臺灣博碩士論文加值系統

中文摘要
沙雷氏菌 Serratia marcescens 為革蘭氏陰性菌，具有生產紅色色素—靈菌紅素 (prodigiosin)與脂胜肽類生物界面活性劑-沙雷溼潤素 (Serrawettin) 之能力。本篇論文擬就 Serratia marcescens SS-1 及其經 quorum sensing 處理之突變株 SMΔR等兩菌株，利用培養基改良以強化其生產靈菌紅素之能力，同時進行靈菌紅素分離、純化及鑑定，以確定其結構。首先，藉由 NMR 及 FAB-MS 證實Serratia marcescens SS-1 及 SMΔR，所生產之靈菌紅素屬於Undecylprodigiosin (UP)，為靈菌紅素衍生物之一。在提升 UP 產量方面，研究證據顯示分別以不同組成比例之 modified LB medium ( MLB)，(SS-1 : yeast extract , 5g/L ; SMΔR : yeast extract , 13.5 g/L, tryptone 22.5 g/L ) 培養兩菌株，其UP產量最高分別可達到 SS-1：700 mg/L，約為以 LB 培養基培養的 23 倍 , SMΔR：150 mg/L， 約為以 LB 培養基培養的 3 倍左右。若針對兩 MLB 培養基分析，再額外添加其適合之受質，則可令UP的產量再次獲得提升。研究顯示，將 SS-1 之 MLB 中再額外添加His, Pro, Asp ( UP 主結構類似的氨基酸)，則可使所生產之 UP 產量大幅提升，分別達到約 1.4、2.5 和 1.4 g/L 左右，最高約為 MLB 培養基的 3 倍，為原始 LB 培養基的 69 倍。同樣若採取相同的策略，在 SMΔR 之 MLB 中再分別額外添加 4% ( v/v ) 大豆油 、6 % ( v/v ) 葵花油及 4 % ( v/v ) 橄欖油 ，則可使所生產之 UP 產量再次提升，分別達到約 525、700 及 490 mg/L 左右，最高為 MLB 培養基的 4.5 倍，為原始 LB 培養基的 13.5 倍。另外，在中、下游回收製程方面，嘗試以不同界面活性劑來改善菌體釋放 UP 的能力以增加 UP 回收率，本研究選用三種界面活性劑 Surfactin、Triton X-100 及 Tween 80之施用，確實可有效將產物UP自菌體細胞套膜上釋放出來，研究進一步顯示其釋放UP的效率，隨著施用界面活性劑濃度之增加而提升，但最後皆達到臨界值。，臨界釋放濃度 (Critical Release Concentration, CRC ) 值在 SS-1 方面，三種界面活性劑之 CRC 值分別為 Surfactin：10000 mg/L，Triton X-100：6000 mg/L，Tween 80：6000 mg/L， SMΔR方面，Surfactin：10000 mg/L，Triton X-100：6000 mg/L，Tween 80：8000 mg/L。研究證實，於Eigen agar固態培養基表面上添加脂胜肽生物界面活性劑—表面素(surfactin)確實有助於菌體之快速滑行，研究顯示：添加界面活性劑濃度越高，菌體表面滑行速度越快，當分別添加100 ppm~1500 ppm之生物界面活性劑表面素，菌體表面滑行速度相較於不添加之控制組約有1.5倍~3倍速率之提升。上述研究證實沙雷氏菌serratia marcescen表面滑行的確與其所分泌的生物界面活性劑沙雷濕潤素相關。

Abstract
Serratia marcescens SS-1and its isogenic mutant Serratia marcescens SMΔR were used to produce a prodigiosin-like pigment. The pigment obtained from these strains is most likely undecylprodigiosin (UP) according to the NMR and MS analysis. Luria- Bertani (LB) broth frequently used for prodigiosin biosynthesis with S. marcescens strains was modified by increasing the concentration of tryptone and yeast extract while completely removing NaCl from the medium. In light of the effect of each LB component on UP production, a modified LB (MLB) medium was formulated. (MLB, SS-1: yeast extract, 5 g/L and SM∆R, yeast extract, 13.5 g/L, tryptone 22.5 g/L). Optimal culture conditions (e.g., temperature, pH, agitation rate) for UP production were also identified. It was found that S. marcescens SS-1 was able to produce 700 mg/L of UP when it was grown with 5 g/L yeast extract alone (MLB for SS-1) under optimal culture conditions of 30oC, 200 rpm, and pH 8. The UP production of 700 mg/L is nearly 23 fold of that obtained from original LB medium. Addition of amino acids containing pyrrole-like structure further enhanced UP production. Nearly 1.4, 2.5 and 1.4 g/L of UP was produced when the SS-1 strain was cultivated with MLB medium supplemented with histidine, proline and aspatic