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研究生:林昌逸
研究生(外文):Chang-Yi Lin
論文名稱:Rheinheimera新菌種抗微生物活性物質之探討
論文名稱(外文):Investigating antimicrobial activity in Rheinheimera sp. nov.
指導教授:陳文明陳文明引用關係
指導教授(外文):Wen-Ming Chen
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:水產食品科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:108
中文關鍵詞:Rheinheimera sp.抗菌活性過氧化氫L-離氨酸氧化酶
外文關鍵詞:Rheinheimera sp.Antimicrobial activityHydrogen peroxideL-lysine oxidase
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本實驗室之分離菌株GR5經由抗菌活性測試結果發現具有抗菌活性能力,初步分析16S rRNA基因序列分析,菌株GR5為Rheinheimera菌屬,此菌屬已發表的菌種中並沒有指出具有抗菌活性的能力,因此菌株GR5的抗菌活性物質值得我們進一步的探討,另外也進行新菌種的鑑定。
菌株GR5具有很好的抗菌能力,對於革蘭氏陽性菌、革蘭氏陰性菌、酵母菌、藻類和GR5本身都具有抗菌(藻)的能力。菌株GR5會合成具有抗菌活性的大分子,此大分子藉由產生過氧化氫來達到抗菌能力,且抗菌能力會受到觸酶的抑制。抗菌活性只能在複合培養基或含有L-離胺酸的化學合成培養基中才呈現。菌株GR5的抗菌活性物質為分子量71 kDa的單體蛋白質,其pI值為3.68。將菌株GR5的抗菌活性物質進行液相層析串聯質譜儀(Liquid chromatography-tandem mass spectrometry)分析比對後,得到一條含有19個胺基酸的序列片段與海洋菌Pseudoalteromonas tunicata D2的抗菌蛋白Alp P和Marinomonas mediterranea的抗菌蛋白marinocine有最高的相似度,此兩種抗菌蛋白屬於L-離胺酸氧化酶。因此菌株GR5的抗菌活性大分子可能為L-離胺酸氧化酶。這是首次發表淡水細菌的抗菌活性是藉由L-離胺酸氧化酶之催化作用進而產生過氧化氫以達到抗菌效應的研究。
菌株GR5菌落為黃綠色,革蘭氏陰性菌,兼性厭氧,桿菌,具有單端單鞭毛,呈高度運動性。生長溫度為15-37 oC,生長pH值為7-8,生長鹽度為0-2 %。經由16S rRNA基因序列與演化分類學分析結果,菌株GR5屬於Rheinheimera菌屬,與最鄰近的已知模式菌株Rheinheimera texasensis A62-14BT有最高相似度98.1 %。主要脂肪酸組成為Sum In Feature 3(C16:1 ω7c/C16:1 ω6c)、C16:0和C12:0 3-OH,主要的醌為泛醌Q-8,DNA G+C含量為51.9 mol%。根據遺傳特性、生理與生化等特性鑑定發現,菌株GR5為Rheinheimera菌屬中的一新種,故將其命名為Rheinheimera aquatica,模式菌株為GR5T(=BCRC 80081T=LMG 25379T)。
In this study, based on the antibiogram assay, strain GR5 possesses potent antimicrobial activity. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GR5 belonged to the genus Rheinheimera. This is the first report on Rheinheimera sp. producing antimicrobial activity. Investigating antimicrobial activity in strain GR5 and characterization of this novel species are further performed.
Based on the antibiogram assay, strain GR5 possesses a broad spectrum of antimicrobial activity including Gram-positive and Gram-negative bacteria, yeast, algae, and strain GR5 itself. Strain GR5 can synthesize a macromolecule with antimicrobial activity due to the generation of hydrogen peroxide and this antimicrobial effect can be inhibited by catalase. This antimicrobial activity is active only in complex culture media or chemically defined culture media containing l-lysine. This antimicrobial macromolecule in strain GR5 is shown to be a monomeric protein with a molecular mass of 71 kDa and isoelectric point of approximately 3.68. Liquid chromatography-tandem mass spectrometry analyses reveal close similarity of a 19-amino acid fragment derived from this protein to the antibacterial protein, AlpP from the marine bacterium Pseudoalteromonas tunicata D2, and to the antibacterial protein, marinocine, from the marine bacterium Marinomonas mediterranea. This study explores the nature of antimicrobial macromolecule such as L-lysine oxidase. This is the first report on a freshwater bacterium producing antimicrobial activity by generating hydrogen peroxide through its enzymatic activity of L-lysine oxidase.
