臺灣博碩士論文加值系統

幾丁質 (chitin) 是典型的海洋多醣，也是豐富的生物質資源，然而，幾丁質因不溶於水之特性，於應用方面受到相對限制，幾丁質經過酵素水解或是鹼處理後會形成幾丁聚醣 (chitosan)，幾丁聚醣又可再透過酵素可生成幾丁寡醣 (chitosan oligosaccharide, COS)，COS 分子量較小，且易溶於水溶液，使 COS發揮有價值的生物活性，已有研究利用幾丁聚糖酶 (chitosanase) 生產 COS 確保更高的產率和產物特異性。 故本次實驗利用 Kitasatospora sp. Root187 來源之幾丁聚醣酶 (CSNK)，利用定位突變以提升重組幾丁聚醣酶的活性及修飾產物特異性。結果顯示原生型於 45ºC、pH 4.75，含有金屬離子 Mn2+ 的反應條件下具有最佳活性，最終產物主要為 (GlcN)2 和 (GlcN)3。突變型與原生型相比，僅 D89A-E117A 之最適溫度上升，且最適 pH 值皆為pH 4.75，此外，D89A、E117A 及 D89A-E117A總活性分別為原生型的 1.98、1.81 及 3.02 倍，而 S53R 活性大幅降低，E65A 與去除 E227- A228-H229-S230 以甘胺酸代替 (replace) 則無測得活性。以 CSNK 生產 COS 時，添加錳離子可增加反應速率及幾丁二醣之產率，此外，D89A 及 D89A-E117A 最終產物幾丁三醣的含量增加 10%，而 S53R 最終產物幾丁二醣佔 75%，且提升S53R 至 10 U 最終產物幾丁二醣佔 90%，成功利用突變將產物特異性提升，從而實現高純度幾丁二糖之成本效益製備，未來可透過結合其他突變以增加活性提升反應效率。

Chitin, a prevalent marine polysaccharide, is a substantial biomass resource. Its insolubility in water, however, restricts its applications. Chitin will be converted to chitosan after enzymatic hydrolysis or alkali treatment. Then, enzymatic processes can degrade chitosan into chitosan oligosaccharide (COS), which has a lower molecular weight and is water-soluble, enhancing its biological activity. This study employed chitosanase (CSNK) from Kitasatospora sp. Root187, utilizing site-directed mutagenesis to enhance enzyme activity and modify product specificity. The wild-type showed optimal activity at 45°C and pH 4.75, in the presence of Mn2+ ions, predominantly producing (GlcN)2 and (GlcN). Among the mutants, only D89A-E117A exhibited an increased optimal temperature, with all mutants maintaining an optimal pH of 4.75. Enzyme total activities for D89A, E117A, and D89A-E117A were 1.98, 1.81, and 3.02 times that of the wild-type, respectively, while S53R activity was significantly reduced. Mutants E65A and E227-A228-H229-S230 replaced by glycine showed no detectable activity. In the production of COS, product analysis indicated that Mn2+ ions increased the reaction rate and yield of chitosan disaccharide. The final products of D89A and D89A-E117A mutants showed a 10% increase in the yield of chitosan trisaccharide, while the S53R mutant produced 75% chitosan disaccharide. When the activity of S53R was increased to 10 U, the final product was composed of 90% chitosan disaccharide. These findings demonstrate that mutagenesis can improve product specificity, facilitating cost-effective production of high-purity chitosan disaccharide. Future research may focus on additional mutations to further enhance enzyme activity and reaction efficiency.

謝誌 I
摘要 II
Abstract II
目錄 III
圖目錄 VI
壹、 研究背景 1
貳、 研究目的 1
參、 文獻整理 2
一、 幾丁質 2
1. 特性 2
2. 製備 2
二、 幾丁聚醣 2
1. 特性 2
2. 製備 3
三、 幾丁寡醣 3
1. 特性 3
2. 製備 3
四、 幾丁聚醣酶 4
五、 不同來源之幾丁聚醣酶之特性 4
1. 來自Kitasatospora sp. Root187 來源之 CSNK 4
2. 來自 Bacillus cereus 之 D-11 5
3. 來自 Bacillus cereus 之 TKU006 5
4. 來自Serratia marcescens 之 TKU011 5
5. 來自Janthinobacterium sp. strain 4239 之 BMS172 5
6. 來自 Gongronella sp. JG 之 Csn2 6
7. 來自 Streptomyces sp. N174 來源之 CSN174 6
六、 立體結構 6
七、 同源結構模擬 (homology modeling) 7
肆、 實驗架構 9
伍、 實驗材料 10
一、 菌株 10
二、 載體 10
三、 抗生素 10
四、 標準品 10
五、 市售套組 10
六、 酵素 10
七、 化學藥品 11
八、 實驗設備 12
九、 軟體 13
陸、 實驗方法 14
一、 電腦模擬 14
二、 定位突變 14
1. 選擇突變點 14
2. 設計引子 14
3. 質體 DNA 抽取 14
4. PCR 反應 14
5. 分析 PCR 產物 15
6. 利用限制酶剪切模板 DNA 16
7. 熱休克轉形 16
8. Colony PCR 16
9. 電穿孔轉形 17
三、 突變型 pET-21b-CSNK 之表現與純化 18
1. 轉形至表現宿主 18
2. 小量誘導表現突變型 CSNK 18
3. 菌種保存 19
4. 蛋白質電泳分析 19
5. 大量誘導表現突變型 CSNK 20
6. 細胞破碎 21
7. 鎳離子親和性層析 (Ni2+ affinity chromatography) 21
四、 突變型 CSNK 之活性分析 22
1. 蛋白質定量 22
2. 酵素活性分析 23
五、 CSNK 產物分析 24
1. 利用突變型 CSNK 生產幾丁寡糖之基質配製 24
2. 利用突變型 CSNK 生產幾丁寡糖 24
3. 以 HPLC 分析酵素產物 24
柒、 結果與討論 26
一、 電腦模擬 26
二、 選擇突變點的過程 26
三、 利用蛋白質工程修飾重組幾丁聚醣酶 27
四、 以酵素活性篩選突變型酵素 27
五、 最適作用溫度 28
六、 最適作用 pH 值 28
七、 純化酵素活性 29
八、 幾丁寡糖之生產 29
捌、 結論 31
玖、 參考文獻 32
壹拾、 圖表 37
壹拾壹、 附錄 I

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