EFFECTIVE PURIFICATION METHOD OF LARGE MOLECULAR WEIGHT PROTEINS USING CONVENTIONAL AOT REVERSE MICELLES
Hydrophilic bulky proteins with molecular weights larger than 60 kDa, such as catalase, <I>β</I>-galactosidase, BSA and hemoglobin, can be easily solubilized into a micro water pool of AOT reverse micelles by the injection method. Those proteins and enzymes solubilized into reverse micelles maintain their activities and native structures, and can be back-extracted effectively to a new aqueous phase when the system <I>pH</I> is kept higher than their isoelectric points, <I>pI</I>, with low salt concentration, i.e. high water content. The back-extraction of hemoglobin is also partially achieved under these conditions. The efficiency of back-extraction is strongly influenced by the <I>pH</I> values in the feed protein solution and in the aqueous solution used for back-extraction, as well as KCl concentration in the solution. The <I>pH</I> values of both solutions should be higher than the protein <I>pI</I> together with high <I>Wo</I> or low salt concentration to preserve native structures and activities, since monomers and oligomers with relatively large molecular weight are likely to be denatured by association with ionic surfactant mediated by salt. In this case, both steric and electrostatic interactions between bulky proteins and micelles play dominant roles in the separation. By reducing interactions with micelles or surfactants, proteins solubitized in micelles can be effectively stabilized, and easily back-extracted to the aqueous phase without inactivation. The present method suggests an another way for the effective bioseparation of bulky proteins with high yields of activity.
- Journal of chemical engineering of Japan
Journal of chemical engineering of Japan 28(6), 803-809, 1995-12-01
The Society of Chemical Engineers, Japan