Bioconversion of Genistein to Orobol by Bacillus subtilis Spore Displayed Tyrosinase and Monitoring the Anticancer Effects of Orobol on MCF-7 Breast Cancer Cells

Orobol (5,7,3′,4′-tetrahydroxyisoflavone) is a highly hydroxylated isoflavone, which is rarely found in natural environment. In this study, orobol was produced due to bioconversion of a soybean frequented isoflavone, genistein (4′,5,7-Trihydroxyisoflavone) by an active, stable, reusable genetically immobilized enzyme, the recombinant Bacillus subtilis spore displayed tyrosinase. Thin layer chromatography and high performance liquid chromatography were used to monitor the reaction. The results revealed that 1 mM orobol was produced from 3′-hydroxylation of 1 mM genistein by spore displayed tyrosinase at 37°C during 90 min incubation. To study on anti-proliferative effects of orobol, MCF-7 breast cancer cell viability was determined by MTT method and flow cytometric analysis. The comparison between reduction in cell viabilities in 50 to 500 µM genistein and orobol treated cells revealed that orobol has more remarkable anticancer effects than genistein. Flow cytometric analysis showed more than 87% cytotoxicity in 500 µM orobol treated cells by flow cytometric analysis. To the best of our knowledge this is the first report of the orobol demonstrated potent anticancer activity against MCF-7 breast cancer cell. It is suggested that enzymatic biotransformation of soybean genistein to orobol will be made a new approach to create highly bioactive products usable in food and pharmaceutical industries.