycoerythrin was purified by DEAE-Sepharose FF column chromatography, and the installed DEAE-Sepharose FF column was equilibrated with 0.02mol/L sodium phosphate (pH7.0) buffer until the pH of the effluent was 7.0. Load the sample on the top of the well-balanced column, wash the column with loading buffer until the absorption peak at 280nm returns to the baseline, then use 1.0mol/L NaCl as gradient elution, the total volume of the eluent is 500mL, and the flow rate is 2.0mL /min, each tube collected about 5mL. Measure the light absorption values at 280nm and 545nm of the collected tubes, and make a 280nm chromatographic curve. According to the absorption peak and the ratio of OD ₅₄₅ /OD ₂₈₀ , a relatively pure sample was collected, dialyzed, concentrated, and freeze-dried to obtain rosea phycoerythrin.

Claims (5)

1. A separation and purification technology for microalgal phycoerythrin using ammonium sulfate fractional precipitation and ion exchange column chromatography, characterized in that: 1. The algae powder is extracted with distilled water overnight in the refrigerator, centrifuged to separate the supernatant, the precipitated algae mud is extracted twice overnight in the refrigerator with distilled water, and the combined supernatant obtained is the crude phycoerythrin extract; II. Add solid ammonium sulfate to the crude phycoerythrin extract, centrifuge overnight, dissolve the precipitate in distilled water and put it into a dialysis bag for dialysis with distilled water, equilibrate dialysis with sodium phosphate buffer, centrifuge the dialyzed sample, and take the supernatant For loading samples; III. Load the sample on the top of the well-balanced DEAE-Sepharose FF ion exchange column, wash the column with buffer until the absorption peak at 280nm returns to the baseline, and perform gradient elution with 0.5-1.0mol/L NaCl. 2. The separation and purification technology according to claim 1, characterized in that the algae powder and the algae mud are leached with distilled water at a ratio of 1:10 to 15 and 1:8.0 to 12.5 respectively, and then placed in a high-speed refrigerated centrifuge at 4°C, 6000r/ centrifuge for 20min. 3. The separation and purification technology according to claim 1, characterized in that the crude phycoerythrin extract is grouped and then solid ammonium sulfate is added, and the saturations are respectively 20%, 40%, 60% and 80% for fractional precipitation. 4. The separation and purification technology according to claim 1, characterized in that the precipitate after the fractional precipitation of solid ammonium sulfate is dissolved in a small amount of distilled water and put into a dialysis bag, dialyzed with distilled water at 4°C for 24 hours, and then using 0.01mol/ Equilibrium dialysis against L sodium phosphate buffer for 24 hours, centrifuge at 4°C, 6000r/min for 20 minutes, and take the supernatant. 5. The separation and purification technology according to claim 1, characterized in that the sample is loaded on the well-balanced DEAE-Sepharose FF ion exchange column top, and the column is washed with 0.01-0.02mol/L sodium phosphate loading buffer until The absorption peak at 280nm returned to the baseline, and then gradient elution was performed with 0.5-1.0mol/L NaCl at a flow rate of 1.5mL/min.