Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase†

doi:10.7503/cjcu20150536

Abstract

Abstract:

Ginsenosides are the principle components that are responsible for the biological and pharmacological activities of ginseng. In this study, biotransformation of ginsenside Rd and CK by recombinant β-glucosidasse(Fpglu1) was studied by high performance liquid chromatography(HPLC). Structure modeling and molecular docking studies were performed to study the interaction between the protein and the ginsenosides. The results showed that ginsenoside Rd and CK could be transformed from major ginsenosides Rb₁, Rb₂ and Rc by Fpglu1. The K_m values for Rb₁, Rb₂ and Rc were 0.318, 1.840 and 5.269 mmol/L, and the k_cat values were 144.191, 0.572 and 0.011 s^-1, respectively. The highest productivity of ginsenoside Rd and CK were 60% and 93% with biotransformation time of 6 and 102 h, respectively. Molecular docking studies show that Glu194 and Glu367 are key active site residues and the predicted inter-energy exhibits linear relations to experimental K_m values and k_cat values.

Key words: β-Glucosidase, Biotransformation, Ginsenoside, Kinetics

CLC Number:

O629
O641

TrendMD:

XU Chunchun, YU Bohao, WANG Honglei, LI Jing, LIU Shuying, YU Shanshan. Transformation of Minor Ginsenoside Rd and CK by Recombinant Thermostable β-Glucosidase^†[J]. Chem. J. Chinese Universities, 2016, 37(2): 281.

Figures/Tables 8

Fig.1 SDS-PAGE analysis of Fpglu1 after purification Lane M: standard protein molecular mass makers;lane 1: Fpglu1 purified by Ni-NTA chromatography.

Fig.2 HPLC analysis of biotransformation of the ginseng root extract at different time (A) Standard ginsenosides; (B)—(H) Fpglu1 reacted with 2 mg/mL ginseng root extract for 0 , 1/6, 2, 6, 66, 78 and 102 h, respectively in 20 mmol/L sodium phosphate buffer(pH=7.5) at 70 ℃.

Fig.3 Transformation pathways of the ginsenosides Rb1, Rb2 and Rc by Fpglu1 The transformation pathways were Rb1→Rd→CK, Rb2→Rd→CK and Rc→Rd/CMc→CK.

Fig.4 Production of Rd and CK from the ginseng root extract by Fpglu1 ●: Ginsenoside Rb1; ○: Ginsenoside Rb2; ▲: Ginsenoside Rc; △: Ginsenoside Rd; ■: CMc; □: CK.

Fig.5 Fitting curve of ginsenosides Rb1(A), Rb2(B) and Rc(C)

Fig.6 Ramachandran plot analysis and predicted 3D structure of Fpglu1 (A) Ramachandran plot showing different regions of the modeled enzyme; (B) the overall structure of the Fpglu1 model.

Fig.7 Docking model of Fpglu1 with ginsenoside Rb1 (A) A zooming view showing the docking model with ginsenoside Rb1. The hydrophobic amino acids around ginsenoside Rb1 and the key catalytic amino acids were indicated. The ginsenoside Rb1 and the surrounding amino acids are colored yellow and blue, respectively; (B) amino acid residues close to ginsenoside Rb1 inside the binding pocket of Fpglu1(2D view).

Table 1 Kinetic parameters of Fpglu1 for Ginsenosides

Substrate	K_m/(mmol·L^-1)	k_cat/s^-1	(k_cat/K_m)/(L·s^-1·mmol^-1)	E/(kJ·mol^-1)
Rb₁	0.318	144.191	453.016	-91.7085
Rb₂	1.840	0.572	0.311	-80.1428
Rc	5.269	0.011	0.002	-74.1413

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