Efficiency of polymer/salt aqueous two-phase electrophoresis system for recovery of extracellular Kytococcus sedentarius TWHKC01 keratinase

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

“…On the other hand, a clear band was observed in Lane 2, indicating that the keratinase was successfully purified in the IL-rich top phase with the removal of other protein contaminants to the salt-rich bottom phase. This is in agreement with the molecular weight of K. sedentarius TWHKC01 stated in the published literature, which was 60 kDa [ 15 ]. The slight discrepancy in the position of band shown in Lane 2 is probably due to the minimal binding of SDS to protein samples as a consequence of the high ionic strength of IL in the top phase sample.…”

“…The concentration of IL and salt for the construction of biphasic system was fixed at 25% ( w / w ) and 15% ( w / w ), respectively, with the loading of 20% ( w / w ) crude feedstock into the biphasic system. Keratinase with isoelectric point of 2.7 acts as a negatively-charged protein in both the alkaline-based biphasic system with pH above its isoelectric point, thus migrating from salt-rich bottom phase to the IL-rich top phase to form electrostatic interaction with IL molecules [ 15 ]. Keratinase possessed a greater negative magnitude in the biphasic system of carbonate salt (pH 11) when compared with that of phosphate salt (pH 9), as increase in pH increases the surface charge negativity, resulting in a stronger electrostatic interaction among the positively-charged imidazolium cations with the negatively-charged keratinase in the IL-rich top phase [ 26 ].…”

“…To identify the operation time for optimal keratinase recovery in the biphasic system incorporated with anode in salt-rich bottom phase and cathode in IL-rich top phase, operation durations differing from 5 min to 20 min were studied ( Figure 3 ). A longer operation time offers sufficient time for complete phase separation and lengthens the duration allowing for the charged target biomolecules to migrate across the phase interface to the IL-rich top phase [ 15 ]. However, a longer operation time was observed to possess an adverse effect on the recovery efficiency whereby the K e , Y T and S recorded were notably decreased.…”

“…For IL-based ABES, IL simultaneously acts as the electrolyte and extractant in the biphasic system, which facilitates the electrophoretic migration of the target biomolecules across the phase interface towards the preferred phase [ 13 , 14 ]. IL-based ABES is feasible at industrial level for the downstream processing of the target biomolecules with shortened processing time because of the enhanced phase separation rate of biphasic system with the imposition of electric fields [ 15 ].…”

“…This is the first paper to date reporting on the feasibility of halide-free IL as the phase component of the ABES in the recovery of enzyme. The Kytococcus sedentarius TWHKC01 is a marine bacteria strain that demonstrates potential for the production of extracellular keratinase in the cultivation medium added with feather waste [ 15 ]. Hence, primary capture of the K. sedentarius TWHKC01 keratinase using biphasic system of alkyl sulphate-based IL was performed with the determination of several system parameters, including types of salt, phase composition and crude feedstock concentration.…”