Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Abstract

Ginsenoside compound K (C-K) is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD)-type ginsenosides in red-ginseng extract (RGE), the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf) was used along with the β-D-glucopyranoside/α-L-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly) because SS-bgly showed very low hydrolytic activity on the α-L-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.

Fig 1. Hydrolytic pathway from ginsenosides Rb₁, Rb₂, and Rc to C-K via C-Mc, ginsenoside Rd, and F₂ by SS-bgly supplemented with CS-abf.

Fig 2. Effect of supplementation of CS-abf to SS-bgly on the conversion of ginsenoside Rc to C-K.

Data represent the means of three experiments and error bars represent standard deviation.

Fig 3. Production of C-K from PPD-type ginsenosides in RGE by SS-bgly supplemented with CS-abf.

Ginsenosides Rb₁ (open circle), Rb₂ (open square), Rc (open triangle), and Rd (open diamond) were converted to C-K (filled circle) via F₂ (open inverted triangle) and C-Mc (filled triangle). Data represent the means of three experiments and error bars represent standard deviation.

Fig 4. Effect of supplementation of CS-abf to SS-bgly on the concentration of C-Mc in RGE.

Data represent the means of three experiments and error bars represent standard deviation.

Fig 5. Effects of (A) pH and (B) temperature on the production of C-K from PPD-type ginsenosides in RGE by SS-bgly supplemented with CS-abf.

Data represent the means of three experiments and error bars represent standard deviation.

Fig 6. Effect of the concentration of (A) SS-bgly supplemented with CS-abf and (B) PPD-type ginsenosides in RGE.

Filled circle and open inverted triangle represent concentration of C-K and conversion yield, respectively. Data represent the means of three experiments and error bars represent standard deviation.

Fig 7. C-K production from PPD-type ginsenosides in RGE.

C-K (filled circle) was produced from ginsenosides Rb₁ (open circle), Rb₂ (open square), Rc (open triangle), and Rd (open diamond) in RGE via F₂ (open inverted triangle) and C-Mc (filled triangle) by (A) SS-bgly. (B) SS-bgly supplemented with CS-abf under the optimized unit ratio of the two enzymes. Data represent the means of three experiments and error bars represent standard deviation.