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研究生:闞公晧
研究生(外文):Kung-Hao Kan
論文名稱:生物界面活性劑—沙雷濕潤素之醱酵生產及相關性質分析
論文名稱(外文):Production and characterization of a lipopeptide biosurfactant-serrawettin from Serratia marcescens SM△R
指導教授:魏毓宏
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技暨生物資訊研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:119
中文關鍵詞:沙雷濕潤素靈菌紅素生物界面活性劑臨界微胞濃度表面滑行
外文關鍵詞:Serratia marcescensSerrawettinSerratamolideProdigiosinbiosurfactant
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沙雷氏菌 Serratia marcescens為革蘭氏陰性菌、短桿菌,能同時代謝生產紅色色素-靈菌紅素(Prodigiosin),與脂胜肽類生物界面活性劑-沙雷溼潤素(Serrawettin)。本篇論文旨在使用Serratia marcescens經 quorum sensing 處理之突變株Serratia marcescens SMΔR,對其所代謝生產之沙雷濕潤素進行分離、純化及鑑定,確立其結構組成,並尋求其增進沙雷溼潤素最適化之發酵培養基,同時探討其相關之界面性質與物/化性質。首先針對發酵液進行分離、純化,利用1H-NMR、FAB-MS及IR等技術分析,結果證實Serratia marcescens SMΔR,所生產的沙雷濕潤素為分子量515 m/z,分類上屬於Serrawettin W1,又名Serratamolide,為一可用作抗癌之代謝產物。沙雷濕潤素發酵生產培養基部份,經碳、氮源篩選,研究顯示以24 g/L Glucose、16g/L Bacto sotyone,可得到最高Serrawettin W1產量達718 mg/L。其次利用RSM(回應曲面)法,探討微量元素最佳組成,研究證實Serratia marcescens SMΔR在2.55μM MgSO4、2.36μM MnSO4添加下,Serrawettin W1產量可達1174 mg/L,與微量元素未最適化之培養基相比,約有1.6倍提昇。在誘發Serrawettin W1產量之發酵策略方面,研究顯示使用3 %(w/v)藻酸鈣顆粒作為固體載體,能進一步提升Serrawettin W1產量至2006 mg/L,相較於未添加固體載體之控制組而言產量約有1.7倍之提升,更為碳氮比最佳化培養基之2.8倍。在界面特性分析部分,利用接觸角探討濕潤性質結果顯示,Serrawettin W1在1500 mg/L濃度下,即可使水接觸角由68度降低至32度,為一不錯之濕潤劑。透過量測界面性質亦證實,僅須添加100 mg/L的Serrawettin W1即可將水溶液與正庚烷之界面張力由40.8 mN/m降至6 mN/m。量測表面滑行現象結果顯示,添加1500 mg/L之Serrawettin W1,菌體滑行速率達0.31cm/h,為未加Serrawettin控制組之3倍。在物/化性質分析方面,研究顯示Serrawettin W1對溫度耐受性強,經100℃加熱2小時、高溫高壓滅菌釜121℃,20分鐘下,仍能維持活性表現,顯見其溫度耐受性高;在酸鹼度耐受性實驗中,發現Serrawettin處在酸性pH 1-7中,仍能維持活性表現,但在pH >7時,對其結構則會造成破壞,故培養條件應維持在酸性pH值較佳;在鹽的耐受性方面,添加鹽度1-7 %,並放置於10-25℃水浴槽中12小時,皆不影響Serrawettin W1活性表現,顯示Serrawettin W1具有應用於海上溢油控制之潛力。
Being biodegradable and less toxic, biosurfactants possess great advantages over synthetic surfactants and hold potential applications in petroleum recovery, and as the emulsifiers and de-emulsifiers in pharmaceutical, cosmetics and food industries. Serrawettin W1, an important lipopeptide-type biosurfactant produced by various strains of Serratia marcescens, is the most powerful biosurfactant ever discovered. This work aimed to develop better culture medium and fermentative strategy enabling more efficient production of serrawettin W1 from S. marcescens SM△R. We shown that the optimal culture media was 24 g/L Glucose; 16g/L Bacto soytone; 2.55μM MgSO4 and 2.36μM MnSO4. Serrawettin W1 production was optimal in batch cultures when the temperature and agitation rate wre controlled at 30℃ and 200 rpm. Per above culture medium, we can be obtained the concentration of serrawettin W1 was 1174 mg/L, which was 1.6-fold over the control LB medium. Addition of a small quantity of solid porous carrier (e.g., activated carbon, sponge and alginate bead) into fermentation broth significantly increased serrawettin W1 production with Serratia macescens SM△R. Culture medium containing 3% (w/v) of alginate bead gave an optimal serrawettin W1 production yield of 2006 mg/L, which was approximately 1.7-fold higher than that obtained from carrier-free liquid culture. Serrawettin W1 in the culture broth was purified primarily via solvent extraction and column chromatography. The NMR and mass spectrometry analysis shows that the purified product is a lipopeptide biosurfacrant, called serrawettin W1. At 1500 mg/L serrawettin W1 decreases the contact angle of water from 68 degrees to 32 degrees. Meanwhile, 100 mg/L serrawettin W1 decreases interface tension and surface tension of water to 6 mN/m and 40.8 mN/m, respectively. Furthermore, we also shown that serrawettin W1 possess higher thermal, saline and acidic tolerance. However, this work was a preliminary feasibility assessment of using the Serratia macescens strains for biosurfactant (serrawettin W1) production in a commercial scale.
