Establishment of High-Throughput Screening Protocol Based on Isomerase Using sp. L-Rhamnose Isomerase.

Directed evolution is a powerful tool in protein engineering that generates diverse variant libraries to enhance enzyme functionalities. However, the identification of desirable variants from large mutant libraries requires an efficient high-throughput screening (HTS) technique. In this study, we established a robust HTS protocol for selecting high-activity isomerase variants, specifically using L-rhamnose isomerase (L-RI) consistently; avoid switching between L-RI and L-RhI. L-RI catalyzes the isomerization of D-allulose to D-allose, which allows for activity detection via the reduction of the ketose D-allulose in a colorimetric assay based on Seliwanoff's reaction. Initial optimization was conducted in a single-tube format, where reaction conditions were refined and interfering factors were minimized. This optimized single-tube protocol demonstrated excellent accuracy when validated against high-performance liquid chromatography measurements, confirming its ability to precisely quantify D-allulose depletion. Subsequently, the protocol was successfully adapted to a 96-well plate format, incorporating further optimizations for protein expression and the removal of denatured enzymes. This involved methods such as cell harvest, supernatant removal, and filtration to reduce assay interference. The analytical quality of the established HTS protocol was evaluated using statistical metrics. The results yielded a Z'-factor of 0.449, a signal window (SW) of 5.288, and an assay variability ratio (AVR) of 0.551. All these values meet the acceptance criteria for high-quality of HTS assay. This HTS protocol is highly reliable and applicable for efficient screening of isomerase activity in various industrial and research areas.