海藻糖 (1-α-D-glucopyranosyl-α-D-glucopyranoside) 是由兩個葡萄糖以 α-1,1 糖苷鍵鍵結所形成的非還原性雙糖，目前已被廣泛應用在許多領域，包括多種生物及器官的抗凍劑、化妝品的保濕成份、臨床製劑的穩定劑、花卉的保鮮劑以及做為食品的甜味劑及防腐劑等。為了使酵素達到重覆使用之目的，本研究將利用融合幾丁質結合區 (chitin binding domain, CBD) 片段及訊息胜肽 (signal peptide, SP) 與目標蛋白結合產生融合蛋白，使酵素可進行胞外分泌，再與幾丁質進行酵素固定化。將本實驗室先前已構築完成之Sulfolobus solfataricus ATCC35092 及 S. acidocaldarius ATCC33909 來源之海藻糖生成相關基因 (treX、treY、treZ) 之 pHT254 質體，利用基因工程技術建立含有 SP 及 CBD 之海藻糖生成相關基因的重組質體，並轉形至枯草桿菌系統進行蛋白質胞外分泌。經 SDS-PAGE 分析及酵素活性測定後發現，含有 SP 及 CBD 之重組酵素未能如預期有效進行胞外分泌。利用 DNA 突變技術進行 DNA 剔除，將啟始密碼子與核糖體結合之間的限制酶剪切位序列進行剔除，以探討限制酶剪切位序列是否會影響核醣體進行轉譯，發現經基因剔除後，蛋白質表現量並無明顯提昇。因此改為利用幾丁聚醣將酵素進行固定化，利用戊二醛做為交聯劑，直接將原生型酵素與幾丁聚醣結合，進行酵素固定化反應。結果發現戊二醛及酵素添加量皆會影響酵素固定化之效率，當 SsMTSase、SaMTSase 及 SsMTHase 之酵素活性為 0.34 U/mL、1.37 U/mL 及 1.22 U/mL，戊二醛濃度為 0.5%、0.25% 及 0.25% 時，其固定化效率分別為 85.49%、18.37% 及 40.49%。利用固定化酵素生產海藻糖，發現幾丁聚醣固定化酵素不耐高溫長時間反應，會發生崩解現象。未來應選用耐高溫之固定化材料進行酵素固定化，以利熱穩定性酵素於工業上之應用。

Trehalose (1-α-D-glucopyranosyl-α-D-glucopyranoside) is a non-reduced disaccharide which is composed of two molecules of glucose with α-1,1 glycosidic bond, it is currently used in many fields, include cryopreservations of many organisms and organs, cosmetic moisturizers, stabilization of clinic reagents, preservations of flowers and foods, sweetner in food industry, and so on. On the purpose of making enzymes to be reused, this study fused a chitin binding domain (CBD) and signal peptides (SP) to target proteins. Target proteins were expected to secret extracellularly, and then could be immobilized on chitin. The trehalose-producing related genes, treX, treY, treZ from Sulfolobus solfataricus ATCC35092 and treY of S. acidocaldarius ATCC33909 were cloned into pHT254 vector. Genetic engineering techniques were used to construct CBD and SP into these targeted genes, then these genes were transformed into Bacillus subtilis to express the enzymes. After SDS-PAGE analysis and enzyme activity measurements, we found that recombinant enzymes couldn’t be secrected extracellularly as expected. Since the ill secretion might arise from the extra restriction recognition sequence between ribosome binding site and start codon, site-directed mutagenesis was performed to delete this extra sequence. After deletion, these enzymes still can’t be expressed well. Therefore, glutaraldehyde was used as cross-linker to immobilize wild-type trehalose-producing related enzymes on chitosan beads. The concentrations of glutaraldehyde and the amounts of enzymes would have influenced the immobilized efficiencies. When the activities of SsMTSase, SaMTSase and SsMTHase were 0.34 U/mL, 1.37 U/mL and 1.22 U/mL respectively, and the concentrations of glutaraldehyde were 0.5%, 0.25% and 0.25% respectively, the immobilized efficiencies were 85.49%, 18.27% and 40.49%, respectively. When these immobilized enzymes were used to produce trehalose from starch under high temperature for longer reaction time, the enzyme-immobilized chitosan beads turned out to be melted. To perform enzyme reaction at high temperature, finding the immobilization materials with thermo-tolerance is needed in the future.

目錄
壹、研究背景與目的 1
貳、文獻整理 2
一、海藻糖 2
1. 海藻糖之發現 2
2. 海藻糖之特性 2
3. 食用安全性 2
4. 海藻糖之應用 3
5. 海藻糖之生產 3
6. 利用澱粉生產海藻糖 5
7. 測定海藻糖產量之方法 6
二、蛋白質表現系統之簡介與比較 6
1. 訊息胜肽 6
三、固定化酵素 8
1. 固定化酵素之簡介 8
2. 利用幾丁質進行固定化 8
3. 利用幾丁聚醣進行固定化 8
參、實驗設計 10
肆、實驗材料與方法 13
一、實驗材料 13
1. 實驗用質體與菌株 13
2. 抗生素 13
3. 標準品 13
4. 市售套組 13
5. 酵素 14
6. 化學藥品 14
7. 實驗設備 15
8. 藥品配製 16
二、實驗方法 21
1. 設計含有訊息胜肽之 CBD 基因 21
2. 抽取質體 DNA 21
3. DNA 電泳 21
4. 限制酶剪切 22
5. 核酸接合作用 (Ligation) 23
6. 定位突變 23
7. 熱休克轉形作用 25
8. Colony PCR 篩選轉形株 25
9. 基因定序 26
10. 以電穿孔轉形重組質體至 B. subtilis WB800N 26
11. B. subtilis WB800N 添加不同濃度之 IPTG 26
12. 異澱粉酶、MTSase 及 MTHase 之大量生產 27
13. 異澱粉酶、MTSase 及 MTHase 之活性測定 27
14. 酵素固定化 31
15. 海藻糖產量試驗 31
伍、結果與討論 33
陸、結論 39
柒、縮寫列表 40
捌、參考文獻 41
玖、圖表 46

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