Characterization of the ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum and bioconversion of major ginsenosides into minor ginsenosides

Abstract

This study focused on the cloning, expression, and characterization of ginsenoside-transforming recombinant β-glucosidase from Actinosynnema mirum KACC 20028(T) in order to biotransform ginsenosides efficiently. The gene, termed as bglAm, encoding a β-glucosidase (BglAm) belonging to the glycoside hydrolase family 3 was cloned. bglAm consisted of 1,830 bp (609 amino acid residues) with a predicted molecular mass of 65,277 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The recombinant BglAm was purified with a GST·bind agarose resin and characterized. The optimum conditions of the recombinant BglAm were pH 7.0 and 37 °C. BglAm could hydrolyze the outer and inner glucose moieties at the C3 and C20 of the protopanaxadiol-type ginsenosides (i.e., Rb(1) and Rd, gypenoside XVII) to produce protopanaxadiol via gypenoside LXXV, F(2), and Rh(2)(S) with various pathways. BglAm can effectively transform the ginsenoside Rb(1) to gypenoside XVII and Rd to F(2); the K (m) values of Rb(1) and Rd were 0.69 ± 0.06 and 0.45 ± 0.02 mM, respectively, and the V (max) values were 16.13 ± 0.29 and 51.56 ± 1.35 μmol min(-1) mg(-1) of protein, respectively. Furthermore, BglAm could convert the protopanaxatriol-type ginsenoside Re and Rg(1) into Rg(2)(S) and Rh(1)(S) hydrolyzing the attached glucose moiety at the C6 and C20 positions, respectively. These various ginsenoside-hydrolyzing pathways of BglAm may assist in producing the minor ginsenosides from abundant major ginsenosides.

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides ( S )-Rh 1 and ( S )-Rg 2

“…When Rb 1 , Rd, Re, and Rg 1 (1.0 mg/ml) were used as substrates, they were biotransformed within 10 min by 10 mg/ml of crude recombinant MBP-BglQM. Several other ginsenoside-hydrolyzing recombinant enzymes have been reported, but most were only able to hydrolyze the outer or inner glucose moiety at the C-3 position of the aglycon (23,24,26). One previously described enzyme from Microbacterium esteraromaticum KCTC 12040BP (32) had the same ability as BglQM, but the authors conducted only a simple enzymatic characterization without further scale up or process engineering.…”

“…strain GL1 ␤-glucosidase (55), which were characterized previously. Several ginsenoside-hydrolyzing ␤-glucosidases in glycoside hydrolase family 3 have previously been cloned, including a ␤-glucosidase (rApy-H11) from Bifidobacterium longum H-1 (8), a ␤-glucosidase (Bgp1) from Microbacterium esteraromaticum (31), a ␤-glucosidase (BgpA) from Terrabacter ginsenosidimutans (23), a ␤-glucosidase (BGL1) from Aspergillus niger (56), a ␤-glucosidase (BglAm) from Actinosynnema mirum (24), and a ␤-glucosidase (BglSk) from Sanguibacter keddieii (26). The relationship between BglQM and these ginsenoside-hydrolyzing ␤-glucosidases is presented in Fig.…”

“…However, these approaches produce nonspecific stereoisomeric mixtures of rare ginsenosides that have low yields and are very difficult to separate. As an alternative, enzymatic methods have been explored as a way to more specifically convert major ginsenosides into pharmacologically active rare ginsenosides (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)52). Furthermore, methods have been developed to efficiently separate a PPT-type ginsenoside mixture (PPTGM) and a PPD-type ginsenoside mixture (PPDGM) from ginseng extracts (34,35).…”

An amino acid at position 512 in β-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII

“…Among the GH3 family, β-glucosidases from Actinosynnema mirum (Cui et al 2013a), Sanguibacter keddieii , and Terrabacter ginsenosidimutans (An et al 2010) have different regioselectivity for hydrolyzing ginsenosides in spite of highly conserved active-site residues (Table 1). These results suggest that the regioselectivity for hydrolyzing ginsenosides can be altered by replacing one residue in the active-site residues.…”

“…Deglycosylated ginsenosides have been mainly obtained with glycoside hydrolase 1 (GH1) and GH3 family β-glucosidases (An et al 2010;Wang et al 2011;Hong et al 2012;Kim et al 2012;Cui et al 2013a;Cui et al 2013b;Quan et al 2013;Shin and Oh 2013;Zhao et al 2013). Among the GH3 family, β-glucosidases from Actinosynnema mirum (Cui et al 2013a), Sanguibacter keddieii , and Terrabacter ginsenosidimutans (An et al 2010) have different regioselectivity for hydrolyzing ginsenosides in spite of highly conserved active-site residues (Table 1).…”