Strain GR5 was greenish-yellow colored, Gram-staining-negative, facultative, rod-shaped, and motile by means of a single polar flagellum. Growth occurred at 15-37oC, at pH 7.0-8.0 and with 0-2% NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GR5 belonged to the genus Rheinheimera and its closest neighbour was Rheinheimera texasensis A62-14BT with sequence similarity of 98.1 %. The major fatty acids were Sum In Feature3(C16:1 7c/C16:1 6c)、C16:0 and C12:0 3-OH. The major respiratory quinone was Q-8. The DNA G+C content of the genomic DNA was 51.9 mol%. The characterization of the genotypy, phenotypy and biochemical property demonstrated that strain GR5 represents a novel species of the genus Rheinheimera. We propose the name Rheinheimera aquatica sp. nov., and type strain is GR5T(=BCRC 80081T=LMG 25379T).
中文摘要 I
Abstract III
誌謝 V
目錄 VI
表目錄 XIII
圖目錄 XV
第壹章、前言 1
ㄧ、細菌 1
二、研究目的 2
第貳章、文獻回顧 3
ㄧ、抗微生物物質簡介 3
(一) 抗微生物物質之定義、來源與種類 3
(二) 抗微生物物質之篩選 5
(三) 抗微生物物質之應用 6
二、Rheinheimera新菌種簡介 7
(一) 菌株來源、分離與初步鑑定 7
(二) 菌株之抗菌活性測試 8
三、Rheinheimera屬簡介 9
四、L-胺基酸氧化酶簡介 10
五、細菌分類學簡介 11
(一) 表現型分類 11
(二) 基因型分類 12
(三) 新菌種的界定 12
六、台灣淡水養殖池簡介 12
第參章、實驗材料與方法 14
一、菌株GR5抗菌活性物質特性探討 15
(一) 菌株之來源與培養 15
(二) 抗菌活性測試 15
(三) 抗菌活性物質製備 16
(四) 抗菌圖譜 16
(五) 抗菌活性物質純化 18
1. 超過濾濃縮 18
2. 硫酸銨沉澱 18
3. 等電點沉澱 18
(六) 抗菌活性蛋白鑑定 19
1. 活性貼片 19
2. 抗菌活性蛋白質胺基酸序列分析與序列比對 19
(七) 抗菌機制探討 20
1. 普魯士藍(Prussian blue)培養基測試 20
2. 觸酶效應測試 21
3. 過氧化氫酶偶合反應分析(Peroxidase-coupled assay) 21
(八) 培養時間與抗菌酵素活性之關係 23
(九) 離胺酸濃度與抗菌能力之關係 23
(十) 抗菌酵素活性與抗菌能力之關係 24
(十一) 抗菌活性酵素之蛋白質含量 24
(十二) 抗菌活性酵素儲存性測試 25
(十三) 抗菌活性酵素溫度穩定性 25
(十四) 抗菌活性酵素pH穩定性 26
(十五) 抗菌活性酵素最小抑菌/殺菌濃度 26
(十六) 抗菌活性酵素的產量與蛋白質含量 26
二、菌株GR5新菌種之鑑定 28
(一) 形態學分析 28
1. 菌落特徵 28
2. 菌體形態特徵 29
(1) 革蘭氏染色 30
(2) KOH反應 31
(3) 運動性觀察 31
a. 懸滴法 31
