Evaluation of recombinant phenylalanine dehydrogenase behavior in aqueous two-phase partitioning Hamid Shahbaz Mohamadi a , Eskandar Omidinia b, * , Rassoul Dinarvand c a Young Researcher Club, Department of Biochemistry, Science & Research Campus, Islamic Azad University, Tehran, Iran b Department of Biochemistry, Pasteur Institute of Iran, Tehran 13164, Iran c Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Received 28 January 2007; received in revised form 30 May 2007; accepted 11 June 2007 Abstract Recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH) partitioning was studied in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The objectives of this work were to investigate influences; varying the molecular mass and concentration of PEG, pH, phase volume ratio (V R ), tie-line length (TLL) and concentration of (NH 4 ) 2 SO 4 on the partition behavior of PheDH. It was revealed that the partitioning was not affected by V R , while PEG molecular mass and concentration and (NH 4 ) 2 SO 4 concentration had significant effects on enzyme partitioning. Longer TLL and higher pH resulted in better partitioning into the top phase. Under the most favorable partition conditions with 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH 4 ) 2 SO 4 and V R = 0.25 at pH 8.0, partition coefficient (K E ), recovery (R%), yield (Y%) and TLL were achieved 58.7%, 135%, 94.42% and 39.89% (w/w), respectively. Overall, the promising results obtained in this research indicated that the ATPS partitioning can be provided an efficient and powerful tool for recovery and purification of recombinant PheDH. # 2007 Elsevier Ltd. All rights reserved. Keywords: Aqueous two-phase systems (ATPS); Ammonium sulfate; Phenylalanine dehydrogenase (PheDH); Partition; PEG-6000 1. Introduction During the last several decades, the development in the field of modern biology such as protein engineering, DNA recombinant technology and biotechnology in general has resulted in increasing demand for novel, fast and cost-effective purification protocols. In fact, today industries desire rapid, simple, efficient and economic methods. As a matter of fact, the traditional procedures including salt or organic precipitation, dialysis, filtration and chromatographic methods or combination of these are expensive and time-consuming which have enormous difficulties in large-scale applications. With regard to these problems, ATPS appears to be a powerful and attractive candidate for separating and purifying the biological products [1–9]. ATPS partitioning is generally obtained by the incompat- ibility between aqueous solutions of two polymers (PEG, dextran, etc.) or a polymer and a salt (phosphate, sulfate, citrate, etc.) at high ionic strength. In comparison with polymer–polymer systems, the polymer–salt systems have higher selectivity, lower cost and viscosity in protein partitioning [1–3,10]. Also it has been found that the desirable biomaterials are usually concentrated into the polymer-rich top phase and the con- taminants are remained in salt-rich bottom phase. Recently, some novel systems such as micro emulsion and affinity phases have been developed [2,3,11]. Partitioning in ATPS is mainly depends on the physiochemical traits of biomolecules such as charge, shape, size, molecular weight, hydrophobicity and specific binding sites. Moreover, the partition profile is also influenced by van der Waals, hydrogen and hydrophobic bonds, static effects and electrostatic interactions between the biomaterial and the phase forming components. As a result, the partition may be affected by altering the system components, the molecular weight and concentration of polymer, the type and concentration of salt, the ionic strength, the system pH and temperature [1–3,12–17]. The causative mechanism of ATPS partitioning is not fully understood. Although, the mathematical models such as response surface methodology (RSM) provide some information about phase behavior and partitioning of target bimolecules, no comprehensive theory currently exists to guide the design of www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 1296–1301 * Corresponding author. Tel.: +98 21 66402770; fax: +98 21 66402770. E-mail addresses: skandar@pasteur.ac.ir (E. Omidinia), dinarvand@tums.ac.ir (R. Dinarvand). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.06.005