L-阿拉伯糖異構酶(L-arabinose isomerase, L-AI)主要功能為催化轉換L-阿拉伯糖和L-核酮糖之間的可逆作用，亦可催化其他醛糖與酮糖的轉換，如D-半乳糖轉換成D-塔格糖。本實驗由Thermoanaerobacterium saccharolyticum NTOU1之基因體中擴增出araA基因，並選殖入大腸桿菌中大量表現酵素，並以鎳離子親和性管柱層析方式純化L-AI 。其最適作用溫度為70 °C，最適作用pH為7−7.5，在溫度55至65 °C狀態下二小時仍可維持完整的活性，其對pH穩定的範圍為pH 6.5 到9.5。此酵素的反應在低濃度的二價金屬離子含量下即具有良好活性，不論是在含有0.1 mM錳離子或0.05 mM 鈷離子狀態下，均可維持在高溫狀態下的穩定性和催化能力。由T. saccharolyticum NTOU1 來源的L-AI已證實為目前反應中添加最少量金屬離子的高溫菌來源L-AI。
此熱穩定T. saccharolyticum NTOU1 之L-AI可用於稀有醣類的生產，例如: 以D-半乳糖生產D-塔格糖，或由L-阿拉伯糖生產L-核糖。D-塔格糖的生產，以L-AI在60°C下反應十小時產量為20%，而在反應十小時可達平衡且產量為41%。而L-核糖的生產，先利用T. saccharolyticum NTOU1 之L-AI先轉換成L-核酮糖，再經由G. obscurus DSM43160 L-核糖異構酶 (L-ribose isomerase, L-RI) 轉換產生L-核糖。所以我們先以L-AI在60°C狀態下反應六小時，再添加L-RI隨即以30°C和40°C反應六小時，最終獲得L-核糖的轉換率分別為15.9% 和 12.5%。

為了能提高L-AI在較低作用温度及較低pH值下的活性，我們建構了六種突變型L-AI，分別為N172H, V259K, V259E, V259Q, V259S 和I402A。其中突變型N172H及I402A L-AIs在40~80℃下，皆可增加對D-半乳糖的活性，N172H L-AI的最適作用溫度為70℃與原生型L-AI相同，而I402A L-AI的最適作用溫度為65℃。另外，突變型V259K L-AI在pH 6.5至7.5範圍下，可增加活性21~33%。

L-Arabinose isomerase (EC 5.3.1.4, L-AI) mainly catalyzes the reversible aldose-ketose isomerization between L-arabinose and L-ribulose. L-AIs can also catalyze other reactions, such as the conversion of D-galactose to D-tagatose. In this study, the araA gene encoding L-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant L-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7−7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55 to 65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn2+ or 0.05 mM Co2+, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 L-AI has the lowest demand for metal ions among all characterized thermophilic L-AIs.
This thermophilic T. saccharolyticum NTOU1 L-AI can used to produce rare sugars, such as the production of D-tagatose from D-galactose and the producton of L-ribose from L-arabinose. The conversion yield of D-tagatose from D-galactose by using the L-AI at 60 oC was around 20% after 10 h, and around 41% when the reaction was approaching equilibrium (50 h). For the production of L-ribose, L-arabinose isomerization was first performed at 60 ºC for 6 h using T. saccharolyticum NTOU1 L-AI. After 6 h of the L-AI reaction, G. obscurus DSM43160 L-RI was added, and the reaction was continued at 30 ºC or 40 ºC for an additional 6 h. The conversion yields of L-arabinose to L-ribose by combining the L-AI and L-RI at 30 ºC and 40 ºC were 15.9% and 12.5% (mol mol-1), respectively.
To increase the activity of L-AI at lower reaction temperatures and lower pH values, we have constructed 6 mutant L-AIs, including N172H, V259K, V259E, V259Q, V259S, and I402A. Compare to wild-type L-AI, mutant N172H and I402A L-AIs have increased their avtivities against D-galactose at 40~80℃. N172H L-AI has an optimum temperature at 70℃ which is same as that of wild-type, while I402A L-AI has an optimum temperature at 65℃. And, mutant V259K L-AI has 21~33% increased activity in the pH range of 6.5~7.5.

