Tools for Directed Evolution of Oxygenases: High Throughput Screening of Epoxidation and Hydroxylation Catalysts

California Institute Of Technology, Pasadena CA

Investigators

Abstract

Chemical oxidations are sloppy processes on an industrial scale, and biological oxidations have been hampered by poor reactivity of enzymes and the need for expensive cofactors. Improvement of these enzymes by directed evolution is likely, but require rapid and sensitive assays. The goal of this project is to develop new technology for the rapid screening of oxygenase libraries. The digital imaging combined with fluorescence-activated cell sorting will be used to screen large collections of mutations for the appropriate activity. Horseradish peroxidase will be coexpressed with the oxygenase to create fluorescent tags from the hydroxylated aromatics formed by the oxygenase. By rapid screening of the large collections, the rare improvement can be detected and further upgraded.