Purification of CBS 819.72 α-amylase by aqueous two-phase systems: Modelling using Response Surface Methodology

Abstract

The partition and purification of α-amylase from a culture supernatant of Aspergillus oryzae CBS 819.72 was made in aqueous two-phase system (ATPS). According to bibliography and preliminary studies, the factors polyethylene glycol (PEG) molecular weight ( M PEG) and concentration ( C PEG), buffer type (BU) and concentration ( C BU), temperature ( T), salt nature (SALT) and concentration ( C SALT), bioligand (BL) and concentration ( C BL) and pH were investigated using a Plackett–Burman design to identify the factors affecting separation. Taking into consideration a simultaneous increase in enzyme recovery ( R Y) and purification factor ( P F), the best performance of the system was obtained at 4 °C and pH 6 using PEG 8000 g/mol, citrate buffer, KCl and sucrose. Experimental Box–Behnken design together with the Response Surface Methodology (RSM) have been used to find optimum C PEG, C Citrate and C SALT. Quadratic models were predicted for P F and R Y in the top phase and a better compromise between these two parameters can be found by superimposing the contour plots of P F and R Y for 8% citrate. A region in the experimental space can be defined where the purification factor is always higher than 3 with yields exceeding 65%.