| Traditionally, the chemical catalyst-based processes were widely used in the productionof bulk chemicals, fine chemicals, pharmaceutical intermediates and even food industry. Inrecent years, there has been an increasing public concerns about―green‖technologies to makethe related processes more safe and non/low toxic, especially for food and drug productions.Due to the mild reaction conditions, environmental friendliness and less by-products,biocatalysis attracts more and more attentions. However, the high cost of enzymatic catalysistechnologies largely limits their industrial applications. As the other kind of biocatalysts,whole-cell catalysts avoid tedious enzyme separation processes and provide a natural cellularenvironment for keeping its high activity. However, compared with the current researchsituations of enzymatic catalysis, studies on whole-cell catalysis in non-aqueous mediumweremuchless and urgently need to be expanded. Moreover, the influence of variousnon-aqueous media on whole-cell catalysts has not been reported yet. In this dissertation, thesynthetic capabilities of microbial whole-cell catalysts in non-aqueous medium were exploredby using the long-chain esters synthesis of nucleosides, a kind of important compounds in thefields of medicine and health care products, as model reactions; The influence of both organicsolvent and ILs on catalytic performances and substrate recognition of fungal and bacterialwhole-cell catalysts were investigated; The effects of the solvents on the surface morphologyof whole-cells and the cell structures in the growth phases were also studied and discussed forthe first time; In addition, we established an efficient whole-cells biocatalysis system forestersynthesis of different nucleoside substrates.Whole-cells from different sources showed varied catalytic properties in synthesis ofaracytidine laurate. Four strains with the catalytic ability were successfully screened out from7bacteria and11fungi. Among them, Pseudomonas fluorescens GIM1.209and Aspergillusoryzae3.5232showed to be the best bacterial and fungal strains, respectively, in terms ofrelatively high catalytic efficiency.The types of non-aqueous medium have an important effect on the catalytic activity ofwhole-cell catalysts. In organic mediums, the whole-cell catalysts showed low activity buthigh regioselectivity. In the optimal organic mixture isopropyl ether/pyridine, the5’-regioselectivity of P. fluorescent whole-cells was higher than99%. The use of pure ILs asreaction media resulted in low reaction efficiency due to their high viscosity. WhenILs-containing mixtures were employed, the catalytic efficiency of whole-cells were greatly improved. It was found that the anionic and cationic species of the ionic liquids had asignificant influence on the reaction. For P. fluorescens whole-cells,[BMI][PF6]-containingsystems gave the highest initial reaction rate (2.34mmol/L h) and product yield (81.05%),followed by [OMI][BF4]-containing system. In halide anion based IL-containing systems,whole-cells showed no catalytic activities. For A.oryzae whole-cell catalyzed reactions, boththe initial reaction rate and product yield declined as the sequence of [BMI][PF6]>[OMI][BF4]>halide ion based ILs. IL content also has a significant effect on the synthesis ofara-C luarate catalyzed by whole-cells. The initial reaction rate and product yield of thesynthesis reaction of ara-C luarate catalyzed by both P. fluorescent and A. oryzae whole-cellswere significantly decreased as the [BMI][PF6] content increasing; When [OMI][BF4] contentwas increased, the initial reaction rate and product yield of the reaction were increased for P.fluorescent whole-cells, while reaches a peak value at the IL content of50%for A. Oryzae.In the non-aqueous medium tested, whole-cell catalyst showed various substraterecognition features on nucleoside substrates of different structures. In ester synthesis of2’-deoxy-nucleoside analogues by P. fluorescence, the initial rate, product yield and5’-regioselectivity of the reaction showed a downward trend as the increase of atomicpolarities of the5-substituent group in the substrates. For those bearing2’-hydroxyl groups,the catalytic efficiency of the whole-cells declined but5’-regioselectivity increased due to thesteric effect of the hydroxyl group.Investigations of the toxic effects of non-aqueous media on the whole-cells showed that,after pre-cultured by organic solvent, P. fluorescens cell surface had a serious shrinkphenomenon. The cell length elongated significantly and the cell surface became little roughwith small depressions or cell damages. It was proven that organic solvents can affect thepermeability of cell membranes by damaging the cell structure. Organic solvents are alsotoxic to the cell growth. Similar effects on both cell surface and growth of the fungus A.oryzae cells were also found.After pre-incubation in ILs, P. fluorescenscell surface also showed serious shrinkphenomenon, indicating that ILs also have toxic effects on the cell morphology. And theirtoxicity is closely related to their types. Rough surface with horizontal pits made the rod cellslook like a bead chain and resulted in significant variation in cell sizes. Among the tested ILs,[OMI][BF4] and [BMI][PF6] showed relatively low inhibition effects on cell growth. ILs alsoaffect the mycelium morphology and growth of A. oryzae significantly.[BF4]--based ILscompletely inhibited the growth of A. oryzae cells. And among other anion-based ILs, halideions showed minimum inhibitory effects on cells.[BMI][PF6] exhibited good biocompatibility to the two kind cells, considering its effects on both the cell morphology and growth.This research not only enriches the theoretical knowledge of whole-cell catalysis, butalso provided new synthetic alternative for synthesis of polyol esters (such as nucleosides). Inaddition, this research has a guiding significance for the whole-cell catalytic biotechnology inthe production of high-quality and safety compounds, especially in food and pharmaceuticindustries. |
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