Ming-Jer Lee

Extraction of an active enzyme by self-buffering ionic liquids: a green medium for enzymatic research

In this work, the extraction of a model enzyme α-chymotrypsin in its active conformation from an aqueous solution by using new biocompatible and self-buffering Goods buffer ionic liquids (GBILs)-based aqueous biphasic systems (ABSs) is demonstrated. The experimental liquid–liquid phase boundary data for these new GBIL-based ABSs were measured at 298.2 K. These new ionic liquids are derived from biological Goods buffers as anions and tetra-alkylammonium as cations. The buffering potential of these GBILs was confirmed by measuring their pH profiles and protonation constants (pKa) in aqueous solution by a potentiometric method at various temperatures. Moreover, these GBILs provide a greater stabilizing effect on the activity of α-chymotrypsin, as compared with the corresponding Goods buffers. Therefore, the new self-buffering GBILs may not only serve as a biocompatible medium for enzymatic research, but also avoid the use of an external buffering agent, which is often needed for pH control in the case of using a conventional ILs-based medium. Therefore, these new biocompatible GBILs are more advantageous over the conventional ILs-based media for enzymatic research.

Self-buffering and biocompatible ionic liquid based biological media for enzymatic research

In the present work, we have designed five new biocompatible, self-buffering ionic liquids (ILs) in which the cationic part is derived from conventional tetra-butylphosphonium (TBP) and the anionic part is derived from common biological buffers TAPS, MOPS, EPPS, CAPS, and BICINE. The new ionic liquid based biocompatible media ([TBP][TAPS], [TBP][MOPS], [TBP][EPPS], [TBP][CAPS], and [TBP][BICINE]) were found to be suitable for overcoming most of the problems associated with enzymatic research including optimum pH range, biocompatibility, and extraction. In comparison with the conventional ionic liquid based biological media, these new media do not involve an external buffering compound and maintain the optimum pH by their self-buffering capability. The buffering nature of these new ILs was confirmed by measuring their pH profiles and protonation constants in aqueous solutions at different temperatures. The biocompatibility of these new ILs was also confirmed by measuring the biological activity of the enzyme α-chymotrypsin (α-CT) in the aqueous media of these ILs. Moreover, these new ionic liquids can form an aqueous two phase system (ATPS) with a common inorganic salt such as sodium sulfate. The excellent extraction efficiency (100%) of these ionic liquid-based ATPS was observed for the extraction of enzyme α-CT, in its active form, in an IL-rich phase via a single stage extraction. Since the selected common biological buffers are biocompatible and nontoxic compounds, the ionic liquids derived from these buffer compounds could be more promising for biological research.