Cell-free biosensor with automated acoustic liquid handling for rapid and scalable characterization of cellobiohydrolases on microcrystalline cellulose.

Engineering enzymes to degrade solid substrates, such as crystalline cellulose from paper sludge or microplastics in sewage sludge, presents challenges for high-throughput screening (HTS), as solid substrates are not readily accessible in cell-based biosensor systems. To address this challenge, we developed a cell-free cellobiose-detectable biosensor (CB-biosensor) for rapid characterization of cellobiohydrolase (CBH) activity, enabling direct detection of hydrolysis products without cellular constraints. The CB-biosensor demonstrates higher sensitivity than conventional assays and distinguishes between CBH subtypes (CBHI and CBHII) based on their modes of action. Integration with the Echo 525 liquid handler enables precise and reproducible sample processing, with fluorescence signals from automated preparations comparable to manual experiments. Furthermore, assay volumes can be reduced to just a few microlitres-impractical with manual methods. This cell-free CB-biosensor with Echo 525 minimizes reagent consumption, accelerates testing, and facilitates reliable large-scale screening. These findings highlight its potential to overcome current HTS limitations, advancing enzyme screening and accelerating the Design-Build-Test-Learn cycle for sustainable biomanufacturing.