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研究生:謝伊金
研究生(外文):Yi-Jin xie
論文名稱:N-乙醯幾丁寡醣生產菌之篩選與幾丁質分解酶之特性分析
論文名稱(外文):Isolation of an N-acetylchitooligosaccharides Producing Bacterium and Characterization of Its Chitinases
指導教授:涂瑞澤涂瑞澤引用關係吳淑姿吳淑姿引用關係
指導教授(外文):Jui-Rze TooShwu-Tzy Wu
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
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:2007
畢業學年度:95
語文別:中文
論文頁數:105
中文關鍵詞:N-乙醯幾丁寡醣幾丁質分解酶酵素分離純化
外文關鍵詞:N-acetyl-chitooligosaccharideschitinaseisolation and purification of enzyme
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本實驗自彰化新寶地區採樣,篩選具分解幾丁質分解酵素之菌株,初步命名為DYU-Too12,經檢驗後得知此菌株為革蘭氏陰性桿菌。
在培養過程中調控起始溶氧量(100%與40%飽和溶氧),以探討其對幾丁質分解酶活性與N-乙醯幾丁寡醣生成之影響。結果顯示,當起始溶氧量為100%與40%飽和溶氧,分別於培養第48與60 h,幾丁質分解酶具最高活性為 833與933 U/L。兩者(100%與40%飽和溶氧)生成之水解產物皆以一~四醣為主,其中N-乙醯葡萄醣胺,最高分別為3.06與0.1 g/L;N-乙醯幾丁二醣,最高分別為1.0 與0.41 g/L;N-乙醯幾丁三醣,最高分別為0.22 與0.40 g/L;N-乙醯幾丁四醣,最高分別為0.19與1.03 g/L。由此結果顯示,於起始溶氧量40%下培養菌株Too12,可生成之N-乙醯幾丁寡醣的聚合度較高。
以不同體積(100與200 mL)培養菌株Too12,探討其對酵素生成之影響。菌株Too12以培養體積(100與200 mL)分別培養72與96 h後所得粗酵素液,經硫酸銨沉澱、透析、DEAE-Sepharose CL-6B及Sephacryl S-100純化步驟後,收集具活性之波峰,酵素之比活性分別為4.70與4.02 U/mg protein,回收率為21.8%與7.23%,純化倍率為3.80與2.77。最適反應溫度為40℃與最適反應pH值為6.0,金屬離子Hg2+、Zn2+、Ag+及Mn2+對於幾丁質分解酶活性皆有抑制作用,而EDTA對幾丁質分解酶之活性則促進作用。
In this study, a strain, named as DYU-Too12, was isolated from the soil of Xin-bao in Changhua County to degrade chitin into N-acetylchitooligosaccharides, and the culture conditions were also examined. This strain was identified to be a Gram’s negative rod shaped bacterium.
The strain was cultivated separately in a 5-L batch fermentor in a CB medium with different levels of initial dissolved oxygen (100% and 40% saturation). The highest activities of chitinase were 833 U/L at 48 h and 933 U/L at 60 h for 100% and 40% saturation, respectively. N-acetyl-chitooligosaccharides with DP (degree of polymerization) 1~4 were produced in these two cases, and highest yields of N-acetyl-glucosamine were 3.06 and 0.1 g/L, N-acetyl-chitobiose was 1.0 and 0.41 g/L, N-acetyl-chitotriose were 0.22 and 0.40 g/L, N-acetyl-chitotetraose were 0.19 and 1.03 g/L, respectively. N-acetyl-chitooligosaccharides with higher DP were produced by strain DYU-Too12 when cultivated at a lower level of dissolved oxygen.
The strain was cultivated separately in a 500-mL flask with each of a volume of 100 or 200 mL of the CB medium. The supernatant of the culture of strain DYU-Too12 cultivated in 100 mL at 72 h and 200 mL at 96 h were further purified. Purification of chitinases was carried out by protein precipitation with ammonium sulfate, dialysis, anion exchange of DEAE-Sepharose CL-6B and gel filtration of Sephacryl S-100 HR. A peak showing chitinase activity was observed, and fractions of this peak were collected for further analysis. The chitinases were purified by 3.8 and 2.77 fold, and the specific activities were 4.7 and 4.02 U /mg protein, and the yields of chitinases were 21.8% and 7.23%, respectively. The optimum reacting temperature was 40℃, and the optimum pH was 6.0 for the chitinases. Metal ions such as Hg2+, Zn2+, Ag+ and Mn2+showed an inhibitive effect on the chitinase activity and EDTA could enhanced the activity.
