The Study On Separation, Purification And Stability Of R-Phycoerythrin From Porphyra Haitanensis

This thesis studied the extraction,separation,purification of R-phycoerythrin from Porphyra haitanensis and its stability.Three methods(repeated freezing-thawing,ultrasonic and H₂O expansion)were applied to extract R-phycoerythrin.The result shows that 3 h for freezing time and six cycles in repeated freezing-thawing reached highest purity;therefore it is the most suitable extracting method.Precipitation,crystal and ultrafiltration were compared to separate R-phycoerythrin from R-phycocyanin and allophycocyanin.35%and 60 %of(NH₄)₂SO₄ for two-step precipitation was better,the first step to remove other protein and the second to precipitate R-phycoerythrin,and then ultra-filtration was applied to wipe off salt and concentrate R-phycoerythrin,which made its purity reach 1.80.Based on one-step hydroxyapatite chromatograph,R-phycocyanin and allophycocyanin were removed completely,the purity achieved 3.20, and after DEAE Sepharose F.F.chromatograph,its purity was 5.30, exceed 4.0(International common standard),the purified R-PE had a maximum absorption peak at 565 nm,a fluorescence emission peak at 575 nm.The purified R-PE denatured by urea,its three subunitsα,β,γwere checked by SDS-PAGE,and the molecular weights were 17.0,18.0 and 37.0 kDa respectively.The study on its stability shows that R-phycoerythrin was relatively stable under such conditions which fits for its process but not for long: the temperature ranges from 4℃to 40℃,pH ranges from 5.0 to 8.0, and in faint light below 2000 lux,the best conditions for preserving R-PE was 4℃,pH 7.0 and in completely dark.And a rule was found for its two phycobilins about their stability on temperature:PUB>PEB.The destruction effects of Fe²⁺,Fe³⁺and Cu²⁺on its stability were obvious, while Ca²⁺,Mg²⁺,Zn²⁺,Mn²⁺had no apparent effect.No addition of lactose neither.Glycerol can not be use as a protect reagent while (NH₄)₂SO₄ can be used as a fluorescence-protect reagent.