An aqueous two-phase flotation (ATPF) composed of polyethylene glycol (PEG) and sodium citrate was. constructed for direct purification of thermostable lipase 42 from recombinant Escherichia coli BL21(DE3) pLysS. The influences of varying phase forming composition, tie-line length (TLL), volume ratio (V-R), crude loading (C-L), system pH, gas nitrogen flow rate (F-R) and flotation time (F-t) upon ATPF partitioning performance were investigated. The optimum purification condition of lipase was achieved at PEG 8000/sodium citrate ATPF comprising TLL of 25.4, V-R of 0.3, C-L of 20% (w/w) at pH 7 with average F-t of 10 min and F-R at 20 mL/min. Lipase was successfully purified using ATPF up to 4.05-fold with a separation efficiency of 99%. Result of this study also demonstrated that recovery yield of lipase in PEG phase of up to 96% was achieved. These results reveal the potential of ATPF partitioning in recovery and purification of recombinant lipase. (C) 2014 Elsevier B.V. All rights reserved.