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

Abstract 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 (Etrue = 5.2 to 6.6) compared to wild-type PFE (Etrue = 3.5).

[1]  H. Sobek,et al.  The metal-ion-free oxidoreductase from Streptomyces aureofaciens has an α/β hydrolase fold , 1994, Nature Structural Biology.

[2]  K. Choi,et al.  Cloning and nucleotide sequence of an esterase gene from Pseudomonas fluorescens and expression of the gene in Escherichia coli. , 1990, Agricultural and Biological Chemistry.

[3]  T. M. Stokes,et al.  Enzyme reactions in apolar solvents: the resolution of (±)-sulcatol with porcine pancreatic lipase. , 1987 .

[4]  QUANTITATIVE ANALYSES OF BIOCHEMICAL KINETIC RESOLUTIONS OF ENANTIOMERS , 1983 .

[5]  D. Goeddel,et al.  A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction , 1989 .

[6]  Manfred T. Reetz,et al.  Creation of Enantioselective Biocatalysts for Organic Chemistry by In Vitro Evolution , 1997 .

[7]  I. Pelletier,et al.  A bacterial esterase is homologous with non-haem haloperoxidases and displays brominating activity. , 1995, Microbiology.

[8]  Frances H. Arnold,et al.  Design by Directed Evolution. , 1998 .

[9]  U. Bornscheuer,et al.  Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens , 1998 .

[10]  T. Yamane,et al.  Purification and Properties of a Thermostable Esterase of Bacillus stearothermophilus Produced by Recombinant Bacillus brevis. , 1992, Bioscience, biotechnology, and biochemistry.

[11]  U. Bornscheuer,et al.  Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay. , 1999, Bioorganic & medicinal chemistry.

[12]  W. Stemmer.  DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  T. Ema,et al.  Enhancement of the Enantioselectivity in Lipase-Catalyzed Kinetic Resolutions of 3-Phenyl-2H-azirine-2-methanol by Lowering the Temperature to −40 °C , 1997 .

[14]  R D Schmid,et al.  Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols. , 1998, Protein engineering.

[15]  M. Reetz,et al.  Superior Biocatalysts by Directed Evolution , 1999 .

[16]  U. Bornscheuer,et al.  Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones. , 1998, Biotechnology and bioengineering.