A GH1 β-glucosidase from the Fervidobacterium pennivorans DSM9078 showed extraordinary thermostability and distinctive ability in the efficient transformation of ginsenosides.

Abstract

A novel GH1 β-glucosidase Fpglu1 from Fervidobacterium pennivorans DSM9078 was successfully cloned and expressed in Escherichia coli. This hyperthermophilic enzyme possesses unique features that make it valuable in biochemistry and pharmacology. It exhibited optimal activity at temperatures exceeding 100 °C, a trait rarely observed in other enzymes, and demonstrated extraordinary thermostability. It displayed multifunctional activity, with the highest activity observed for p-nitrophenyl-β-d-glucopyranoside (pNPGlu) at 92.47 U/mg. Furthermore, the distinctive capacity of Fpglu1 to transform ginsenosides (Rb1, Rb2, and Rc) into Compound-K (C-K) sets it apart from the other enzymes. It effectively cleaved the external β-(1-6) glycosidic linkage at the C-20 position of ginsenosides Rb1, Rb2, and Rc, followed by hydrolysis ofthe internal glycosidic bond connected to the C-3 position. The k_cat/K_m value of Fpglu1 for Rb1 was 453 ± 1.27 mM^-1/s, significantly higher than those of Fpglu1 for other ginsenosides. The crystal structure of Fpglu1, determined at 1.85 Å resolution, provided a deeper understanding of its catalysis and substrate specificity. The evaluation of the binding conformation, hydrogen bond, and key amino acids of β-glucosidase Fpglu1 with different ginsenosides (Rb1, Rb2, and Rc) further elucidated the structural basis of its substrate-binding preference. In summary, Fpglu1, which had excellent thermostability and unique ginsenoside-transforming ability, was a highly promising catalyst for the industrial production of ginsenoside C-K. Additionally, structural studies have laid a theoretical foundation for further improving the catalytic properties of the enzyme through directed evolution in the future.