acid, respectively. On the other hand, S. marcescens SM∆R was able to produce 150 mg/L of UP when it was grown with MLB medium (yeast extract, 5 g/L). The resulting MLB medium achieved nearly 3.0 fold increase in UP yield (150 mg l-1) when compared with original LB broth. The best medium for UP production with the S. marcescens SMΔR strain was sunflower oil (6%)-supplemented MLB medium, which attained approximately 14-fold higher UP yield than that of the original LB broth. Furthermore, this work also tried to enhance the release of UP by surface active agents. The critical concentrations of surfactants for the best UP release from SS-1 and SM∆R strains were as follows: For SS-1, Surfactin: 10000 mg/L, Triton X-100: 6000 mg/L, Tween 80: 6000 mg/L, respectively. On the other hand, For SM∆R, Surfactin: 10000 mg/L,Triton X-100: 6000 mg/L and Tween 80: 8000 mg/L. For sliding test, this work shows that adding lipopeptide biosurfactant to Eiken agar medium surface enhanced sliding ability of S. marcescens SS-1 and SM∆R. The data indicate that the higher surfactant concentration was added, the faster sliding ability was obtained. Addition of surfactin (100- 1500 mg/L) into agar surface markedly enhances the sliding rate. The resulting surfactin-containing surface achieved nearly 1.5- 3.0 fold increase in sliding rate when compared with surfactin-free surface.

目錄
封面內頁 頁次
目錄
表目錄
圖目錄
第一章 前言………………………………………………………….….1
第二章 文獻回顧………………………………………………….….…3
2-1. Serratia marcescens 之分類…………………………………..…3
2-2. Serratia 屬及 Serratia marcescens 之生化特性…………….....4
2-3. 靈菌紅素............…………………………………………………5
2-3-1 靈菌紅素的生產.......................................................................7
2-4. 表面滑行.......…………………………………………………...11
第三章 材料與方法……………………………………………………20
3-1. 菌株……………………………………………………………..20
3-2. 培養基…………………………………………………………..20
3-3. 前培養…………………………………………………………..21
3-4. Serratia marcescens SMΔR 之關鍵培養基……………………21
3-5. Serratia marcescens SS-1 之關鍵培養基………………….......22
3-6. Undecylprodigiosin ( UP ) 之純化與分離..................................23
3-7. UP之化學結構鑑定及分子量分析.............................................24
3-8. UP之定量分析.............................................................................24
3-9. 微生物生長之分析......................................................................25
3-10. 乳化指數分析............................................................................25
3-11. 表面活性之分析........................................................................25
3-12. 界面活性劑之選擇……………………………………………26
3-13. 界面活性劑對細胞釋放 UP 之影響......................................26
3-14. 菌體表面滑行測量……………………………………………28
3-14-1. 表面滑行培養基………………………………………......28
3-14-2. 表面滑行步驟………………………………………...…...28
第四章 結果與討論……………..……………………………………33
4-1. 靈菌紅素之化學結構及分子量鑑定……………………………33
4-2. 環境因子對 S. marcescens SS-1 及 SMΔR 生產UP 之影響...
