Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/28006
DC FieldValueLanguage
dc.contributor譚鎮中zh_TW
dc.contributorChen-Chung Tanen_US
dc.contributor簡宣裕zh_TW
dc.contributor劉瑞美zh_TW
dc.contributor蔡珊珊zh_TW
dc.contributorShiuan-Yuh Chienen_US
dc.contributorRey-May Liouen_US
dc.contributorSan-San Tsayen_US
dc.contributor.advisor楊秋忠zh_TW
dc.contributor.advisorChiu-Chung Youngen_US
dc.contributor.author陳天來zh_TW
dc.contributor.authorChen, Tien-Laien_US
dc.contributor.other中興大學zh_TW
dc.date2008zh_TW
dc.date.accessioned2014-06-06T07:29:03Z-
dc.date.available2014-06-06T07:29:03Z-
dc.identifierU0005-0702200720550100zh_TW
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dc.identifier.urihttp://hdl.handle.net/11455/28006-
dc.description.abstractDuring screening for protease-producing bacteria, a strain designated Ⅰ1-1T was collected from a Sih-Chong-Si hot spring at Pingtung, located in the southern Taiwan. This strain formed light-yellow pigmented colonies. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. The sizes of rods were approximately 0.4-0.5 μm in diameter and 0.8-2.0 μm in length. Strain Ⅰ1-1T grew at temperatures ranging from 35 to 60℃, pH values between 6.0 and 10, and NaCl concentrations between 0-0.5%. Optimal growth condition was around 55℃, pH 7-8 and 0% NaCl. Comparison of the 16S rRNA gene sequence similarity of strainⅠ1-1T with those collected in public databases reveals that strain Ⅰ1-1T belonged to the comamonadaceae within Burkholderiales group of the β-Proteobacteria. According to the results from the phylogentic analysis, the 16S rDNA sequence similarty of strain Ⅰ1-1T clustered together with genus Tepimonas which contained species, On the basis of DNA-DNA similarity data, physiological and biochemical characteristics of API, Biolog, antibiotic susceptibility, fatty acid compositions, whole cell protein profile comparisons, as well as G+C content, the organism belonged to the protease genus Tepimonas and represented a novel species within this genus. We propose to classify Ⅰ1-1T strain as Tepidimonas taiwanesis sp.nov. The 16S rDNA sequence of strain Ⅰ1-1T clustered together with T. ignava(97.2﹪similarity)and T. aquatica.(96.8﹪similarity). Whole-genome DNA-DNA hybridization experiment was performed between strain Ⅰ1-1T and two type strains of it's nearest phylogenetic neighbors, T. ignava and T. aquatica strain. The binding levels of strain Ⅰ1-1T twords T. ignava and T. aquatica were 38.3﹪and 40.8﹪, respectively. The whole-cell protein profile of strain Ⅰ1-1T was compared with that of T. ignava(92.1﹪similarity) and T.aquatica (89.8﹪similarity). The G+C content of it's DNA is 68.1mol﹪. The predominant cellular fatty acids of strain Ⅰ1-1T were 16: 0 ( about 41﹪), 18:1 ω7 c ( about 13﹪), and summed feature 3a (16:1ω or 15:0 iso 2OH or both) (about 26﹪). A zymogram analysis of the strain Ⅰ1-1T in the alkaline medium reveales the presence of protease activity near the molecular weights of 32, 60, and 75 kDa. The condition of protease activity from the strain I1-1T in this research were assayed, and the optimal temperature was at 55℃, and optimal pH was at 8.en_US
