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研究生:陳靜怡
研究生(外文):Jing-Yi Chen
論文名稱:醱酵生產環狀糊精葡萄糖苷轉移酶及生產環狀糊精反應條件之探討
論文名稱(外文):Production of Cyclodextrin Glucanotransferase and Reaction Conditions to Produce Cyclodextrins
指導教授:段國仁段國仁引用關係
指導教授(外文):Kow-Jen Duan
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:100
中文關鍵詞:噴霧乾燥環狀糊精環狀糊精葡萄糖苷轉移酶冷凍乾燥
外文關鍵詞:Spray dryingcyclodextrinCyclodextrin glycosyltransferase (CGTase)Freeze dried
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環狀糊精葡萄糖苷轉移酶Cyclodextrin glucanotransferase (CGTase)是具有將澱粉轉換成環狀糊精分子的酵素,本研究使用alkalophilic Bacillus sp. (ATCC 21595)菌種以麥芽糊精(DE10)為碳源進行醱酵生產環狀糊精轉移酶,在搖瓶培養中,以2%麥芽糊精(DE10)(C:N=1:1)為碳源,得到CGTase活性為6099 unit/ml,用醱酵槽醱酵則得到活性為19540 unit/ml。將培養基的碳源麥芽糊精(DE10)(C:N=2:1)進一步提升到10%,得到的CGTase活性為48580 unit/ml。
以環狀糊精轉移酶將澱粉進行環化反應生成環狀糊精,以2000 unit/g-starch CGTase進行反應,對於5%、10%、15%玉米澱粉轉換率分別為42.8%、27.3%、20.4%,以低濃度澱粉反應有高轉換率。15%玉米澱粉以1000 unit/g-starch CGTase反應,在75℃反應轉換率最高達16.3 %,為了降低環狀糊精生產的成本,先在澱粉液化的階段使用α-amylase進行玉米澱粉的水解,澱粉水解後再加入環狀糊精葡萄糖苷轉移酶,酵素的劑量為CGTase 1000 unit/g-starch,轉換率則可達20 %。
在環狀糊精轉移酶之乾燥方面,以冷凍乾燥方式,環狀糊精轉移酶可達84%殘餘活性。以噴霧乾燥方式,出口溫度分別設定在90 ℃仍保有80%殘餘活性。
Cyclodextrins can be synthesized by cyclodextrin glycosyltransferase (CGTase) from starch. The present investigation employed an alkalophilic Bacillus sp. (ATCC 21595) to produce CGTase using soluble starch (DE10) as carbon source in the fermentation process. The activity of 6099 by the shaking culture and 19540 unit/ml by the batch fermentation were achieved using 2% (w/v) DE10 as carbon source (C:N=1:1). Furthermore, another fermentation using 10% (w/v) DE10 (C:N=2:1) as carbon source was conducted to achieve 48580 unit/ml.

In the production of cyclodextrin, 5, 10, 15 % (w/v) of the corn starch (2000 unit/g starch) was used, and the conversion yield of 0.43, 0.27, 0.20 respectively (g ��-cyclodextrin/g corn starch) was obtained. Higher conversion yield was obtained in lower starch concentration. To reduce the cost of CGTase, 1000 unit/g starch was used to achieve the conversion yield of 0.16. However, the conversion yield could increase to 0.2 using 1000 unit/g starch when ��-amylase was used in the starch liquefaction process.

The CGTase was freeze dried to retain 84% of the residual activity. Spray drying was also conducted remove water content of the CGTase, 80% residual activity was obtained when the outlet gas temperature was set at 90 �aC.
