Study On The Purification Of Phycocyanin By Aqueous Two Phase Extraction And Related Mechanism

Phycocyanin(PC) is a kind of water soluble chromoprotein existing in rhodophyta,cyanophytes. It has been extensively used in many fields, such as food, cosmetic, dye,medicine. The traditional processes for obtaining purified phycocyanin were complicated and inefficient, refraining the wide applications of PC. In this study, phycocyanin was purified by applying aqueous two-phase method. In order to rise the purity of PC product to drug level(>2.5), the optimization of conditions for extraction was performed. Besides, the molecular structure of extracting polymer was modified according to the results of investigation of the interaction between PC and PEG, for improving the extraction efficiency.1.One-factor experiment design was used to determine which molecular weights of PEG and which kinds of salts were optimal in the aqueous two-phase extraction. After that, the amounts of PEG and salt were optimized using response surface analysis. When ω(PEG1000)=14.06 %, ω(Na2SO4)=13.81 %, the purity of PC reached 2.25 and the recovery ratio of PC were99%. Moreover, 25~30 ℃ and p H=6.0 were determined as the optimal temperature and p H value for obtaining the highest value of PC purity and partition coefficient. The purity of PC can be increased to 2.8 by a two-step extraction strategy with a recovery ratio of 88.2 %.2. The mechanism for the interaction of phycocyanin and PEG was studied using florescence spectrum. The fluorescence of PC can be quenched by the addition of PEG no matter the variation in molecular weights of PEGs. The thermodynamic study found that the binding constant of PEG1000 and PC was higher than that of PEG2000 and PEG4000. The binding between PEG and PC was through hydrophobic interaction driven by the entropy.Furthermore, compared to other inorganic salts tested, the addition of Na2SO4 showed the most positive impact on the entropy increase, promoting the combinative interaction between PEG and PC. Moreover, the investigations of synchronous fluorescence spectroscopy found that the hydrophobicity of micro-environment around tryptophan residues was enhanced by the presence of PEG1000, and further increased by the addition of Na2SO4. Finally, the result of aqueous two-phase extraction demonstrated that PEG1000/Na2SO4 is the optimum extraction system for obtaining the highest purity of phycocyanin.3.The trimethylamino-PEG was synthesized by the reaction of PEG with phosphorus tribromide and trimethylamine sequentially. The purity and the recovery ratio of PC reached2.8 and 99 % respectively in the extraction system composed of double polymers and salts. In addition, the fluorescence analysis showed that the fluorescence of PC can be quenched by the addition of TMA-PEG. Meanwhile, the thermodynamic parameters showed that the binding force between TMA-PEG and PC was interaction of hydrophobic and electrostatic.Finally, the results illustrated that the electrostatic attraction in the process of extraction canbe more effectively, which was beneficial for the purification of PC.