dc.description.abstract本研究之菌種來自南臺灣屏東地區四重溪溫泉水中之一株具分解蛋白能力的好氧性嗜熱桿菌,名為Ⅰ1-1T 菌株。Ⅰ1-1T 菌株經培養後菌落顏色呈現半透明淡黃色,菌體形態大小為0.4-0.5μm x 0.8-2.0μm之革蘭氏陰性菌,具單鞭毛運動能力。Ⅰ1-1T 菌株生長溫度範圍為35-60 ℃( 最適55℃),生長pH值範圍為6-10(最適pH7-8)‚生長鹽度範圍為0-0.5%的NaCl。Ⅰ1-1T 菌株經由16S rDNA 序列分析與資料庫比對,Ⅰ1-1T 菌株屬於β-Proteobacteria 綱、Burkholderiable 目、Comamonadaceae 科。經由系統分類學分析,確認Ⅰ1-1T 菌株與Tepidimonas 菌屬中的菌種之16S rDNA 序列相似度,再經由DNA-DNA 雜合反應、生化分析之API 系統、Biolog 系統、抗生素感受性、脂肪酸組成、全細胞蛋白質電泳、G+C content分析比對結果, Ⅰ1-1T 菌株屬於Tepidimonas 菌屬中的另一新種,因此命名為Tepidimonas taiwanesis。 Ⅰ1-1T 菌株與Tepidimonas 菌屬中之二菌種T. ignava 及T. aquatica 之16S rDNA 序列相似度分別為97.2﹪及96.8﹪,而Ⅰ1-1T 菌株與T. ignava 及T. aquatica DNA-DNA 雜合結果,其相似度分別為38.3﹪、40.8﹪。另全細胞蛋白質電泳結果Ⅰ1-1T 菌株與T. ignava 、T. aquatica 之序列相似度分別為92.1﹪、89.8﹪。此外, Ⅰ1-1T 菌株脂肪酸成分以16:0 ( 約41﹪)、18:1 ω7 c ( 約13﹪)和summed feature 3a (16:1ω or 15:0 iso 2OH or both) ( 約26﹪)為主。以Ⅰ1-1T菌株之胞外粗酵素液進行SDS-PAGE電泳分離,證明Ⅰ1-1T菌株具有在鹼性下生產蛋白酶的能力,其蛋白酶分子量約為32、60與75 kDa。本研究菌株粗酵素液的蛋白酶活性最適反應溫度為55℃ ,而最適作用pH值為8之鹼性條件。zh_TW
dc.description.tableofcontents中文摘要………………………………………………………………………………… Ⅱ 英文摘要………………………………………………………………………………… Ⅲ 壹、前言 .…………………………………………………………………………………1 貳、前人研究 .……………………………………………………………………………3 一、蛋白酶的種類與應用.…………………………………………………………………3 (一)蛋白酶的種類 …………………………………………………………………... 3 (二)蛋白酶在工業上之應用 …………………………………………………………4 二、嗜熱性細菌之基礎與應用 ……………………………………………………….…5 三、細菌分類概況 …………………………………………………………………….…7 (一)表現型分類法……………………………………………………………….…..8 (二)基因型分類方法 ……………………………………………………………….9 参、材料與方法 …………………………………………………………………………14 一、研究架構流程…………………………………………………………………………14 二、嗜熱性鹼性蛋白酶生產菌種之來源、鑑定與保存 ……………………………15 (一)分離菌種來源 …………………………………………………………………15 (二)細菌培養基 ………………………………………………………………......15 (三)菌種之純化培養…………………………………………………...…………...15 三、菌落與菌體形態之特徵 ……………………………………………………………...17 (一)菌落形態之特徵 ……………………………………………...………………17 (二)菌體形態之特徵 ………………………………………………...……………17 (三)菌種形態之鑑定 ………………………………………………...……………17 1.革蘭氏染色 …………………………………………………………….…… 17 2.孢子染色 ….………………………………………………………………….18 3.鞭毛染色 ………………………………………………………………….… 18 4.運動性觀察 …………………………………………………………….… 19 5.分離菌株之掃描式電子顯微鏡 ………………………………………….… 19 (四)菌株之生理測試……………………………………………………..………….22 1.生長NaCl濃度範圍之測定 ………………………………………….……… 22 2.生長溫度範圍測定 ………………………………………………….……… 22 3.生長pH值範圍之測定 ……………………………………………….……… 22 4.氧氣生長需求之測定……………………………………………….………….22 (五)菌株之生化特徵 …………………………………………………………….25 1.細胞色素氧化酵素測定……………………………………………………….25 2.觸酶測定(Catalase test) ……………………………………………….…25 3.API-20NE系統之鑑定 ……………………………………………………… 25 4. API-ZYM系統之鑑定 ………………………………………………….…28 5.Biolog GN系統之鑑定 …………………………….………………………30 6.抗生素感受性測試…………………………………………….……………… 30 (六)嗜熱性菌16S rDNA之序列分析 ………………………………...……………34 1.I1-1T 菌株DNA萃取 ……………………………………………..………… 34 2.16S rDNA片段序列分析………………………………………….………...… 36 3.16S rDNA PCR擴增…………………………………………….