b. 半固體法 32
(4) 鞭毛染色 32
a. 乾片染色法 32
b. 濕片染色法 32
(二) 菌株生理生化特徵分析 33
1. 生長溫度範圍測定 33
2. 生長鹽度範圍測定 33
3. 生長pH範圍測定 33
4. 生長氧氣需求測定 34
5. 培養基需求之測試 35
6. 抗生素感受性測試 35
7. 水解能力測試 36
(1) 澱粉水解 36
(2) 核苷酸水解 36
(3) 酪蛋白水解 36
(4) 脂肪水解 36
(5) Tweens (20、40、60、80)脂質水解能力 37
(6) 卵磷脂水解能力 37
(7) 幾丁質水解能力 37
8. 細胞色素氧化酵素 38
9. 觸酶測定 38
10. API 20NE系統鑑定 38
11. API ZYM系統鑑定 41
12. BIOLOG GN II系統鑑定 42
13. 脂肪酸組成分析 44
14. 醌分析 45
(1) 菌體前處理 45
(2) 醌萃取流程 45
(3) 高效液相層析之分析 46
15. 色素萃取 46
(三) 菌株遺傳特徵分析 47
1. 16S rRNA基因序列分析 47
(1) 抽取DNA 47
(2) 16S rRNA基因序列擴增 48
(3) 洋菜膠體水平電泳 49
(4) 16S rRNA基因序列定序 50
(5) 16S rRNA 基因序列之資料比對與演化樹建構 50
2. DNA G+C mol% 51
(1) 菌株培養 51
(2) Genomic DNA抽取與純化 51
(3) DNA G+C mol%分析 51
3. DNA雜交分析 52
(1) 探針的製備 52
(2) 探針測試 52
(3) DNA雜合反應 53
第肆章、結果與討論 55
一、菌株GR5抗菌活性物質特性探討 55
(一) 菌株之培養 55
(二) 抗菌活性物質製備 55
(三) 抗菌(藻)圖譜 56
(四) 抗菌活性物質純化 58
1. 超過濾濃縮 58
2. 硫酸銨沉澱 58
3. 等電點沉澱 59
(五) 抗菌活性蛋白鑑定 60
1. 活性貼片 60
2. 抗菌活性蛋白質序列定序與序列比對 61
(六) 抗菌機制探討 63
1. 普魯士藍(Prussian blue)培養基測試 63
2. 觸酶效應測試 63
3. 過氧化氫酶偶合反應分析(Peroxidase-coupled assay) 64
(七) 培養時間與細菌細胞數和抗菌酵素活性之關係 65
(八) 離胺酸濃度與抗菌能力之關係 66
(九) 抗菌酵素活性與抗菌能力之關係 67
(十) 抗菌活性酵素儲存性測試 68
(十一) 抗菌活性酵素溫度穩定性 69
(十二) 抗菌活性酵素pH穩定性 70
(十三) 抗菌活性酵素最小抑菌/殺菌濃度 71
(十四) 抗菌活性酵素的產量與蛋白質含量 72
(十五) 抗菌活性酵素產品開發與未來發展 73
二、菌株GR5之分類鑑定 75
(一) 菌株GR5之形態觀察 75
1. 菌落形態 75
2. 菌體形態 75
(二) 菌株GR5之生理生化特徵 76
1. 生長溫度範圍之測定 76
2. 生長鹽度範圍之測定 77
3. 生長pH範圍之測定 78
4. 生長氧氣需求測定 78
5. 培養基需求之測試 78
6. 抗生素感受性測試 79
7. 水解能力測試 79
8. 細胞色素氧化酶測定 79
9. 觸酶測定 79
10. API 20NE系統鑑定 79
11. API ZYM系統鑑定 81
12. BIOLOG GN II系統鑑定 82
13. 脂肪酸分析 83
14. 醌分析 84
15. 色素萃取 84
(三) 菌株GR5之遺傳特徵 85
1. 16S rRNA基因序列分析 85
2. 親緣樹建構 85
3. DNA G+C mol% 86
(四) 菌株GR5與Rheinheimera屬相關菌種之特性比較 86
(五) 菌株GR5新種命名 88
第伍章、結論 90
第陸章、參考文獻 92
附錄 103
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