Solubilization of subtilisin in CO2 using fluoroether-functional amphiphiles

Carbon dioxide is a naturally abundant, environmentally benign solvent whose use, like water, in a process is not regulated by either EPA or FDA. Unfortunately, polar compounds such as amino acids and proteins are essentially insoluble in carbon dioxide. Further, alkyl-functional surfactants, which have been shown to allow extraction of proteins into conventional organic solvents, exhibit very poor or negligible solubility in CO2 at pressures below 50 MPa. Consequently, highly CO2-soluble fluoroether-functional surfactants have been generated and used to solubilize subtilisin Carlsberg from aqueous buffer and cell culture medium into CO2, with recovery accomplished by depressurization. Both the amount of protein solubilized in the emulsion and the extent of activity retention by the protein following recovery are functions of the initial protein concentration in the buffer. This, plus the observation that the presence of protein affects the stability of the emulsion, suggests that some of the protein is sacrificed to act as a stabilizer in these systems. In addition to solubilization via an inverse emulsion, it has also been shown that one can strip protein-surfactant aggregates from a middle phase emulsion using pure CO2, suggesting an ion-pairing type mechanism.