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

The partition and purification of alpha-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 degrees 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%. (C) 2009 Elsevier B.V. All rights reserved.