目錄 Ⅰ
圖目錄 ⑻Ⅱ
表目錄 Ⅲ
一、實驗背景與目的 1
二、文獻整理 2
1. 稀有醣類(rare sugars) 2
1.1. 簡介 2
1.2. 來源及生產方式 2
1.3. 核糖(ribose) 3
1.4. 塔格糖介紹 3
1.4.1. 特性 3
1.4.2. 發現 4
1.4.3. 功能 4
1.4.4. 代謝途徑 5
2. 塔格糖之生產方式與L-阿拉伯糖異構酶(L-AI)的發展 6
2.1. 生物轉換法 6
2.2. 化學生產方法 6
2.3. 酵素生產方法 7
2.4. 固定化L-AI 7
3. L-AI之來源與特性 7
3.1. L-AI的來源 7
3.2. L-AI的特質 8
3.3. 木糖異構酶(XI)與L-AI之比較 8
4. L-核糖異構酶(L-RI) 9
4.1. L-RI的來源 9
4.2. L-RI的生產方式 9
5. L-核糖的生產方法 10
5.1化學合成法 10
5.2酵素生產法 10
6. L-AI的進展 11
6.1. 發展從E.coli生產之固定化L-AI 11
6.2. 發展工業高溫酵素 12
6.3. 輔因子對耐熱L-AI的影響 12
6.4. 定點突變對特性的影響 13
6.4.1. 最適作用pH值的改變 13
6.4.2. 最適作用溫度的改變 14
6.4.3. 催化部位的探討 14
三、實驗材料與設備 16
1. 質體、菌株和酵素 16
2. 試液與化學藥品 17
3. 儀器設備 18
四、T. saccharolyticum NTOU1所產L-AI之基因選殖、表現、純化及
特性分析 19
1. 前言 19
2. 實驗設計 19
2.1. 擴增、選殖 T. saccharolyticum NTOU1之araA基因 19
2.2. L-AI之表現及純化 20
2.3. 特性分析 22
3. 實驗方法 22
4. 結果與討論 37
五、利用T. saccharolyticum NTOU1 L-AI搭配G. obscurus DSM 43160 L-RI由阿拉伯糖生產L-核糖 41
1. 前言 41
2. 實驗方法 41
3. 結果與討論 45
六、T. saccharolyticum NTOU1所產L-AI之蛋白質工程 47
1. 前言 47
2. 實驗設計 47
3. 實驗方法 48
4. 結果與討論 52
七、結論 55
八、縮寫及中英對照 56
九、參考文獻 57
圖目錄
圖一、L-AI轉換三種單糖示意圖 64
圖二、以洋菜膠電泳分析T. saccharolyticum NTOU1 araA基因PCR擴增之產物 65
圖三、以洋菜膠電泳分析 colony PCR後之產物 66
圖四、各菌種來源L-AI之比對演化樹 67
圖五、以SDS-PAGE分析在不同濃度IPTG誘導T. saccharolyticum NTOU1 L-AI 於 E.coli BL21-CodonPlus®(DE3)-RIL中之表現 68
圖六、以SDS-PAGE分析T. saccharolyticum NTOU1 L-AI粗酵素液之熱處理 69
圖七、以SDS-PAGE分析T. saccharolyticum NTOU1 L-AI之各純化步驟 70
圖八、以MALDI-TOF 質譜儀分析T. saccharolyticum NTOU1-L-AI的分子量 71
圖九、以HPLC分析T. saccharolyticum NTOU1 L-AI之分子量 72
圖十、錳和鈷離子含量對於T. saccharolyticum NTOU1 L-AI活性影響 73
圖十一、溫度與pH對T. saccharolyticum NTOU1 L-AI活性和穩定影響 74
圖十二、 以HPLC分析T. saccharolyticum NTOU1 L-AI轉換成塔格糖的轉換率 75
圖十三、以HPLC分析不同溫度T. saccharolyticum NTOU1 L-AI轉成塔格糖的轉換率 76
圖十四、以HPLC分析由T. saccharolyticum NTOU1 L-AI和G. obscurus DSM4316 L-RI作用之產物 77
圖十五、L-ribulose與 L-ribose之產量試驗 78
圖十六、溫度對於原生型及突變型N172H和I402A L-AIs的影響
79
圖十七、pH對於原生型及突變型N172H和I402A L-AIs的影響
80
圖十八、pH對於原生型及突變型V259K、V259S、V259Q、V259E L-AIs的影響 81
圖十九、溫度對於原生型及突變型V259K、V259S、V259Q、V259E L-AIs的影響 82
表目錄
表一、T. saccharolyticum NTOU1 與其他來源 L-AIs 胺基酸序列比對結果 83
表二、以E. coli BL21-CodonPlus (DE3)-RIL 表現T. saccharolyticum NTOU1 L-AI 各純化步驟之純化表 84
表三、L-AI對不同單糖的活性 85
表四、糖醇對酵素活性的抑制性 86
表五、各種來源L-AIs的動力學參數和金屬離子需求 87
表六、重組G. obscurus DSM 43160 L-RI之純化表 88
表七、重組G. obscurus DSM 43160 L-RI對不同單糖的活性
89
表八、原生型與突變型T. saccharolyticum NTOU1 L-AI對D-galactose之比活性和動力學數據 90

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