Enzymatic Production of 3-OH Phlorizin, a Possible Bioactive Polyphenol from Apples, by Bacillus megaterium CYP102A1 via Regioselective Hydroxylation.

ABSTRACT

Phlorizin is the most abundant glucoside of phloretin from the apple tree and its products. Phlorizin and its aglycone phloretin are currently considered health-beneficial polyphenols from apples useful in treating hyperglycemia and obesity. Recently, we showed that phloretin could be regioselectively hydroxylated to make 3-OH phloretin by Bacillus megaterium CYP102A1 and human P450 enzymes. The 3-OH phloretin has a potent inhibitory effect on differentiating 3T3-L1 preadipocytes into adipocytes and lipid accumulation. The glucoside of 3-OH phloretin would be a promising agent with increased bioavailability and water solubility compared with its aglycone. However, procedures to make 3-OH phlorizin, a glucoside of 3-OH phloretin, using chemical methods, are not currently available. Here, a biocatalytic strategy for the efficient synthesis of a possibly valuable hydroxylated product, 3-OH phlorizin, was developed via CYP102A1-catalyzed regioselective hydroxylation. The production of 3-OH phlorizin by CYP102A1 was confirmed by HPLC and LC-MS spectroscopy in addition to enzymatic removal of its glucose moiety for comparison to 3-OH phloretin. Taken together, in this study, we found a panel of mutants from B. megaterium CYP102A1 could catalyze regioselective hydroxylation of phlorizin to produce 3-OH phlorizin, a catechol product.