Lessons learned on how to reengineer enzymes for improved performance in ionic liquids: insights from BSLA saturation mutagenesis library

Abstract

Ionic liquids (ILs) are attractive reaction media in biocatalysis due to their excellent substrate solubilization properties and their promotion of mild and often environmentally friendly reaction conditions. However, enzyme activity is typically reduced at IL concentrations above 10%. In recent decades, continuous efforts in enzyme engineering have aimed to improve enzyme resistance to ILs, yet achieving robust enzymes remains challenging. This review summarizes research efforts over the past decades aimed at improving IL resistance of enzymes, spanning mechanistic insights and engineering strategies. Analyses of enzyme–IL interactions revealed that the primary effect of ILs is the stripping of water molecules from the enzyme surface. Subsequently, a comprehensive site-saturation mutagenesis (SSM) library of Bacillus subtilis lipase A (BSLA), covering all 181 positions, provided a systematic basis for understanding IL tolerance. Screening this library in the presence of four [BMIM]-based ILs ([BMIM]Cl, [BMIM]Br, [BMIM]I, and [BMIM][TfO]) revealed a general engineering principle: the hydration shell of enzymes is a key determinant of IL resistance. Finally, strategies to identify functional positions associated with improved IL resistance and to efficiently recombine beneficial substitutions are discussed. These engineering approaches minimize experimental effort while maximizing enzyme performance in ILs, providing a powerful and broadly applicable framework for the future design of IL-tolerant enzymes.