Rapid activity-based colorimetric CoFi blot for screening of engineered FAD-dependent oxidoreductases

The rapid expansion of protein variant libraries, facilitated by advanced computational design, has created an urgent need for efficient functional validation, as experimental screening of catalytic activity remains a major bottleneck compared to binding-based assays. Conventional high-throughput methods often rely on tedious purification or activity-blind antibody detection, which fail to assess actual enzymatic performance. Here, we report an ultra-fast, tag-free, activity-based colorimetric colony filtration (CoFi) blot to bridge the gap between sequence exploration and functional validation. Unlike traditional expression-based protocols, our system visualizes enzymatic turnover within minutes via a 2,6-dichlorophenolindophenol (DCIP)-based redox coupling reaction directly on a membrane. Using FAD-dependent glucose dehydrogenase (FAD-GDH)—a gold-standard enzyme for continuous glucose monitoring—as a model system, we demonstrated simultaneous assessment of solubility and activity directly from E. coli colonies. Our platform successfully identified high-activity variants from engineered libraries, with the lead variant exhibiting a 16-fold increase in decolorization halo area compared to the wild-type. This approach provides a robust, cost-effective tool for the rapid functional validation of a wide range of oxidoreductases capable of DCIP reduction, regardless of the specific design methodology. • A novel activity-based colorimetric CoFi blot was developed for the screening of engineered FAD-dependent oxidoreductases. • The system enables tag-free, in situ detection of catalytic activity within minutes. • DCIP-mediated decolorization facilitates rapid visual identification of high-performance variants. • A redesigned FAD-GDH lead variant was identified with a 16-fold increase in halo area. • The platform bridges the gap between computational design and functional enzyme validation.