Directed evolution of an esterase from Psueudomonas fluorescens.: Random mutagenesis by error-prone PCR or a mutator strain and identification of mutants showing enhanced enantioselectivity by a resorufin-based fluorescence assay

The gene encoding an esterase from Pseudomonas fluorescens (PFE) was subjected to random mutagenesis by error- prone PCR or by using the mutator strain Epicurian coli XL1-Red. Enzyme libraries were then created in microtiter plates by expression of PFE-variants in E. coli. These were assayed for improved enantioselectivity in a Beckman robot system using optically pure (R)- or (S)-3-phenylbutyric acid resorufin esters, resulting in the identification of several mutants showing up to almost two-fold enantioselectivity (E-true = 5.2 to 6.6) compared to wild-type PFE (E-true = 3.5).