Klebsiella oxytoca SYSU-011是一株由南台灣電鍍工廠廢水中分離出之氰化物分解菌。K. oxytoca可以利用九種不同之腈化合物[包括acetonitrile (100 mM)、benzonitrile (1 mM)、butyronitrile (100 mM)、glutaronitrile (50 mM)、methacrylnitrile (100 mM)、phenylacetonitrile (1 mM)、propionitrile (25 mM)、succinonitrile (25 mM) 及valeronitrile (50 mM)]作為其生長所需的氮源。其中又以acetonitrile及propionitrile最容易被此菌分解。本研究係以氣相層析法(GC) 和蛋白質體分析來探討K. oxytoca分解acetonitrile及propionitrile的代謝途徑。由GC分析結果發現分解腈化合物的產物中，有amide、carboxylic acid和ammonia的產生；其中amide為中間產物，而carboxylic acid和ammonia則為分解腈化物的最終產物。推測K. oxytoca是以nitrile hydratase及amidase路徑來分解腈化物。此外，培養在含有25mM succinonitrile環境下的菌體，利用雙向膠體電泳（two-dimensional polyacrylamide gel electrophoresis）來分析比對本菌基因表現出蛋白質的種類與數量之不同，確定有明顯被誘導出現變化的蛋白質共23種。經由質譜分析後blast所比對出來的蛋白質共有11種，其餘的蛋白質點並沒有比對到合適的蛋白質。Succinonitrile所誘導出蛋白質如：superoxide dismutase、glutathione s-transferase、dyp-type peroxidase、metal binding protein PsaA（主要是在傳遞金屬離子）、LraI及FepA（無機離子的傳遞），防止菌體受到氧化性傷害。另外還有三個蛋白質glutamine synthetase、methylenetetrahydrofolate reductase和dihydroxyacid dehydratase具有合成胺基酸的功能，另外ribosomal protein L9目前功能尚不清楚，而nucleoside triphosphates kinase則是催化nucleoside diphosphates轉換成nucleoside triphosphates的nonsubstrate specific conversion。另外超氧岐化酶活性（superoxide dismutase, SOD）及glutathione S-transferase（GST）酵素活性測定結果顯示，培養在25mM succinonitrile環境下SOD 酵素及GST酵素活性皆提分別升為2.5倍及1.8倍，而且也發現ROS濃度有上升現象。這些結果顯示出succinonitrile可能會造成氧化性傷害而誘導出一些抵抗氧化性傷害的蛋白質來保護菌體不受傷害。

The cyanide-degradation bacteria Klebsiella oxytoca SYSU-110 was isolated from the waste water of a metal-plating plant in southern Taiwan. K. oxytoca can utilize many nitrile compounds [including acetonitrile (100 mM), benzonitrile (1 mM), butyronitrile (100 mM), glutaronitrile (50 mM), methacrylnitrile (100 mM), phenylacetonitrile (1 mM), propionitrile (25 mM), succinonitrile (25 mM) and valeronitrile (50 mM)] as its sole nitrogen source. In this study, we found out that K. oxytoca was capable of degrading acetonitrile and propionitrile. Frome GC analysis, we recognized amide was an intermediate compound, while the carboxylic acid and ammonia were the final end-products. Therefore, we presume that K. oxytoca biodegraded nitrile compounds by two enzymes, the nitrile hydratase and amidase. We also analyzed the total cell proteins by 2-D polyacrylamide gel electrophoresis after the cells were cultured in medium containing 25mM succinonitrile. There were 23 proteins could be induced or overexpressed by nitrile and we had identified 11 by Mascot Peptide mass Fingerprint and Blast. Six proteins that can protect the cells from oxidative damage are: superoxide dismutase, glutathione s-transferase, dyp-type peroxidase, metal binding protein PsaA (that can transport metal ions into the cells), LraI, and FepA (used to transport inorganic ions into the cells). Three enzymes glutamine synthetase, methylenetetrahydrofolate reductase,　and dihydroxyacid dehydratase were used to synthesize amino acids. One protein was identified as ribosomal protein L9. The last identified protein is nucleoside triphosphates kinase which can convert nucleoside diphosphates to nucleoside triphosphates non-specifically. From the activity analysis, superoxide dismutase and glutathione S-transferase activities were escalated when the cells were cultured in 25mM succinonitrile, and the concentration of ROS has rise. These results suggested that succinonitrile could cause oxidative damage to the cells and induce some anti-oxidative damage proteins to protect them.

中文摘要………………………………………………………………………II
英文摘要………………………………………………………………………IV
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表目錄…………………………………………………………………IX
前言……………………………………………………………………1
材料與方法………………………………………………………………………14
結果與討論………………………………………………………………………22
參考資料………………………………………………………………………30
圖………………………………………………………………………46
表………………………………………………………………………61
附錄……………………………………………………………………62

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