臺灣博碩士論文加值系統

幾丁質是自然界中一種含氮多醣類生物性高分子，其蘊藏量僅次於纖維素，主要為甲殼類動物外殼上的主要構造成分，具有保護及支持生物的作用。一直以來無法被食用，大量的甲殼類廢棄物造成汙染問題，所以可利用幾丁質酵素產生具有活性的幾丁寡醣。
幾丁質酵素廣泛存在於自然界不同生物體，在任何含有幾丁質之動物、植物及微生物身上可發現。從過去研究中了解到這些寡醣中的幾丁六醣產物對於免疫反應與抗腫瘤有關。近年來，在台灣南部紅樹林的土壤裡分離出菌株Aeromonas schubertii，可從A.schubertii分離純化得到產物為幾丁六醣之幾丁質酵素AsChi61。
由於利用重組蛋白質量產目標蛋白質以滿足在生物醫學、農業或是工業應用上逐漸增加之需求為近年來之趨勢，故利用大腸桿菌生產重組蛋白質是一種快速及滿足經濟效應的方式。本研究以大腸桿菌BL21 (DE3)株及大腸桿菌DH5α株作為重組AsChi61表現His-tag及其GST融合蛋白質之菌株，進行生產重組型幾丁質酵素AsChi61。藉由界面活性劑對其大腸桿菌的包涵體中之AsChi61於cobalt resin及GSH agarose 分別進行純化，以達到重組AsChi61之大量表現。

Chitin is a biological polymer of nitrogenous polysaccharide. It is the abundant polysaccharide next to cellulose in world. Chitin distributes in many organisms for protection and support mainly. It is the main component of exoskeleton of crustaceans and the key molecule in physiology such as immune response and lubricant in the cartilage. However, chitin is also the main solid waste in the seafood processing industry. By chemical and biological process, chitin can be isolated for application.
Chitin analogues, including polysaccharides, oligosaccharides and monosaccharides, are biodegradable, histocmpatible and nontoxic. The chitin-oligosaccharides have been found to enhance the functions of immunological system and they might display as tumoricidal agents. Among the derivates, the N-acetylchitohexaose with strong biological medical function has been demonstrated. Recently, a microorganism, Aeromonas schubertii was isolated and its chitinase, AsChi61, was identified. The hexameric chitinoligosaccharide is the main product of the enzyme.
A plenty of recombinant proteins expressed for industrial, agricultural or biomedical applications have elevated dramatically. The recombinant AsChi61 was expressed in GST-fusion protein or His tagged one. For large scale expression, the detergents were subjected for purifying the recombinant protein from inclusion in Escherichia coli.

