海藻糖(trehalose)是一種非還原性雙糖，由兩分子的葡萄糖以α,α-1,1-糖苷鍵鏈結構成，可作為生物體的碳源與能量來源，亦具有保護蛋白質與脂質的功能，協助細胞抵抗脫水與結凍等極端環境壓力。海藻糖在食品、化妝品、醫藥產業上都有廣泛的應用。海藻糖合成酶(Trehalose synthase, TS)可以將低價的麥芽糖直接轉化為高價的海藻糖，而耐熱的海藻糖合成酶在海藻糖工業化生產中具有應用潛力。本實驗室已從食品工業發展研究所生物資源保存及研究中心(BCRC)購得之菌株Thermus sp. BCRC17551分離出一種新型嗜熱性海藻糖合成酶(TTS)，其最適作用溫度為65℃，並已選殖到其蛋白質N端區域(nTTS)的基因片段，該片段所轉譯出之胺基酸序列(538 a.a.)，經過NCBI protein blast比對收尋，發現與其他已知12種Thermus genus TS N端區域有極高的保守性，序列相同度平均為90.4 %。已知嗜熱性菌株Thermus thermophilius ATCC33923之TS結構內具有一特殊C端區域(C-terminal domain, cTtTS)，該特殊結構對酵素之熱穩定性及高溫下酵素的異構化活性有重大影響。本研究發現以大腸桿菌中表達之重組nTTS的最適作用溫度(40℃)較原菌株所純化出的TTS (65℃)為低，藉由蛋白質工程將nTTS基因與cTtTS基因融合成nTTS-cTtTS基因，結果顯示該融合重組酵素最適作用溫度由40℃提高為60℃，活性提升6714倍。熱穩定性測試結果顯示，nTTS-cTtTS的熱變性自由能(ΔG°)較nTTS為高，顯示其熱變性所需之能量較大，較為穩定。上述結果顯示將nTTS結合cTtTS可以提升nTTS的嗜熱性與熱穩定性。受質專一性測試結果顯示，nTTS與nTTS-cTtTS皆可催化maltose、trehalose與sucrose，然而，nTTS對maltose與trehalose的異構化活性相近，但對trehalose的水解副反應活性高於對maltose，而nTTS-cTtTS對maltose的異構化活性高於對trehalose，但對maltose與trehalose的水解副反應活性則相近。此外，nTTS尚可以利用lactose作為受質，其產物可能是一種海藻糖類似物(galactosyl trehalose analogue, G-TA)，nTTS-cTtTS則無法作用lactose。海藻糖轉化率分析顯示，nTTS在其最適溫時海藻糖轉化率為31.9 %，但其水解成葡萄糖的副反應轉化率則高達26.8 %，nTTS-cTtTS在其最適溫的海藻糖轉化率為50.3 %，副反應葡萄糖轉化率則僅為14.7 %，顯示nTTS-cTtTS較nTTS具有高海藻糖合成效率與低水解活性。金屬離子與化學試劑對活性的影響測定結果顯示，nTTS與nTTS-cTtTS的活性均能被Ca2+促進，但是受Zn2+、Fe2+、Fe3+、Ni2+、Co2+或Cu2+抑制，而nTTS-cTtTS異構化活性會被Tris所抑制，但Tris能促進nTTS異構化活性高達4倍，顯示Tris可能會改變並穩定nTTS結構，使其適於生產海藻糖。酵素動力學結果顯示，在最適溫度40℃下，nTTS無法在接近受質的飽和濃度(2 M 麥芽糖或海藻糖)下達到Vmax，其Km值可能相當高。在最適溫度60℃下，nTTS-cTtTS以麥芽糖為受質Vmax為0.1028 (μmol / min)，Km為128.1 (mM)，kcat/Km為1.5 (s-1*mM-1); 以海藻糖為受質Vmax為0.1165 (μmol / min)，Km為270.0 (mM)，kcat/Km為0.8 (s-1*mM-1)，顯示nTTS-cTtTS對麥芽糖親和力較高，而且以麥芽糖為受質的催化效率較以海藻糖為受質高。當maltose和xylose同時存在下作為受質時，nTTS與nTTS-cTtTS均能合成新的醣類，這種醣類可能是一種海藻糖類似物(xylosyl trehalose analogue, X-TA)。另外，本研究藉由蛋白質工程技術構築nTTS-cTtTS與cellulose binding domain (CBD)融合基因，使酵素固定化於價格便宜之重製纖維素(regenerated amophous cellulose, RAC)上，重複使用實驗結果顯示，nTTS-cTtTS-CBD-RAC能於8次的重複使用後仍保持50 %以上的活性，且每次重複使用的轉化率分析結果顯示，nTTS-cTtTS-CBD-RAC能在重複使用中保持一致的高海藻糖轉化率 (皆約56 %)，顯示其酵素轉化特性沒有因重複使用而改變。在最適溫度測定結果顯示，nTTS-cTtTS-CBD-RAC的最適溫度較nTTS-cTtTS-CBD低約5℃，顯示CBD與RAC結合的構型可能會略為影響到nTTS-cTtTS-CBD在高溫下的活性。但nTTS-cTtTS-CBD-RAC的pH穩定性較nTTS-cTtTS獲得提升。本研究發現cTtTS對nTTS的嗜熱性、熱穩定性、減少水解反應與受質專一性有顯著的影響。而重組nTTS有許多一般海藻糖合成酶所沒有的特殊催化功能，後續蛋白質工程研究，應可提升這些催化功能的效率，並應用於海藻糖類似物的合成。固定化nTTS-cTtTS-CBD在價格便宜之RAC上能多次重複使用，有助於降低海藻糖工業生產成本，具有極大的工業應用價值。

Trehalose is a non-reducing disaccharide, formed by two glucose units with an α,α-1,1-glycosidic linkage. It has many important physiological functions such as carbon source, energy storage, and protectant of proteins and lipids. It prevents cells from damage due to environmental extreme stresses such as desiccation and freezing. Trehalose has been widely applied in food, cosmetic, and pharmaceutical industries. Trehalose synthase (TS) can convert inexpensive maltose into high-value trehalose. Thermostable TS has the potential for the industrial production of trehalose. A novel thermostable TS is purified from a cell-free extract of the thermophilic bacterium Thermus sp. BCRC17551 (TTS) which is purchased from Bioresource Collection and Research Center (BCRC). The optimal temperature of TTS is 65℃. The DNA fragment encoding the N-terminal domain of TTS (nTTS) is cloned from the genome of Thermus sp. BCRC17551 and the deduced amino acid sequence (538 residues) is highly conserved to the other twelve Thermus genus TSs (with an average sequence identity of 90.4% ). It is known that the C-terminal domain of Thermus thermophilius ATCC33923 (cTtTS) may play a key role in maintaining its thermostability and isomerization activity at high temperature. In this study, we observed the optimal temperature of the recombinant nTTS (40℃) expressed in Escherichia coli was lower than that of native TTS (65℃). A fusion protein (nTTS-cTtTS) was created by fusing cTtTS with nTTS at its C-terminal. It was found that the recombinant nTTS-cTtTS had a higher optimal temperature (60℃) and 6714 times higher specific activity than those of the recombinant nTTS. The thermostability analysis revealed that the thermal inactivation energy (ΔG°) of nTTS-cTtTS