Biocatalysis in organic solvents (OSs) enables more efficient routes to the synthesis of various valuable chemicals. However, OSs often reduce enzymatic activity, which limits the use of enzymes in OSs. Herein, we report a comprehensive understanding of interactions between surface polar substitutions and DMSO by integrating molecular dynamics (MD) simulations of 45 variants from Bacillus subtilis lipase A (BSLA) and substitution landscape into a “BSLA‐SSM” library. By systematically analyzing 39 structural‐, solvation‐, and interaction energy‐based observables, we discovered that hydration shell maintenance, DMSO reduction, and decreased local flexibility simultaneously govern the stability of polar variants in OS. Moreover, the fingerprints of 1631 polar‐related variants in three OSs demonstrated that substituting aromatic to polar amino acid(s) hold great potential to highly improve OSs resistance. Hence, surface polar engineering is a powerful strategy to generate OS‐tolerant lipases and other enzymes, thereby adapting the catalyst to the desired reaction and process with OSs.

Biocatalysis in ionic liquids (ILs) gained substantial interest due to solvent properties of the ILs, such as near-zero vapor pressure, high thermal stability, and wide tunability. Enzymes are often not catalytically active in ILs; therefore, understanding and improving enzyme resistance in ILs are essential to enable efficient biocatalysis in ionic liquids. Surface charge engineering has repeatedly been reported to enhance enzyme resistance toward ILs. However, the molecular knowledge about how substitutions to charged amino acids improve enzyme activity in an IL is far from being understood. Here, we report a comprehensive study to provide some principles of how surface charged amino acid substitutions (negatively and positively) strengthen the IL resistance of the Bacillus subtilis lipase A (BSLA) in [BMIM]Cl. Twenty typical BSLA substitutions (ten beneficial and ten nonbeneficial, obtained from the BSLA-SSM library) were studied by molecular dynamics (MD) simulations in the [BMIM]Cl system. The BSLA-IL interaction patterns were printed by analyzing several structural-and solvation-based observables. Lessons learned by analyzing the SSM library of BSLA comprise the following: (i) A general trend was found where both negatively and positively charged substitutions increased the essential water molecules locally at the substitution site, thereby contributing to the overall protein hydration shell. (ii) Electrostatic repulsion of both IL ions and the refined hydration shell are ultimately the two main drivers to enhanced IL resistance. The analysis of 20 BSLA substitutions and the identified common interactions reveals that surface charge engineering is very likely to be a general protein engineering strategy to enhance lipase/enzyme activity in ILs. Moreover, this study also suggests that MD is a valuable technique to screen for beneficial substitutions that repel/recruit surface solvation.

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