Analytical Chemistry

Rapid Screening of Diverse Biotransformations for Enzyme Evolution

Abstract

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

Version notes

version one

Content

Thumbnail image of Kempa_Galman_Rapid_Screen_Sub.pdf

Supplementary material

Thumbnail image of Kempa_Galman_Supplementary dataset S1.pdf
Kempa Galman Supplementary dataset S1
Thumbnail image of Kempa_Galman_Supplementary dataset S2.pdf
Kempa Galman Supplementary dataset S2
Thumbnail image of Kempa_Galman_Rapid_Screen_Sub_SI.pdf
Kempa Galman Rapid Screen Sub SI