was higher than that of the nTTS, and the nTTS-cTtTS was more stable than nTTS. These results suggest that the thermophilicity and thermostability are improved by fusing cTtTS with nTTS. The substrate specificity analysis revealed that both nTTS and nTTS-cTtTS could use maltose, trehalose and sucrose as their substrate. The isomerization activities of nTTS toward maltose and trehalose were similar, and the hydrolysis activity toward trehalose was higher than that toward maltose. However, the isomerization activity of nTTS-cTtTS toward maltose was higher than that toward trehalose, and the hydrolysis activities toward maltose and trehalose were similar. In addition, the nTTS could use lactose as its substrate to synthesize a putative galactosyl trehalose analogue (G-TA). At optimal temperature, the trehalose conversion rate of nTTSwas 31.9 % with a by-product glucose conversion rate of 26.8 %. In contrast, the nTTS-cTtTS presented a trehalose conversion rate of 50.3 % with a glucose conversion rate of 14.7 % at its optimal temperature. These results suggest that the nTTS-cTtTS has higher trehalose production efficiency and lower hydrolysis rate than nTTS. The effects of metal ions and chemical reagents showed that Ca2+ improved, but Zn2+、Fe2+、Fe3+、Ni2+、Co2+ or Cu2+ inhibited the activities of both nTTS and nTTS-cTtTS. In the presenting of Tris, the isomerization activity of nTTS-cTtTS was inhibited, but it significantly enhanced that of nTTS 4 times higher. These results suggest that the Tris may change and stabilize the conformation of nTTS for the trehalose production. The enzyme kinetic analysis of nTTS at the optimal temperature (40℃) revealed that the Vmax of nTTS could not be measured, even at nearly the saturated concerntrations of the substrates (2 M maltose or trehalose). These results suggest the Km of nTTS may be very large. The enzyme kinetic analysis of nTTS-cTtTS at the optimal temperature (60℃) revealed that its Vmax was 0.1028 (μmol / min), Km was 128.1 (mM) and kcat/Km was 1.5 (s-1*mM-1) using maltose as substrate. When using trehalose as substrate, its Vmax was 0.1165 (μmol / min), Km was 270.0 (mM) and kcat/Km was 0.8 (s-1*mM-1). These results suggest nTTS-cTtTS had higher affinity and catalytic efficiency toward maltose than toward trehalose. When using a mixture of maltose and xylose as substrates, a novel sugar was formed by nTTS and nTTS-cTtTS. The novel sugar might be a xylosyl trehalose analogue (X-TA). For the industrial application, we fused nTTS-cTtTS with cellulose binding domain (CBD) by protein engineering. The recombinant enzyme nTTS-cTtTS-CBD was immobilized on low-cost regenerated amophous cellulose (RAC). The result of reusability analysis of nTTS-cTtTS-CBD -RAC revealed that during first 8 reuse cycles, the activity of nTTS -cTtTS-CBD-RAC was maintained above 50 % of the first cycle activity and its trehalose conversion rate was maintained above 56.0 %. These results suggest that the properties of nTTS-cTtTS-CBD-RAC do not change during each reuse. It was found that the optimal temperature of nTTS-cTtTS-CBD-RAC was 5℃ lower than that of free nTTS-cTtTS -CBD. This result suggests that the interaction between CBD and RAC may cause conformational changing of nTTS-cTtTS-CBD and slightly interfere the activity of nTTS-cTtTS-CBD at higher temperature. The pH stability of nTTS-cTtTS-CBD was improved when comparing with that of nTTS-cTtTS. Our study indicates that the cTtTS may play important role in modulating the thermophilicity, thermostability, hydrolysis activity and substrate specificity of the recombinant nTTS-cTtTS. The recombinant nTTS has many unusal properties that do not appear in many other TSs. These knowledges may help the further protein engineering to improve the activity of nTTS and to be applied in the synthesis of trehalose analogues. The reusiblilty of the immobilized nTTS-cTtTS -CBD-RAC may lead to more economic trehalose production and wider application of trehalose synthase.

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