Development and Application of Aqueous Two-Phase Partition for the Recovery and Separation of Recombinant Phenylalanine Dehydrogenase

Abstract

Aqueous two-phase systems (ATPS) have emerged as a powerful extraction method for the downstream processing of bio-molecules . The aim of this work was to investigate the possibility of utilizing ATPS for the separation of recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH). Polyethylene glycol (PEG) and ammonium sulfate systems were selected for our experiment. The effect of different elements such as; type and concentration of PEG, concentration of (NH4)2SO4, pH, phase volume ratio (VR) and tie-line length (TLL) on the extraction behavior and selective separation was also studied. Desirable conditions for differential partitioning was obtained in 8.5 % (w/w) PEG-6000, 17.5 % (w/w) (NH 4)2SO4 and VR 0.25 at pH 8.0. PheDH was mainly concentrated into the upper PEG-rich phase in all tested systems. The partition coefficient (K), recovery (R %), yield (Y %), TLL and selectivity were found to be 58.7, 135 %, 94.42 %, 39.89 % (w/w) and 2174, respectively. From the experimental results, it was revealed that the PEG molecular weight, (NH4)2SO4 concentration, TLL and pH of system had strong impacts on partition features. The extraction efficiency was increased with elevation of pH and TLL values. In this paper, we described the partitioning behavior in PEG/(NH 4)2SO4 ATPS in order to evaluate the applicability of ATPS for partitioning and recovery of PheDH.