Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study (sequences 5′→3′).
Table 3.
Total activities of β-glucosidase in cell-free extracts of induced E. coli and GRAS host strains which were constructed with different vectors, and induced by different promoters used in this study.
The GRAS strain, which was well-expressed and showed high enzyme activity with recombinant E. coli, is indicated in bold.
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.
(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.
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.
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.
Schematic view of transformation pathways for Rh2-Mix production and the relative structures of ginsenosides.