Short communication Practical implementation of aqueous two-phase processes for protein recovery from yeast Marco Rito-Palomares 1 * and Andrew Lyddiatt 2 1 Centro de Biotecnologı ´ a, Instituto Tecnolo ´ gico y de Estudios Superiores de Monterrey (ITESM), Campus Monterrey, Sucursal de Correos J, Monterrey, NL 64849, Me ´ xico 2 Biochemical Recovery Group, Centre for Biochemical Engineering, School of Chemical Engineering, University of Brimingham, B15 2TT, UK Abstract: A two-stage extraction process for the recovery of intracellular proteins from brewers' yeast was selected as a practical model system to study the implementation of polyethylene glycol (PEG)± phosphate aqueous two-phase systems (ATPS). Disrupted all suspensions generated by homogenisa- tion and bead milling were used to study the impact of cell debris upon the partition behaviour of the intracellular products (bulk protein, fumarase and pyruvate kinase). Regardless of their origin debris particles did not signi®cantly in¯uence the partition behaviour of the intracellular products in selected ATPS distant from the binodal and at volume ratios greater than one. Recycling of used PEG into the initial extraction stage did not signi®cantly in¯uence the protein partition behaviour in batch ATPS. In the polymer recycling studies in continuous ATPS using spray columns, the addition of fresh materials to make up the de®cits of phase-forming chemicals compensate any negative effect of the continuous recycling of the top PEG-rich phase. The ®ndings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. # 2000 Society of Chemical Industry Keywords: aqueous two-phase partitioning; protein recovery; disrupted yeast; polymer recycle 1 INTRODUCTION Combinations of hydrophilic solutes (polymers or a polymer and certain salts) may display incompatibility in aqueous solution above critical concentrations, such that two aqueous phases form. In polymer±polymer systems, it has been suggested that the hydrated surface of each species are suf®ciently incompatible to generate phase separation 1,2 but the descriptive mechanisms in systems composed of polymer and salt remain unclear. It has been suggested that phase separation is associated with differing interactions with the ether dipoles of the poly-(ethylene glycol) (PEG) chain. 3 The density of the phases in these systems de®nes the salt-rich fraction as the lower phase and the polymer-rich fraction as the upper phase. Current research in aqueous two-phase systems (ATPS) can be readily divided into two areas. One is strongly concerned with a mechanistic molecular understand- ing of solute partition in ATPS, whilst the other focuses upon practical implementation. The current focuses on generating a practical understanding of a two-stage process for the recovery of bulk protein from waste brewers' yeast using PEG- phosphate. Such a process will produce bulk protein fraction from a variety of biological suspensions in a state suited for further puri®cation and extraction of selected products (eg target enzymes). An `ideal' ATPS process is characterised by two stages (see Fig 1), in which the First Extraction eliminates the bottom phase particles (cells or cell debris) and contaminants (eg RNA, carbohydrates, lipid) from the feedstock and generates a top phase enriched in the target soluble protein fraction. The high concentration of PEG in the top phase compromises the value of the product in that state and presents practical problems of handling such a viscous phase. Consequently, in the second step (Back Extraction) the protein is partitioned to a more suitable environment (bottom phosphate-rich phase) which enables re-use of the polymer-rich top phase. We have previously reported 4 the sequential optimisa- tion and development of a two-extraction stage ATPS to process biological suspensions and recover bulk protein from unclari®ed, milled brewers' yeast using PEG of average molecular weight (MW) 1000 daltons (referred to as PEG alone) and di-potassium phos- phate (referred to as phosphate). The selection of PEG MW 1000 was derived from previous work. 5,6 This PEG promoted the partition of the majority of yeast (Received 4 June 1999; accepted 13 February 2000) * Correspondence to: Marco Rito-Palomares, Centro de Biotecnologı ´a, Instituto Tecnolo ´gico y de Estudios Superiores de Monterrey (ITESM), Campus Monterrey, Sucursal de Correos J, Monterrey, NL 64849, Me ´xico E-mail: mrito@campus.mty.itesm.mx Contract/grant sponsor: CONACyT Contract/grant sponsor: International Foundation for Science; contract/grant number: IFS E/2620-2 # 2000 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/2000/$17.50 632 Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:632±638 (2000)