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. In this review, we outline our endeavours over the past decades aimed at improving IL resistance of enzymes. Initially, we conducted systematic research on the interplay between enzymes and ILs, revealing that the primary effect of ILs is to strip water molecules from the enzyme surface. Subsequently, we generated a site saturation mutagenesis (SSM) library of Bacillus subtilis lipase A (BSLA), covering all 181 positions. Based on this library, screening in presence of four [BMIM]-based ILs ([BMIM]Cl, [BMIM]Br,[BMIM]I, and [BMIM][TfO]) revealed the general engineering principle: the hydration shell of enzymes determines their IL resistance. Finally, we designed strategies to identify functional positions for improved IL resistance and to effectively recombine beneficial substitutions. 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.