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

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 24 h; 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.

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