…………… 40 4.洋菜膠體水平電泳……………………………………………….…………… .43 5.16S rDNA定序……………………………………………………………….…43 6.16S rDNA之序列資料與演化樹建構……………………………………….…43 7.G+C content之分析…………………………………………………………….44 (七)嗜熱性菌株之菌種比較 …………………………………………………… 45 1.菌種萃取脂肪酸之方法………………………………………………….…… 45 2.脂肪酸樣品分析 …………………………………………………….……….46 3.DNA 相似度分析 …………………………………………………………….46 (1)DNA轉漬 ………………………………………………………… 46 (2)探針製備……………………………………………………………… 47 (3)測試探針製備的效果 …………………………………………………47 (4)DNA-DNA雜合反應……………..……………………….……………48 4.蛋白質SDS-PAGE 電泳圖譜分析…………………………………….………50 (八)嗜熱菌的保存 ……………………………………………………………….53 1.菌株的活化培養 ……………………………………………………..………53 2.菌種保存: …………………………………………………………..……… 53 (1)菌種短期保存方法……………………………………………...………53 (2)菌種長期保存方法………………………………………….………… 54 (九)資料分析(Data analysis)…………………………………………………… 55 (十)酵素活性分析(Assay of enzyme activity) ……………………………….55 1.胞外粗酵素液之製備(Preparation of crude enzyme solution) ………………….…55 2.粗酵素液之透析及濃縮 ..…………………………………………..………... 56 3.酵素活性檢測…...……………………………………………………....………56 4.酵素生化特性之分析…………………………………………….…………….57 (1)作用最適溫度的測定…………………………………………..…….… 57 (2)酵素熱安定性的測定……………………………………………………57 (3)I1-1T菌株粗酵素作用最適pH的測定…………………………………57 (4)酵素pH安定性的測定……………………………………………….….58 5.基質特異性………………………………………………………………….… 58 6.酵素之活性染色 …….……………………………………………………….. 58 7.酵素SDS-PAGE-聚丙醯胺膠體電泳法……………………………………… 59 8.膠片-玻璃紙三明治組合包紮乾燥法 …………………………………….…63 肆、結果與討論 …………………………………………………………….……..…64 一、嗜熱性蛋白酶生產菌株之分離與形態觀察 …………………………….……..…64 (一)菌株之來源 ………………………………………………………………..… 64 (二)菌株形態觀察………………………………………………………………...…64 二、嗜熱性鹼性蛋白酶生產菌株生理及生化特徵………………………….…….…... 67 (一)嗜熱性鹼性蛋白酶生產菌株之生理特徵 ………………………….….…… .67 1.生長NaCl濃度範圍之測定………………………………………………..… 67 2.生長溫度範圍測定 ……………………………………………………….67 3.生長pH值範圍之測定………………….…………………………………..… 69 4.氧氣生長需求之測定……………………………………………………………...……71 5.培養基需求之測試…………………………………………………………...…71 (二)Ⅰ1-1T菌株之生化特徵 ……………………………………………………..…71 1.細胞色素氧化酵素測定………………………………………………………...71 2.觸酶測定………………………………………………………………………...71 3.API-20NE系統之鑑定 ………………………………………………….…..…72 4.API-ZYM系統之鑑定 …………………………………………………….…72 5.Biolog GN系統之鑑定 …………………………………………………..…72 6.抗生素感受性測試…………………………………………………………...…77 三、嗜熱性菌株之序列分析與演化樹建構 ………………………………………..…… 79 (一) 16S rDNA序列分析………………………………………………………… 79 (二) G+C content之分析……………….. …………………………………………80 (三)16S rDNA之序列資料比對與演化樹建構…………………………...……… 80 四、Ⅰ1-1T與Tepidimonas ignava、Tepidimonas ignava菌種之特性比較…………..…82 (一) DNA-DN A雜合分析 .…………………………………………………….…83 (二) 全細胞蛋白質電泳圖譜分析………………………………………………...83 (三)脂肪酸組成差異比較………………………………………………………… 86 (四)生理生化差異比較…………………………………………………………… 88 五、菌種命名與寄存…………………………………………………………………..… 90 六、Ⅰ1-1T菌株之酵素活性分析……………………………………………………..… 91 (一)胞外粗酵素之活性染色……………………………………………………… 91 (二)Ⅰ1-1T菌株對基質之作用特性……………………………………………… 93 (三)粗酵素最適作用溫度…………………………………………………….….. 93 (四)胞外粗酵素液熱安定性……………………………………………………….95 (五)蛋白酶最適作用pH值………………………………………………………..95 (六)蛋白酶pH值之安定性……………………………………………………… 97 (七)Ⅰ1-1T蛋白酶之酵素活性分析結論…………………………………………. 99 伍、結論………………………………………………………………………………….… 100 陸、參考文獻…………………………………………………………………………..… 105 表目錄 表1、本研究培養基配方表……………………………………….