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Cytochrome c is a small water-soluble protein that is abundantly found in the mitochondrial intermembrane space of microorganism, plants and mammalians. Ionic liquids (ILs)-based aqueous two-phase electrophoresis system (ATPES) was introduced in this study to investigate the partition efficiency of cytochrome c to facilitate subsequent development of two-phase electrophoresis for the separation of cytochrome c from microbial fermentation. The 1-Hexyl-3-methylimidazolium bromide, (C 6 mim)Br and potassium citrate salt were selected as the phase-forming components. Effects of phase composition; position of electrodes; pH and addition of neutral salt on the partition efficiency of cytochrome c in the ATPES were evaluated. Highest partition coefficient (K = 179.12 ± 0.82) and yield of cytochrome c in top phase (Y T  = 99.63% ± 0.00) were recorded with IL/salt ATPES composed of 30% (w/w) (C 6 mim)Br and 20% (w/w) potassium citrate salt of pH 7 and 3.0% (w/w) NaCl addition with anode at the bottom phase and cathode at the top phase. The SDS-PAGE profile revealed that cytochrome c with a molecular weight of 12 kDa was preferably partitioned to the IL-rich top phase. Present findings suggested that the single-step ATPES is a potential separation approach for the recovery of cytochrome c from microbial fermentation. Graphical Abstract.

• Incorporation of electric fields reduces the phase settling time and facilitates mass transfer of solutes. • Ionic liquids is the excellent phase-forming component because of the good electrical conductivity. • The purification fold of keratinase was enhanced from 5.94 ± 0.22 to 8.22 ± 0.44. • High keratinase recovery yield of 95.82% ± 0.17 was attained in optimized condition. The purification of keratinase is essential to enhance the catalytic efficiency of keratinase enzyme for industrial applications. Biphasic system incorporated with electric fields is an effective purification tool for the recovery of biomolecules. Ionic liquids (IL) with good conductivity are ideal phase-forming components for biphasic system which promotes the separation of charged biomolecules via electromigration. This study aimed to evaluate the effect of electric fields on enhanced recovery of Kytococcus sedentarius TWHKC01 keratinase using IL-based biphasic system. Keratinase was recovered with biphasic system incorporated with anode in salt-rich bottom phase and cathode in IL-rich top phase operated at 5 V for 5 min. A total of 95.82% ± 0.17 of keratinase was attained with the biphasic system at pH 9 comprising of 20% (w/w) 1‑butyl‑3-methylimidazolium tetrafluoroborate ((Bmim)BF 4) and 25% (w/w) ammonium sulphate loaded with 15% (w/w) K. sedentarius TWHKC01 crude feedstock. The selectivity (S) and purification fold (PF T) of keratinase were increased from 7.25 ± 1.50 to 10.87 ± 0.61 and 5.94 ± 0.22 to 8.22 ± 0.44, respectively with the incorporation of electrodes, demonstrating the efficiency of electric fields in enhancing the purity of recovered keratinase. [Display omitted] [ABSTRACT FROM AUTHOR]

• Ionic-liquids aqueous biphasic system was used to recover cyt c from simulated S. cerevisiae cells culture. • Phase composition, pH, temperature and NaCl addition determines the recovery of cyt c. • Cyt c was optimally recovered in top phase of (C 6 mim)Br/citrate system with K c of 364.00 ± 1.09. Ionic liquid-based aqueous biphasic system (ILABS) has emerged as an attractive green approach for the extraction and separation of various biomolecules. The growing market demands of cytochrome c (cyt c) due to its vast uses in medical applications have urged the search for cost-efficient approaches for the production and purification of cyt c. In this study, the feasibility of ILABS to recover cyt c from simulated Saccharomyces cerevisiae cell cultures was investigated by evaluating the effects of phase composition, pH, and additives concentration on the recovery efficiency of cyt c. The ILABS was developed using the hydrophilic ionic liquids, 1-hexyl-3-methylimidazolium bromide ((C6mim)Br) and potassium citrate. The optimal separation conditions for recovery of cyt c from simulated cells cultures were attained with ILABS of pH 9 comprising of 28% (w/w) of (C6mim)Br and 24% (w/w) of potassium citrate and the addition of 0.2% (w/w) NaCl at room temperature (25 °C). Cyt c was recovered in the IL-rich phase with partition coefficient (Kc) of 364.00 ± 1.09, recovery yield (Y) of 99.76% ± 0.03 and selectivity (S) of 223.31 ± 0.56. The results suggest that ILABS can efficiently recover cyt c from microbial fermentation broth with high recovery yields and separation efficiency. [ABSTRACT FROM AUTHOR]

• Ionic liquids-based aqueous biphasic flotation was applied for keratinase recovery. • Highest recovery efficiency was attained with airflow rate of 20 cc/min for 5 min. • Keratinase was recovered in IL-rich phase with recovery yield of 91.28% ± 0.64. Ionic liquids (IL) is a unique environmental benign solvent with attractive characteristics which can be used in the formation of aqueous biphasic system. Aqueous biphasic flotation (ABF) system can improve the recovery yields of target biomolecules by incorporating the gas bubbles into the aqueous biphasic system (ABS). Keratinase has been widely applied in numerous industries due to the capability to degrade the keratinous wastes and convert them into valuable products via bioconversion. The feasibility of IL-based ABF system to recover the Kytococcus sedentarius TWHKC01 keratinase from crude feedstock was investigated in this study. IL-based ABF system formed with 25.0% (w/w) 1-ethyl-3-methylimidazolium ethyl sulphate, (EMIM)(ESO 4), 12.5% (w/w) sodium carbonate and 20% (w/w) crude feedstock demonstrated the optimum yield (Y) of 91.28% ± 0.64 and partition coefficient (K e) of 8.39 ± 0.64. Keratinase was transferred to the IL-rich top phase of the ABF system which was operating at an airflow rate of 20 cc/min for 5 min. The keratinase selectivity (S) of 2.48 ± 0.31 and purification fold (PF) of 2.12 ± 0.04 were recorded. The IL-based ABF system is therefore highly recommended as a one-step operation to recover and purify the microbial keratinase due to the cost-saving and environmentally friendly properties. [Display omitted] [ABSTRACT FROM AUTHOR]

Aqueous biphasic electrophoresis system (ABES) incorporates electric fields into the biphasic system to separate the target biomolecules from crude feedstock. Ionic liquid (IL) is regarded as an excellent candidate as the phase-forming components for ABES because of the great electrical conductivity, which can promote the electromigration of biomolecules in ABES, and thereby enhances the separation efficiency of the target biomolecules from crude feedstock. The application of electric fields to the conventional biphasic system expedites the phase settling time of the biphasic system, which eases the subsequent scaling-up steps and reduces the overall processing time of the recovery process. Alkyl sulphate-based IL is a green and economical halide-free surfactant when compared to the other halide-containing IL. The feasibility of halide-free IL-based ABES to recover Kytococcus sedentarius TWHK01 keratinase was studied. Optimum partition coefficient (Ke = 7.53 ± 0.35) and yield (YT = 80.36% ± 0.71) were recorded with IL-ABES comprised of 15.0% (w/w) [EMIM][ESO4], 20.0% (w/w) sodium carbonate and 15% (w/w) crude feedstock. Selectivity (S) of 5.75 ± 0.27 was obtained with the IL-ABES operated at operation time of 5 min with 10 V voltage supplied. Halide-free IL is proven to be a potential phase-forming component of IL-ABES for large-scale recovery of keratinase. [ABSTRACT FROM AUTHOR]