Differential partitioning of β-galactosidase and β-glucosidase using aqueous two phase extraction

Abstract

Aqueous two phase extraction (ATPE) is used for the first time for simultaneous separation and purification of β-galactosidase and β-glucosidase from barley (Hordeum vulgare). The influence of various process parameters such as polymer molecular weight and its concentration, salt type and its concentration, system pH, tie line length, phase volume ratio and neutral salt addition on separation and purification of these two enzymes was evaluated. The β-galactosidase and β-glucosidase were selectively partitioned to top and bottom phases, respectively. Suitable conditions for purification were found in aqueous two phase system, having 14% (w/w) polyethylene glycol 1500/13% (w/w) ammonium sulphate, at tie line length of 19.65% (w/w). Single stage of ATPE resulted in an activity recovery of 98.26% with purification of 2.1 fold of β-galactosidase and an activity recovery of 92.58% with purification of 3.3 fold of β-glucosidase. Second stage of ATPE with respective new phases increased the purification of β-galactosidase and β-glucosidase to 2.4 and 4.1 fold, respectively. During ATPE, conditions which enabled a balance between yield and purification of both the enzymes were selected. Further ultrafiltration in diafiltration mode increased the purification of enzymes (β-galactosidase 6.8 fold and β-glucosidase 7.7 fold) besides the removal of the phase components.