Mediating electrostatic binding of 1-butyl-3-methylimidazolium chloride to enzyme surfaces improves conformational stability.

We have recently developed a general approach to improve the utility of enzymes in ionic liquids (ILs) via tuning of the ratio of enzyme-containing positive to negative surface charges. In this work, the impact of enzyme surface charge ratio on the biophysical interaction of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) with chymotrypsin and lipase was investigated to understand this approach at the molecular level. Results of fluorescence quenching assays indicated that the extent of binding of the [BMIM] cation decreased (7- and 3.5-fold for chymotrypsin and lipase, respectively) as a function of increasing ratio of positive to negative surface charges. Conformational stability assays further showed a close correlation between thermodynamic stabilization and enzyme surface charge ratio as well as [BMIM] binding. As evidence of this correlation, succinylation and acetylation resulted in the stabilization of chymotrypsin in 10% (v/v) [BMIM][Cl] by 17.0 and 6.6 kJ/mol, respectively, while cationization destabilized chymotrypsin by 3.6 kJ/mol. Combined, these results indicate that altering the surface charge ratio mediates the organization of IL molecules, namely, [BMIM] and [Cl], around the enzymes. Preferential exclusion of [Cl], in particular, via lowering of the ratio of positive to negative surface charges, correlated with increased enzyme stability. Accordingly, these results more broadly provide insight into the mechanism of stabilization in ILs via charge modification.