In the current study, reverse micellar extraction (RME) for the purification of stem bromelain was successfully achieved using the sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane system. A maximum forward extraction efficiency of 58.0% was obtained at 100 mM AOT concentration, aqueous phase pH of 8.0 and 0.2 M NaCl. Back extraction studies on altering stripping phase pH and KCl concentration, addition of counter-ion and iso-propyl alcohol (IPA) and mechanical agitation with glass beads indicated that IPA addition and agitation with glass beads have significant effects on extraction efficiency. The protein extraction was higher (51.9%) in case of the IPA (10% v/v) added system during back extraction as compared to a cetyltrimethylammonium bromide (100 mM) added system (9.42%). The central composite design technique was used to optimize the back extraction conditions further. Concentration of IPA, amount of glass beads, mixing time, and agitation speed (in rpm) were the variables selected. IPA concentration of 8.5% (v/v), glass bead concentration of 0.6 (w/v), and mixing time of 45 min at 400 rpm resulted in higher back extraction efficiency of 45.6% and activity recovery of 88.8% with purification of 3.04-fold. The study indicated that mechanical agitation using glass beads could be used for destabilizing the reverse micelles and release of bromelain back into the fresh aqueous phase.
Keywords: central composite design; enzyme activity; glass beads; protein recovery; purification; reverse micelles.
© 2014 American Institute of Chemical Engineers.