A High-ThroughputCell-Free Enzyme Screening SystemUsing Redox-Responsive Hydrogel Beads as Artificial Compartments

We have developed a rapid, simple, and high-throughput screening system for recombinant enzymes using disulfide-bonded hydrogel beads (HBs) produced via a microfluidic method. These redox-responsive HBs were compatible with the biosynthesis of enzyme mutants via cell-free protein synthesis, fluorescent staining through an enzymatic reaction, and genetic information recovery after fluorescence-activated droplet sorting (FADS). The expression of microbial transglutaminase zymogen (MTGz) using cell-free protein synthesis and the cross-linking-reactivity-based staining of HBs with a fluorescent product were validated. Next-generation sequencing (NGS) analysis of the genes recovered from highly fluorescent HBs identified novel mutation sites (N25 and N27) in the propeptide domain. The introduction of these mutations allowed for the design of an engineered active MTGz, demonstrating the potential of HBs as artificial compartments for the FADS-based selection of enzymes that catalyze peptide and protein cross-linking reactions.