Journal article
A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts
Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high-throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput.
We present an enzyme activity assay that allows the high-throughput mass-spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click-assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria.
Using this approach, a cytochrome P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.
Language: | English |
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Year: | 2019 |
Pages: | 10114-10119 |
ISSN: | 15213773 and 14337851 |
Types: | Journal article |
DOI: | 10.1002/anie.201901782 |
ORCIDs: | 0000-0002-5203-4662 , 0000-0001-8263-9198 , 0000-0001-8404-3259 and 0000-0003-4170-6088 |