Abstract
Gut digestion of gluten releases immunogenic peptides. Among the digestion products, a proline and glutamine-rich 33-mer peptide from wheat α-gliadin triggers celiac disease. Despite multiple proteases from different families being investigated for degradation of the peptide, no FDA-approved protease treatment towards celiac disease is available to date. Here we identified three naturally occurring proteases from the aspartic, cysteine, and serine protease family as model enzymes for directed evolution campaigns: pepsin from human, EP-B2 from barley, and POPB from G. marginata. We expressed and purified these recombinant model enzymes in E. coli strains, determined their respective activity towards a fluorogenic model substrate resembling the 33-mer immunogenic epitope, designed a droplet microfluidics screening workflow, and prepared mutagenic libraries for high-throughput droplet fluorescence sorting. We aim to develop a gluten-detoxifying protease through directed evolution that is active at gastric pH and capable of degrading gluten immunogenic peptides.