Mixed reverse micellar systems for extraction and purification of β-glucosidase

Abstract

Mixed reverse micellar (MRM) systems of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and nonionic surfactants (Tween 20, Tween 80, Tween 85 and Triton X-100) in iso-octane were used for the extraction and primary purification of β-glucosidase (EC 3.2.1.21) from the aqueous extract of barley (Hordeum vulgare) for the first time. Studies are carried out with both phase transfer as well as injection mode of reverse micellar extraction, among which, injection mode is observed to be more suitable for β-glucosidase extraction. The process parameters such as concentration of surfactants and their molar ratio, type of solvent, volume of sample injected and its protein concentration, pH and ionic strength of the initial aqueous phase for forward extraction, buffer pH, concentration of hexanol and salt during back extraction are varied to maximize the extraction efficiency. The molar ratio of AOT:nonionic above 4.5:1.0 only resulted in clear phase formation and below this ratio precipitation was observed at the interface during forward extraction. Mixed reverse micelles found to solubilize almost three times more volume of water and protein than AOT alone under otherwise similar conditions. Activity recovery was found to be in the order of AOT/Tween 20 > AOT/Tween 80 > AOT/Triton X-100 > AOT/Tween 85 > AOT. MRM system of 150 mM AOT/15 mM Tween 20 has resulted in maximum activity recovery of 95.18% and degree of purification of 4.8-fold.