Site-directed mutagenesis of α-L-rhamnosidase boosts its deglycosylation activity for the production of bioactive flavonoid glycosides.

Abstract

α-L-Rhamnosidases are valuable biocatalysts for the synthesis of bioactive flavonoids, however, their industrial application is often restricted by limited catalytic efficiency and suboptimal thermal stability. This study reports the rational engineering of an α-L-rhamnosidase (DthRha) to address these limitations and enhance its performance for flavonoid production. A library of mutants was constructed via substrate-binding pocket modeling and site-directed mutagenesis. Among them, the R783A variant exhibited a 3.56-fold increase in enzymatic activity relative to the wild-type enzyme (WT), along with remarkable thermal stability, retaining over 92% of its initial activity after 2 h incubation at 60-90 °C， whereas the WT rapidly lost activity above 60 °C. Kinetic assays revealed a 1.46-fold increase in kcat/Km over the WT, coupled with enhanced substrate specificity. Molecular docking and MD simulations suggested that the enhanced catalytic performance of R783A may arise from favorable steric conformation and a more accessible catalytic tunnel. The practical applicability of the R783A was demonstrated at a 50 mL laboratory scale, affording prunin and isoquercitrin in >98% yield with space-time yields of 3.58 and 1.92 g/L/h, respectively. These findings highlight the R783A mutant as a robust and thermally stable biocatalyst with great potential for the sustainable production of bioactive flavonoids.