How to Engineer Organic Solvent Resistant Enzymes: Insights from Combined Molecular Dynamics and Directed Evolution Study

Abstract

Expanding synthetic capabilities to routinely employ enzymes in organic solvents (OSs) is a dream for protein engineers and synthetic chemists. Despite significant advances in the field of protein engineering, general and transferable design principles to improve the OS resistance of enzymes are poorly understood. Herein, we report a combined computational and directed evolution study of Bacillus subtlis lipase A (BSLA) in three OSs (i. e., 1,4‐dioxane, dimethyl sulfoxide, 2,2,2‐trifluoroethanol) to devise a rational strategy to guide engineering OS resistant enzymes. Molecular dynamics simulations showed that OSs reduce BSLA activity and resistance in OSs by (i) stripping off essential water molecules from the BLSA surface mainly through H‐bonds binding; and (ii) penetrating the substrate binding cleft leading to inhibition and conformational change. Interestingly, integration of computational results with “BSLA‐SSM” variant library (3439 variants; all natural diversity with amino acid exchange) revealed two complementary rational design strategies: (i) surface charge engineering, and (ii) substrate binding cleft engineering. These strategies are most likely applicable to stabilize other lipases and enzymes and assist experimentalists to design organic solvent resistant enzymes with reduced time and screening effort in lab experiments.

CompassR Yields Highly Organic‐Solvent‐Tolerant Enzymes through Recombination of Compatible Substitutions

“…Additionally, there were no differences in terms of solvation performance in the substrate binding site (see more details in Figures S13–S15 and Table S6 in the Supporting Information). All results confirmed that the hydration shell played a critical role in influencing enzyme activity and resistance in organic (co‐)solvent environments [11b, 15c] …”

“…A similar definition to that of the hydration level is also used for the organic‐solvent layer. A 6.8 Å cutoff was employed for DOX, as in a previous study [11b] . Number of water (d) or OS (e) molecules around the substituted residue positions in water and DOX.…”

“…reported that, in comparison with WT monooxygenase P450 BM‐3, variant T235A/R471A/S1024T, obtained from iterative saturation mutagenesis, achieved a 7.9‐fold increase in specific activity in the presence of 2 % (v/v) THF [9] . Many successful enzyme engineering studies were reported to improve OS resistance (e.g., on Candida antarctica lipase B evolution, [10] BSLA‐SSM, [3a, 11] esterase, [12] subtilisin, [13] and protease [14] ) and several protein engineering principles have been proposed, regarding their structural features (e.g., surface region, [11b, 15] access tunnel, [16] binding cleft, [11b, 17] disulfide bridges, [18] and hydrophobic core [16, 18b] ). Multiple recombinations will deepen our understanding of OS resistance and enable general design principles to be systematically studied [11b] .…”

Less Unfavorable Salt Bridges on the Enzyme Surface Result in More Organic Cosolvent Resistance

“…Indeed, very few reports have been conducted looking at the effect of naturally occurring substitutions on OS resistance of enzymes, but Bacillus subtilits Lipase A (BSLA) is a rare exception [9b, 34, 35] . A site saturation library (BSLA‐SSM) was constructed in our previous study and screened for variants with improved resistance toward three water‐miscible OSs (1,4‐dioxane (DOX), DMSO, 2,2,2‐trifluoroethanol (TFE)) [9b, 34–36] .…”

“…Indeed, very few reports have been conducted looking at the effect of naturally occurring substitutions on OS resistance of enzymes, but Bacillus subtilits Lipase A (BSLA) is a rare exception [9b, 34, 35] . A site saturation library (BSLA‐SSM) was constructed in our previous study and screened for variants with improved resistance toward three water‐miscible OSs (1,4‐dioxane (DOX), DMSO, 2,2,2‐trifluoroethanol (TFE)) [9b, 34–36] . This library covers the full natural diversity at each of 181 amino acid positions consisting in total of 3440 BSLA variants (19×181 + wild‐type(WT)=3440) and is therefore well‐suited to study a putative correlation between salt bridges and OS resistance, thus opening the way to guide salt bridge engineering for obtaining OS and thermal resistance.…”