Construction of a high-throughput cell-free system for screening thermostable laccase with redox-responsive hydrogel...

Thermostable enzymes are highly desirable for industrial applications. Conventional thermostability screening typically relies on microplate assays; however, this approach becomes impractical as the library size increases exponentially with the number of mutated sites, resulting in excessive reagent use and high costs. Small droplet-based screening systems have recently emerged as an attractive alternative, offering reduced reagent consumption and scalability. However, constructing a heat-compatible screening system requires careful optimization of the reaction conditions under elevated temperatures. In this study, we applied our recently developed high-throughput screening system, which integrates hydrogel beads with cell-free protein synthesis (CFPS), to the selection of thermostable enzymes. Using two laccases with distinct thermostabilities, we showed that the hydrogel bead-based high-throughput screening system reliably discriminates the enzymes based on their thermal stability. The results highlight the potential of this platform as a cost-effective and scalable CFPS-based method for the directed evolution of thermostable enzymes.