NMR-Guided Rational Engineering of an Ionic-Liquid-Tolerant Lipase

Biocatalysis in ionic liquids (ILs) is largely limited by the instabilities of enzymes in these solvents, thus negating their auspicious solvent properties. Here, we have engineered an IL-tolerant variant of lipase A (lipA) from Bacillus subtilis by examining the site-specific interactions of lipA with 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Results of NMR analysis found that [BMIM][Cl] induced structural perturbations near the active site of lipA, underscoring the importance of mediating direct ion interactions with the IL in this region. Mutation of G158 near the active site to glutamic acid resulted in a 2.5-fold improvement in tolerance of lipA to 2.9 M (or 50% v/v) [BMIM][Cl], which correlated with the retention of active site structure. The effect of the G158E mutation was likely the result of inhibition of hydrophobic interactions with the [BMIM] cation. Further analysis of the electrostatic surface of lipA led to the mutation of K44 to glutamic acid to diminish the attraction of chloride anions, which also improved lipA tolerance to [BMIM][Cl]. Beneficial point mutations, which had an additive effect on lipA stability, were combined, resulting in a super stable lipA quadruple mutant (G158E/K44E/R57E/Y49E) with a 7-fold improvement in stability. In comparison, nonspecific charge modification via acetylation and succinylation resulted in only a 1.2- and 1.9-fold improvement in stability, respectively, over wild-type lipA. Ultimately, these results, while providing insight into the nature of the structural effects of ILs on enzymes, highlight the utility of combining NMR and charge engineering to rationally optimize enzyme stability for biocatalysis in ILs.