臺灣博碩士論文加值系統

壹、研究背景與目的 1
一、研究背景 1
二、研究目的 2
貳、文獻整理 3
一、醣類 3
1. 幾丁質 3
2. 幾丁聚醣 3
3. 幾丁寡醣 (COS) 3
二、幾丁聚醣水解物之製備 3
1. 化學法 4
2. 物理法 4
3. 酵素法 4
三、幾丁聚醣酶 4
1. 來源與分類 4
2. 內切和外切型 5
四、不同來源幾丁聚醣酶的特性 5
1. Streptomyces albolongus ATCC 27414 來源之 Sa-CSN 5
2. Amycolatopsis sp. CsO-2 來源之 Am-CSN 5
3. Streptomyces sp. N-174 來源之 St-CSN 6
五、Kitasatospora sp. Root187 來源之 CSNK 與 Streptomyces xanthocidicus 來源之 CSN 簡介 6
六、GH 46 家族之 chitosanase 晶體結構 6
1. Streptomyces sp. N-174 6
2. Bacillus circulans MH-K1 7
七、利用分子伴護蛋白增加異源蛋白之表現 7
八、大腸桿菌表現宿主 8
1. E. coli BL21 (DE3) 8
2. E. coli BL21 StarTM (DE3) 8
3. ClearColiTM BL21 (DE3) 8
4. CodonPlus-RIL 9
參、實驗設計與流程 10
一、Kitasatospora sp. Root187 來源之 CSNK 的表現及特性探討 10
二、Streptomyces xanthocidicus 來源之 CSN 的表現及特性探討 10
三、CSNK / CSN 與 chaperone 共表現後於 E. coli BL21 (DE3) 宿主之表現 11
肆、實驗材料與方法 12
一、 材料 12
1. 菌株與載體 12
2. 抗生素 12
3. 標準品 12
4. 市售套組 12
5. 培養基 12
6. 化學藥品 12
7 . 實驗設備 14
二、 藥品配置 16
1. LB-agar 16
2. Terrific broth (TB) 培養液 16
3. SOB 培養液 16
4. SOC 培養液 16
5. Ampicillin (100 mg/mL) 17
6. Chloramphenicol (34 mg/mL) 17
7. 1M IPTG 17
8. 1M Benzamidine 17
9. Chitosan 基質製備 17
10. 測活性之反應液 17
11. DNS reagent 17
12. 4× Dye-binding reagent 17
13. Bovine serum albumin (BSA) stock solution 18
14. SDS-PAGE 相關藥劑製備 18
三、 方法 19
1. Kitasatospora sp. Root187 來源之 CSNK 及 Streptomyces xanthocidicus 來源之 CSN 基因合成 19
2. CSNK 與 CSN 之表現與純化 21
3. CSNK 與 CSN 之特性探討 24
4. CSNK/CSN 與分子伴護蛋白共表現之影響 28
5. 幾丁聚醣去乙醯度測定 28
伍、結果與討論 30
一、 CSNK 與 CSN 的基因挑選 30
二、 CSNK 與 CSN 的表現 30
三、 CSNK 的純化及特性探討 30
1. CSNK 之純化 30
2. CSNK 之特性探討 31
四、 CSN 的純化及特性探討 32
1. CSN 之純化 32
2. CSN 之特性探討 32
五、 CSNK/CSN 與分子伴護蛋白共表現之影響 34
陸、結論 36
柒、參考文獻 37
捌、圖表 42
玖、附錄 80

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