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

✍ Scribed by Henke, E.; Bornscheuer, U. T.

• Book ID: 120571137
• Publisher: Walter de Gruyter GmbH & Co. KG
• Year: 1999
• Tongue: English
• Weight: 165 KB
• Volume: 380
• Category: Article
• ISSN: 1431-6730
• DOI: 10.1515/BC.1999.128

✦ Synopsis

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).