Direct recovery of polyhydroxyalkanoates synthase from recombinant Escherichia coli feedstock by using aqueous two-phase systems

Abstract

Polyhydroxyalkanoates (PHAs) are intracellular bacterial polyesters which play a role as carbon and energy storage materials. The types of polymers produced depend on the available carbon sources, the flexibility of the organism's intermediary metabolism, and the substrate specificity of the PHA biosynthetic enzymes. The recombinant Escherichia coli harboring phaC gene obtained from Cupriavidus necator H16 was constructed and conducted in this work. The overexpressed proteins in the cell broth were directly recovered in the aqueous two-phase systems (ATPS). The optimized concentration of ATPS for separation of PHA synthase from unclarified feedstock was found to compose polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) in the absence of NaCl at pH 8.7 and 4°C. The results indicated that the partition coefficient of activity and protein content are 8.30 and 0.23, respectively. The specific activity, selectivity, purification factor and recovery of phaCRe can achieve 1.82Umg−1, 35.30, 16.62 and 96.42%, respectively. The increment of concentration of loading biomass and temperature demonstrated a decreasing trend in terms of selectivity and purification factor. The present results demonstrated that ATPS can be applied to directly recover PHA synthase from crude feedstocks and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro.