Jon D. Stewart, Department of Chemistry, University of Florida, 127 Chemistry Research Building, Gainesville, FL 32611
Recent work from our laboratory has focused on ways to utilize proteins discovered in genome sequencing projects for asymmetric synthesis. Beyond our desire to expand the biocatalytic toolbox, we also aimed to develop methods that allow us to discover the most suitable enzyme catalyst(s) for specific reactions rapidly, then use the same enzymes in whole cells for preparative-scale reactions. These points will be illustrated by our recent work in asymmetric alkene reductions. We have identified a number of candidates by computational genome analysis, then overproduced the most promising enzymes as fusions with glutathione S-transferase to facilitate their isolations. Individual proteins were used for rapid screening, then whole Escherichia coli cells expressing the same proteins were employed for preparative-scale reactions. These points will be illustrated by some synthetic examples, including substituted 2-cyclohexenones and β2-amino acids. We have also used both rational and random mutagenesis strategies to improve the substrate- and stereoselectivities of these enzymes, and examples of these studies will also be discussed.
