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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).

biological chemistry

Directed Evolution of an Esterase from  Pseudomonas fluorescens . Random Mutagenesis by Error-Prone     PCR or a Mutator Strain and Identification of Mutants Showing Enhanced     Enantioselectivity by a Resorufin-Based Fluorescence Assay