海藻糖是由兩個葡萄糖中間以α,α-1,1-糖苷鍵結的非還原性雙醣類。海藻糖廣泛存在於植物、昆蟲、酵母菌及細菌中。海藻糖可以保護生物體抵抗各種壓力，例如冷、熱、乾燥等。海藻糖可以應用在許多方面，例如當作食品的甜味劑、防腐劑、穩定劑、使用在化妝品或藥品。在工業生產海藻糖，可以利用maltooligosyltrehalose synthase (MTS) 和 maltooligosyltrehalose trehalohydrolase (MTH) 酵素系統，將低價的澱粉轉化為高價的海藻糖以降低成本。原核生物的藍綠菌Cyanobacteria Nostoc可以生長在極端的環境下，並且在乾燥的狀況下細胞可以累積海藻糖。在Nostoc punctiforme IAM M-15 及 Nostoc flagelliforme已被證明了在乾燥的壓力下，MTS及MTH的RNA表現量會增加。因此，推測Nostoc在乾燥的壓力下，MTS和MTH會參與反應以促進海藻糖的合成，提升細胞內海藻糖的濃度，以度過乾燥的環境。2001年，Nostoc punctiforme PCC 73102的完整基因體序列已經被定序，但它的MTS和MTH性質尚未被研究。除此之外，我們由台灣的烏來採集了Nostoc sphaeroides當作研究對象。本論文由Nostoc punctiforme PCC 73102 (NP) 及 Nostoc sphaeroides選殖MTS及MTH的基因，並以大腸桿菌表達重組酵素。並針對NPMTS作生化特性之分析，NPMTS重組酵素的最適溫度為30℃，最適pH值為8。NPMTS放置在10~30℃ 1小時酵素仍可維持穩定。額外加入1 mM的Ca2+、K+、Mg2+、Na+或Mn2+離子可略為增加NPMTS的活性，然而添加Cu2+及Zn2+離子則會抑制酵素活性。NPMTS以maltohexaose當受質時的轉糖活性較使用maltopentaose、maltotetraose、maltotriose和amylase高。然而，當使用maltotriose當受質時，NPMTS之水解活性會升高。NPMTS使用maltohexaose當作受質時的Km為57.9 mM，Vmax為1.9 U/mg. 以上結果闡述了此麥芽寡糖苷海藻糖合成酶之特性，並提供未來在生物技術的應用上，改進NPMTS的線索。

Treholose is a non-reducing disaccharide composed of two glucose residues connected by an α,α-1,1-glucosidic linkage. It is widely present in plants, insects, fungi, and bacteria. Trehalose protects organisms against various environmental stresses, such as freezing, heat, and desiccation. This sugar has many applications such as a sweetener component, preservative, or stabilizer for food, cosmetics, and medicines. It has been produced on an industrial scale by the maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase (MTS-MTH) enzymatic system, and the cost has been reduced by using inexpensive starch to produce the expensive trehalose. Cyanobacteria Nostoc can grow under extreme conditions, and these cells accumulated trehalose upon desiccation. It has been proven that MTS and MTH was upregulated during drought stress in Nostoc punctiforme IAM M-15 and Nostoc flagelliforme. It is theoretically possible that MTS and MTH are involved in dehydration stress response in Nostoc by enhancing the synthesis of trehalose, and the intracellular accumulation of trehalose thus increases the resistances to desiccation. Recently, the complete genome of Nostoc punctiforme PCC 73102 (NP) has been sequenced, and there exist putative MTS and MTH genes whose products have not been characterized yet. We cloned the NPMTS, NPMTH, and NSMTH genes from the genomic DNA of Nostoc punctiforme PCC 73102 and Nostoc sphaeroides which was collected from Wulai of Taiwan. These recombinant enzymes was expressed in Escherichia coli. The recombinant NPMTS showed an optimal temperature of 30℃ and an optimal pH of 8.0. The enzyme was stable after one hour incubation at 10~30℃. The addition of Ca2+, K+, Mg2+, Na+, or Mn2+ had slightly activating effect on NPMTS activity, while the addition of Cu2+ and Zn2+ inhibited the enzyme activity at 1 mM. The transglycosylation activity of NPMTS was higher when using maltohexaose as a substrate than maltopentaose, maltotetraose, maltotriose, and amylase. However, the hydrolysis activity of NPMTS only appeared when using maltotriose as substrate. The Km of NPMTS for maltohexaose was 57.9 mM, and the Vmax was 1.9 U/mg. These results revealed the characterization of NPMTS, and provide important clues for further engineering of NPMTS in biotechnological applications.

