Selective enzymes separation from the fermentation broth of Yarrowia lipolytica using aqueous two-phase system based on quaternary ammonium compounds

Abstract

Aqueous two-phase systems (ATPS) are considered efficient and sustainable downstream processing techniques in extracting and separating enzymes. In this work, we evaluated the use of ATPS based on quaternary ammonium (tetrabutylammonium bromide ([N4444]Br), tetrabutylammonium chloride ([N4444]Cl), choline chloride (ChCl), and betaine) + potassium phosphate buffer (pH 7) to separate selective lipase and protease from the fermentation broth with Y. lipolytica. Cultivation in YPD medium supplemented with 200 mM potassium phosphate buffer (pH 7) in the preparation of the culture medium positively influenced the production of lipase and protease, whereby led to lesser pH variations and better values of biomass production (15.29 g p.s. L-1), lipolytic (455.96 U L-1), and proteolytic activity (23.70 U L-1). The presence of the culture medium enhanced the two-phase region, and the phase separation followed the order of ammonium compounds hydrophobicity: [N4444]Br > [N4444]Cl > betaine > ChCl. Lipase was partitioned mainly to the ammonium-rich phase and protease migrates preferentially to the salt-rich phase in all systems studied. Remarkable extraction efficiencies of 100% for lipase and 96.87% for protease were achieved in a single step for [N4444]Cl-based ATPS. Furthermore, a high level of purification was achieved with values of 10.55 and 1.69 for lipase and protease, respectively. According to the remarkable results, quaternary ammonium-based ATPS can be considered an alternative and efficient platform to separate lipase from protease, obtaining two high-value-added compounds selectively.