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

Abstract

<em>Aqueous two-phase systems (ATPS) have emerged as a powerful extraction method for the downstream processing of bio-molecules</em><em>.</em><em> 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 (V_R) 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) (NH₄)₂SO₄andV_R 0.25at 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/(NH₄)₂SO₄ ATPS in order to evaluate the applicability of ATPS for partitioning and recovery of PheDH.</em>