Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

Abstract

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml⁻¹ Rf, and 10.6 U ml⁻¹ enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l⁻¹ h⁻¹ after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l⁻¹ h⁻¹ after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml⁻¹ APPT from 2.29 mg ml⁻¹ PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l⁻¹ h⁻¹, and 0.62 mg ml⁻¹ APPT from 1.35 mg ml⁻¹ PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l⁻¹ h⁻¹. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.

Complete conversion of all typical glycosylated protopanaxatriol ginsenosides to aglycon protopanaxatriol by combined bacterial β-glycosidases

“…The optimal temperature and pH for APPT production from R1 using DT-bgl were previously determined to be 80 °C and 6.0, respectively (Lee et al 2014 ). APPT production was investigated at enzyme concentrations ranging from 1.0 to 8.0 mg ml −1 at 1.0 mg ml −1 R1 as a substrate for 1.5 h (Additional file 1 : Figure S1a).…”

“…Genes of β-glycosidases from D. turgidum DSM 6724 (DSMZ, Brauschweig, Germany) and P. furiosus DSMZ 3638 were cloned as described previously (Lee et al 2014 ; Oh et al 2014 ). Recombinant Escherichia coli ER2566 (New England Biolabs, Herfordshire, UK) expressing DT-bgl (GenBank Accession Number YP_002352162) or PF-bgl (GenBank Accession Number AAC25555) was cultivated at 37 °C in a 2000 ml flask containing 500 ml Luria–Bertani medium mixed with 20 µg ml −1 kanamycin with rotation at 200 rpm in a shaker.…”

“…Recently, β-glycosidase from Dictyoglomus turgidum (DT-bgl) was reported to be the most efficient APPT producer. However, this enzyme cannot hydrolyze the outer rhamnose residue at C-6 in PPT (Lee et al 2014 ). Thus, DT-bgl does not produce APPT from Re, Rg2, and F3.…”

Synergistic production of 20(S)-protopanaxadiol from protopanaxadiol-type ginsenosides by β-glycosidases from Dictyoglomus turgidum and Caldicellulosiruptor bescii

“…The content of total major PPD-type ginsenosides was 6.2-fold higher than that of total major PPT-type ginsenosides. The concentration ratios of PPD-type to PPT-type ginsenosides for Korean red ginseng, American ginseng ( Panax quinquefolius ) root and seed, P. ginseng root, and Panax notoginseng root were reported as 1.6, 1 and 2.7, 0.8, and 0.7, respectively (Ko et al 2008 ; Lee et al 2014 ; Schlag and McIntosh 2006 ; Shin et al 2015 ). Commercial ginseng extract powder is the best substrate for APPD production based on the utilization efficiency of ginsenosides because the ratio of PPD-type to PPT-type ginsenosides in the powder form is the highest as 6.2 among the different types of ginseng extracts.…”

Study on Transformation of Ginsenosides in Different Methods

“…The optimal reaction conditions for APPT production using D. turgidumβ -glycosidase were pH 6.0, 80°C. The transformed pathways of PPT-type ginsenosides using D. turgidumβ -glycosidase were as follows: R1 → R2 → Rh2 → APPT, Rg1 → Rh1 → APPT, Rf → Rh1 → APPT, Re → Rg2 [ 38 ].…”