Rapid Screening of Diverse Biotransformations for Enzyme Evolution

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification active and selective biocatalysts, with number variants often exceeding capacity traditional analytical platforms to assess their activity practical time scale. Here, we show application direct infusion biotransformations mass spectrometer (DiBT-MS) variety enzymes, different formats, achieving sample throughputs equivalent ∼40 s per sample. heat map output allows rapid selection enzymes within 96-well plates facilitating industrially relevant biocatalysts. This DiBT-MS workflow has been applied directed evolution phenylalanine ammonia lyase (PAL) as case study, enhancing its toward electron-rich cinnamic acid derivatives which are lignocellulosic biomass degradation. Additional benefits platform include discovery biocatalysts (kinases, imine reductases) novel activities incorporation ion mobility technology for product hits increased confidence.