Enzymatic Production of 3-OH Phlorizin, a Possible Bioactive Polyphenol from Apples, by <i>Bacillus megaterium</i> 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.

Keywords

• bacterial CYP102A1
• dihydrochalcone
• glucoside
• regioselective hydroxylation
• phloretin
• phlorizin