Application Of Three-phase Partitioning In Purification Of C-phycocyanin

C-phycocyanin(C-PC)is a bright blue fluorescent protein with high commercial value.It has been widely used in the fields of nutrition,health care,pharmaceutical,food and cosmetics due to its antioxidant,antibacterial,anti-inflammatory and fluorescent properties.The value of C-PC tends to increase with the improvement of its purity.However,C-PC is sensitive to some external conditions,such as light and temperature,and easy to degrade.Therefore,how to obtain high purity C-PC quickly and efficiently is of great significance.Three-Phase Partitioning(TPP)is a fast,simple and efficient non-chromatographic protein extraction technique,which has been used in the purification process of upstream and downstream biomolecules.Although the traditional separation and purification procedures such as chromatography can obtain C-PC with high purity,they are usually of high cost,complicated operation,long processing time and difficult to be produced on a large scale.TPP method can provide a more favorable process environment for natural protein structure and meet the needs of green chemistry.Based on this background,this paper based on ammonium sulphate-tert-butanol three-phase extraction system,carried out its application research in the separation and purification of C-PC,the main research contents are as follows:The algal sludge of Spirulina platensis was treated by repeated freeze-thaw method,and the obtained C-PC freeze-thaw solution was salted out in two steps to obtain C-PC crude extract(CE).A three-phase extraction system of ammonium sulfate and tert-butanol was established.The optimized process conditions for TPP extraction of C-PC were ammonium sulfate salt with 90%saturation,and the volume ratio of crude extract to tert-butanol was 1:1,pH 7.0,10?,the purity of C-PC could reach 3.458 under the extraction conditions,the total recovery of 52.41%.The C-PC samples before and after extraction were characterized,and SDS-PAGE electrophoresis confirmed that the purified samples had?and?subunits of the protein.FTIR,UV and fluorescence spectra showed that the structure and function of the protein samples before and after purification were intact,which proved that the extraction conditions were mild.The tert-butanol phase of the system was recovered by evaporation and reused into TPP extraction C-PC system,and the results showed that the recovered tert-butanol still had purification ability.Nine factors affecting the extraction process were screened by PB experiment,and the three most influential factors were selected as temperature,pH and ammonium sulfate salt saturation.The climbing experiment design of three significant influencing factors was carried out,and the optimal extraction conditions were screened out and verified by using the CCD experimental results fitting model with three factors and five levels.A ten-fold magnification study was conducted under the optimal extraction conditions,and the results showed that the extraction system is stable and has the possibility of large-scale preparation.The phase formation between different salts and tert-butanol was determined.The results showed that ammonium sulfate salt has the strongest phase formation ability.The extraction was carried out at different temperatures to explore the thermodynamics of the three-phase extraction system,and the fitting linear equation was obtained as Y=6535.51+5.95*x,with good fitting degree.By calculating the formula,the value of?H was-6.536 k J/mol,and the value of?S was 5.95 J/(mol·K).The value of?G and?S were both negative,indicating that the reaction can be spontaneous,the reaction is exothermic and the temperature is not conducive to the reaction.Kinetic simulation was carried out for the extraction process.According to Fick's second law,the fitting equation was Ct=-0.237*e^-0.212*t+0.527,R² was 0.9869.The fitting kinetic equation of Peleg model was Ct=0.283+t/(13.348+3.652*t),and its R² was 0.9873.The second-order rate law fitting equation was Ct=t/(1.484+0.178*t),R² was 0.334.Peleg's law was the best fit to describe the extraction process.In this study,the conditions for the purification of C-PC by TPP were optimized through the combination of experiments and models,and the changes of proteins in the extraction process were explored through kinetic modeling,etc.,which proved that the TPP extraction method was rapid,mild and efficient,and had potential application prospects.