Bioconversion of Genistein to Orobol

Orobol was produced as described in our previous study^{31 (link)} via the bioconversion of genistein using tyrosinase which leads to ∂-hydroxylation^{42 (link),58 (link)} in Bacillus megaterium. Polyethylene glycol 200 (PEG 200), 3-isobutyl-1-methylcanthine (IBMX), dexamethasone (DEX), human insulin, genistin, genistein, daidzin, daidzein, and equol were purchased from Sigma-Aldrich (St. Louis, MO). 6,7,4′-Trihydroxyisoflavone (6-ODI) was obtained from Chromadex™ (Irvine, CA). 7,3′,4′-Trihydroxyisoflavone (3′-ODI) and 7,8,4′-trihydroxyisoflavone (8-ODI) were purchased from Indofine Chemical (Hillsborough, NJ). 3-[4,5-Diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from USB (Cleveland, OH). CK1ε and eIF4E antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against phosphorylated 4E-BP1 and 4E-BP1 were purchased from Cell Signaling Biotechnology (Danvers, MA). The antibody against phosphorylated eIF4E was purchased from EPITOMICS (Burlingame, CA).