One-step separation of beta-galactosidase from beta-lactoglobulin using water-in-oil microemulsions

Solubilisation of beta-galactosidase from Kluyveromyces lactis in Aerosol-OT water-in-isooctane microemulsions was measured as a function of buffer type and concentration, pH, and protein concentration. For buffer concentrations above similar to 40 mM, the enzyme was largely excluded from the droplets. Based on these results, a one-step separation was developed. A protein-containing aqueous feed was injected into an AOT/isooctane solution, with the feed volume slightly in excess of the predicted water solubility. Impurity proteins were entrapped inside the microemulsion droplets that then formed in the organic phase, while the high MW target protein was excluded and entered a newly formed, excess aqueous phase. The separation of beta-galactosidase from the test protein beta-lactoglobulin was most complete at 100 mM KCl salt concentration, where the droplets were large enough to carry beta-lactoglobulin but too small for beta-galactosidase. At 100 mM [KCI], 92% of the total enzyme activity was recovered in a concentrated and virtually pure form. (C) 2011 Elsevier Ltd. All rights reserved.