臺灣博碩士論文加值系統

Pseudomonas putida SH1能夠利用與酚作為唯一的碳源與能源而生長，本研究則在探討P. putida SH1以及酚分別當作唯一的碳源生長時，轉化之產物分析與生成途徑。P. putida SH1以為唯一碳源生長在MSB（minimal salts basal）培養基的最適濃度為0.1%，而以酚為唯一碳源生長的最適濃度則為0.05%；當以0.1%的為唯一碳源生長時，依其生物轉化之主要產物indigo量最多時，加入的最適濃度為2.5 mM，相同的實驗，碳源改為0.05%酚時，轉化的最適濃度則為1 mM。
為了使不同碳源的生物轉化反應能夠互相比較，在本研究中以薄層液相層析及高壓液相層析的實驗中，碳源的濃度皆為0.05%，濃度為1 mM；P. putida SH1以為碳源在50毫升MSB的生物轉化中，產物有2-hydroxy indole（oxindole）、indigo、isatin及七個有顏色的未知化合物，隨著時間的增長的量越少，反應大約十小時用完，在25小時內產物的量則隨時間增長而增加；當碳源為酚時菌株轉化的產物有2-hydroxy indole、indigo、isatin及三個有顏色的未知化合物，其有顏色的產物種類比以為碳源的生物轉化至少少四種，在50小時內產物的量隨著時間增長而增加，但的量在轉化100小時後尚未用完；顯示P. putida SH1以為唯一生長碳源的培養基中轉化的效率比含酚的培養基快20倍。
P. putida SH1以為碳源參與生物轉化的酵素可能是naphthalene dioxygenase，推測其路徑可能為naphthalene dioxygenase催化進行hydroxylation反應，使接上了兩個hydroxy group轉變為2,3-dihydroxy-2,3-dihydroindole，經過脫水反應形成hydroxy indoles，indigo由兩個3-hydroxy indole（indoxyl）分子經由空氣的氧化聚合形成或isatin的氧化形成，indirubin則由2-hydroxy indole及3-hydroxy indole聚合而成，isatin 由2,3-dihydroxy-2,3-dihydroindole、2-hydroxy indole及3-hydroxy indole的氧化及indigo或indirubin的分解形成，至於有顏色的產物（Unk. 1、Unk. 2、Unk. 3、Unk. 6、Unk. 7及Unk. 7-1）並不清楚其真正生成的途徑。P. putida SH1以酚為碳源的生物轉化反應中推測經由phenol hydroxylase的催化進行hydroxylation，使接上一個hydroxy group形成hydroxy indoles，indigo及isatin的生成則可能亦依上述途徑，有顏色的產物（Unk. p2、Unk. p3及Unk. p4）的結構與生成尚不清楚。
與文獻比較，P. putida SH1與其他微生物的生物轉化確實有所不同，在其他文獻中生物轉化的產物，不外乎為2-hydroxy indole、3-hydroxy indole、藍色的indigo、紅色的indirubin以及黃色的isatin，而P. putida SH1的生物轉化之產物除了上述以外還有許多其他有顏色的產物，但因不知這些新生成產物之化學結構，故其生成途徑與機制則需進一步研究才能確認。

Pseudomonas putida SH1 was capable to utilize naphthalene and phenol as sole carbon source and energy to grow. This study was focused on the transformation of indole by P. putida SH1 grown from naphthalene or phenol. The major intermediates and products from indole tranaformation were characterized and the pathways were proposed. The optimal concentration of naphthalene in MSB (minimal salts basal medium) for P. putida SH1 to grow was 0.1% and that for phenol was 0.05%. According to the major products, indigo, the optimal biotransformation concentration of indole in the preseuce of 0.1% naphthalene was 2.5 mM. When the carbon source was changed to 0.05% phenol, the optimal concentration of indole was 1 mM.
In order to compare the intermediates and products of indole transformation induced by naphthalene or phenol, the concentration of carbon source and indole was 0.05% and 1 mM, respectively, in both the analysis by thin layer chromatography and high pressure liquid chromatography. The products of indole transformation by P. putida SH1 grown in naphthalene were oxindole, indigo, isatin, and seven other unknown colored compounds. The indole was used up at 10 hours and the products increased up to 25 hours. The products of indole transformation by P. putida SH1 induced by phenol were 2-hydroxy indole (oxindole), indigo, isatin, and three other unknown color compounds. The varieties of the color products induced under the presence of phenol were less than those induced by naphthalene. The indole was not used up at the transformation experiment for 100 hours.
The proposed enzyme induced by naphthalene for indole transformation was naphthalene dioxygenase in P. putida SH1. Indole may be oxidized by naphthalene dioxygenase to form 2,3-dihydroxy-2,3-dihydroindole, which was then spontaneously dehydrated to give hydroxy indoles. Condensation of two molecules of 3-hydroxy indole (indoxyl) followed by air oxidation or oxidation of isatin led to the production of indigo. The condensation of 2-hydroxy indole and 3-hydroxy indole yield indirubin. Isatin may be formed by the oxidiation of 2,3-dihydroxy-2,3-dihydroindole, 2-hydroxy indole, or 3-hydroxy indole or by the decomposition of indigo or indirubin. No evidence was found for the formation of other color products and they may be formed from hydroxy indoles. Indole may be oxidized by phenol hydroxylase to form hydroxy indoles in the phenol-grown SH1 cells. The formation of indigo and isatin may go through the same pathway as descirbed above.

中文摘要……………………………………………………………………Ⅰ
英文摘要……………………………………………………………………Ⅲ
目錄…………………………………………………………………………Ⅴ
圖目錄………………………………………………………………………Ⅶ
表目錄………………………………………………………………………Ⅸ
論文名詞對照表……………………………………………………………Ⅹ
壹、緒論……………………………………………………………………1
（一） 芳香族化合物……………………………………………………1
（二） 芳香族化合物的微生物分解……………………………………2
（三） 的生物轉化與加氧酵素……………………………………3
（四） 生物轉化的應用……………………………………………5
（五） 研究動機與大綱…………………………………………………6
貳、材料與方法……………………………………………………………8
（一） 微生物與培養基…………………………………………………8
（二） 生長碳源測試……………………………………………………8
（三） 碳源與濃度測試……………………………………………9
（四） 細菌培養…………………………………………………………9
（五） 生物轉化反應與產物………………………………………10
（六） TLC分析…………………………………………………………10
（七） HPLC分析…………...…………………………………………..10
（八） 純化生物轉化之產物………………………………………11
（九） 生物轉化產物性質測定…………………………………………12
（十）化學藥品與耗材……..…………………………………………..13
（十一）儀器設備………………………………………………………13
參、結果……………………………………………………………………14
（一） 生長測試…………………………………………………………14
（二） 碳源及濃度測試……………………………………………15
（三） 生物轉化反應與產物………………………………………15
（四） 以TLC片分析生物轉化產物……...……………………...16
（五） 以HPLC分析生物轉化產物…………………..………….17
（六） 純化生物轉化之產物………………………………………18
（七） 生物轉化產物的性質測定………………………………………18
肆、討論……………………………………………………………………19
伍、參考資料………………………………………………………………22
陸、圖………………………………………………………………………29
柒、表………………………………………………………………

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