Partitioning and purification of α-amylase in aqueous two-phase systems

Abstract

The partition behavior of pure α-amylase (1,4-α- d-glucan glucanohydrolase, E.C. 3.2.1.1) in aqueous two-phase systems was examined in order to investigate the effects of changes in type of phase components on the partition coefficient K. Polyethylene glycol (PEG)/dextran, PEG/phosphate, and PEG/sulfate systems were evaluated. Factors such as PEG molecular weight (MW), pH, and concentration of NaCl were all found to influence K. At low values (<2 kUml −1 phase system), enzyme concentration had no effect on K. At higher concentrations (up to 20.40 kUml −1 phase system was used, corresponding to ca. 12.3 gl −1, the phases became saturated and a twofold increase in K was observed, but also lower recovery. Subsequently, the separation and purification of α-amylase from typical contaminants from supernatant and whole broth of Bacillus subtilis fermentation was examined. The best partition conditions were found in PEG 4000/phosphate systems with 8.8% w/w NaCl where the K can be increased 78 times from K = 1.3 to K = 100. These conditions were then used to study the effect of phase volume ratio ( R) on the partition coefficient, the purification factor (PF), and recovery of α-amylase from industrial fermentations. R was found to influence the purification factor and the recovery, but not the partition coefficient. However, both α-amylase and contaminants partitioned to the top phase, leading to relatively poor separation (PF = 3.2 for R = 1). In a PEG/sulfate system, the addition of NaCl had an extreme effect on the partition behavior of α-amylase, giving an extreme K for α-amylase ( K = 6,800) at a high concentration of NaCl 8.8% w/w) and an extremely low K ( K < 0.005) at a lower concentration of NaCl. By exploiting this extreme partition behavior and manipulating R in a two-stage strategy, a 53-fold purification (with 86% w/w purity) could be calculated of a maximum possible of 63 at 100% w/w pure α-amylase.