To improve the applicability of halohydrin dehalogenase as a catalyst for reactions in the presence of organic cosolvents, we explored a computational library design strategy (Framework for Rapid Enzyme Stabilization by Computational libraries) that involves discovery and in silico evaluation of stabilizing mutations. Energy calculations, disulfide bond predictions and molecular dynamics simulations identified 218 point mutations and 35 disulfide bonds with predicted stabilizing effects. Experiments confirmed 29 stabilizing point mutations, most of which were located in two distinct regions, whereas introduction of disulfide bonds was not effective. Combining the best mutations resulted in a 12-fold mutant (HheC-H12) with a 28°C higher apparent melting temperature and a remarkable increase in resistance to cosolvents. This variant also showed a higher optimum temperature for catalysis while activity at low temperature was preserved. Mutant H12 was used as a template for the introduction of mutations that enhance enantioselectivity or activity. Crystal structures showed that the structural changes in the H12 mutant mostly agreed with the computational predictions and that the enhanced stability was mainly due to mutations that redistributed surface charges and improved interactions between subunits, the latter including better interactions of water molecules at the subunit interfaces.

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso ‐epoxides. Three substrates of different sizes were targeted: cis ‐2,3‐butene oxide, cyclopentene oxide, and cis ‐stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis ‐stilbene oxide, and enantiocomplementary mutants produced ( S , S )‐ and ( R , R )‐stilbene diol with >97 % enantiomeric excess. An ( R , R )‐selective mutant was used to prepare ( R , R )‐stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee ). Some variants displayed higher catalytic rates ( k cat ) than the original enzyme, but in most cases K M values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening.