niger produced a less polar metabolite, with an Rf value similar to that of ginsenoside Rh2 and compound K. This result suggests that the microbial conversion of ginsenoside Rb1 using A. niger KCCM 11239 induced the production of diverse PPD-type ginsenosides. We estimated that this result was induced by different types of β-glucosidase from Cobimetinib mouse A. niger KCCM 11239. Aspergillus species are known to produce different types of β-glucosidase. For example, Aspergillus sp. g48p produces two types of ginsenoside-hydrolyzing β-glucosidases: ginsenosidase type II hydrolyzes 20-O-glycosides of PPD-type ginsenosides and ginsenosidase type I hydrolyzes 3-O

and 20-O glycosides of PPD-type ginsenosides [25]. Sorafenib The enzymatic conversion of ginsenoside Rb1 over a time-course was conducted using a crude enzyme. Ginsenoside Rb1 was reacted with the same volume of crude enzyme for 48 h at 30°C and 50°C; the TLC results are shown in Fig. 2. When ginsenoside

Rb1 was reacted at 30°C, the levels of ginsenoside Rd were increased within 30 min and the levels of ginsenoside Rg3 were increased after 4 h. All of the ginsenoside Rb1 was converted to ginsenoside Rd and ginsenoside Rg3 after 24 h of incubation. Ginsenoside Rb1 was reacted with crude enzyme at 50°C to compare the effects of the reaction temperature. The results demonstrate that high reaction temperatures accelerate the reaction to produce ginsenoside Rg3. In addition, the conversion activity of A. niger after 48 h of a reaction time were compared by using three commercial enzymes ( Fig. 3). When ginsenoside Rb1 was reacted with Celluclast 1.5L and Cellulase 12T, the content of ginsenoside Rb1 was reduced and a productivity of Rd increased after 48 h. However, these enzymes were not converted further into active minor ginsenosides Rg3. In case of β-glucosidase from almond, ginsenoside hydrolyzing activity was not detected. Various products of Rb1 transformed by the crude enzyme isolated

from A. niger KCCM 11239 were confirmed via HPLC analysis. The profile of the reaction mixture of ginsenoside Rb1 at 30°C for 24 h of reaction is Pyruvate dehydrogenase lipoamide kinase isozyme 1 shown in Fig. 4. HPLC analysis yielded results similar to the findings of TLC. In addition, the amount of ginsenoside Rb1 was reduced with the extension of the reaction time, whereas other hydrolysis products including ginsenoside Rd and S-Rg3 increased after 24 h of incubation at 30°C. Ginsenoside Rb1 harbors four β-glucosidic linkages, including a 20-C, β-(1→6) and a 3-C, β-(1→2) linkage. Fig. 4 shows that a peak area of Rd in a sample increased after 8 h, but decreased after 24 h. In the meantime, ginsenoside Rg3 was detected in a 24 h-reaction mixture and a ratio of the peak area was approximately 48.5%. By contrast, ginsenoside Rb1 was not detected in a 24-h reaction mixture.