Study On Acylation Of Troxerutin Catalyzed By Whole-Cell Biocatalyst In Non-Aqueous Phase

Flavonoids are natural polyhydroxy compounds with multiple biological functions.However,their use is greatly restricted due to their low solubility in oils.As one kind of flavonoids,troxerutin and its derivatives have various pharmacological and physiological activities,such as anti-erythrocyte and platelet aggregation,anti-microbial,scavenging free radical and treatment of diabetes.Because of the polyhydroxyl structural limitation,they also have low solubility in oils,thus limiting their development and application.Previous reports showed that selective acylation of flavonoids could not only improve their solubility in oils,but also enhance their bioactivity.In this dissertation,we developed a new biocatlytic strategy for the acylation of troxerutin using whole-cell biocatalyst in non-aqueous medium.The absorptivity and antioxidant activities of troxerutin and its esters were evaluated.Besides,the cellular localization of microbial cell-bound lipases with acylation activity was investigated and the effects of different conditions on their catalytic activity were also investigated.Using Pseudomonas stutzeri as representative bacterial cells,the cellular localization of cell-bound lipase with acylation activity was investigated.Results indicated that 47.3%,22.8% and 29.9% of lipases with acylation activity were localized in the cytoplasm,cell membrane and cell wall of P.stutzeri cells,respectively.Both the reaction time,the culture time and the types of inducers influenced the acylation activitiesof lipases in different cell components.The reaction equilibrium times by lipases in the separated cell components were longer than that by whole-cells.The highest acylation activities of different components lipase were obtained at culture time of 48 h with soybean oil as the inducer.According to these results,the lipase production pattern in P.stutzeri cells was established,which provided a theoretical reference for the development of whole-cell biocatalysts in the modification of related natural products.Fifteen strains were tested for catalyzing the acylation of troxerutin.Among them,the cells of Pseudomonas stutzeri GIM1.273 and Pseudomonas aeruginosa GIM 1.46 showed relatively high catalytic activities,giving two kinds of products(monoesters and diesters).The acylation occurs on only on hydroxyethyl group of B ring of troxerutin,giving troxerutin monoester and the acylation sites of diester is on the hydroxyethyl groups of A and B ring of troxerutin.Pseudomonas stutzeri preferred to catalyze the reaction to produce diersters with the regioselectivity of 69.1%,while Pseudomonas aeruginosa catalyzed the reaction with the dominant product of a monoester(regioselectivity of 80.2%).Both the type of non-aqueous medium and the reaction conditions markedly influenced the acylation of troxerutin catalyzed by P.aeruginosa cells.The optimum reaction medium,mole ratio of substrates,dosage of whole-cell catalysts,initial water content and temperature and reaction time were 35%(v/v)n-heptane-pyridine,30:1,50 mg/mL,0%,40 ?,120 h,respectively,for P.aeruginosa cells-catalyzed acylation of troxerutin.Under the above reaction conditions,conversion of troxerutin reached above 94% and the yield of monoester was 81%.Conclusively,the physiological activities of of the troxerutin esters were investigated using the Caco-2 cell model.Results showed the acylation for troxerutin improved its solubility in oils,thus increased the absorptivity of small intestine.The absorptivities of troxerutin monoester and diester were 12.6% and 23.7%,respectively,much higher than that of the original troxerutin(1.1%).Due to the enhancement of lipophilicity,troxerutin esters could be more easily gathered in the cell surface and get inside the cells,so as to better protect cells avoided the attack of free radicals.This research not only possesses certain academic significance,but also shows promising industrial application prospective.A new efficient strategy fort the acylation of troxerutin was provided based on whole-cell biocatalysis,which was helpful to solve the complex problem of protecting and deprotecting procedures used in the traditional chemical methods for acylation of flavonoids.Consequently,whole-cell catalyst can be widely applied in the modification and reconstruction of natural products due to the simple preparation and the low cost.