目錄

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、 幾丁質及其相關醣類 3
1. 幾丁質 (chitin) 3
2. 幾丁聚醣 (chitosan) 4
3. 幾丁寡醣 (COS) 5
二、 幾丁聚醣酶 (chitosanase) 7
三、 不同來源之幾丁聚醣酶的特性 8
1. Streptomyces albolongus ATCC 27414 來源之幾丁聚醣酶 8
2. Streptomyces sp. N174 來源之幾丁聚醣酶 8
3. Streptomyces griseus HUT 6037 來源之幾丁聚醣酶 8
4. Streptomyces coelicolor A3 (2) 來源之幾丁聚醣酶 8
5. Streptomyces sp. no. 6 來源之幾丁聚醣酶 9
6. Streptomyces roseolus 來源之幾丁聚醣酶 9
7. Amycolatopsis sp. CsO-2 來源之幾丁聚醣酶 9
四、 Kitasatospora sp. Root187 來源之 CSNK 介紹 9
五、 提升酵素熱穩定性 10
1. 氫鍵 (hydrogen bond) 10
2. 鹽橋 (salt bridges) 10
3. 疏水性交互作用 (hydrophobic interactions) 11
4. 芳香基團之交互作用 (aromatic interactions) 11
5. 導入雙硫鍵 (disulfide bonds) 11
6. 增加脯胺酸 (proline, Pro, P) 或減少甘胺酸 (glycine, Gly, G) 12
7. 酵素表面導入精胺酸 (arginine, Arg, R) 12
六、 電腦輔助軟體介紹 13
1. SWISS-MODEL 13
2. PyMOL 13
3. Disulfide by Design 2 13
參、 實驗設計與流程 15
肆、 材料與方法 16
一、 實驗材料 16
1. 菌株與載體 16
2. 抗生素 16
3. 培養基 16
4. 標準品 16
5. 市售套組 16
6. 酵素 16
7. 化學藥品 17
8. 實驗設備 19
9. 電腦輔助軟體 20
二、 實驗步驟與方法 21
1. 選擇突變點 21
2. 定位突變 21
3. 轉形至表現宿主 E. coli BL21 (DE3) 26
4. 菌種保存 26
5. 突變型酵素熱穩定篩選 27
6. CSNK表現與純化 28
7. 蛋白質特性探討 30
伍、 結果與討論 33
一、 突變點選擇及引子設計並進行定位突變 33
二、 以酵素活性及熱穩定性篩選突變型酵素 33
三、 探討純化突變型 CSNK 特性 34
1. 大量表現與純化 34
2. 熱穩定性 34
3. 最適作用 pH 值 35
4. 最適作用溫度 35
陸、 結論 37
柒、 參考文獻 38
捌、 圖表 46
玖、 附錄 65

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