.......................................................................................................38
4-2-1. 溫度對S. marcescens SS-1 及 SMΔR 生產UP之影響.....38
4-2-2. 攪拌速度對S. marcescens SS-1 及 SMΔR 生產UP之影響..............................................................................................39
4-2-3. pH 值對 S. marcescens SS-1 及 SMΔR 生產UP之影
響..............................................................................................39
4-3. Serratia marcescens SMΔR 之培養基改良………………......…43
4-3-1. LB 成分對S. marcescens SMΔR 生產 UP 的影響………..43
4-3-2. 不同比例之 Trypton 提升 UP 產量的影響………...…….44
4-3-3. 不同比例之 Yeast extract 提升 UP 產量的影響………..45
4-3-4. 不同比例之NaCl提升UP 產量的影響……………………..45
4-3-5. LB關鍵成分比例之最適化…………………………………..46
4-3-6. 植物油添加對 UP 生產之影響…………………………….47
4-4. Serratia marcescens SS-1 之培養基改良…………..…………….56
4-4-1. LB成分對S. marcescens SS-1生產UP的影響………….......56
4-4-2. 不同比例 NaCl 提升 UP 產量之影響…..………………...57
4-4-3. 不同比例 Tryptone 提升 UP產量之影響..……………......58
4-4-4. 不同比例 Yeast extract 提升UP產量之影響…….……......60
4-4-5. LB關鍵成分比例之最適化…………………………………..60
4-4-6. 氨基酸添加對生產 UP 之影響…………………………….61
4-4-7. 氨基酸添加濃度之最適化………………………………......63
4-5. 菌種比較..........................................................................................74
4-6. 界面活性劑釋放UP之影響………………………………………79
4-6-1. 臨界釋放濃度（Critical Release Concentration）…………..79
4-6-2. 界面活性劑種類對釋放UP之影響…………………………79
4-7. 表面滑行………………………………………………………….88
4-7-1. 添加界面活性劑對菌體滑行之影響……………………......88
4-7-2. 溫度對菌體滑行之影響…………………………………......89
第五章 結論……………………………………………………………95
第六章 未來展望………………………………………………………97
參考文獻………………………………………………………………..98
自述……………………………………………………………………105
論文著述………………………………………………………………106











表目錄
頁次
表2-1 Serratia 屬之生化特性……………………………………...….13
表2-2 Serratia marcescens之生化特性………………………………..14
表2-3 續前表……………………………………………………….… 15
表3-1 Composition of the media used to grow Serratia marcescens
SM∆R for the production of the Undecylprodigiosin…………...30
表3-2 Composition of the media used to grow Serratia marcescens
SS-1 for the production of the Undecylprodigiosin……………..31
表4-1 Production of Undecylprodigiosin and cell-free surface activities
for batch cultures of Serratia marcescens SMΔR cultivated in
oil-supplemented and oil-free modified LB (MLB) medium…..50
表4-2 野生株Serratia marcescens SS-1與突變株Serratia marcescens
SM∆R之比較...........................................................................76
表4-3 為Serratia marcescens之醱酵培養液中，上清液與pellet 所含
色素的百分比，本研究與文獻記載相符。…………………..83



圖目錄
頁次
圖2-1 為以PPM為主幹的各種 prodigiosin之衍生物。……………...16
圖2-2 為靈菌紅素之生合成路徑。……………………………..…….17
圖2-3 為靈菌紅素衍生物 Undecylprodigiosin生合成路徑…………18
圖2-4 為在螢光顯微鏡下BHK 細胞用 acridine orange染色以觀察是否有細胞凋亡的現象 (a) 沒有添加靈菌紅素之BHL 細胞(b) 添加靈菌紅素10 μM 之 BHK細胞…………………………..19
圖2-5 為Serratia marcescens 進行於Eiken agar plate上進行swarming 與 sliding的情形..……………………………………………...19
圖3-1為Serrstia marcescens SS-1之OD600-細胞乾重之校正曲
