摘要

L-鼠李糖異構? (L-rhamnose isomerase, L-RhI) 是屬於一種醛酮糖異構? (aldose-ketose isomerase or ketol isomerase)。能夠催化醛糖與酮糖間之轉換，並產生大自然中稀少的糖類。
將 Thermoanaerobacterium sp. NTOU2 之 L-RhI 基因進行選殖構築於表現載體 pET-21b 上，並於宿主 Escherichia coli BL21(DE3)-CodonPlus-RIL 表現酵素。重組之酵素則經由陰離子層析管柱進行純化，純化後可得重組之 L-RhI 其比活性為 109.8 U/mg，純化倍率為 4.26，而活性回收率為 28.6%。 L-RhI 基因全長含有 1278 個鹼基，可轉譯出 426 個胺基酸，重組型之 L-RhI 其分子質量為 49 kDa。重組型之 L-RhI 的最適作用溫度為 80oC，而最適作用 pH 為 7.5，於 40-70oC 與 pH 5.0-9.0 之間具有良好的穩定性，而經含有1 mM EDTA 之 20 mM Tris-HCl buffer (pH 7.5) 透析處理後之重組 L-RhI 活性會完全喪失，經 EDTA 處理後之 L-RhI 若於酵素反應液中添加 Mn2+ 與 Co2+，則可使酵素活性恢復。
經由無限制?剪切位基因選殖法 (restriction-Free cloning) 成功將 L-RhI 基因自 pET21b-L-rhi 表現載體選殖入酵母菌表現載體 pPICZα B，並將 pPICZα B-L-rhi 載體成功送入 Escherichia coli (DH5α)。選殖成功之載體再以電穿孔作用 (electroporation) 之方式轉形至 Pichia pastoris，已有轉型株生長，有待後續分析。

目錄 i
圖目錄 iii
表目錄 iv
壹、前言 1
貳、文獻整理 3
1. L-rhamnose isomerase 3
1.1 L-rhamnose isomerase 背景 3
1.2 L-rhamnose isomerase 催化機制 4
1.3 L-rhamnose isomerase 結構 4
1.4 不同來源 L-RhI 之特性比較 5
2. 稀有糖類簡介與應用 6
3. 異源表現系統 7
3.1 真核微生物細胞 7
3.2 嗜甲基酵母菌 P. pastoris 表現生產蛋白質的優點 9
3.3 P. pastoris 之甲醇帶謝途徑 10
3.4 驅動異源蛋白質表現之 AOX 啟動子 10
3.5 表現載體嵌入 P. pastoris 染色體之作用 11
3.6 酵母菌之轉殖與基因重組 12
3.7 重組蛋白之表現型式 12
參、實驗設計、材料與方法 13
1. 實驗設計 13
2. 實驗流程 14
2.1 L-RhI 在大腸桿菌的表現與純化 14
2.2 將 L-rhi 選殖至酵母菌 15
偵測粗酵素液活性 15
肆、材料與方法 16
1. 實驗材料 16
1.1 L-rhi 基因來源 16
1.2 質體來源 16
1.3 菌株來源 16
2. 標準品 17
3. 市售套組 17
4. 酵素與 PCR 試劑 17
5. 化學藥品 18
6. 實驗設備 20
2. 實驗方法 22
2.1 大腸桿菌中 L-RhI 之蛋白質表現與純化 22
2.1.1 Terrific Broth (TB) 培養基之製備 22
2.1.2 L-RhI 在大腸桿菌中之表現 22
2.1.3 於大腸桿菌中表現之 L-RhI 之純化 23
2.2 選殖、擴增 Thermoanaerobacterium sp. NTOU2 之 L-rhi 基因 23
2.2.1 製備小量質體 DNA 24
2.2.2 DNA濃度之定量 25
2.2.3 Low salt Luria-Bertani Broth (LLB Broth) 培養基之製備 25
2.4 Resteiction-Free (RF) cloning之PCR反應 25
2.4.1 設計RF cloning之引子 25
2.4.2 TE buffer 之製備 25
2.4.3 RF cloning之 PCR 反應 26
2.4.3.1 第一階段 PCR 反應 26
2.4.3.2 第二階段 PCR 反應 27
2.4.3.2 PCR 反應產物純化 27
2.4.3.3 第三階段 PCR 反應 28
2.5 模板股 DNA 之剪切 29
2.6 製備電穿孔勝任細胞 E. coli DH5α 29
2.7 電穿孔轉形作用 (electroporation) 30
2.8 以 Colony PCR 篩選轉形株 30
2.8.1 Colony PCR 反應之溶液組成 30
2.9 膠體電泳分析 31
2.10 序列分析 31
2.11 菌種保存 32
3. L-rhi 轉殖至酵母菌 32
3.1 酵母菌之菌種保存與活化 32
3.2 Yeast Extract Peptone Dextrose (YPD) 培養基製備 32
3.3 大量質體 DNA 製備 32
3.4 酵母菌電穿孔勝任細胞製備 33
3.5 酵母菌電穿孔轉形作用 34
4. 酵素 L-RhI 表現量測定 34
4.1 蛋白質定量 34
4.1.1 定量蛋白質相關試劑之製備 35
4.1.2 測定蛋白質濃度 35
4.2 蛋白質電泳分析 35
4.2.1 膠體成份 35
4.2.2 膠體製備 36
4.3 膠電泳之操作方法 37
4.3.1 2X SDS sampling buffer之製備 37
4.3.2 膠片之染色與脫色 38
4.3.3 製備染色液 38
4.3.4 製備脫色液 38
5. 酵素性質分析 38
5.1 L-RhI 酵素反應 38
5.1.1 Fructose 標準曲線 38
5.1.2 Cysteine-Cabrzole 法偵測 L-RhI 活性 39
5.1.3 製備 Cysteine-Cabrzole reagent 39
5.2 L-RhI特性分析 40
5.2.1 L-RhI的最適作用溫度探討 40
5.2.2 L-RhI 的最適作用 pH 值探討 40
5.2.3 L-RhI 的熱穩定性探討 40
5.2.4 L-RhI 的 pH 值穩定性探討 40
5.2.5 金屬離子對 L-RhI 的影響 41
5.2.6 基質特異性分析 41
伍、結果與討論 41
1. 重組型酵素 L-RhI 之蛋白質表現與純化 41
2. 重組型 L-RhI 之特性探討 42
2.1 最適作用溫度 42
2.2 最適作用 pH 43
2.3 熱穩定性 43
2.4 pH 穩定性 44
2.5 金屬離子之影響 44
2.6 不同基質之特異性 45
2.7 序列比對 45
3. Thermoanaerobacterium sp. NTOU2 之 L-RhI 基因的擴增與選殖 46
陸、結論 48
柒、參考文獻 49

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