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A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts.
de Rond, Tristan; Gao, Jian; Zargar, Amin; de Raad, Markus; Cunha, Jack; Northen, Trent R; Keasling, Jay D.
Afiliación
  • de Rond T; College of Chemistry, University of California, Berkeley, Berkeley, CA, 94270, USA.
  • Gao J; Joint Bioenergy Institute (JBEI), Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, USA.
  • Zargar A; Current address: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92037, USA.
  • de Raad M; Department of Energy Joint Genome Institute (DOE JGI), Lawrence Berkeley National Laboratory, USA.
  • Cunha J; Joint Bioenergy Institute (JBEI), Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, USA.
  • Northen TR; Department of Energy Joint Genome Institute (DOE JGI), Lawrence Berkeley National Laboratory, USA.
  • Keasling JD; Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory, USA.
Angew Chem Int Ed Engl ; 58(30): 10114-10119, 2019 07 22.
Article en En | MEDLINE | ID: mdl-31140688
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bioensayo / Sistema Enzimático del Citocromo P-450 / Ensayos Analíticos de Alto Rendimiento Idioma: En Revista: Angew Chem Int Ed Engl Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Alemania
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bioensayo / Sistema Enzimático del Citocromo P-450 / Ensayos Analíticos de Alto Rendimiento Idioma: En Revista: Angew Chem Int Ed Engl Año: 2019 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Alemania