The Study Of Environmental Extraction Technology Of Phycobiliprotein In Aqueous Two-phase System

Phycobiliprotein is a kind of water soluable chromoprotein exsting in rhodophyta, cyanophytes, and photosynthesis of algae can be achieved by its light-harvesting structure which can be used in the study of environmental function-material. Therefore, the extraction of phycobiliprotein is a hot research area now. This study is to look for a method to extract and purify phycobiliprotein efficiently and environmentally. Aqueous two-phase system (ATPS) has some special advantages at the extraction of organisms compared to the traditional organic solvent extraction, because it has the features that the extraction condition is mild, mass transfers quickly, the cost is low and the operation is simple and controllable, etc. Apply aqueous two-phase extraction technique to separate and purify the phycobiliprotein can achieve the goal of efficient extraction of phycobiliprotein, which satisfies the goals and requirements in this study, and also has importance in environmental protection.R-phycoerythrin (R-PE) from porphyra haitanensis and C-phycocyanin (C-PC) from spirulina platensis is selected as the objects of study in this dissertation to learn the knowledge of breaking cell method and aqueous two-phase system method of extraction and separation of phycobiliprotein.The methods of repeated freezing and melting, liquid nitrogen gliding, cell dissolving-bulging, CaCl₂ solution of low concentration, SDS solution extraction, cellulose extraction and ultrasonication are used to extract the phycobiliprotein. The study results illustrate that, the repeated freezing and melting method is the optimal method to extract R-PE with the conditions that between -20℃and 4℃, use 0.01M phosphate buffer as solvent, freezing it for 2 hours, then repeated freezing and melting the sample 6 times, which can obtain the purity 0.63. For C-PC, cell dissolving-bulging is the best way, and can reach the purity 0.57 when extract 120 h by using distilled water at 4℃. Through analyzing the spectrum of two extraction solution of the algae, we find that the peak absorption of R-PE is at 560 nm, and the peak of fluorescence emission is at 579 nm at indoor temperature. The peak absorption of C-PC is at 620 nm, and the peak of fluorescence emission is at 665 nm. These results show that the extraction solution contains the protein with some characteristic fluorescence and bioactivity.PEG/ammonium sulfate ATPS is applied to extract the phycobiliprotein and compared with the traditional precipitate. The study results about the traditional precipitate illustrate that, the purity of R-PE extraction is higher than 1.36, with the recovery over 70%, when the saturation of ammonium sulfate is about 45⁵5%, and the purity of R-PE extraction is higher than 0.80, with the recovery over 40%, when the saturation of ammonium sulfate is about 40⁴5%, because the high salinity condition leads to the denaturation of the protein and the low recovery of obtaining the phycobiliprotein. PEG/ammonium sulfate ATPS has the optimal extraction, the purity of R-PE is 0.91 and recovery is 98.5% when W_PEG1000= 10%,W_(NH4)2SO4=15%, and the purity of C-PC is 1.00 and recovery is 97.2% when W_PEG1000= 10%,W_(NH4)2SO4=12.6%. The advantages of PEG/ammonium sulfate ATPS are that the operation is simple, the recovery is high, and PEG plays sTab. effect on the phycobiliprotein. Therefore, PEG/ammonium sulfate ATPS is better than the traditional precipitate.Use different PEG/phosphate ATPS to extract the phycobiliprotein and study the effect of molecular weight of PEG, mass fraction of PEG, mass fraction of phosphate and pH value to the extraction result through orthogonal test, to determine the priority effect of each factor, and compare the effect of NaHP and KHP on the extraction of ATPS. The results show that, in the ATPS of PEG2000/KHP, R-PE and C-PC have better extraction results that the purity of R-PE is 1.07 and the recovery is 98.6% when W_PEG=14%,W_KHP=18% and pH6.8, and the purity of C-PC is 1.16 and the recovery is 98.2% when W_PEG=12%,W_KHP=20% and pH 5.6.There is high recovery of R-PE and C-PC through the extraction of ATPS, which states that, the extraction process is mild and the loss of phycobiliprotein is low, which avails to the industrial production. The purity of C-PC is higher than that of R-PE through ATPS, which shows that the ATPS in this study is appropriate for the extraction of C-PC from prokaryotic cell with simple cell structure, but for the extraction of R-PE in eukaryocyte, the ATPS needs to be improved.