β-Glucosidase plays a pivotal role in transforming ginsenosides into specific minor ginsenosides. In this study, total ginsenosides from
Panax notoginseng
leaves were used as substrates to stimulate the growth of
Aspergillus niger
NG1306. Transcriptome analysis identified a β-glucosidase gene,
Anglu
04478 (1455 bp, 484 amino acids, 54.5 kDa, pI = 5.1), as a participant in the ginsenosides biotransformation process. This gene was cloned and expressed in
Escherichia coli
BL21
Transetta
(DE3). The
An
Glu04478 protein was purified using a Ni
2+
column, and its enzymatic properties were characterized. Purified
An
Glu04478 exhibited a specific activity of 32.97 U/mg when assayed against
p
NPG. Under optimal conditions (pH 4.5, temperature 40 °C), the kinetic parameters,
K
m
and
V
max
, for
p
NPG were 1.55 mmol/L and 0.014 mmol/min, respectively. Cu
2+
displayed an inhibitory effect on
An
Glu04478, whereas Ca
2+
, Co
2+
, and Ni
2+
ions had minimal impact. The enzyme showed tolerance to ethanol and was largely unaffected by glucose feedback inhibition. Testing with ginsenosides as substrates revealed selective hydrolysis at the C3 position of ginsenosides Rb1, Rb2, Rb3, and Rc, with the metabolic pathway delineated as Rb1 → GypXVII, Rb2 → C–O, Rb3 → C-Mx1 → C-Mx, and Rc → C-Mc1. The conversion rates of ginsenosides Rb1, Rb2, Rb3, and Rc varied from 2.58 to 20.63%. With 0.5 U purified enzyme and 0.5 mg total ginsenosides, incubated at 40 °C for 12 h, the conversion rates were 42.6% for GypXVII, 10.4% for C–O, 6.27% for C-Mx1, 26.96% for C-Mx, and 90% for Rc. These results suggest that
An
Glu04478 displays substrate promiscuity as a β-glucosidase, thus broadening the potential for ginsenoside biotransformation.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00284-024-04012-0.
