Transcriptome profiling, cloning, and characterization of AnGlu04478, a Ginsenoside Hydrolyzing β-glucosidase from Aspergillus niger NG1306

Abstract

Abstract Minor ginsenosides exhibit superior pharmacological activity compared to major ginsenosides, yet their presence in plants is limited. Therefore, it is crucial to efficiently obtain minor ginsenosides. Specific glycoside hydrolases offer the advantage of converting major ginsenosides into specific minor counterparts under mild reaction conditions while minimizing structural damage. In this study, we utilized total ginsenosides extracted from Panax notoginseng leaves as substrates to stimulate the growth of Aspergillus niger NG1306. Transcriptome analysis revealed that Anglu04478 potentially participates in the biotransformation process of ginsenosides. Subsequently, it was cloned and expressed in Transetta (DE3). The AnGlu04478 protein was purified by Ni2+ column and its enzymatic properties were characterized. The results show that the optimum pH was 4.5 and the optimum temperature was 40°C, Cu2+ had a certain inhibitory effect on AnGlu04478, while other metal ions had little effect on it. AnGlu04478 had a certain tolerance to ethanol, and it was not significantly affected by product (glucose) feedback inhibition. Using pNPG as a substrate, the kinetic parameter Km of AnGlu04478 was 1.55 mmol/L, the Vmax was 0.014 mmol/min. The test with ginsenosides as substrate showed that it could selectively hydrolyze glucose of ginsenoside Rb1, Rb2, Rb3 and Rc at C3, and the putative metabolic pathway was Rb1 → GypXVII, Rb2 → C-O, Rb3 → C-Mx1 → C-Mx, Rc →C-Mc1.These findings indicate that AnGlu04478 exhibits substrate promiscuity as a β-glucosidase, thereby expanding the options for ginsenosides biotransformation.