Transcriptome Profiling, Cloning, and Characterization of AnGlu04478, a Ginsenoside Hydrolyzing β-Glucosidase from Aspergillus niger NG1306

Abstract

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, Anglu04478 (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 AnGlu04478 protein was purified using a Ni²⁺ column, and its enzymatic properties were characterized. Purified AnGlu04478 exhibited a specific activity of 32.97 U/mg when assayed against pNPG. Under optimal conditions (pH 4.5, temperature 40 °C), the kinetic parameters, K_m and V_max, for pNPG were 1.55 mmol/L and 0.014 mmol/min, respectively. Cu²⁺ displayed an inhibitory effect on AnGlu04478, whereas Ca²⁺, Co²⁺, and Ni²⁺ 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 AnGlu04478 displays substrate promiscuity as a β-glucosidase, thus broadening the potential for ginsenoside biotransformation.

Fig. 1

a The phylogenetic tree results from analysis of β-glucosidase of amino acid sequences using Neighbor-Joining (NJ) method. Numbers on nodes correspond to percentage bootstrap values for 1000 replicates. b SDS-PAGE of AnGlu04478 protein. (1: control; 2: crude enzyme solution; 3: purified AnGlu04478)

Fig. 2

Effects of pH (a) and temperature (b) on the activity of recombinant AnGlu04478 determined using pNPG as a substrate, AnGlu04478 tolerance to ethanol (c) and glucose (d). The maximum activity obtained was defined as 100%. Results are presented as means ± standard deviations (n = 3)

Fig. 3

TLC detection of different ginsenoside conversion products. Ginsenoside reference substances: Rb1, GypXVII, Rb2, CO, Rb3, C-Mx1, C-Mx, Rc, C-Mc1. a S1: Rb1 conversion product detection, b S2: Rb2 conversion product detection, c S3: detection of Rb3 conversion products, d S4: detection of Rc conversion products

Fig. 4

Detection of ginsenoside conversion products by HPLC. Red peak was reference substance, blue peak was sample. a Detection of Rb1 conversion products, b detection of Rb2 conversion products, c detection of Rb3 conversion products, d Detection of Rc conversion products

Fig. 5

Diagrammatic representation of the AnGlu04478 to ginsenoside conversion pathway (Rb1 → GypXVII, Rb2 →  C–O, Rb3 → C-Mx1 → C-Mx, Rc → C-Mc1)