明志科技大學碩士學位論文指導教授推薦書 i
明志科技大學碩士學位論文口試委員審定書 ii
誌謝 iii
中文摘要 iv
Abstract v
縮寫表 vi
目錄 ii
圖目錄 viii
表目錄 ix
第一章 緒論 1
1.1 前言 1
1.2 誘導機制 2
第二章 文獻回顧 4
2.1 幾丁質介紹 4
2.1.1 幾丁質 4
2.1.2 幾丁聚醣 4
2.2 幾丁質結構 5
2.2.1 化學結構 5
2.2.2 晶體結構 5
2.3 幾丁質在自然界的分佈 6
2.3.1 環節動物 6
2.3.2 節肢動物 6
2.3.3 軟體動物 6
2.3.4 腔腸動物 7
2.3.5 海藻類 7
2.3.6 真菌類 7
2.4 幾丁質及其衍生物之製備 7
2.4.1 化學修飾法 8
2.4.2 超音波降解法 8
2.4.3 酸水解法 8
2.5 幾丁質酵素 8
2.5.1 幾丁質酵素用途、分佈與分類 8
2.5.2 動物幾丁質酵素 9
2.5.3 植物幾丁質酵素 9
2.5.4 微生物幾丁質酵素 10
2.6幾丁質酵素之命名 10
2.6.1 Family 18 10
2.6.2 Family 19 11
2.6.3 Family 20 12
2.7幾丁質酵素水解模式之分類 12
2.7.1 內切型幾丁質酵素(endo-chitinase) 12
2.7.2 外切型幾丁質酵素(exo-chitinase) 12
2.7.3 任意型幾丁質酵素(random chitinase) 12
2.7.4 轉醣型幾丁質酵素(transglycosylation-catalyzingchitinase) 12
2.8 幾丁質酵素之純化 13
2.8.1 野生型幾丁質酵素之純化 13
2.8.2 重組型幾丁質酵素之純化 13
2.8.2.1 His-tag融合重組蛋白質純化 13
2.8.2.2 GST 融合重組蛋白質純化 14
2.9幾丁質酵素活性分析方法 14
2.9.1 還原醣測定法 14
2.9.2 DNS測定法 14
2.9.3 幾丁質藍測定法(chitin azure) 15
2.9.4 膠態幾丁質藍測定法(colloidal chitin azure) 15
2.9.5 CM-chtitn-RBV測量法 15
2.9.6 N-乙醯葡萄糖胺生成量測定法 15
2.9.7 黏度計測定法 16
2.9.8 混濁度計測定法 16
2.9.9 螢光光度測定法 16
2.9.10 放射性測定法 16
2.9.11 電子掃描測定法 17
2.10 以大腸桿菌表現重組蛋白質的系統 17
2.10.1 pET表現系統 17
2.10.2 GST表現系統 17
2.11 蛋白質的鑑定 18
2.11.1 蛋白質之N端胜肽定序 18
2.11.2 質譜法 18
2.12幾丁質酵素AsChi61 19
2.12.1 於pET表現系統表現 19
2.12.2 於GST表現系統表現 20
第三章：實驗規劃 21
3.1 實驗目的 21
3.2 實驗器材 22
3.2.1 儀器 22
3.2.2 藥品 23
3.2.3 材料 24
3.3 實驗方法 25
3.3.1 菌種來源 25
3.3.2 1/3TB培養基的配製 25
3.3.3 1/3TB(Amp+)培養基的配製 25
3.3.4 1/3TB培養液的配製 25
3.3.5 1/3TB培養液(Amp+)的配製 25
3.3.6 轉型(transformation) 26
3.3.7 液態培養 26
3.3.8 IPTG誘導重組蛋白生產 26
3.3.9 小量表現(small scale expression ) 26
3.3.10 SDS-PAGE 27
3.3.10.1 鑄膠玻璃板之裝備 27
3.3.10.2 分離性膠體的配製 27
3.3.10.3 堆積性膠體的配製 27
3.3.10.4蛋白質電泳樣品的製備 27
3.3.10.5 蛋白質電泳操作方法 28
3.3.10.6 蛋白質電泳膠體CBB染劑 28
3.3.10.7 蛋白質電泳膠體CBB染色 28
3.3.10.8 蛋白質電泳膠體CBB褪染 28
3.3.10.9 蛋白質電泳膠體銀染色法(silver stain) 28
3.3.11蛋白質大量表現 29
3.3.11.1試劑之製備 29
3.3.11.1.1濃度10 毫克/毫升 溶菌酶的配製 29
3.3.11.1.2濃度10 毫克/毫升DNase I的配製 29
3.3.11.1.3濃度10 毫克/毫升RNase A的配製 29
3.3.11.1.4 PB溶液的配製 29
3.3.11.1.5 緩衝溶液PBS的配製 29
3.3.11.1.6 咪唑(imidazole)的配製 30
3.3.11.1.7 緩衝溶液STE的配製 30
3.3.11.1.8 濃度 1 M 二硫蘇糖醇的配製 30
3.3.11.1.9濃度10% 月桂醯肌氨酸鈉的配製 30
3.3.11.1.10 濃度1 M HEPES,pH7的配製 30
3.3.11.1.11 濃度100 mM 還原型穀胱甘肽的配製 31
3.3.11.1.12 GST沖洗緩衝溶液 31
3.3.11.1.13 GST沖提緩衝溶液 31
3.3.11.2 大量表現 31
3.3.11.2.1 菌體發酵 31
3.3.11.2.2 pET系統 31
3.3.11.2.2.1 樣品製備 31
3.3.11.2.2.2 純化 32
3.3.11.2.3 GST系統 32
3.3.11.2.3.1樣品製備 32
3.3.11.2.3.2 純化 32
3.3.12 蛋白質轉漬法 33
3.3.12.1試劑之製備 33
3.3.12.1.1 CAPS轉漬緩衝液 33
3.3.12.1.2 染色液 33
3.3.12.1.3 褪色液 33
3.3.12.2 轉漬操作程序 33
3.3.12.3 自PVDF膜回收蛋白質 34
3.3.12.4 TCA蛋白質純化 34
第四章：實驗結果 35
4.1 菌種培養 35
4.2 於pET系統中重組蛋白AsChi61 35
4.2.1 小量表現 35
4.2.2 大量表現 35
4.2.3 蛋白質轉漬 35
4.3 於GST系統中重組蛋白AsChi61 36
4.3.1小量表現 36
4.3.2大量表現 36
第五章：結論與展望 37
參考文獻 56

圖目錄
圖一、幾丁質、幾丁聚醣、纖維素之化學結構。 38
圖二、α-chitin與β-chitin的分子鏈排列情形。 39
圖三、自然界中幾丁質常見三種晶體型態之結構。 40
圖四、四種幾丁質酶水解模式。 41
圖五、表現載體pGEX2T轉型至E.coli (DH5α)之三區劃線。 42
圖六、以IPTG誘導E.coli BL21(△DE3)於胞內生產重組蛋白質AsChi61。 43
圖七、大量表現蛋白質純化pET15b-AsChi61膠體電泳 44
圖八、大量表現蛋白質純化pET15b- AsChi61膠體電泳 45
圖九、pET15b-AsChi61大量表現之蛋白質轉漬 46
圖十、以IPTG誘導E.coli DH5α於胞內生產重組融合蛋白質GST- AsChi61。 47
圖十一、大量表現蛋白質純化pGEX2T-AsChi61膠體電泳 48
圖十二、大量表現蛋白質純化pGEX2T-AsChi61膠體電泳 49

表目錄
表一、各個濃度的SDS-聚丙烯醯胺膠片製作劑量表 50
表二、SDS-PAGE電泳試劑配置表 51
表三、1/3TB固態培養基配置表 52
表四、1/3TB培養液配置表 53
表五、CBB染色液及染色液配置表 54
表六、硝酸銀染色試劑配方與簡易操作表 55

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