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研究生:余宛儒
研究生(外文):Wan-Ju Yu
論文名稱:以SerratiamarcescensC3生產天然抗癌藥物-靈菌紅素之醱酵製程開發
論文名稱(外文):Development of fermentation process for the production of a natural anti-tumor drugs-prodigiosin by Serratia marcescens C3
指導教授:魏毓宏
指導教授(外文):Yu-Hong Wei
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:99
中文關鍵詞:靈菌紅素Serratia macescens C3最適化培養基多孔性
外文關鍵詞:ProdigiosinSerratia macescens C3Medium optimizationSolid porous carrier
相關次數:
  • 被引用被引用:12
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本研究主要探討Serratia macescens C3生產靈菌紅素( Prodigiosin )相關製程之開發,靈菌紅素結構的特徵為具有pyrrolylpyrromethene(PPM)骨幹及有相近烷基取代物鍵結在主要骨幹上,因而形成不同結構之色素分子。目前靈菌紅素在免疫抑制、抗真菌和抗增生的能力中,尤其是抗癌和防止癌細胞轉移等功能上具有不錯的效果,所以其目前在醫學臨床上的應用極具潛力。為了提升靈菌紅素在商業上應用的潛力,需要從如何提高產量和降低成本兩大方向來進行研究,因此本研究針對Serratia macescens C3生產靈菌紅素計劃開發出較佳之培養基和醱酵策略,使其能有效提高靈菌紅素產量。
本研究首先証實Serratia macescens C3醱酵產生之紅色色素經分離純化後,藉由1H-NMR及FAB-MS經証實為靈菌紅素無誤。其次,是探討不同的環境因數:溫度、攪拌速度和酸鹼值和不同的碳氮源對Serratia macescens C3代謝靈菌紅素之影響,並以回應曲面實驗設計法( RSM )來探討微量元素之間交互關係和最適化濃度。結果顯示,培養Serratia macescens C3之最適化培養基組成為:Starch : 16 g/L、peptone : 10.67 g/L、FeSO4.7H2O : 0.56 mM、MnSO4.4H2O : 3.25 mM、MgSO4.7H2O : 2.5 mM、CaCl2 : 60 mM。而最適化靈菌紅素條件為:培養溫度:30℃,攪拌速率:200 rpm,pH 7.0。以最適化培養基及培養條件可得靈菌紅素最大產量約為7.05 g/L,為LB培養基控制組之8倍。
另外,本研究亦以細胞固定化和多孔性固體載體添加策略,探討其對靈菌紅素醱酵之影響。結果顯示,細胞固定化會阻礙Serratia macescens C3釋放出靈菌紅素於培養基,而使得定量不易,所以不適用;而在固體載體添加策略方面,活性碳、海綿和菜瓜布對靈菌紅素之生合成均無明顯之提升,但是在未包覆菌體之藻珠的添加上卻有明顯的效果,以添加入60 g/L的藻珠,可將靈菌紅素產量提高到15 g/L,為未加藻珠載體之控制組的2倍左右,更是一般LB基礎培養基之16倍。証實了以添加固體載體來量產靈菌紅素之醱酵策略之可行性。
Prodigiosin (PG), a secondary metabolite (red pigment) produced by Serratia marcescens and other gram-negative bacteria, possesses a common pyrrolylpyrromethene (PMM) skeleton and has a series of close relatives bearing the same PPM core with different alkyl substituents. PG is of great interest due to its immunosuppressive, antifungal, and antiproliferative properties, as well as the ability to induce apoptosis in certain cancer cells. It seems to have the potential for applications in medical industries. In light of potential commercial values, there is a demand to develop high-throughput and cost-effective bioprocess for the production of these prodigiosin-like pigments (such as prodigiosin or undecylprodigiosin). This study aimed to develop better culture medium and fermentative strategy enabling more efficient production of PG from S. marcescens C3. We shown that the optimal culture media was 15.6 g/L starch; 8 g/L peptone; 0.56 mM FeSO4.7H2O; 3.25 mM MnSO4.4H2O; 2.5 mM MgSO4.7H2O and 60 mM CaCl2 . PG production was optimal in batch cultures when the temperature and agitation rate were controlled at 30℃ and 200 rpm. From the above condition, we can be obtained the concentration of PG was 7.05 g/L, which was eight times 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 PG production with Serratia macescens C3. Culture medium containing 60 g/L of alginate bead gave an optimal PG production yield of 15 g/L, which was approximately 2-fold higher than that obtained from carrier-free liquid culture. Furthermore PG production by Serratia macescens C3 was purified and characterized by 1H-NMR and FAB-MS to determine its chemical structure and molecular weight. The 1H-NMR and mass spectrometry analysis shows that the purified product is a possess pyrrolylpyrromethene skeleton, called prodigiosin.
中文摘要 I
英文摘要 III
誌 謝 V
目錄 VII
表目錄 X
圖目錄 XI
第一章 研究動機與目的 1
第二章 文獻回顧 3
2-1 Serratia marcescens之基本特性 3
2-2 Serratia 屬及 Serratia marcescens 之生化特性 4
2-3 靈菌紅素 5
2-4 固定化細胞 7
2-5 實驗設計—回應曲面法(Response surface methodology) 8
2-5-1 二水準因數設計(Two-Level Factorial Design) 9
2-5-2 陡升路徑法( method of path steepest ascent ) 10
2-5-3 中心混成設計(Central Composite Design,CCD ) 11
2-5-4 回應曲面模式適切性之統計試驗 11
2-5-5 檢定係數R2 11
第三章 材料與方法 21
3-1 菌株 21
3-2 活化培養基 21
3-3 Serratia marcescens C3 最適化培養基配方 21
3-4 靈菌紅素( Prodigiosin, PG)的分離與純化 22
3-5 靈菌紅素之化學結構鑑定及分子量分析 23
3-6 靈菌紅素之定量分析 23
3-7 微生物生長之分析 24
3-8 細胞固定化之分析 25
第四章 結果與討論 28
4-1 論文研究架構 28
4-2 靈菌紅素之化學結構和分子量鑑定 29
4-3 環境因數對S. marcescens C3生產PG之影響 32
4-3 環境因數對S. marcescens C3生產PG之影響 32
4-3-1 溫度對S. marcescens C3生產PG之影響 32
4-3-2 攪拌速度對S. marcescens C3 生產PG之影響 32
4-3-3 pH值對 S. marcescens C3 生產PG之影響 33
4-4 S. marcescens C3之最適化醱酵培養基開發 35
4-4-1 碳源和氮源之探討 35
4-4-2 碳氮比之最適化探討 36
4-4-3 以實驗設計法進行培養基中微量元素組成之最佳化探討 37
4-4-3-1 二水準因數設計( Two-level factorial design ) 37
4-4-3-2 陡升路徑法(Method of path of steepest ascent) 38
4-4-3-3 回應曲面法( Response surface methodology, RSM ) 39
4-5 醱酵策略對於S.marcescens C3合成PG之影響 67
4-5-1 固定化細胞醱酵策略之探討 67
4-5-2 多孔性固體載體添加策略 67
4-5-3 醱酵槽培養 68
第五章 結論 76
第六章 未來展望 78
第七章 參考文獻 79
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