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.
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).
Henke
E
E
Institute for Technical Biochemistry, University of Stuttgart, Germany.
Bornscheuer
U T
UT
D019020:Directed Molecular Evolution; D004950:Esterases / Q000235:genetics*; D016296:Mutagenesis; D010078:Oxazines / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D010078:Oxazines; C014180:resorufin; D004950:Esterases