Regioselective Hydroxylation of Naringin Dihydrochalcone to Produce Neoeriocitrin Dihydrochalcone by CYP102A1 (BM3) Mutants

Naringin dihydrochalcone (DC) is originally derived from the flavonoid naringin, which occurs naturally in citrus fruits, especially in grapefruit. It is used as an artificial sweetener with a strong antioxidant activity with potential applications in food and pharmaceutical fields. At present, enzymatic and chemical methods to make products of naringin DC by hydroxylation reactions have not been developed. Here, an enzymatic strategy for the efficient synthesis of potentially valuable products from naringin DC, a glycoside of phloretin, was developed using Bacillus megaterium CYP102A1 monooxygenase. The major product was identified to be neoeriocitrin DC by NMR and LC-MS analyses. Sixty-seven mutants of CYP102A1 were tested for hydroxylation of naringin DC to produce neoeriocitrin DC. Six mutants with high activity were selected to determine the kinetic parameters and total turnover numbers (TTNs). The kcat value of the most active mutant was 11 min−1 and its TTN was 315. The productivity of neoeriocitrin DC production increased up to 1.1 mM h−1, which corresponds to 0.65 g L−1 h−1. In this study, we achieved a regioselective hydroxylation of naringin DC to produce neoeriocitrin DC.