致謝 I
中文摘要 II
Abstract V
目錄 VIII
表目錄 XIV
圖目錄 XV
第一章 前言 1
第二章 文獻回顧 3
2-1 界面活性劑的簡介 3
2-2 生物界面活性劑 3
2-3 生物界面活性劑之種類與應用 4
2-4 沙雷氏菌所產之脂胜肽生物界面活性劑 8
2-5界面活性劑之活性分析 13
2-6薄層層析法(Thin-layer Chromatography) 16
2-6-1 吸附薄層層析 16
2-6-2 展開液選擇 17
2-6-3 TLC(Thin-layer Chromatography)顯色 18
2-7 試驗設計法 19
2-7-1 回應曲面實驗設計法 20
2-7-2 回應曲面法最適化步驟 23
2-7-3 二水準因子設計 25
2-7-4 陡升路徑法 26
第三章 實驗材料與方法 28
3-1 實驗藥品 28
3-2實驗儀器 30
3-3菌種培養 32
3-3-1實驗菌株 32
3-3-2培養基組成 32
3-3-2-1固體培養基配方 32
3-3-2-2 搖瓶醱酵培養基配方 32
3-4培養基最適化 33
3-4-1碳源篩選 33
3-4-2氮源篩選 34
3-4-3微量元素之添加 34
3-5 實驗步驟與分析方法 36
3-5-1 SMΔR生產Serrawettin定性分析 36
3-5-1-1 樣品前處裡 36
3-5-1-2 薄層層析法 37
3-5-2 SMΔR生產Serrawettin之分離 38
3-5-3 SMΔR生產Serrawettin之純化 38
3-5-4 Serrawettin 之定性分析 39
3-5-4-1 Serrawettin 之化學結構分析 39
3-5-4-2 Serrawettin 之分子量測定 39
3-5-4-3 Serrawettin 之吸收全波長掃描 40
3-5-5 Serrawettin之定量分析 40
3-5-6 固體載體製備 41
3-5-7 表面/界面張力之測定 41
3-5-8 臨界微胞濃度測定(CMC) 42
3-5-9 S. marcescens SMΔR乳化指數分析 42
3-5-10 臨界乳化指數之測定 43
3-5-11 菌體表面滑行測量 43
3-5-11-1 表面滑行培養基 43
3-5-11-2 表面滑行步驟 44
3-5-12 Serrawettin W1 (Serratamolide)性質分析 45
3-5-12-1 熱穩定性分析 45
3-5-12-2 pH值穩定性分析 45
3-5-12-3 鹽耐受性分析 46
第四章 結果與討論 47
4-1論文整體架構 47
4-2-1 Serrawettin 之發酵生產 49
4-2-1-1碳源篩選 49
4-2-1-2氮源篩選 51
4-2-1-3碳氮比最適化 52
4-2-2利用回應曲面法最佳化培養基微量元素組成 54
4-2-2-1二水準因子設計 55
4-2-2-2陡升路徑法 58
4-2-2-3回應曲面法 60
4-2-3固體載體添加策略 65
4-3 Serrawettin 發酵及分離純化 67
4-3-1 Serrawettin分離 67
4-3-2 Serrawettin純化 68
4-4 Serrawettin 定性定量分析 69
4-4-1化學結構分析 69
4-4-2分子量鑑定 69
4-4-3 TLC定性分析 70
4-4-4 Serrawettin吸收全波長掃描 70
4-4-5 CMC定量分析 77
4-5 Serrawettin 界面特性分析 78
4-5-1濕潤活性(Wetting activity)分析 78
4-5-2表面/界面張力分析 80
4-5-3 Serrawettin幫助菌體滑行分析 82
4-6 Serrawettin 物/化性質分析 85
4-6-1臨界乳化指數之測定 85
4-6-2溫度耐受性分析 87
4-6-3酸鹼耐受性分析 87
4-6-4鹽類耐受性分析 87
第五章 結論 91
第六章 未來展望 93
參考文獻 94
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