Cultivation And Organic Solvent Tolerance Of Microbial Whole Cells With Catalytic Activities For Nucleoside Ester Synthesis

As a typical kind of biocatalyst, microbial whole-cells can catalyze conversions of specific substrates to desired products. This kind of catalysts has sinilar advantages to enzymes, such as high selectivity and mild reaction conditions. In addition, the use of microbial whole-cell as a catalyst could avoid the tedious enzymes purification and immobilization thereby greatly reducing industrial cost. Thus whole cell biocatalysis has gained much more attentions in recent years especially in the synthesis field of non-natural nucleoside esters with anticancer and anti-viral activities. In order to enhance the catalytic activity and stability of the whole cell catalysts in organic media, the culture conditions on the biomass and catalytica activity of microbial whole-cell in nucleoside ester synthesis were investigated in this dissertation. 5-Fluorouridine ester synthesis was adopted as a model reaction. Pseudomonas fluorescens and Aspergillus Oryzae, the common microbe in food industry, were employed as the model bacteria and fungi, respectively. The culture conditions were optimized used statistical method. And the impact of organic solvent on whole-cell catalyst were also evaluated and the screening of solvent-tolerant strains with catalytic activity in acylation was explored.Effects of seven factors in cultivation of Pseudomonas fluorescens, including yeast extract, soybean, K and Mg salts, shaking speed, p H and inoculums, on biomass and catalytic activity of the cells were investigated. The contents of yeast extract, soybean and Mg salts were showen to be the important ones by Plackett-Burman(PB) design analysis through 5-fluorouridine ester synthesis. The culture conditions of Pseudomonas fluorescens were further optimized by Response Surface Methodology(RSM). Taking into account the energy consumption and operation possibility in actual process, the optimal culture conditions for Pseudomonas fluorescens were 0.42% soybean oil, 0.07% yeast extract, 0.06% Mg salt, 0.5% ammonium sulfate, 0.4% dipotassium hydrogenphosphate, 3% inoculums, p H 7.8, 200 rpm shaking speed,. Under the optimized culture conditions, Pseudomonas fluorescens had the maximum biomass of 0.0716 g and showed the highest activity with the acylation yield of 27.4%, which were much higher than the the biomass of the cells(0.0423 g) and acylation yield catalyzed by the cells(17.9%) cultured under the initial conditions.Fungi were another important source of whole-cell catalysts. The influence of Aspergillus oryzae 3.5232 culture conditions including yeast extract, Tween 80, K/Mg salts, shaking speed, p H and inoculums, on its biomass and the corresponding acylation yield catalyzed by the cells were investigated. The contents of yeast extract, Mg salts and inoculums were found to be the important ones by Plackett-Burman(PB) design analysis. Further optimization of culture conditions of Aspergillus oryzae 3.5232 were made by Response Surface Methodology(RSM) and the optimal culture conditions of Aspergillus oryzae 3.5232 were 0.07% yeast extract, 0.6% Tween 80, 0.03% Mg salt, 0.5% dipotassium hydrogenphosphate, 4% inoculums, p H 7.0, 210 rpm shaking speed. Under the optimized culture conditions, Aspergillus oryzae 3.5232 had the maximum biomass of 0.2899 g and the acylation yield catalyzed by the cells reached the highest value of 5.87%, which were much higher than those by the cells cultivated without cultivation(0.2284 g and 4.36%, respectively).Biocatalysis in nonaqueous medium showed that the catalyst stability and efficiency were influenced by their organic solvent tolerance. To obtain strains with organic sovlent tolerance, four bacteria strains, Pseudomonas stutzeri, Pseudomonas putida coli, Pseudomonas aeruginosa and Pseudomonas fluorescens, was treated with solvent-shock methods. The result showed that Pseudomonas fluorescens gained good toluene tolerance after cultivations by toluene shock method, with a better catalytic activity compared to the original strain. In addition, this strain can also be tolerant of many different kinds of organic solvents, such as acetonitrile, acetone and t-butanol and the like.This study established new culture systems for preparation of whole-cell biocatlaysts with acylation activities, thus providing an effective route for synthesis of nucleoside esters in industrial scale. In addition, it expands the application ranges of microbial commonly in food production to biocatalzed organic synthesis.