Aqueous two-phase extraction coupled with ultrafiltration for purification of amyloglucosidase

Aqueous two phase extraction (ATPE) in combination with ultrafiltration was employed for concentration and purification of amyloglucosidase produced by solid state fermentation. After extraction (with water) from dry moldy bran the dilute enzyme extract was concentrated by ATPE in a polyethylene glycol (PEG)/maltodextrin (MDX) system. The enzyme in the top PEG rich phase was then extracted into a Na2HPO4 rich bottom phase and further concentrated by ultrafiltration. The partitioning behavior of amyloglucosidase was examined in PEG/MDX, PEG/Na2SO4, PEG/Na2HPO4, PEG/KH2PO4 aqueous two phase systems. Effect of buffering salts such as NaCl, Na2HPO4, KH2PO4 and Na2SO4 on the partitioning behavior of enzyme was studied in PEG/MDX system. Maximum partitioning of amyloglucosidase was seen with KH2PO4 (m = 18.1). A two stage ATPE employing PEG/MDX (buffered with KH2PO4) and PEG/Na2HPO4 systems, followed by ultrafiltration has resulted in an overall recovery of 78.4% with 3.1 fold purification and 9.4 fold concentration of the enzyme.