Aqueous two-phase extraction (ATPE): An attractive and economically viable technology for downstream processing of Aspergillus oryzae alpha-galactosidase

Abstract

Aqueous two-phase systems (ATPSs) have many applications for protein purification in biochemistry and biotechnology. One of the major challenges in the biotechnology industry is the large-scale purification of a desired protein from a fermentation broth containing a wide variety of biomolecules. One possible strategy for addressing this challenge is to use an aqueous two-phase system as the initial primary downstream processing (DSP) for partial purification of industrial enzymes. The aim of this work is to extract and purify Aspergillus oryzae ?-galactosidase using an aqueous two-phase system. The ?-galactosidase was extracted by partitioning in ATPS composed of polyethylene glycol (PEG) and phosphate. The effects of phase composition, molecular weight of the PEG, PEG concentration, phosphate salt concentration, pH, temperature and neutral salt (sodium chloride) concentration on enzyme partition and purification were studied. The optimal system was found at pH 5.0, containing 12% (w/w) PEG 4000 and 11.9% (w/w) phosphate with a K?-galactosidase of 0.156, purification factor of 3.6 and 87.71% of yield of enzyme activity in the bottom phase. After purification by ATPS, the ?-galactosidase optimum pH and temperature were not altered. © 2008.