Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia con harboring phaC gene derived from Raistonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000(30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4 degrees C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg(-1), 29.05, 16.23 and 9532%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37 degrees C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.