Pichia stipitis OYE 2.6 variants with improved catalytic efficiencies from site-saturation mutagenesis libraries.

ABSTRACT

An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes. Several substrates were sterically demanding, and as hoped, creating additional active site volume yielded better conversions for these alkenes. The most impressive improvement was found for 2-butylidenecyclohexanone. The wild-type provided less than 20% conversion after 24h; a triple mutant afforded more than 60% conversion in the same time period. Moreover, even wild-type OYE 2.6 can reduce cyclohexenones with very bulky 4-substituents efficiently.