| Most of the nucleoside analogs, such as1-β-D-arabinofuranosylcytosine (ara-C), are ofhigh hydrophilicity and thus have major clinical shortfalls in the treatment of solid tumors,since they cannot easily transfer across the cell membrane by passive diffusion and mightundergo a rapid enzymatic deactivation by deaminas in plasma. To improve the clinicalefficiency of those analogs, lipophilic modification has gained much attention as a promisingstrategy. Compared with enzymatic modifications, whole cell-mediated acylation ofnucleoside has higher efficiency and lower cost due to reduced processing steps. In ourpervious researches, Aspergillus oryzae3.5232were proved to be a new efficient whole cellbiocatalyst with high regioelectivity to acylation of3’-hydroxy group in nucleoside. But theenzymatic mechanism of the whole cell biocatalysis for nucleoside acylation still remainsunexplored. In this dissertation, the localization of lipases with synthetic activity inAspergillus oryzae was investigated and the effects of different inducers on the lipaseproduction during cultivation of the cells were studied. In addition, the lipase with acylationactivity in different fractions of the cells were purified and identified.Through ultrasonication and triton X-100extraction, cell fractions (cell wall, cellmembrane and cytoplasm) were prepared. The acylation of ara-C was regarded as anacylation model of nucleoside analogs. HPLC analysis showed that the three cell fractions hadenzyme activities towards ara-C acylation. Substrate conversions of the reaction catalyzed bycell fractions were lower than that by Aspergillus oryzae whole cells. Highest conversion(19.55%) was achieved by using cytoplasm after a36h-reaction, which was higher than thatby cell wall (7.35%) and cell membrane factions (6.89%). Compared with whole cellmycelium, total enzyme activity loss of the cell fractions was42.43%. The cell fractionsshowed5’-regioselectivity (about82%), which was rather different from whole cell (about27%).Analysis of the hydrolyzing activity of the cell fractions showed that cytoplasmic fractionpossessed the highest activity, followed by the cell wall and cell membrane. Culture timeshowed obvious effect on the hydrolyzing activity of the lipase in cell fractions. Enzymaticactivities of cell membrane and cell wall reached the highest level at84h, being of314.48 U/g and443.53U/g, respectively. The highest enzymatic activity of cytoplasm (2180.66U/g)was obtained at72h. Analysis of the synthetic activity of the cell fractions in acylation ofara-C showed that total conversion rate of ara-C catalyzed by cell fractions reached thehighest of36.50%after cultivation for48h with tween80. The cytoplasmic synthetic activityincreased with increasing the cultivation time. But the activity of the enzymes located in cellwall and membrane firstly increased but then decreased, the highest values of which were10.50%(36h) and12.11%(72h), respectively. Based on these data, a model was establishedto help understand the dynamic metabolic of synthetic lipase in Aspergillus oryzae mycelium.The contents of substrate and products were monitored by HPLC. Results showed that Ara-C,3’-O-vinyl ara-C and5’-O-vinyl ara-C existed not only on cell wall and cell membrane, butalso in cytoplasm, which strongly suggested that cytoplasmic lipase also catalyzed theacylation of ara-C. A model of dynamic concentration changes of ara-C,3’-O-vinyl ara-C and5’-O-vinyl ara-C was thus constructed.Synthetic and hydrolytic activities of the lipases in Aspergillus oryzae were not coincident.The sequence of hydrolytic activity of different cell fractions were: cytoplasm>cellmembrane>cell wall. For synthetic activity of the enzymes, the order was: cytoplasm>cellmembrane>cell wall. The changing tendency of two activities was not coincident.Analysis of the hydrolytic activity of the lipases of the cells cultured with various carbonsources showed that glucose can inhibit the lipase with hydrolytic activity in cell wall andcytoplasm, but showed little effect on the lipase located in cell membrane. The lipase withhydrolytic activity was produced during the growth of mycelium. When the myceliumstopped growing, the detected hydrolytic activity of lipase declined in all cell fractions. Thesurfactant tween80can induce or maintain lipase activity towards acylation of ara-C in allcell fractions. The substrate conversion of the reaction catalyzed by cell wall of washedmycelium reached67.95%, only a little lower than that by the whole cell.By SDS-PAGE analysis, the effects of different carbon sources on induced expressions oflipase on cell wall, cell membrane and in cytoplasm were studied. The types and theconcentrations of surfactants had different effects on the expressions of lipases in differentcell fractions. When glucose was added into the culture media, however, the production of thelipase with synthetic activity was repressed. Expression level of the lipase on the membrane maintained a permanent level, however, the level on cell wall decreased following the time.The cytoplasmic synthetic activities increased as culture time extended. A simple dynamicmodel for this lipase metabolism within mycelium was constructed.Using hydrolytic activity as index, a lipase with molecular weight of36kDa was isolatedand purified to single band on SDS-PAGE, which showed high hydrolytic activity but littlesynthetic activity. The lipase with activity towards acylation of ara-C was characterized byTOF-MS, being matched as an unnamed protein product [Aspergillus oryzae RIB40], accessNo: gi|837646493, molecular weight:21474.9kDa, isoelectric point:6.01.This research not only enriches the theories of the whole cell biocatalysis, but also providesa new green route for synthesis of bioactive nucleoside derivatives. |
PDF Full Text Request