In this paper, aqueous two-phase flotation (ATPF) composed of thermo-sensitive ethylene oxide-propylene oxide (EOPO) copolymer and ammonium sulfate was developed for direct recovery of Burkholderia cepacia (B. cepacia) strains ST8 lipase from fermentation broth. The effect of varying polymer molar mass, concentration of ammonium sulfate, pH, amount of loaded crude feedstock, initial volume of EOPO phase, concentration of EOPO, initial volume of aqueous phase, nitrogen flow rate and flotation time upon ATPF performance were investigated. Under the optimal conditions of ATPF, the average separation efficiency and purification fold are 76% and 13%, respectively. The recycling of phase components was introduced to minimize the use of organic solvent and salt in ATPF. It was demonstrated EOPO phase in the ATPF system was recovered up to 75%. There was no significant difference in selectivity, purification fold, separation efficiency and recovery yield of lipase obtained between ATPF using fresh and recycled chemicals. B. cepacia lipase was successfully purified by using ATPF, which is composed of copolymer EOPO/ammonium sulfate in a single downstream processing step.
Recycling hydrophilic organic solvent/inorganic salt aqueous two-phase flotation (ATPF) is a novel, low cost, green and high efficient technique for recovery of biomolecules. Recycling ATPF composed of 2-propanol and potassium phosphate was developed for sustainable separation, concentration and purification of Burkholderia cenocepacia ST8 lipase from liquid fermentation broth. Thirteen parameters upon recycling hydrophilic organic solvent/inorganic salt ATPF performance were investigated. The optimum conditions for this recycling ATPF were determined to be 40 mL volume of 50% (w/w) 2-propanol, 1.0 L of 250 g/L of potassium phosphate, pH 8.5, 100% (v/v) of crude feedstock, 30 mL/min of N2 flow rate for 30 min in a 8 cm radius of colorimeter tube with G4 porosity (5-15 μm) sintered glass disk. A purification factor of 14.4 ± 0.04 and a lipase yield of 99.2 ± 0.03% were achieved in this optimized ATPF. The recycling of phase-forming components employed at the end of recovery process was based on the principals of green chemistry, with high efficiency and economical viability. There was no gross variation of results during the process of scaling-up. Therefore, this novel recycling ATPF is feasible to be applied at industrial-scale.
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