Regioselective hydroxylation of phloretin by tyrosinase using Bacillus subtilis spore display system
摘要
In this study, we tried the biotransformation of phloretin using Bacillus subtilis spore-displayed tyrosinase from Bacillus megaterium. Phloretin is mainly found in the root bark of apple trees and in apples where it acts as a natural antibacterial plant defense metabolite. The cell surface expression of tyrosinase was verified by flow cytometry using FITC-labeled anti-His6tag antibody. After 7 h of reaction at 37 °C, high-performance liquid chromatography analysis confirmed that all the phloretin was completely transformed into new product. And it was further analyzed using liquid chromatography with tandem mass spectrometry and 1H-NMR to be revealed as 3ʹ-hydroxyphloretin. 3ʹ-Hydroxyphloretin was expected to have increased antioxidant efficacy due to the increased number of hydroxyl groups compared to phloretin, and because of measuring the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, it was confirmed that the electron-donating ability increased by about 54%. In addition, the spores were washed with Tris–HCl buffer [pH9] and reused in the repeated reaction. The biotransformation proceeded through 7 recycling reactions without significant decrease of the displayed enzymatic activity.