Improved back extraction of papain from AOT reverse micelles using alcohols and a counter-ionic surfactant

The extraction of papain (MW 23 kDa, pI 9.6) from water was studied using reverse micelles containing sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Forward extraction of papain was carried out in the pH range of 4.0-6.5 and a maximum extraction of 65-70% was observed at pH 6.3. Papain extracted into the micelles could be back extracted to only 30% into a conventional aqueous phase (high pH and high KCl concentrations). In order to improve the back extraction of papain, 8-10 vol% alcohols were added because it was known that alcohols have an effect on the micelle-micelle interactions. However, this way resulted in the loss of papain activity inspite of good back extraction yields (80-90%). The back extraction of papain encapsulated in AOT reverse micelles was then carried out by adding a counter-ionic surfactant tri-n-octylmethyl-ammonium chloride (TOMAC). This technique resulted in faster back extraction compared to conventional method. Around 80-90% of activity recovery was obtained after back extraction. The improved backward transfer was likely caused by electrostatic interaction between the oppositely charged surfactant molecules, which led to the collapse of reverse micelles.