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

RESEARCH ARTICLE Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-LArabinofuranosidase from Caldicellulosiruptor saccharolyticus Kyung-Chul Shin, Hye-Yeon Choi, Min-Ju Seo, Deok-Kun Oh* Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea * Abstract OPEN ACCESS Citation: Shin K-C, Choi H-Y, Seo M-J, Oh D-K (2015) Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus. PLoS ONE 10 (12): e0145876. doi:10.1371/journal.pone.0145876 Editor: Kyung-Jin Min, Inha University, REPUBLIC OF KOREA Received: September 24, 2015 Accepted: December 9, 2015 Published: December 28, 2015 Copyright: © 2015 Shin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data are all contained within the paper and its Supporting Information files. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Ginsenoside compound K (C-K) is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and antiskin 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 PPDtype 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. Introduction Ginseng (Panax ginseng C. A. Meyer), one of the most valuable herbs, has been used in traditional medicine in Asian countries for over 2000 years [1]. The active components of ginseng are ginsenosides, which possess diverse biological and pharmaceutical activities, including anti-fatigue [2], anti-allergic [3], anti-oxidant [4], anti-inflammatory [5], anti-cancer [6], and anti-skin aging [7] activities. Ginsenosides are classified into three groups on the basis of the types of the aglycone structures, namely, oleanane, protopanaxadiol (PPD), and PLOS ONE | DOI:10.1371/journal.pone.0145876 December 28, 2015 1 / 16 Enzymatic Compound K Production from Red Ginseng Extract protopanaxatriol (PPT). Oleanane, containing oleanolic acid aglycone, exists in only one ginsenoside, that is, Ro. PPD- and PPT-type ginsenosides exist in glycosylated compounds consisting of a non-sugar component with PPD or PPT aglycone and a sugar component with 1−4 glycoside molecules [8]. Glycosylated major ginsenosides (Rb1, Rb2, Rc, Rd, Rg1, and Re) constitute more than 80% of total ginsenosides in wild ginseng [9]. Deglycosylated minor ginsenosides exhibit higher biological activity than major glycosylated ginsenosides because of their smaller size, higher bioavailability, and better permeability across the cell membrane [10]. Commonly, wild ginseng is processed into white ginseng and red ginseng to improve its preservation and efficacy. White ginseng is produced by sun drying, while red ginseng is heated by steaming and then dried. The ginsenoside content in red ginseng differs from that in white ginseng because of the heating process. Red ginseng contains higher content of PPD-type ginsenosides in total ginsenosides than PPT-type ginsenosides, whereas white ginseng contains higher content of PPT-type ginsenosides than PPD-type ginsenosides. The concentration of PPD-type ginsenosides in red ginseng is higher than that in white ginseng. Because red ginseng exerts stronger activities than white ginseng toward anti-skin aging [7], skin damage protection [11], and burn wound healing [12], it has also been used in the production of cosmetics. Red ginseng is fermented using Lactobacillus species to enhance the anti-aging potential by [13–15]. Fermented red ginseng increases the content of ginsenosides such as Rg3, Rh1, F2, Rg2, and compound K (C-K) [15]. The minor ginsenoside C-K is one of the most biologically and pharmaceutically active PPD-type ginsenosides [16]. C-K has attracted attention in recent years because of hepatoprotective activity [17], inhibition of tumor invasion [18], and induction of tumor cell apoptosis [19]. C-K is also effective against wrinkling and skin damage [20, 21]. Therefore, C-K has been used as the principal ingredient of skin care products. Because C-K is not present in ginseng, the production of C-K from major PPD-type ginsenosides have been intensively tried by biotransformations, including cell conversion [22–24], fermentation [25], and enzymatic conversion [26–28]. Of these methods, enzymatic conversion showed the highest selectivity, yield, and productivity for C-K production. For the industrial production of C-K, ginseng extract should be used as the substrate, instead of purified ginsenosides. However, most studies on C-K production have used purified ginsenosides; only some have used ginseng extracts [29– 33]. For the effective application of ginseng extracts to the manufacture of cosmetics, PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion because C-K is an effective agent for anti-wrinkling and anti-skin aging. In this study, the PPD-type ginsenosides in red ginseng extract (RGE) were completely converted to C-K by the enzymatic reaction involving the β-D-glucopyranoside/α-L-arabinopyranoside-hydrolyzing β-glucosidase from Sulfolobus solfataricus (SS-bgly) supplemented with the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf). Materials and Methods Materials The ginsenoside standards Rb1, Rb2, Rc, Rd, F2, compound Mc (C-Mc), and C-K were purchased from BTGin (Daejon, Korea). Red ginseng was purchased from a local ginseng market (Geumsan, Korea (...truncated)