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研究生:蕭欣培
研究生(外文):Hsiao, Hsinpei
論文名稱:利用Streptomyces hygroscopicus發酵生產免疫抑制劑 Rapamycin之探討
論文名稱(外文):The Fermentation Process Development Of Rapamycin Production By Streptomyces Hygroscopicus
指導教授:顏宏偉
指導教授(外文):Yen, Hongwei
口試委員:楊芳鏘劉永銓
口試委員(外文):Yang, FanchiangLiu, Yungchuan
口試日期:2012-06-15
學位類別:碩士
校院名稱:東海大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:76
中文關鍵詞:放線菌免疫抑制劑
外文關鍵詞:rapamycinS.hygroscopicus
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本研究利用放線菌Streptomyces hygroscopicus發酵生產rapamycin免疫抑制劑之探討,目的希望找出較佳的發酵環境以及發酵控制,提高產物rapamycin的生產濃度與生產速率。1975年,放線菌S. hygroscopicus在復活節島上的土壤被發現,而菌體的發酵液中分離出rapamycin這物質,早期一直被當做治療真菌之感染,相繼研究出具有免以抑制劑、抗腫瘤、防止衰老延長壽命等多種效能。
本研究先以三角搖瓶進行較佳發酵環境的選定,再放大至5L發酵槽進行發酵控制與監測並探討與改良。在三角搖瓶方面,結果指出在種菌培養時間的探討,以培養48小時後再進行發酵培養,會有較佳的rapamycin產量。培養基中的碳源方面以葡萄糖(glucose)較佳,而氮源則以酵母萃取物(YE)有較佳的產物生成。在胺基酸的添加裡,採用胺基酸L-Lysine進行實驗測試,結果並無預期之效果提高產物rapamycin生產,在發酵培養基裡可能就含有L-Lysine成分,故猜測再額外添加反而會產生抑制效果。
然而在5L發酵槽的培養方面,發現採用兩階段溶氧策略控制,有助於提升產物產量,從空白組的最大產量104 mg/L增加至288 mg/L,提升了177%;DO50%高溶氧下,更能使產物產量增加,從空白組的最大產量104 mg/L增加至782 mg/L,提升了652%。然而在pH的變化中,發現對於產物生成似乎極為重要,並做了兩段式調控pH來進行實驗測試,發現第二階段控制pH為5.5時,可以提高產量至570 mg/L,相較於控制組濃度104 mg/L增加了約448 %;放大至15 L發酵槽下,以同樣條件培養,可以提高產量至683 mg/L,相較於控制組濃度104 mg/L增加了約557 %。由以上結果得知,在S.hygroscopicus的發酵培養裡,產物的生成與溶氧量和pH值的改變有相當大的關聯,在未來實驗裡將對溶氧與pH的影響性進行更深入的探討。

Rapamycin is a 31-member ring macrolide produced by Streptomyces hygroscopicus and has many applications in clinical medicine. S.hygroscopicus isolated from an Easter Island soil sample. This natural product possesses various bioactivities including antifungal (Vezina et al. 1975), antitumor, immunosuppressant and extends the life activities (Eng et al. 1984; Sehgal et al. 1994).
This study will develop fermentation process to harvest rapamycin by S. hygroscopicus. The purposes want to find a better fermentation environment and fermentation control, and improve product rapamycin. First selected better fermentation environment from the flask experiment, and then transferred to a 5L fermentor for fermentation control and monitoring, and to explore and improved.
In flask experiment, the result pointed out glucose was to be carbon source, yeast extract be nitrogen source, and seed culture time for 48 hours before fermentation, there will be better of rapamycin production. However, the amino acid added experiment, added L-Lysine can increase rapamycin production (Cheng et al. 1995), but in this study didn’t expect the effect to improve production. Guess that medium of yeast extract may contain L-Lysine, so further to add but will have inhibitory effects.
However, in 5L fermentor found using the two-stage dissolved oxygen control strategy help to improve the product rapamycin. The yield was reach 288 mg/L, increased about 177%, compared with control group which concentration of 104 mg/L; using DO50% high concentration of oxygen control strategy also help to improve the product rapamycin. The yield was reach 782 mg/L, increased about 652%, compared with control group which concentration of 104 mg/L. And found that the pH change is very interesting and very important to produce rapamycin in fermentation. And bold design a two-stage regulation of pH to ferment in 5L fermentation. Found that the second phase control to pH 5.5 which can increase production to 570 mg/L, increased about 448%; enlarged to a 15 fermentor in the same conditions can increase production to 683 mg/L, increased about 557%.
These results suggest that, in fermentation product formation, oxygen content change and pH change have relationships very much, so will to in-depth discussion in the future experiment.

中文摘要 I
Abstract III
目錄 V
圖目錄 IX
表目錄 XIII
第一章 緒論 1
第二章 文獻回顧 3
2.1菌種介紹(Streptomyces hygroscopicus) 3
2.1.1放線菌門(Actinobacteria)介紹 4
2.1.2鏈黴菌屬(Streptomyces)介紹 5
2.2 Rapamycin介紹 7
2.2.1抗真菌 8
2.2.2免疫抑制劑 8
2.2.3抗腫瘤 9
2.2.4延長壽命 10
2.3 rapamycin合成路徑 11
2.4培養條件 12
2.4.1碳源影響 13
2.4.2氮源影響 13
2.4.3胺基酸添加的影響 14
2.4.4初始pH值的影響 14
2.5發酵策略 14
2.5.1 Batch 15
2.5.2 Fed-batch 16
2.6文獻產值 18
第三章 實驗材料與方法 19
3.1實驗材料 19
3.1.1實驗菌株 19
3.1.2實驗藥品 19
3.2實驗儀器 21
3.3分析方法 23
3.3.1 Biomass分析方法 23
3.3.2 Glucose分析方法 23
3.3.3 Rapamycin分析方法 23
3.4 實驗方法 24
3.4.1 菌種保存 24
3.4.2 種菌培養基 25
3.4.3 發酵培養基 26
3.4.3 接菌 26
3.6 菌落型態與篩菌 28
3.7.1 三角瓶液態培養實驗 29
3.7.1.1 種菌培養時間的影響 29
3.7.1.2 不同碳源的影響 29
3.7.1.3 不同氮源的影響 29
3.7.1.4 添加胺基酸的影響 30
3.7.1.5 不同轉速下培養的影響 30
3.7.2 發酵槽5 L培養實驗 31
3.7.2.1 DO控制之影響 31
3.7.2.2 pH控制之影響 32
3.7.3 發酵槽15 L培養實驗 32
第四章 結果與討論 34
4.1 菌落型態與篩菌 34
4.2 發酵實驗培養 38
4.2.1 三角瓶液態培養實驗 38
4.2.1.1 種菌培養時間的影響 38
4.2.1.2 不同碳源與濃度的影響 40
4.2.1.3 不同氮源的影響 42
4.2.1.4 添加胺基酸的影響 44
4.2.1.5 不同轉速下培養的影響 46
4.2.2 發酵槽培養實驗 48
4.2.2.1 DO控制之影響 49
4.2.2.2 DO控制之比較 52
4.2.2.3 pH控制之影響 56
4.2.2.4 pH控制之比較 60
4.2.2.5 發酵槽15 L放大培養 63
第五章 結果與未來展望 66
5.1 結論 66
5.2 未來展望 67
參考文獻 68
附錄 74
附錄1: 產物rapamycin標準曲線 74
簡歷 75


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