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.
Biol Chem
; 380(7-8): 1029-33, 1999.
Article
em En
| MEDLINE
| ID: mdl-10494857
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).
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pseudomonas fluorescens
/
Evolução Molecular Direcionada
/
Esterases
Tipo de estudo:
Clinical_trials
/
Diagnostic_studies
Idioma:
En
Revista:
Biol Chem
Ano de publicação:
1999
Tipo de documento:
Article