A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts.
Angew Chem Int Ed Engl
; 58(30): 10114-10119, 2019 07 22.
Article
em En
| MEDLINE
| ID: mdl-31140688
ABSTRACT
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 cytochromeâ
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.
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Base de dados:
MEDLINE
Assunto principal:
Bioensaio
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Sistema Enzimático do Citocromo P-450
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Ensaios de Triagem em Larga Escala
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article