Incorporation of electric fields to ionic liquids-based aqueous biphasic system for enhanced recovery of extracellular Kytococcus sedentarius TWHKC01 keratinase

Evaluation of Aqueous Biphasic Electrophoresis System Based on Halide-Free Ionic Liquids for Direct Recovery of Keratinase

“…High crude feedstock concentration often corresponds to high impurity content which gives rise to the alteration of phase composition and electrostatic potential of biphasic system [22]. The impurities might transfer to the IL-rich top phase forming electrostatic interaction with the IL molecules, consequently minimizing the free space available in the IL-rich top phase to accommodate keratinase molecules [21]. The biphasic system added with 15% (w/w) crude feedstock demonstrated the highest recovery efficiency, indicating that the maximum solubility of keratinase in IL-rich top phase of the developed biphasic system has been achieved.…”

“…The keratinase enzyme activity and total protein content in the phase sample were assessed using keratinolytic activity assay and bicinchoninic acid (BCA) assay, respectively, by referring to the procedure mentioned in previous studies [15,21]. Briefly, the keratinolytic activity assay was performed by incubating the mixture of enzyme-containing sample and 0.5% (w/v) keratin substrate solution in a water bath (40 • C, 30 min).…”

“…where p T represents the total protein content in the top phase and p B represents the total protein content in the bottom phase, respectively. Selectivity (S) was evaluated using Equation (3) [21]:…”

“…Trichloroacetic acid (TCA) precipitation method was employed to precipitate and concentrate the protein samples prior to the SDS-PAGE analysis [21]. SDS-PAGE analysis was carried out by referring to the procedure mentioned in a previous study using ATTO electrophoresis unit (Tokyo, Japan) [15].…”

“…Previous studies reported that IL-based ABES demonstrated better recovery yields and purity of keratinase when compared with the polymer/salt ABES [21]. In the present study, halide-free IL was used as the phase-forming component for the IL-based ABES with the aim to further reduce the cost and environmental hazards of the overall processing of the keratinase recovery.…”