Increased enantioselectivity by engineering bottleneck mutants in an esterase from Pseudomonas fluorescens.

Four hydrophobic and bulky amino acid residues (F126, F144, F159, and I225) were identified to form a bottleneck guarding the entrance to the active site of an esterase from Pseudomonas fluorescens (PFE I). Hence, a range of nonpolar amino acids were introduced into PFE I to broaden the substrate range and to increase enantioselectivity while preserving the hydrophobicity of the tunnel. First, single variants were created and then the most enantioselective ones were combined to find cooperative effects. This resulted in several mutants, which showed substantially enhanced enantioselectivity; for instance, in the kinetic resolution of 1-phenyl-1-propyl acetate, with which the wild type only showed E=1.2, two mutants gave E>46. For 1-phenyl-1-ethyl acetate enantioselectivity increased from approximately 50 to >100 for all mutants studied. Furthermore, higher conversions could be found at shorter reaction times; this indicates that the mutations not only enhanced selectivity, but that also the entrance into the active site was indeed facilitated by these mutations. The experimental results could be explained by computer modeling.