376297 Effect of Site-Specific Mutations on Solvent-Induced Inactivation of Lipase in Ionic Liquids: Towards the Rational Design of Ionic Liquid Tolerant Enzymes

Wednesday, November 19, 2014
Galleria Exhibit Hall (Hilton Atlanta)
Erik Nordwald, Geoffrey Armstron and Joel L. Kaar, Department of Chemical & Biological Engineering, University of Colorado, Boulder, CO

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.

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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division