Deep eutectic solvents (DESs) are gaining rapid prominence in numerous biocatalysis processes due to their green characteristics, biodegradability, low cost, and simple preparation compared to harsh organic solvents and toxic ionic liquids. However, natural enzymes tend to show activity reduction, even inactivation in the presence of many DESs. Here, we present the first rational design approach to achieve enzymes resistant to both DESs and high temperatures. Using the interaction pattern between DESs and BSLA (Bacillus subtilis lipase A, our model enzyme) derived from all-atom molecular dynamics (MD) simulations, we formulated a solvation-guided engineering strategy. This was established after assessing 33 structural, solvation, and energy observables. We rationally designed and experimentally tested 36 single substitutions, of which 28 (a 77.78% success rate) exhibited improvements in at least two DES cosolvents: choline chloride (ChCl) : acetamide, tetrabutylphosphonium bromide (TBPB) : ethylene glycol (EG), and ChCl : EG. Additionally, through stepwise recombination, we identified two robust BSLA variants, D64H/R107L/E171Y and D64H/R142L, showing stability improvements of up to 4.4-fold and 3.2-fold in three DESs and at 50 °C, respectively. Further MD studies demonstrated that (i) the restricted overall structure but fine-tuned local flexibility and (ii) increased water but decreased DES molecules at substituted sites are two main factors contributing to the enhanced multiple DES resistance. Overall, solvation-guided engineering offers an efficient and rational approach for designing lipases tolerant to DES cosolvents and elevated temperatures, with a considerable potential for adaptation to other enzymes.