摘要 i
Abstract iii
目錄 v
表目錄 xiii
第一章 緒論 1
1.1 前言 1
1.2 研究動機 2
1.3 組織章節 3
第二章 文獻回顧 4
2.1 細菌生產環狀糊精葡萄糖苷轉移酶 4
2.2 環狀糊精葡萄糖苷轉移酶(Cyclodextrin Glucanotransferase, CGTase) 8
2.3 環狀糊精(Cyclodextrin, CD)的種類、特性 13
2.4 環狀糊精(Cyclodextrin, CD)之應用 19
第三章 實驗材料、設備及方法 23
3.1 材料 23
3.1.1 菌株 23
3.1.2 藥品 23
3.1.3 酵素 24
3.2 設備 24
3.3 方法 26
3.3.1 菌種的保存、配方 26
3.3.2 醱酵系統,如圖3.1.所示。 29
3.3.2.1 培養基滅菌 29
3.3.2.2 pH值 30
3.3.2.3 DO值 30
3.3.2.4 電腦控制系統 31
3.3.2.5通氣 32
3.3.3 環狀糊精反應 32
3.3.4 乾燥 34
3.3.4.1 冷凍乾燥 35
3.3.4.2 噴霧乾燥 35
3.3.5 分析方法 36
3.3.5.1菌體濃度測定 36
3.3.5.1.1 alkalophilic Bacillus sp.菌體乾重之標準曲線 37
3.3.5.2 β-環狀糊精葡萄糖苷轉移酶的活性測定 37
3.3.5.3 殘餘還原糖測定 40
3.3.5.4 葡萄糖含量測定 42
3.3.5.5 β-環狀糊精之含量測定 42
3.3.5.6 α-、β-及γ-CD比例分析 43
3.3.6 環狀糊精葡萄糖苷轉移酶醱酵實驗 44
3.3.6.1 搖瓶試驗 44
3.3.6.2 以不同濃度的Na2CO3進行醱酵槽醱酵試驗 44
3.3.6.3 使用等量的碳氮源進行饋料醱酵及批次醱酵之比較 45
3.3.6.4 以不同濃度的DE10進行醱酵槽醱酵試驗 47
3.3.7 環狀糊精反應 48
3.3.7.1 用2000 unit/g-starch CGTase對不同濃度玉米澱粉所形成β-環狀糊精之轉換率 48
3.3.7.2 於不同溫度以1000 unit/g-starch CGTase對15%玉米澱粉所形成β-環狀糊精之轉換率 48
3.3.7.3 以1000 unit/g-starch CGTase對於15%澱粉反應生成β-環狀糊精之轉換率 49
3.3.7.4 澱粉先以α-amylase水解再加入1000 unit/g-starch CGTase生成β-環狀糊精之轉換率 50
3.3.8 環狀糊精葡萄糖苷轉移酶乾燥 50
3.3.8.1 冷凍乾燥的環狀糊精葡萄糖苷轉移酶 51
3.3.8.2 噴霧乾燥的環狀糊精葡萄糖苷轉移酶 51
3.3.9 環狀糊精乾燥 52
3.3.9.1 冷凍乾燥的環狀糊精 52
3.3.9.2 噴霧乾燥的環狀糊精 53
第四章 結果與討論 55
4.1 環狀糊精葡萄糖苷轉移酶醱酵實驗 55
4.1.1 搖瓶試驗 55
4.1.2 以不同濃度的Na2CO3進行醱酵槽醱酵試驗 57
4.1.3 使用等量的碳氮源進行饋料醱酵及批次醱酵結果之比較 64
4.1.4 以不同濃度的DE10進行醱酵槽醱酵試驗 69
4.2 環狀糊精反應 78
4.2.1 用2000 unit/g-starch CGTase對不同濃度玉米澱粉所形成β-環狀糊精之轉換率 78
4.2.2 於不同溫度以1000 unit/g-starch CGTase對15%玉米澱粉所形成β-環狀糊精之轉換率 78
4.2.3 以1000 unit/g-starch CGTase對於15%可溶性澱粉反應生成β-環狀糊精之轉換率 79
4.2.4 澱粉先以α-amylase水解再加入1000 unit/g-starch CGTase生成β-環狀糊精之轉換率 80
4.3 環狀糊精葡萄糖苷轉移酶乾燥 93
4.4 環狀糊精乾燥 95
第五章 結論 96
參考文獻 97
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