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 (∼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.

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However,n ative enzymes often have lowa ctivity and resistance in OSs and at elevated temperatures.H erein, we report as mart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme,B acillus subtilits Lipase A( BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems.I terative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance towardt hree OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and halflife up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 8 8C. The salt bridge redesign provides protein engineers with ap owerful and likely general approach to design OSsand/or thermal-resistant lipases and other a/b-hydrolases.

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However,n ative enzymes often have lowa ctivity and resistance in OSs and at elevated temperatures.H erein, we report as mart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme,B acillus subtilits Lipase A( BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems.I terative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance towardt hree OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and halflife up to 3.3-fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50-100 8 8C. The salt bridge redesign provides protein engineers with ap owerful and likely general approach to design OSsand/or thermal-resistant lipases and other a/b-hydrolases.

Application of ionic liquids (ILs) as media in biocatalysis has enormous potential for synthesizing valuable compounds and bulk products in pharmaceuticals and bioenergy due to their unique solvent properties such as volatility, flammability, and solubility. However, ILs as reaction media are often limited by poor enzymatic activity and stability in ILs. We printed a comprehensive IL−enzyme interaction map by studying 45 molecular observables of 30 lipase A from Bacillus subtilis (BSLA) variants in four ILs and a substitutional landscape with 1504 BSLA variants. The results demonstrated that the enzyme hydration shell is the deciding and independent factor determining the enzyme's IL resistance. A universal positive correlation (up to R 2 = 0.96 in 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM][TfO]) and R 2 = 0.85 in 1-butyl-3methylimidazolium chloride ([BMIM]Cl)) was verified, and an experimentally derived ranking of amino acid substitutions is summarized in a list to provide benefits for better protein engineering practice. Hydration-guided engineering yielded a supremely tolerant BSLA variant I12R/D34K/A132K with 8.1-fold, 8.6-fold, 6.6-fold, and 4.6-fold improved tolerance toward [BMIM]Cl, [BMIM]Br, [BMIM]I, and [BMIM][TfO], respectively, when compared to the wild-type BSLA. The obtained knowledge provides a lesson learned on forecasting enzyme stability in ILs and simplifies a rational design of the IL-tolerant enzymes.