Stabilization of substilisin E in organic solvents by site-directed mutagenesis

Abstract

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 → Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface‐charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 → Asn + Asn218 → Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.

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