幾丁質酶可分為幾丁質內切酶及幾丁質外切酶兩種，常應用於幾丁質的水解，以生成 N-乙醯幾丁寡醣及幾丁寡醣，這些產物為幾丁質的衍生物且有多元性的生物多活性，而使用酵素之製程汙染低且水解方式和產物分佈易控制品質穩定，本實驗透過不同酶譜法對幾丁質酶酵素活性測驗，分別以灰鏈酶菌之chitinase 對內切型酶及自 Chitinibacter tainanenis 所得具水解幾丁質酵素活性之複合物進行活性之測試以建立幾丁質水解酵素之活性篩選模式。
以乙二醇幾丁質酶譜法分析內切型幾丁質酶之活性及其靈敏度，並使用 pNP-NAG 酶譜法分析 C. tainanenis 外切型幾丁質酶之活性並可於膠體中看到活性並確認其位置，作為分析幾丁質酶體之基礎。

Chitin and its derivatives are versatile and applied broadly. The analogues are usually produced by chemical or biological technique. The quality of products is stable and homogeneous with biological procedure, enzyme degradation. There are a bunch of enzymes distributed worldwide. According to the degradation mechanism, there are two types of chitinases, endochitinase and exochitinase. To identify the chitinolytic enzymes is the issue for biological procedure.
The enzyme activity was monitored with glycol chitin for endochitinases and p-nitrophenyl-N-acetyl-glucosamine for exochitinases. With the substrates, the enzymes cab be identified in situ.

Keyword:zymography、chitinase、glycolchitin、pNP-NAG

明志科技大學碩士學位論文口試委員審定書 i
誌謝 ii
摘要 iii
Abstract iv
目錄 v
圖目錄 ix
表目錄 xi
第一章 緒論 1
第二章 文獻回顧 3
2.1 幾丁質類物質 3
2.1.1 幾丁質 3
2.1.2 幾丁聚醣 3
2.1.3 N-乙醯葡萄糖胺 4
2.2 幾丁質酵素 5
2.2.1 幾丁質分解酵素 5
2.2.2 分類 6
2.2.2.1 幾丁質分解酵素 7
2.2.2.2 幾丁聚醣酶 7
2.2.2.3 幾丁二醣酶 8
2.2.2.4 幾丁質去乙醯基酶 8
2.2.2.5 β-N-乙醯葡萄糖胺酶 8
2.3 幾丁質酵素水解之作用機制 8
2.4 幾丁質分解酵素活性測定方法 9
2.4.1 還原醣測定法 9
2.4.2 濁度測定法 10
2.4.3 黏度測定法 10
2.4.4 放射線測定法 10
2.4.5 螢光測定法 10
2.4.6 N-乙醯葡萄糖胺生成量測定法 11
2.4.7 DNS測定法 11
2.4.8 CM-chitin-RBV測量法 11
2.5 十二烷基硫酸鈉聚丙烯醯胺膠體電泳 12
2.6 酶譜法 12
2.7 浸潤型酶譜法 13
第三章 實驗規劃 14
3.1 實驗目的 14
3.2 實驗器材 15
3.2.1 儀器 15
3.2.2 藥品 16
3.3 實驗步驟 17
3.3.1 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) 17
3.3.1.1 鑄膠玻璃板組合 17
3.3.1.2 10%分離性膠體的配置 17
3.3.1.3 堆積性膠體的配置 17
3.3.1.4 電泳操作方法 17
3.3.2 含受質乙二醇幾丁質之分離膠片的製備 18
3.3.2.1 0.01% 乙二醇幾丁質之10% 分離性膠體 18
3.3.2.2 膠體活性反應程序 18
3.3.3 蛋白質CBB染色及褪色 18
3.3.3.1 膠體之染色 18
3.3.3.2 膠體之褪色 18
3.3.4 蛋白質硝酸銀染色 19
3.3.5 乙二醇幾丁質製備 19
3.3.6 含1% triton X-100 之0.1M pH6醋酸鈉緩衝液配製 19
3.3.7 蛋白質含量測定 20
3.3.7.1 1 mg/ml 標準蛋白溶液製備 20
3.3.7.2 Dye Reagent concentrate 20
3.3.7.3 標準曲線製作 20
3.3.7.4 蛋白質未知濃度樣品測定 20
3.3.8 乙二醇幾丁質之酶譜法 21
3.3.9 乙二醇幾丁質之浸潤型酶譜法 21
3.3.10 pNP-NAG之酶譜法 21
3.3.10.1 原生膠體電泳 21
3.3.10.2 分離性凝膠溶液製備 21
3.3.10.3 堆積性凝膠溶液製備 22
3.3.10.4 電泳操作 22
3.3.10.5 受質緩衝液反應 22
3.3.11 pNP-NAG之浸潤型酶譜法測定 22
第四章 實驗結果與討論 23
4.1 傳統型酶譜法活性的測定 23
4.2 幾丁質酵素定量 23
4.3 傳統型酶譜法靈敏度 23
4.3.1 灰鏈酶菌幾丁質酶 24
4.3.2 Lysozyme傳統型酶譜法活性測定之靈敏度 24
4.3.3 RC蛋白質複合物 24
4.4 浸潤型酶譜法活性測定 24
4.4.1 以乙二醇幾丁質為受質 24
4.4.2 以pNP-NAG為受質 25
第五章 未來展望 26
第六章 參考文獻 51
圖 一、幾丁質分解酵素水解幾丁質之路徑 27
圖 二、幾丁質分解酵素之作用機構 28
圖 三、纖維素、幾丁質、幾丁聚醣之化學結構 29
圖 四、傳統型酶譜法步驟 30
圖 五、浸潤型酶譜法步驟 31
圖 六、乙二醇幾丁質成品 32
圖 七、傳統型酶譜法測定幾丁質酵素 33
圖 八、BSA標準曲線製作 34
圖 九、BSA標準曲線製作 35
圖 十、以SGCTS測定傳統酶譜法之靈敏度 36
圖 十一、SGCTS之銀染 37
圖 十二、以Lysozyme測定傳統型酶譜法 38
圖 十三、lysozyme浸潤型酶譜法浸潤2小時 39
圖 十四、lysozyme浸潤型酶譜法浸潤18小時 40
圖 十五、lysozyme浸潤型酶譜法浸潤24小時 41
圖 十六、pNP-NAG傳統型酶譜法活性測定 42
圖 十七、pNP-NAG浸潤型酶譜法活性測定 43
表 一、各種酶譜法的比較 44
表 二、SDS-PAGE各濃度製作劑量表 47
表 三、SDS-PAGE電泳試劑配置表 48
表 四、CBB染色褪色試劑配方 49
表 五、硝酸銀染色試劑配方與簡易操作表 50

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