Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block

A triple mutant of on esterose from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enontioselectivity (E = 89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high electivity Back mutations gave a variant (double mutant PFE IIe76Val/ Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).