CompassR-guided recombination unlocks design principles to stabilize a lipase in ILs with minimal experimental efforts

Biocatalysis in ionic liquids (ILs) has gained enormous attention for the production of biodiesel, sugar esters, and pharmaceuticals. However, hydrophilic IL interaction with enzymes often results in reduced activity or even inactivation. In this report, we prove that intrinsic lipase stability and preservation of hydration shells of Bacillus subtilis lipase A (BSLA) are two synergistic design principles to retain enzymatic activity in ILs. After in silico screening of nine beneficial amino acid positions by the CompassR rule (in total, 172 variants), we rationally designed two variants, to be constructed by site-directed mutagenesis, and three libraries by site-saturation mutagenesis. With minimal experiment effort, we identified three all-around variants towards four [BMIM]-based ILs. Remarkably, the variant M1a F17S/V54K/D64N/D91E/G155N had 6.7-fold higher resistance against 40% (v/v) [BMIM]Cl, 5.6-fold in 80% (v/v) [BMIM]Br, 5.0-fold in 30% (v/v) [BMIM][TfO], and 2.7-fold in 10% (v/v) [BMIM]I compared to wild-type BSLA, respectively, while showing 1.9-fold improvement in specific activity. Computational analysis of molecular dynamics and thermodynamic stability analysis of the variants revealed the molecular basis for the resistant variants M1a and M1b as the synergistic enhancement of protein stability (ΔΔGfold ranging from −4.26 to −4.80 kcal mol−1) and increased hydration shells around the substitutions in the four ILs (up to 1.7-fold). These design principles and the gained molecular knowledge not only open the door to direct experimentalists for rationally designing promising IL-resistant enzymes, but also provide new insights into enzymatic catalysis in ILs.