Extraction And Purification Of R-phycoerythrin From Porphyra Haitanensis And Its Analysis

As an important physiological active substance of Porphyra haitanensis, R-phycoerythrin is widespread applied in food, cosmetics and pharmaceutical industry with a very high economic value. At present phycoerythrin is mainly separated from the red algae. There are many reports of the separation and purification about it at home and abroad.Conventional protein purification procedures involve three steps:pretreatment of the sample to allow the intracellular material to become liberated, making a crude extract by ammonium sulfate precipitation, and combination of different chromatographies to purify further. A lot of shortages occur in this process, such as the low extraction efficiency, operation tediously, high cost and so on, causing the price of high-purity phycoerythrin is very expensive and limiting phycoerythrin widespread application enormously. In the present thesis, we set up a new extraction and purification procedures of R-phycoerythrin, mainly including two steps:Knief stirring to prepare the crude extact and combinations of ion exchange chromatography and gel filtration chromatography to separate and purify R-phycoerythrin.R-phycoerythrin of Porphyra haitanensis is intracellular protein, so cell breakage technology as the first step of isolation and purification process is very important. Because it concerns raw material utilization and production costs of R-phycoerythrin. In this research, four cell wall-breaking methods Swelling, Chemical permeabilization, Ultrasonication and Knief stirring with yield, purity and extracting time as indexes are carried out to extract R-phycoerythrin from Porphyra haitanensis. Overall comparison, with a very high extracting efficiency and work capacity, Knief stirring is an effective method for phycoerythrin extraction on a large scale. The conditions is 0.01mol/L Tirs-HCl (pH8.0) as extract solvent, the ratio 1:30 and stiring rate 18000rpm, within only 6min we get the yield of phycoerythrin is 23.11mg/g and the purity is 0.30.In order to solve problems on salting out and chromatographies, we choose two step combination of ion-exchange chromatography with media Q Sepharose FF and gel filtration chromatography with media Sephacryl S-300 HP to purify the crude extract of R-phycoerythrin. Both of medium are easy to scale up. The conditions of chromatographies are optimized. With 50mmol/L sodium phosphate buffer (pH6.0) as the start buffer, a gradient elution of ionic strength (0-0.10-0.35-1.00mol/L NaCl) is used and the colour fraction under 0.35mol/L NaCl is collected. And then we use UF membrane with MWCO 50kDa to concentrate it. At last, Sephacryl S-300 HP chromatography is used with 50mmol/L sodium phosphate buffer (pH6.0,0.15mol/L NaCl) as mobile phase and flow rate 0.7mL/min and we get the fraction of peak 1 with A565/A280 11.23.The fraction of peak 1 collected from gel filtration chromatography was not only examined with UV-visible absorption spectrum and fluorescence spectrum but was also evaluated by polyacrylamide gel electrophoresis (PAGE) performed under native and denaturing conditions. The results are the maxima absorption peak at 565nm, a strong secondary absorption peak at 498nm and an shoulder peak at 540nm in the UV-visible absorption spectrum. Purity of this fraction is also estimated by means of the following indices: A565/A280>4, A620/A565<0.02 and A565/A280<1.5 and all of them are conformed. Fluorescence emission peak is at 576nm under three fixed excitation wavelengths. The fraction shows one band in Native PAGE gel and two bands in SDS PAGE gel in accordance with the previous reports. So it is proved that the fraction obtained by the new extraction and purification process is high-quality R-phycoerythrin.