BODIPY-Glucosinolates for Rapid, High-throughput Screening Compatible Assays of Myrosinase

The pursuit of high-performance myrosinase (MYR) systems for efficient production of bioactive isothiocyanates (ITCs) requires advanced enzymatic activity assays. Herein, we developed two dipyrromethene boron difluoride (BODIPY)-based fluorogenic probes for rapid and reliable monitoring of MYR activity. Upon hydrolysis by MYR, the probe BDP-GSL2 undergoes a specific cascade reaction involving glucosinolate (GSL) cleavage, Lossen rearrangement, and intermolecular cyclization, leading to a 110-fold fluorescence enhancement within 10 min at 37 °C or within 90 s at 50 °C. By BDP-GSL2, we demonstrated that Rmyr exhibited the highest catalytic efficiency among all tested microbial MYRs, both in vitro and in vivo. Furthermore, we established a high-throughput screening (HTS)-compatible, fluorescence-based method for rapid profiling of <i>Pichia pastoris</i> libraries expressing recombinant Rmyr. The ranking of top-performing clones from the HTS-compatible approach correlated well with results from glucose-based assays, validating the reliability of our method for identifying high-expressing clones.