Less unfavorable salt bridges on the enzyme surface result in more organic cosolvent resistance

22/03/2021
  Haiyang "Ocean" Cui Copyright: Bio VI

Cui, H., Eltoukhy, L., Zhang, L., Markel, U., Jaeger, K. E., Davari, M. D., Schwaneberg, U., Angewandte Chemie, doi.org/10.1002/ange.202101642

Removing unfavorable surface salt bridges increased the organic solvent and thermal resistance of enzymes.

The application of organic (co-)solvents (OSs) as reaction media for biocatalysts is mandatory for a large number of applications in the chemical industries. OSs are needed to solubilize hydrophobic substrates and products, they allow for easy product recovery and shift the reaction equilibrium into the desired direction. However, native enzymes often suffer from low activity and/or sensitivity in the presence of OSs, constraining their extended application for biocatalysis. 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°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 α/β‐hydrolases.

Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship. This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0169 and JARA0187).

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Cui, H., Eltoukhy, L., Zhang, L., Markel, U., Jaeger, K. E., Davari, M. D., Schwaneberg, U., Angewandte Chemie International Edition, doi.org/10.1002/ange.202101642

  Rational salt bridge  design strategy: Enzyme surface interacts with organic solvents. Copyright: Angewandte Chemie International Edition By removing unfavorable surface salt bridges, the organic solvent and thermal resistance of enzymes can be significantly improved. This design strategy results in locally refined flexibility and strengthened hydration.