OLD YELLOW ENZYME ENGINEERING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We are applying sstate-of-the-art protein engineering techniques to alkene reductases so that they become first-choice catalysts for preparative organic synthesis.We have chosen iterative saturation mutagenesis (ISM) for most of the protein improvement studies since this methodology requires relatively small libraries (ca. 100 for a given amino acid) and correspondingly simple technology. We are using X-ray crystallography to assess the results of protein engineering studies and guide future efforts. Two relevant structures have been solved with several others nearing completion. Our first choice alkene reductase is Pichia stipitis OYE 2.6; should this prove unsuitable, we are also prepared to use Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis) OYE1.