Exploring the Effect of Choline-Based Ionic Liquids on the Stability and Activity of Stem Bromelain.

Enzymes are very important components which are vital for the existence of every cellular life. There is significant interest in the use of structurally stable and catalytically active enzymes in pharmaceutical, food, fine chemicals industries, and in various industrial processes as catalysts. Stem bromelain (BM) is a proteolytic enzyme which is widely used in chemical, medical, and pharmaceutical fields. However, harsh process conditions are the main barriers to the effective use of this enzyme in different applications. To overcome these drawbacks, biocompatible bio-based ionic liquids (ILs), composed of the choline cation (an essential nutrient) and different anions are used. The ILs namely choline chloride [Ch]+[Cl]-, choline acetate [Ch]+[Ac]-, choline dihydrogen phosphate [Ch]+[Dhp]-, choline bitartrate [Ch]+[Bit]-, choline iodide [Ch]+[I]-, and choline hydroxide [Ch]+[OH]- are chosen for the current work. Therefore, in the present study, structural stability and activity of BM have been evaluated in the presence of choline-based ILs using various biophysical techniques at different concentrations. The present work demonstrated that [Ch]+[OH]- is the strongest destabilizer, whereas [Ch]+[Cl]- is the best stabilizer for the native structure of BM among all studied ILs. This work revealed the suitability of some choline-based ILs as potential media for sustained stability and activity of BM.

Effect of Imidazolium-Based Ionic Liquids on the Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect.

In the present work, changes in the structure and stability of stem bromelain (BM) are observed in the presence of a set of four imidazolium-based ionic liquids (ILs) such as 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]), and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]), using various biophysical techniques. Fluorescence spectroscopy is used to observe the changes taking place in the microenvironment around the tryptophan (Trp) residues of BM and its thermal stability because of its interactions with the ILs at different concentrations. Near-UV circular dichroism results showed that the native structure of BM remained preserved only at lower concentrations of ILs. In agreement with these results, dynamic light scattering revealed the formation of large aggregates of BM at higher concentrations of ILs, indicating the unfolding of BM. In addition to this, the results also show that higher alkyl chain length imidazolium-based ILs have a more denaturing effect on the BM structure as compared to the lower alkyl chain length ILs because of the increased hydrophobic interaction between the ILs and the BM structure. Interestingly, it is noted that low concentrations (0.01-0.10 M) of short alkyl chain ILs only alter the structural arrangement of the protein without any significant effect on its stability. However, high concentrations of all five ILs are found to disrupt the structural stability of BM.

Does 1-Allyl-3-methylimidazolium chloride Act as a Biocompatible Solvent for Stem Bromelain?

The broader scope of ILs in chemical sciences particularly in pharmaceutical, bioanalytical and many more applications is increasing day by day. Hitherto, a very less amount of research is available in the depiction of conformational stability, activity, and thermal stability of enzymes in the presence of ILs. In the present study, the perturbation in the structure, stability, and activity of stem bromelain (BM) has been observed in the presence of 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) using various techniques. This is the first report in which the influence of [Amim][Cl] has been studied on the enzyme BM. Fluorescence spectroscopy has been utilized to map out the changes in the environment around tryptophan (Trp) residues of BM and also to discuss the variations in the thermal stability of BM as an outcome of its interaction with the IL at different concentrations. Further, the work delineates the denaturing effect of high concentration of IL on enzyme structure and activity. It dictates the fact that low concentrations (0.01-0.10 M) of [Amim][Cl] are only changing the structural arrangement of the protein without having harsh consequences on its activity and stability. However, high concentrations of IL proved to be totally devastating for both activity and stability of BM. The observed decrease in the stability of BM at high concentration may be due to the combined effect of cation and anion interactions with the protein residues. The present work is successful in dictating the probable mechanism of interaction between BM and [Amim][Cl]. These results can prove to be fruitful in the studies of enzymes in aqueous IL systems since the used IL is thermally stable and nonvolatile in nature thereby providing a pathway of alteration in the activity of enzymes in potentially green systems.