Partitioning of Geotrichum candidum Lipase from fermentative crude extract by aqueous two-phase system of polyethylene glycol and sodium citrate

Abstract

The partitioning behaviour of the crude extracellular lipase of Geotrichum candidum (GCL) were studied in aqueous two-phase systems (ATPS) formed by mixtures of poly(ethylene oxide) (PEO) (1500 or 4000 or 6000 g mol−1) or triblock copolymer [(EO)11(PO)16(EO)11] (L35) (1900 g mol−1), where EO and PO are ethylene oxide and propylene oxide, respectively, + sodium citrate + water. The partition ratio for the proteins present in the crude broth (which is predominantly lipase) in the different ATPS were investigated as a function of the pH, PEO molar mass, phases hydrophobicity, tie-line length and temperature. The proteins were preferentially partitioned into the salt-rich phase in all of the systems studied with partition ratios ranging between 0.025 and 0.7. The thermodynamic parameters of transfers of the proteins between phases were obtained and shows that the transfer of proteins to the bottom phase is entropic driven, however negative values obtained for the enthalpy of the transfer of proteins the bottom to the top phase (ΔtrHo = −12.12 to −7.10 kJ mol−1 in the temperature range of 5.0–45.0 °C, respectively) constitutes evidence of the strong interactions between the proteins presents in the crude broth and the PEO chains in the top phase of the ATPS. The ATPS formed for a mixture of PEO 1500 g mol−1 + sodium citrate + water at pH 7.0 was optimised using the response-surface methodology. The optimized conditions for the partition of proteins of crude broth were: 19.0% w/w + 12.0% w/w and 17.6% w/w + 14.0% w/w of PEO 1500 g mol−1 and sodium citrate, respectively at pH 7.0. In these conditions were obtained the highest enzymatic concentrations in the bottom phase, with Kp < 0.15.