niger, which has been LY294002 in vitro generally regarded as safe by the Food and Drug Administration. Ginsenosides Rb1, Rd, 20(S)-Rg3, 20(R)-Rg3, Rh2, and CK were purchased from Vitrosys, Inc.
(Yeongju, Korea). Ginsenoside Rb1, Rd, 20(S)-Rg3, and 20(R)-Rg3. ρ-Nitrophenyl-β-D-glucopyranoside (PNPG), ρ-nitrophenol (PNP), and β-glucosidase from almond were purchased from Sigma-Aldrich (St Louis, MO, USA). Potato dextrose broth was purchased from Difco (Miller, Becton Dickinson, and Co., Sparks, MD, USA). Celluclast 1.5L and Cellulase 12T were purchased from Novozymes (Bagsværd, Denmark) and Bioland Co. (Chungnam, Korea), respectively. High performance liquid chromatography (HPLC; Agilent 1100 series; Agilent Technologies, Palo Alto, CA, USA) was conducted using a UV/vis detector and a gradient pump. All solvents used in chromatography were of HPLC grade and all other chemicals were of analytical reagent grade. A. niger KCCM 11239 was purchased
from the Korean Culture Center of Microorganisms selleck chemicals llc (KCCM, Seoul, Korea). The fungus was cultured on potato dextrose agar at 30°C for 4 d, and the stock cultures were maintained at 4°C. Erlenmeyer flasks were filled to 20% of their volume with potato dextrose broth, and subsequently inoculated with 5-d cultures. The cultures were grown for 16 d under shaking conditions at 200 rpm at 30°C. During the shake flask culturing, a few glass beads were added to prevent mycelial clumping and thus to achieve homogeneous growth. After incubation, the culture broth was centrifuged at 9,000 × g at 4°C for 10 min, and a crude enzyme was obtained by precipitation with 70% of (NH4)2SO4 of the supernatant. The specific activity of crude enzyme was detected to 91 U/mg. Beta-glucosidase activity was evaluated via a colorimetric method using PNPG as a substrate. The reaction mixture, which contained 1 mL of 5 mM PNPG and 100 μL of enzyme solution, was incubated at 50°C for 10 min. The reaction
was subsequently terminated via the addition Histidine ammonia-lyase of 1 mL of 0.5 M NaOH, and the absorption of the released PNP was measured at 400 nm. One unit of β-glucosidase activity was defined as the quantity of enzyme required to liberate 1 μM of PNP/min under standard conditions . Microbial transformation was conducted via a modified Cheng’s method . In brief, suspensions of the 5 d-old cultures were mixed with an equal volume of 1 mM ginsenoside Rb1 dissolved in 0.5 M sodium phosphate buffer (pH 5.5) and were shaken for 16 d, 200 rpm, at 30°C. Enzymatic transformation was conducted with 200 μL of a 16-d culture supernatant (centrifuged at 14,400 × g for 30 min at 4°C) and the same volume of 1 mM ginsenoside Rb1 was reacted for 48 h at 30°C and 50°C. Aliquots were withdrawn at suitable time intervals (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h).