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 degrees 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 mu M 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 mu M 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.