臺灣博碩士論文加值系統

本研究針對台灣地熱區進行嗜熱性微生物之研究，由台灣東部的上里溫泉中，篩選出一株可生長於50 oC的黃色菌落菌株 NTU-1589。藉細菌16S rDNA 保守片段的擴增及序列的比對，發現與已發表菌株 Thermomonas haemolytica A50-7-3T 相似度為 94.5 %，與 Pseudoxanthomonas taiwanensis CB-226T 和 Lysobacter gummosus LMG 8763T 相似度均為 94.2 %。利用形態觀察、生理、生化以及遺傳特性分析，發現有不同於標準菌株的地方。其菌株呈桿狀、為格蘭氏陰性細菌、具有單一端鞭毛；生長溫度範圍為 30 oC 到 50 oC，最適生長溫度為50 oC；生長酸鹼值範圍為 pH 6-10，最適酸鹼值為 pH 8；耐鹽度為 1 %，在培養基含2 % NaCl則無生長現象；能利用、D-Galactose、D-Melibiose、myo-Inositol 當作生長的單一碳源；具有 a-glucosidase、elastinase 活性；脂肪酸組成主要為 C16:0 、C16:0 iso 和 C18:0；對於抗生素 Polymyxin B 具有抗性：DNA G+C 含量為 68.7 mol %。藉由上述差異及親緣關係樹的建立，推測 NTU-1589 為 Pseudoxanthomonas 屬之新種菌株。

This study is aimed to research the thermophile from hot springs in Taiwan. One yellow pigmented organism with growth temperature around 50 oC, was isolated from hot springs named Shang-Li in Taitung. According to the resμlt of 16S rDNA sequence analysis, the strain showed 94.5 % to Thermomonas haemolytica A50-7-3T and 94.2 % sequence similarity to Pseudoxanthomonas taiwanensis CB-226T and Lysobacter gummosus LMG 8763T. There are differences between the strain NTU-1589 and type strains on morphological observation, physiological, biochemical and genetic analyses. The strain was gram-negative rods and had a polar flagellum. Growth occurs between 30 oC and 50 oC, with optimal temperature at 50 oC. The pH range for growth was 6-10, with an optimal at pH 8. Growth does not occur in modified thermus plate contain 2 % NaCl. The isolate can utilize D-Galactose, D-Melibiose and myo-Inositol, and have α-glucosidase and elastase activities. The major composition of fatty acids are C16:0, C16:0 iso and C18:0. It is resistant to polymyxin B. The DNA G+C contain is 68.7 mol %. Based on these differences and phylogenic tree, the strain NTU-1589 coμld be considered as novel species related to the genus of Pseudoxanthomonas.

中文摘要 I
英文摘要 II
縮寫對照表 III
表目錄 VI
圖目錄 V
第一章 前言 1
Ⅰ. 嗜熱性微生物 1
Ⅱ. 嗜熱性微生物之研究 2
Ⅲ. 嗜熱性微生物具熱穩定酵素 3
Ⅳ. PROTEOBACTERIA 5
V. 研究目的與架構 6
第二章 材料與方法 8
一、材料 8
(一) 菌種 8
(二) 藥品與酵素 8
(三) 培養基 8
(四) 儀器 9
二、方法 10
(一) 樣本採集 10
(二) 嗜熱菌之分離、純化與保存 10
1. 菌種之分離與純化 10
2. 菌種之保存 11
(三) 菌種鑑定 12
1. 菌種形態觀察 12
1.1 菌落形態觀察 12
1.2 格蘭氏染色 12
1.3 移動性測試 13
1.4 穿透式電子顯微鏡觀察 13
2. 菌種生理生化特性分析 13
2.1 溫度生長範圍測試 13
2.2 酸鹼度生長範圍測試 13
2.3 耐鹽度測試 14
2.4 單一碳源的利用能力 14
2.5 澱粉水解能力測試 15
2.6 蛋白酶 (Protease) 水解測試 15
2.7 白明膠水解酶( Gelatinase )活性測試 15
2.8 幾丁質 (Chitin) 水解測試 16
2.9 甲殼素 (Chitosan) 水解測試 16
2.10 Tween 20 及 Tween 80 水解測試 16
2.11 彈力蛋白酶 ( elastinase ) 活性測試 17
2.12 氧化酶 (Oxidase) 活性測試 17
2.13 觸酶 (Catalase) 活性測試 17
2.14 硝酸鹽及亞硝酸鹽還原 17
2.15 API-ZYM 酵素活性檢測 18
2.16 脂肪酸組成分析 18
2.17 抗生素測試 19
3. 菌種遺傳分析 19
3.1 16S rDNA 序列分析 19
3.1.1 細菌 genomic DNA 之抽取 19
3.1.2 PCR 反應 20
3.1.3 PCR 反應 21
3.1.4 16S rDNA 片段序列分析 22
3.2 DNA G+C 含量分析 22
3.2.1 細菌 genomic DNA 的抽取 22
3.2.2 高效率液相層析分析 23
3.3 DNA相似度分析 23
3.3.1 探針製備 23
3.3.2 DNA 轉漬 24
第三章 研究結果 26
1. 菌種取得與保存 26
2. 菌種形態分析 26
2.1 菌落形態觀察 26
2.2 格蘭氏染色 26
2.3 移動性測試 27
2.4 穿透式電子顯微鏡觀察 (負染法) 27
3. 菌種生理特性分析 27
3.1 溫度生長範圍測試 27
3.2 酸鹼度生長範圍測試 28
3.3 耐鹽度測試 28
3.4 單一碳源利用測試 28
3.5 澱粉水解能力測試 29
3.6 蛋白酶活性測試 29
3.7 白明膠水解測試 29
3.8 幾丁質水解測試 29
3.9 甲殼素水解測試 30
3.10 解脂酶活性測試 30
3.11 彈力蛋白水解測試 30
3.12 氧化酶活性測試 30
3.13 觸酶活性測試 31
3.14 硝酸鹽及亞硝酸鹽還原 31
3.15 API-ZYM 酵素活性檢測 31
3.16 脂肪酸組成分析 32
3.17 抗生素測試 32
4. 菌種遺傳特性分析 33
4.1 DNA G+C 含量分析 33
4.2 16S rDNA 序列分析 33
4.3 DNA - DNA 雜合反應 33
4.4 16S rDNA 序列系統演化之分析 34
第四章 討論 35
1. NTU-1589 與對照菌株的差異性 35
2. 酵素應用潛力 36
3. 脫氮反應 37
4. DNA 相似度與親緣演化樹之差異 38
參考文獻 40
圖表 53
附錄一 77
附錄二 78
附錄三 79
附錄四 80
附錄五 82

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陳懋彥 2002 臺灣嗜熱性細菌之研究。國立臺灣大學植物學研究所博士論文

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