Selection of Horseradish Peroxidase Variants with Enhanced Enantioselectivity by Yeast Surface Display

SummaryWe report a method for in vitro selection of catalytically active enzymes from large libraries of variants displayed on the surface of the yeast S. cerevisiae. Two libraries, each containing ∼2 × 106 variants of horseradish peroxidase (HRP), were constructed; one involved error-prone PCR that sampled mutations throughout the coding sequence, whereas the other involved complete combinatorial enumeration of five positions near the active site to non-cysteine residues. The enzyme variants displayed on the yeast surface were allowed to modify it with a fluorescently labeled substrate. A combination of positive and negative selection applied to the active-site-directed library resulted in variants with up to an 8-fold altered enantioselectivity, including its reversal, toward l/d-tyrosinol. In contrast, the library constructed by using error-prone PCR yielded no HRP variants with a significantly improved enantioselectivity.