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E. Henke, U. T. Bornscheuer

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).

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 380, 07/1999
Pages: 1029 - 1033

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