| Deinococcus radiodurans is one of the most radioresistant organisms with extraordinary resistance to extreme stresses, and is used as a model bacterium for research in the field of adaptation mechanism of organisms to exteme environments. The extreme anti-oxidative ability of D. radiodurans plays an important role in its extraordinary resistance to the extreme environments. Antioxidant system related enzymes and their products have a great potential in the areas of medicine, dietary supplement and other human health applications. Our previous work has revealed the strong antioxidant activity of carotenoids in D. radiodurans and identified several pivotal carotenoid biosynthetic genes (enzymes). According to the results of homologue sequence analysis, there exists a predicted CrtE homologue gene in D. radiodurans, which encodes geranylgeranyl diphosphate synthase (GGPPS). Geranylgeranyl diphosphate is a key precursor for the carotenoid and other isoprenoids biosynthesis.In this thesis, the functional identification of the CrtE gene is carried out by bioinformnatics and molecular technology. By homologue analysis, dr1395and dr0932are the two possible genes for encoding the GGPPS, and the dr1395is more relative to the identified GGPPS gene in T. thermophilus. Furthermore, the dr1395is identified as the CrtE gene encoding GGPPS in D. radiodurans by knockout mutation, compensational mutation, protein expression, and analysis of carotenoid products by HPLC.Cryogel chromatography is a novel bioseparation technology, owning both advantages of expanded bed and fixed bed chromatography, and integrating the processes of centrifugation, filtration, concentration, and chromatography into a profile. Objective biomolecules could be recovered directly by cryogel chromatography for the merits of low pressure drop, short residence time, high selectivity and good separation efficiency. Characterization of cryogel was normally by experiments, while the methods of combining experiments and mathematical model are focused by most researchers. The homogenate of His-tagged expression enzyme GGPPS was directly purified by this novel chromatographic technology with a Cu2+-IDA metal chelated cryogel. The average purity of the GGPPS by this one step chromatography profile is about91.4%. The Cu2+-IDA cryogel was characterized by experiments and capillary model. The data calculated by the model agree well with the experimental ones, indicating the effectiveness of the model.As the two typical antioxidants in D. radiodurans, carotenoid and superoxide dismutase (SOD) have prosperous applications in the future. In this thesis, effiecient and effective preparation methods were investigated. For carotenoids, light radiation was introduced to induce their biosynthesis. Low dose of light radiation effectively enhanced the biosynthesis of carotenoid. An light radiation with intensity of100-150μmol·m-2·s-1and duration of0.5hr, and post-induction cultivation of2hr benefits for the biosynthesis of carotenoid, while the over-dose of light radiation may cause converse results. As for metal ions, Fe3+, Zn2+and Cu2+induce the biosynthesis of carotenoids apparently, while Fe2+and Mn2+act as negative regulators. Generally, Mn-SOD is the main SOD type in D. radiodurans. Mn2+is introduced as inducing reagent to enhance the productivity of Mn-SOD, and Cu2+-IDA cryogel is also used as chromatographic matrix for protein separation purpose.In a word, the main point of this thesis includes identification of the key gene in the carotenoid biosynthetic pathway, capture of the expressed protein GGPPS with a novel cryogel chromatographic technology, promotion of the carotenoid biosynthetic process by inducer factors of light and metal ions, and purification of the Mn-SOD directly from D. radiodurans. |
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