目錄 i
表次 vi
圖次 vii
附錄 ix
摘要 x
Abstract xi
壹、緒論 1
一、海藻糖的簡介 1
二、海藻糖的歷史 1
三、海藻糖的特性 2
四、海藻糖的應用 4
五、海藻糖的生合成路徑 5
六、海藻糖的生產 6
七、Cyanobacteria Nostoc之海藻糖生成相關研究 7
八、MTS與MTH之結構及催化機制 10
1.MTS之結構 10
2.MTH之結構 12
3.MTS與MTH催化機制 14
九、各物種之MTS及MTH特性比較 17
貳、研究目的 18
參、材料與方法 20
一、N. punctiforme PCC73102之NPMTS及NPMTH基因選殖與表達 20
1.引子之設計 20
2.PCR反應 20
3.插入片段的建構 21
4.載體的建構 22
5.接合反應 22
6.BL21(DE3)勝任細胞之製備 22
7.NPMTS及NPMTH基因在BL21(DE3)的表達 23
二、N. sphaeroides菌種純化 24
1.菌種採集 24
2.菌種培養 24
3.菌種純培養 (pure culture) 27
三、N. sphaeroides之NSMTS及NSMTH基因選殖與表達 27
1.基因體DNA製備 27
2.以Genome Walking的方法選殖NSMTS及NSMTH 28
2.1建構Genomic Libraries 28
2.2GenomeWalker DNA Walking 29
3.利用NPMTS及NPMTH之基因選殖引子來選殖NSMTS及NSMTH 32
4.質體之構築及NSMTH和NSMTS基因的表達 32
四、MTS及MTH之純化 33
1.蛋白質粗萃取液 33
2.固定化金屬離子親和性層析法 (Immobilized Metal Affinity Chromatography，IMAC) 純化蛋白質 33
五、MTS酵素特性分析 34
1.MTS酵素活性分析 34
2.最適作用溫度分析 35
3.最適作用pH分析 35
4.熱穩定性分析 35
5.金屬離子對活性的影響 36
6.受質專一性分析 37
7.酵素動力學分析 37
8.MTS的水解副反應分析 38
9.不同溫度對MTS之maltooligosyltrehalose轉化率及副反應的影響 39
六、MTH酵素活性分析 40
七、NPMTS胺基酸序列分析及三級結構預測 40
肆、結果 42
一、N. punctiforme PCC73102之NPMTS及NPMTH基因選殖與表達 42
二、N. sphaeroides之菌種純化 42
三、N. sphaeroides之NSMTS及NSMTH基因選殖與表達 43
四、NPMTS及NPMTH的純化 44
五、NSMTH的純化 45
六、NPMTS的特性分析 45
1.NPMTS酵素活性分析 45
2.NPMTS之最適作用溫度分析 46
3.NPMTS之最適作用pH值分析 46
4.NPMTS之熱穩定性分析 46
5.金屬離子對NPMTS活性的影響 47
6.NPMTS受質專一性分析 48
7.酵素動力學分析 49
8.探討NPMTS的maltooligosyltrehalose轉化率及其副反應 50
七、MTH酵素活性分析 52
八、NPMTS之胺基酸序列分析及三級結構預測 52
伍、討論 55
1.NPMTS之最適作用溫度分析 55
2.NPMTS之最適作用pH值分析 55
3.NPMTS之熱穩定性分析 55
4.金屬離子對NPMTS活性的影響 56
5.NPMTS受質專一性分析 58
6.酵素動力學分析 58
7.探討NPMTS的maltooligosyltrehalose轉化率及其副反應 60
8.NPMTS之胺基酸序列比對及三級結構預測 62
陸、參考文獻 63

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