………………………..16 表2、 Luria-Bertani( LB)培養基配方…………………………………………………… 16 表3、菌體系列脫水之酒精濃度與浸泡時間………………..………………….……..… 21 表4、菌種之API 20NE系統之反應……………………………………………….………27 表5、 API-ZYM○R套組中之試驗項目與判讀依據……………………………..………… 29 表6、 抗生素之種類、濃度及抑菌機制……………………………………….…………33 表7 、16S rDNA PCR反應所需之試劑………………………………………….………. 42 表8、 PCR之加熱程式…………………………………………………………………… 42 表9 、SDS-PAGE分析方法中之各種製備物成分…………………………………….….52 表10、 SDS膠體溶液的配方…………………………………………………..….……… 62 表11、 I1-1T菌株在不同NaCl濃度培養基中生長的情形……………………..…………68 表12、 I1-1T菌株在不同溫度下生長的情形……………………………………..……… 68 表13、 I1-1T菌株在不同酸鹼度培養基中生長的情形 ………………………..……… 70 表14、Ⅰ1-1T菌株API-20NE生化分析之結果…………………………………………... 74 表15、Ⅰ1-1T菌株API-ZYM生化分析之結果…………………………………………... 75 表16、Ⅰ1-1T菌株在Biolog GN生化分析之結果…………………………………………76 表17、Ⅰ1-1T菌株之抗生素感受性分析…………………………………..…………...… 78 表18、Tepidimonas taiwanensis Ⅰ1-1T與T. ignava SPS- 1037T、T. aquatica CLN-1T 之脂肪酸組成差異比較………………………………………………………… 87 表19、T. ignava SPS- 1037T、T. aquatica CLN-1T與T. taiwanensis Ⅰ1-1T菌株之生理 生化差異比較……………………………………………………………...……… 89 圖目錄 圖1、 本研究之實驗流程………………………………………………………………14 圖2、 厭氧培養瓶………………………………………………………………………24 圖3 、Biolog GN2 Micro PlateTM碳源分析種類………………………………………32 圖4、Ⅰ1-1T菌株在20% LB培養基之生長情形…………………………………… 65 圖5、Ⅰ1-1T菌株經革蘭氏染色後之顯微鏡觀察(1000×)…………………………… 65 圖6、Ⅰ1-1T菌株之掃描式電子顯微鏡圖…………………………………………… 66 圖7、Ⅰ1-1T菌株在API ZYM的酵素分析……………………………………..…… 73 圖8、Ⅰ1-1T菌株在Biolog GN的生化分析………………………………………..… 73 圖9、Ⅰ1-1T菌株之16S rDNA序列……………………………………………………79 圖10、Ⅰ1-1T菌株之16S rDNA序列親緣關係圖………………………………..……81 圖11、以SDS-PAGE分析T. taiwanensis Ⅰ1-1T全細胞蛋白質的結果……………..85 圖12、以SDS-PAGE分析T. taiwanensis Ⅰ1-1T粗酵素液蛋白酶電泳活性染色的結 果.…………………………………………………………………………………………..92 圖13、Tepidimonas taiwanensis Ⅰ1-1T粗酵素對不同基質水解作用之活性比較……..……………94 圖14、 I1-1T菌株粗酵素液中蛋白酶活性與溫度之關係…………………………..… 94 圖15、 I1-1T菌株粗酵素液中蛋白酶安定性與溫度之關係……………………..…… 96 圖16、 I1-1T菌株粗酵素液中蛋白酶活性與酸鹼度之關係…………………………...96 圖17、 I1-1T菌株粗酵素液中蛋白酶安定性與酸鹼度之關係 …………….………… 98 附錄 附錄一Ⅰ1-1T菌株之16S rDNA序列以NCBI中的BLAST(blastn)之比對結果…. 115 附錄二 Ⅰ1-1T菌株之16S rDNA序列以RDPΠ資料庫進行比對結果…………………116 附錄三、Tepidimonas taiwanensis Ⅰ1-1T 與T. ignava SPS-1037T 、T. aquatica CLN-1T 之16S rDNA序列(1429 bp)比較 …………………117 附錄四、臺灣溫泉地熱區與地質分佈圖 ………………………………………………120 附錄五、嗜熱性細菌在細菌界之分佈圖…………………………………………………121 附錄六、高溫原核生物最佳生長溫度與最佳生長酸鹼度分佈圖………………………122 附錄七、生長溫度與微生物分佈之關係圖...…………………………………………….123zh_TW
dc.language.isoen_USzh_TW
dc.publisher土壤環境科學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0702200720550100en_US
dc.subjectTepidimonas taiwanesisen_US
dc.subjectTepidimonas taiwanesiszh_TW
dc.subjectβ-Proteobacteriaen_US
dc.subjectHot springen_US
dc.subjectAlkaline proteaseen_US
dc.subjectTaxonomyen_US
dc.subjectβ-Proteobacteriazh_TW
dc.subject溫泉zh_TW
dc.subject鹼性蛋白酶分類學zh_TW
dc.titleThermophilic Alkaline-Protease-Producing Bacterium - Tepidimonas taiwanensis sp.nov. from Southern Taiwanen_US
dc.title南臺灣溫泉之嗜熱性鹼性蛋白酶生產菌Tepidimonas taiwanensis sp.nov.之研究zh_TW
dc.typeThesis and Dissertationzh_TW
Appears in Collections:土壤環境科學系
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