Natural Diversity to Guide Focused Directed Evolution

@article{Jochens2010NaturalDT,
  title={Natural Diversity to Guide Focused Directed Evolution},
  author={Helge Jochens and Uwe T. Bornscheuer},
  journal={ChemBioChem},
  year={2010},
  volume={11}
}
Simultaneous multiple site‐saturation mutagenesis was performed at four active‐site positions of an esterase from Pseudomonas fluorescens to improve its ability to convert 3‐phenylbutyric acid esters (3‐PBA) in an enantioselective manner. Based on an appropriate codon choice derived from a structural alignment of 1751 sequences of α/β‐hydrolase fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivalent positions. Thus, the… Expand
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A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the buildingExpand
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To increase the enantioselectivity of a Pseudomonas fluorescens esterase toward methyl 3-bromo-2-methylpropionate, mutagenesis into the substrate binding site was focused at Trp28, Val121, Phe198, and Val225, and five of the catalytically active mutants showed better enantiosity than wild-type PFE. Expand
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Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri to show increased thermostability with no loss of catalytic function at room temperature. Expand
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