Extraction of Proteins by Winsor III Microemulsion Systems

Purification of proteins by microemulsion was improved through use of a 3-phase (“Winsor-III”) microemulsion system instead of the traditionally employed 2-phase (“Winsor-II”) water-in-oil microemulsion system. As a consequence, two of the main problems of the traditional method were improved: the low and slow rate of recovery of proteins encapsulated by the microemulsions and the low protein solubilization capacity of the microemulsions.

Microemulsion systems employed a mixture of surfactants, two pH-degradable “cyclic ketal” alkyl ethoxylates (O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl) methoxy]–O’- methoxy poly(ethylene glycol)_n , where n, the average degree of polymerization for the ethoxylate group, equaled 3.0 or 5.45, CK3 and CK7, respectively) and Aerosol-OT (AOT). CK7’s ethoxylate size has broad molecular weight distribution. Partitioning behavior of CK7 molecules as a function of their ethoxylate size, temperature, and the addition of a second surfactant (AOT, CK3, or octyl β-D-glycoside) were investigated. A semi-empirical thermodynamic mathematical model was developed to calculate the phase inversion temperature (PIT) for a surfactant mixture of a specified composition. This information is useful to find the optimal Winsor-III for protein extraction.

Finally different Winsor-III systems formed by water, isooctane and surfactant mixtures of CK7, CK3 and AOT were tested in forward extraction of α-chymotrypsin, cytochrome-c, lysozyme, BSA and pepsin. Three approaches were used to obtain Winsor- III systems suitable for forward extraction: employment at 40°C, addition of a more hydrophobic surfactant (CK3, 25°C), and the addition of 1.5 wt. % NaCl (aq) to increase the ionic strength (25°C). Protein concentrations achieved in microemulsion phase were 10 times higher than values reported in the literature for extraction by Winsor-II microemulsion systems.

Back extraction was tested for α-chymotrypsin, cytochrome-c, and lysozyme. Aqueous striping solutions used for back extraction contained either a high ionic strength (5 wt. % NaCl) or a high pH (12.0). Back-extraction was fast and total recovery of the activity for α-chymotrypsin was achieved. Other proteins have smaller percentage of mass recovery in the conditions tested but comparable with yields reported in the literature for microemulsion-based extraction.