Development of different deep eutectic solvent aqueous biphasic systems for the separation of proteins

In this work, aqueous biphasic systems (ABSs) formed by different deep eutectic solvents (DESs) were prepared and applied to extract proteins. The five kinds of DESs comprised amino acids and polyols ([amino acids][polyols]). They were combined with another DES resulting from tetrabutylammonium chloride and polypropylene glycol 400 ([TBAC][PPG400]) to form ABSs. The phase-forming abilities of [TBAC][PPG400]/[amino acids][polyols] were compared with those of [TBAC][PPG400]/amino acids and [TBAC][PPG400]/polyols. The results exhibited that the biphasic formation ability of [amino acids][polyols] lies between those of amino acids and polyols when [TBAC][PPG400] acts as the other phase in ABSs. The systems comprising [TBAC][PPG400] and [l-proline][xylitol] ([Pro][Xyl]) were further investigated to optimize the extraction performance. It was found that 97.30% chymotrypsin tended to distribute into the [Pro][Xyl]-rich phase under optimum conditions. The practical application of the system was demonstrated by the extraction of chymotrypsin from porcine pancreas. Besides, UV-Vis spectrophotometry (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), and circular dichroism (CD) spectroscopy proved that the conformation of proteins remained unchanged during the extraction process. The extraction mechanism of the formation of DES-protein aggregates was investigated via conductivity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The overall results suggest that the DES/DES-based ABSs have outstanding potential in the green extraction of proteins.