High-throughput mass-spectrometric enzyme activity determination with PECAN and its application to the directed evolution of cytochrome P450s

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst, biomarker and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations. On the other hand, more universal enzyme characterization methods usually require chromatography, greatly diminishing throughput. We have developed a novel enzyme activity assay termed “Probing Enzymes with ‘Click’-Augmented NIMS” (PECAN), which allows for the high-throughput mass-spectrometric detection of enzyme activity in crude cell lysate or whole cell culture, without the need for a chromatographic step. Here, we demonstrate the applicability of this technology to the rapid determination of acetyltransferase and cytochrome P450 activity in both lysate and whole cells. We successfully apply PECAN to a directed evolution campaign in which we improve the ability of a cytochrome P450 to perform the notoriously challenging oxidation of the sequiterpene valencene into the flavoring compound and bug-repellent nootkatone, with high product specificity.