Extraction of a-chymotrypsin using sodium bis(2-ethylhexyl) phosphate(NaDEHP) reverse micellar system

Abstract

Recently, liquid-liquid extraction using reverse micelles has been proposed as an effective method to separate, concentrate, and purify proteins from a complex biomedium such as fermentation broth with scaled-up bility. In this study, the reverse micellar system of sodium b is (2-ethylhexyl) phosphate (NaDEHP)/isooctane/brine was used to extract a-chymotrypsin from an aqueous phase. The influence of factors affecting the extration efficiency such as pH, salt concentration, protein loading, and type of cosurfactants were examined. The resuls showed that extraction efficiency is strongly affected by pH and salt concentration in the aqueous phase. At near neutral pH and low salt conentration, high extraction efficiency (>90%) was obtained. At pH above pI of the protein (pH 8.5), extraction of the protein into the reverse micelles decreased dramatically. Increasing salt concentration resulted in a decline in the proteins transferred into the micellar phase due to lessening attractive interaction. Back extraction of proteins was performed by contacting the micellar phase with a divalent cation aqueous solution, which caused the reverse micelles to destabilize, thus releasing a quantitative amount of protein back into the aqueous solution. After back extraction, the enzymatic activity of the recovered proteins was also examined using a simple hydrolysis reaction.