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

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co-)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6-fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137-fold, and half-life 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 degrees C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs- and/or thermal-resistant lipases and other alpha/beta-hydrolases.

Automated summary

A smart salt bridge design strategy was used to improve the organic solvent resistance and thermostability of Bacillus subtilits Lipase A (BSLA), resulting in variants with significantly enhanced resistance towards organic solvents and improved thermal stability. Molecular dynamics simulations showed that locally refined flexibility and strengthened hydration played key roles in the increased resistance in organic solvents and at elevated temperatures. This salt bridge redesign approach could be a powerful tool for protein engineers to design lipases and other alpha/beta-hydrolases with enhanced resistance to organic solvents and/or thermal stability.