Applications Of Deep Eutectic Aqueous Two-phase System In Separation Of C-PC And Recombinant Grx

Deep Eutectic Solvents?DES?are a kind of solvents with similar physicochemical properties and phase behaviors compared with ionic liquids?IL?that synthesized from hydrogen bond donors and acceptors.These green solvents with the advantages of low vapour pressure,excellent designability,low cost and strong recyclability have been widely applied in the fields of catalytic reactions,electrochemistry and extraction as environmentally friendly solvents.Although some traditional separation and purification technologies can be utilized in the separation of target substances with high-quality,there are still many problems such as high cost,complexed operations and environmental pollution,which hinder their applications in the separation and purification of other active substances and make them difficult to meet the requirements of green chemistry.Therefore,the construction of a recyclable,economic and green system has important scientific research significance and practical application values for the separation of high-quality bioactive substances.In this study,Deep Eutectic Solvents-Aqueous two-phase systems?DES-ATPS?were constructed based on the physicochemical properties of DES,and applied in the extraction and separation of C-phycocyanin?C-PC?and recombinant glutaredoxin?Grx?.The research contents include the following aspects:Five kinds of DES that synthesized from choline chloride?hydrogen bond acceptor?and glycerol,urea,D-?-?-fructose,D-?+?-glucose,ethylene glycol?hydrogen bond donors?,respectively,were characterized by infrared spectroscopy.The result revealed that all the DES had hydrogen bond structures.Five kinds of DES-ATPS were then constructed by K₂HPO₄ and synthesized DES.The equilibrium phase behavior results showed that the volume ratio of upper and lower phases increases linearly with the increase of DES content,and the slope is 1.3878,and an opposite trend was obtained when the concentration of salt increased.These results laid a foundation for the establishment of stable DES-ATPS for the extraction of bioactive substances.The C-PC crude extract with the purity of 1.275 was isolated from Spirulina platensis through the repeated freezing and thawing method.Different kinds of DES-ATPS were used of the extraction of C-PC,and [choline chloride-urea]/K₂HPO₄ was the best extraction system with the purity and recovery of C-PC of 3.383 and 65.64 %,respectively,under the optimal extraction parameters(DES 3.25 g K₂HPO₄ 0.90 g m L^-1 extraction time 2 h and Na Cl content 2 %)obtained by single factor experiments.After four times extractions,the purity of C-PC reached analysis grade?4.1?and the recovery was 64.93 %,and the protein was consisted of alpha and beta subunits detected by SDS-PAGE.Addtionally,the DES recyclability consequence exhibited that C-PC could still be separated by DES after the fifth cycle.Various characterization methods were utilized for the exploration of the mechanism of extraction of C-PC by DES-ATPS,the results turned out that DES-ATPS could provide a mild extraction environment,and aggregation was the main driving force of the extraction of C-PC.Using the conbinant strain that constructed by our laboratory as raw materials,the crude suspension of target protein was prepared by ultrasonication.The [choline chloride-glycerol]-ATPS was selected as the best extraction system through the calculation of enzyme activity of recombinant Grx,partition coefficient and its selectivity of upper and lower phases,and recombinant Grx was found in bottom phase,while the cell fragments were in the DES-rich phase.Moreover,the optimum extraction conditions were DES 3.25 g,K₂HPO₄ 1.00 g m L^-1 of 5 m L,extraction time 12 h with 0 % Na Cl,according to the results of the single factor experiments,which made the selectivity to the lower phase of the recombinant Grx was indentified to be 15.50.SDS-PAGE results demonstrated that the molecular weight of the extracted recombinant Grx was about 9.8 k Da,indicating that DES-ATPS could recover recombinant Grx with enzyme activity from cell fragments and inclusion bodies.In this study,two kinds of marine bioactive proteins were effectively extracted by DES-ATPS preliminarily,involving the characteristics of economic,mild,simple and green separation technology.These results indicated that the DES-ATPS established in this study would have potential application values in the fields of the separation of bioactive substances.