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

化学氧化在工业规模上是草率的过程，而生物氧化由于酶的反应性差和需要昂贵的辅因子而受到阻碍。 通过定向进化来改进这些酶是可能的，但需要快速和灵敏的测定。 本项目的目标是开发新的技术来快速筛选加氧酶文库。 数字成像与荧光激活细胞分选相结合将用于筛选大量突变的适当活性。 辣根过氧化物酶将与加氧酶共表达，以从由加氧酶形成的羟基化芳族化合物产生荧光标记。 通过快速筛选大集合，可以检测到罕见的改进并进一步升级。

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.