(599u) Effect of Site-Specific Mutations on Solvent-Induced Inactivation of Lipase in Ionic Liquids: Towards the Rational Design of Ionic Liquid Tolerant Enzymes

Authors: 
Kaar, J. L., University of Colorado Boulder
Nordwald, E., University of Colorado
Armstron, G., University of Colorado

Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ionic liquids for biocatalysis. One strategy to improve the functionality of enzymes in these environments is to mitigate denaturing interactions with the enzyme caused by either the anion or the cation of ILs. The anion in particular, which may compete for hydrogen bonding with the enzyme backbone, may be repelled electrostatically by  negatively charged amino acids. Also, denaturing interactions between enzymes and IL cations, which can disrupt hydrophobic packing interactions, may be mitigated with polar and charged amino acids. As such, we examined sites where ILs induce changes in local chemical environment of lipase A from Bacillus subtilis using 2D HSQC NMR. Site-directed mutagenesis of surface residues near the identified sites led to a reduction in local perturbations as well as an increase in activity retention ( i.e., half-life) in buffer in the presence of the IL [BMIM][Cl] . Additionally, highly exposed positively charged residues were also mutated to reduce potential anion interactions. Overall, the combination of beneficial single mutations allowed improvement of the half-life of lipase by greater than ~7fold. We propose that this approach may be general and thus applied to increase the tolerance of other enzymes to ILs.