封面內頁
簽名頁
授權書.....................iii
中文摘要...................iv
英文摘要...................vi
誌謝.......................viii
目錄.......................ix
圖目錄.....................xiii
表目錄.....................xvii

1. 緒論..........................................1
2. 文獻回顧.......................................2
2.1幾丁質.........................................2
2.1.1幾丁質的製備..................................2
2.1.3幾丁質的功能與應用.............................5
2.2幾丁質分解酶....................................5
2.2.1幾丁質分解酶之分類.............................5
2.2.2幾丁質分解酶之應用.............................8
2.2.3幾丁質分解酶之活性分析..........................9
2.2.4幾丁質分解酶之純化與特性........................11
2.3 N-乙醯幾丁寡醣..................................17
2.3.1 N-乙醯幾丁寡醣之製備...........................17
2.3.2 N-乙醯幾丁寡醣之應用...........................18
3. 材料與方法.......................................20
3.1實驗儀器.........................................20
3.2實驗藥品.........................................21
3.3培養基與試劑......................................22
3.3.1培養基組成......................................22
3.3.2膠態幾丁質製備..................................22
3.3.3 McIlvaine buffer之配製........................24
3.3.4還原醣呈色劑之配製...............................24
3.4實驗方法..........................................24
3.4.1菌株篩選、保存及鑑定..............................24
3.4.2菌株生長曲線測定.................................26
3.4.3還原醣測定......................................26
3.4.4幾丁質分解酶活性測定..............................26
3.4.5蛋白質濃度測定...................................26
3.4.6幾丁質水解產物分析................................27
3.5幾丁質分解酶之分離純化...............................27
3.6純化酵素之特性分析..................................28
3.7批次發酵培養.......................................30
3.7.1操作步驟..........................................30
3.7.2培養條件..........................................30
3.8 SDS-聚丙醯胺膠體電泳分析.............................31
4. 結果與討論...........................................35
4.1幾丁質分解菌之篩選....................................35
4.1.1幾丁質分解酶之活性分析與還原醣量......................35
4.1.2水解產物分析........................................39
4.1.3菌株DYU-Too12之生長型態.............................39
4.1.4菌株DYU-Too12之生長曲線.............................41
4.1.5菌株DYU-Too12之幾丁質分解酶、還原醣量、蛋白質量及pH值.................41
4.1.6菌株DYU-Too12之初步鑑定.............................41
4.2幾丁質粗酵素液之特性分析..............................41
4.2.1最適反應pH值.......................................45
4.2.2最適反應溫度.......................................45
4.3不同培養體積培養菌株DYU-Too12.........................45
4.3.1幾丁質分解酶之活性分析...............................48
4.3.2還原醣及蛋白質量分析.................................48
4.3.3幾丁質水解產物分析...................................51
4.4批次發酵培養..........................................55
4.4.1幾丁質分解酶之活性分析...............................60
4.4.2還原醣及蛋白質量分析.................................60
4.4.3幾丁質水解產物分析...................................63
4.4.4生質量分析..........................................67
4.4.5殘留幾丁質分析......................................67
4.5幾丁質分解酶之分離純化.................................70
4.5.1培養於100 mL之幾丁質分解酶的純化......................70
4.5.2培養於200 mL之幾丁質分解酶的純化......................72
4.5.3比較不同條件下生產酵素之不同..........................76
4.6幾丁質分解酶之特性分析..................................81
4.6.1培養於100 mL之幾丁質分解酶的特性分析..................81
4.6.2培養於200 mL之幾丁質分解酶的特性分析..................86
4.6.3比較不同條件下生產酵素之特性分析......................92
5. 結論.................................................96
5.1結論.................................................96
5.2展望.................................................97
參考文獻.................................................98
附錄....................................................103
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