Enzyme Hydration Determines Resistance In Organic Cosolvents

Biocatalysis in organic solvents (OSs) has found various important applications, particularly in organic synthesis and for the production of pharmaceuticals, flavors, and fragrances. However, the use of enzymes in OSs often results in enzyme deactivation or a dramatic drop in catalytic activity. Herein, we have developed a comprehensive understanding of the interactions between enzymes and OSs based on numerous observables obtained from molecular dynamics simulation of 32 variants of Bacillus subtilis lipase A (BSLA). We have tested the wild-type enzymes and variants carrying single and multiple substitutions toward the organic cosolvent 2,2,2-trifluoroethanol (TFE, 12% (v/v)). After analyzing the distribution of 35 structural and dynamic observables, we uncovered that increased enzyme surface hydration of substituted sites is the predominant factor to drive the improved resistance in OS. The iterative recombination of four surface substitutions revealed that the extent of hydration in BSLA variants correlates strongly with its OS resistance (R-2 = 0.91). Remarkably, the substitutions recombination led to a highly resistant BSLA variant (I12R/M137H/N166E) with a 7.8-fold improved resistance in 12% (v/v) TFE, while retaining comparable catalytic activity (similar to 92%) compared to the wild-type enzyme. Our findings prove that strengthening protein surface hydration via surface charge engineering is an effective and efficient rational strategy for tailoring enzyme stability in OSs.