Whole Cell Mediated Acylation Of Esculin And Naringin

As kinds of important polyphenolic compounds, flavonoids contain a serious of physiological functions including anti-tumor, antioxidant and antiviral activities, thus playing an important role in food, cosmetics and pharmaceutical industries. It was found that the flavonoids bioactivities can be heavily limited by their low stability and weak solubility in aqueous solutions. Selective acylation of the hydroxyl groups in flavonoids have been proved an efficient strategy, which can improve the solubility of flavonoid and thus enhance their bioactivities. The chemical acylation methods need tedious steps to reach high selectivity and are environmentally-unfriendly. The enzymes-based methods showed high selectivity but their application was greatly hampered by their high cost. In this dissertation, whole-cells was, for the first time, tested as catalysts in acylation of flavonoids.Inducers in culture media showed significant effects on biomass and catalytic ability of the whole-cells. Among 12 strains tested, P.aeruginosa GIM 1.46 and P.stutzeri GIM 1.273 cultured in SM-1 medium containing soy oil can catalyze acylation of esculin in pyridine, with conversions of 47.9% and 19.7%, respectively. By using MS and 13 C NMR, the acylation products of esculin and naringin were identified as esculin-6’-O-propionate and naringin-6’’-O-propionate with highly regioselectivities above 99%.Both solvent polarity and substrate solubility play an important role in the acylation. No products were detected when P.stutzeri was applied in strong polar solvents that naringin can be hardly dissolved. Higher products yield were found in solvents with lower polarity(t-butanol and t-pentanol). In t-pentanol, the best reaction conditions were 20 mg/m L whole cell catalysts dosage, 0μL/m L water content, 20:1 of substrate molar ratio, 40℃, and 180 r/m, under which conversion and 6’’-regioselectivity of the reaction reached 70.8% and > 99%, respectively.The problem between high solubility of esculin and low enzyme inactivity in polar solvents can be well solved by adding hydrophobic solvent to the polar ones, forming binary solvent mixtures. In the best binary mixture isooctane-pyridine, the optimum dosage of whole cell catalysts, water content, mole ratio of substrates, temperature and shaking speed were 20 mg/m L, 0 μL/m L, 20:1, 40 and 150 r/m, ℃ respectively, for P.stutzeri whole cell catalyzed acylation of esculin. The highest conversion and 6’-regioselectivity of this reaction were 97% and 99%, respectively.Preparative-scale experiment of P.stutzeri whole cell acylation of naringin or esculin showed the possibility of the whole-cell method for industrial production of flavonoids.In addition, P.stutzeri whole cell can be recycled and showed well operation stability.