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

► W/o microemulsions extract impurity proteins from β-galactosidase (β-gal) in one-step. ► Solubilisation studies determine microemulsion conditions for excluding β-gal. ► Mixtures contained β-gal and impurity protein (β-Lg) with similar pI but lower MW. ► Microemulsions with [buffer] >40mM and added KCl extract β-Lg, expel purified β-gal. ► At 100mM [KCl], process yields virtually pure β-gal with 92% activity recovery. Solubilisation of β-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 ∼40mM, 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 β-galactosidase from the test protein β-lactoglobulin was most complete at 100mM KCl salt concentration, where the droplets were large enough to carry β-lactoglobulin but too small for β-galactosidase. At 100mM [KCl], 92% of the total enzyme activity was recovered in a concentrated and virtually pure form.