Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2017 Apr 19;12(4):e0176098.
doi: 10.1371/journal.pone.0176098. eCollection 2017.

Comparative Analysis of the Expression Level of Recombinant Ginsenoside-Transforming β-Glucosidase in GRAS Hosts and Mass Production of the Ginsenoside Rh2-Mix

Affiliations
Free PMC article
Comparative Study

Comparative Analysis of the Expression Level of Recombinant Ginsenoside-Transforming β-Glucosidase in GRAS Hosts and Mass Production of the Ginsenoside Rh2-Mix

Muhammad Zubair Siddiqi et al. PLoS One. .
Free PMC article

Abstract

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A β-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. (A and B). SDS-PAGE analysis of recombinant E. coli and GRAS host strains.
A: Lane 1, molecular weight standard; lane 2, soluble crude extract of recombinant E. coli without induction; lane 3, BglPm of recombinant E. coli after induction; lane 4, purified soluble fraction of recombinant E. coli (BglPm); lane 5, non-inducible fraction of Corynebacterium glutamicum harboring pCES208; lane 6, inducible BglPm_C; lane 7, purified BglPm_C (C. glutamicum); lane 8, molecular weight standard. B: lane 9, molecular weight standard; lane 10, non-inducible fraction of Saccharomyces cerevisiae; lane, 11 inducible BglPm_S; lane 12, BglPm_S protein of S. cerevisiae after purification; lane, 13–14, non-inducible and inducible fraction of Lactococcus lactis; lane 15, molecular weight standard.
Fig 2
Fig 2. (a, b, c and d) shows the effect of sonication on the enzyme activity of; recombinant BglPm (E. coli), BglPm_C (C. glutamicum), BglPm_S (S. cerevisiae) and BglPm_L (Lactococcus lactis), respectively.
From the sonication analysis, these results clearly show that enzymes lose their activities after a specific time interval for all recombinant enzymes used in this study.
Fig 3
Fig 3. TLC analyses of time course of ginsenosides by acid and enzyme (BglPm_C) treatment.
(A) Transformation of ginsenoside PPD-Mix. (B) Biotransformation of Rg3-Mix to Rh2-Mix after 24 h. Developing solvent: CHCl3-CH3OH-H2O (65:35:10, lower phase). Lane S represents PPD-Mix (A) and Rg3-Mix (B). PPD, protopanaxadiol.
Fig 4
Fig 4. HPLC analysis of the transformation of the ginsenosides (PPD-Mix and Rg3-Mix) by acid and enzyme treatments.
(A) Ginsenosides standard. (B) PPD-Mix as a starting substrate. (C) Rg3-Mix after 15 min at 121°C by acid treatment of PPD-Mix. (D) Rh2-Mix after 24 h of the reaction of BglPm_C with Rg3-Mix. PPD-Mix, protopanaxadiol-type ginsenoside mixture (Rb1, Rb2, Rb3, Rc and Rd).
Fig 5
Fig 5. Schematic view of transformation pathways for Rh2-Mix production and the relative structures of ginsenosides.

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

References

    1. Sengupta S, Toh SA, Sellers LA, Skepper JN, Koolwijk P, Leung HW, et al. Modulating angiogenesis: The yin and the yang in ginseng. Circulation 2004; 110(10):1219–1225. 10.1161/01.CIR.0000140676.88412.CF - DOI - PubMed
    1. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: Multiple constituents and multiple actions. Biol Pharm Bull. 1999;58(11):1685–1693. - PubMed
    1. Yuan CS, Wu JA, Osinski J. Ginsenoside variability in American ginseng samples. Am J Clin Nutr. 2002;75(3):600–601. 10.1016/S0006-2952(99)00212-9 - DOI - PubMed
    1. Lee CH, Kim JH. A review on the medicinal potentials of ginseng and ginsenosides on cardiovascular diseases. J Ginseng Res. 2014;38(3):161–166. 10.1016/j.jgr.2014.03.001 - DOI - PMC - PubMed
    1. Christensen LP. (2009). Ginsenosides chemistry, biosynthesis, analysis, and potential health effects. Adv Food Nutr Res. 2009;55:1–99. 10.1016/S1043-4526(08)00401-4 - DOI - PubMed

Publication types

MeSH terms

Grant support

This work was supported by the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2014M3A6A8066437) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01060255).
Feedback