Protein extraction by Winsor-III microemulsion systems

Proteins (bovine serum albumin (BSA), α-chymotrypsin, cytochrome c, and lysozyme) were extracted from 0.5 to 2.0 g L(-1) aqueous solution by adding an equal volume of isooctane solution that contained a surfactant mixture (Aerosol-OT, or AOT, and a 1,3-dioxolane (or cyclic ketal) alkyl ethoxylate, CK-2,13-E5.6), producing a three-phase (Winsor-III) microemulsion with a middle, bicontinuous microemulsion, phase highly concentrated in protein (5-13 g L(-1)) and small in volume (12-20% of entire volume). Greater than 90% forward extraction was achieved within a few minutes. Robust W-III microemulsion systems were formulated at 40°C, or at 25°C by including a surfactant with shorter ethoxylate length, CK-2,13-E3 , or 1.5% NaCl (aq). Successful forward extraction correlated with high partitioning of AOT in the middle phase (>95%). The driving force for forward extraction was mainly electrostatic attractions imposed by the anionic surfactant AOT, with the exception of BSA at high ionic strength, which interacted via hydrophobic interactions. Through use of aqueous stripping solutions of high ionic strength (5.0 wt %) and/or pH 12.0 (to negate the electrostatic attractive driving force), cytochrome c and α-chymotrypsin were back extracted from the middle phase at >75% by mass, with the specific activity of recovered α-chymotrypsin being >90% of its original value.