線..................................................................................................32
圖3-2為Serrstia marcescens SMΔR之OD600-細胞乾重之校正曲
線……………………………………………………………......32
圖4-1 (a)為由Serratia marcescens SS-1及SMR所萃取出來之色
素，在以NMR 分析所得之圖譜。 (b) 為prodigiosin 之結
構，結構上之數字對應到(a)圖為 peak 1~peak 7 的部份。…35
圖4-2 為由Serratia marcescens SS-1及SMR所萃取出來之色素，在以FAB-MS分析所得之圖譜。………………………………36
圖4-3 為於MS分析下所得到之UP 片段。………………………......37
圖4-4 為以 LB 培養下，環境因子對 Serratia marcescens SS-1 生
產 UP 之影響。……………………………………………….41
圖4-5 為以 LB 培養下，環境因子對 Serratia marcescens SMΔR
生產 UP 之影響。……………………………………………42
圖4-6 為Serratia marcescens SMΔR 在各個關鍵成分培養基中菌體的生長情形 與代謝生產UP之情形。………………………....51
圖4-7 為Serratia marcescens SMΔR 在不同比例yeast extract 培養基中菌體的生長情形與代謝生產UP之情形。………………….52
圖4-8 為Serratia marcescens SMΔR 在不同比例Tryptone培養基中菌體的生長情形與代謝生產UP之情形。……………………..53
圖4-9 為Serratia marcescens SMΔR 在不同比例NaCl培養基中菌體的生長情形與代謝生產UP之情形。…………………………..54
圖4-10 為Serratia marcescens SMΔR 在已修飾過培養基中培養菌體
的生長情形與代謝生產UP之情形。…………………….…...55
圖4-11 為Serratia marcescens SS-1 在各個關鍵成分培養基中菌體的
生長情形與代謝生產UP之情形。…………….……………..64
圖4-12 為Serratia marcescens SS-1 在不同比例NaCl培養基中菌體
的生長情形與代謝生產UP之情形。………………………....65
圖4-13 為Serratia marcescens SS-1 在不同比例Tryptone培養基中菌
體的生長情形與代謝生產UP之情形。……………………....66
圖4-14 為Serratia marcescens SS-1以不同廠牌之Trptone所配之LB培養基，菌體的生長情形與代謝生產UP之情形。…………..67
圖4-15 為Serratia marcescens SS-1 在不同比例yeast extract培養基中菌體的生長情形與代謝生產UP之情形。…………………68
圖4-16 為以不同濃度之yeast extract，試圖找出最適化之LB培養基，圖中所示為菌體的生長情形與代謝生產UP之情形。………69
圖4-17 為Serratia marcescens SS-1 在不同種類之胺基酸添加至MLB中為氮源時 ，菌體的生長情形與代謝生產UP之情形。
………………………………………………………….…….70
圖4-18 為Serratia marcescens SS-1 以在不同比例之His中，菌體的
生長情形與代謝生產UP之情形。…………………………..71
圖4-19 為Serratia marcescens SS-1 以在不同比例之Pro中，菌體的
生長情形與代謝生產UP之情形。…………………………..72
圖4-20 為Serratia marcescens SS-1 以在不同比例之Asp中，菌體的生長情形與代謝生產UP之情形。…………………………....73
圖4-21 野生株 S. marcescens SS-1 在各種疏水性植物油培養所得到
最大的 UP 產量。.....................................................................77
圖4-22 為 Serratia marcescens SMΔR 以在不同比例Asp中，菌體的生長情形與代謝生產UP之情形。…………………………....78
圖4-23 分別為 (a) 為以1000 mg/L DB 作用後， (b) 為以100 mg/L
DB作用後， 明顯與(a)有極大差別，顯示 DB 促使 UP 吸
附住 pellet，因而使釋放的效率變差………………………...84
圖4-24 分別為(a)醱酵液離心後biomass中色素堆積的情形、 (b) 為
以Triton X-100作用後色素堆積的情形及 (c) 以過量甲醇作
用後色素堆積的情形。………………………………………..85
圖4-25 為S. marcescens SS-1經界面活性劑作用後，由細胞套膜釋
放色素的表現。………………………………………………..86
圖4-26 為 S. marcescens SMΔR 經界面活性劑作用後，由細胞套
膜釋放色素的表現。………………………………………..…87
圖4-27 為Surfactin、Triton X-100及Tween 80三種界面活性劑之表
面張力。………………………………………………………..88
圖4-28 為不同界面活性劑添加對Serratia marcescens SS-1表面滑行之影響。………………………………………………………....91
圖4-29 為不同界面活性劑添加對Serratia marcescens SMΔR表面滑
行之影響。……………………………………………………....92
圖4-30 界面活性劑種類與其表面活性之關係。…………………….93
圖4-31 (a), (b) 分別於25℃及30℃之表面滑行。由左而右添加表面素
濃度依次為0 mg/L, 100 mg/L, 500 mg/L, 1000 mg/L, 1500
mg/L……………………………………………………………..94

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