A Novel Ginsenoside-Transforming α-L-Rhamnosidase from Bifidobacterium: Screening, Characterization and Application

Despite the rapid advancement of glycosidase biotechnology, ginsenoside-transforming rhamnosidases remain underexplored due to a lack of research. In this study, we aimed to bridge this gap by evaluating eight putative rhamnosidases for their ability to transform ginsenosides. Among them, a novel rhamnosidase (C118) from Bifidobacterium was identified as being efficient at hydrolyzing ginsenoside Re. This enzyme was expressed well in Escherichia coli and exhibited optimal activity at pH of 6.0 and 45 °C. Protein structural predictions revealed that the potential active hydrophobic area near an active pocket may influence the ginsenoside-transforming activities compared to non-active screened rhamnosidases. This enzyme's thermal stability exceeded that of the only previously known ginsenoside-transforming rhamnosidase, BD890. Additionally, the k_cat/K_m value of C118 was 1.45 times higher than that of BD890. Using recombinant C118 from E. coli, all ginsenoside Re in a PPT-type ginsenoside mixture (2.25 mg/mL) was converted after 12 h of reaction. To the best of our knowledge, this is the most efficient ginsenoside Re-transforming α-L-rhamnosidase reported to date, enhancing our understanding of rhamnosidase-substrate interactions and potentially improving the efficiency and specificity of the conversion process. These findings offer promising implications for the production of pharmacologically active ginsenosides in the pharmaceutical, cosmetic, and functional food industries.