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

摘要

A triple mutant of on esterase from Pseudomonas fluorescens(PFE)that was created by directed evolution exhibited high enan-tioselectivity(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 selectivity.Back mutations gave a variant(double mutant PFE lle76Val/ Val175Ala)that showed excellent selectivity(E = 96)and activity(20 min for 50% conversion,which corresponds to 1.25 U per mg of protein).