確認序列之突變株依方法五.1.反應蔗糖之海藻酮糖最大轉化率 與反應麥芽糖所得產物組成結果合併(表九)，可發現能異構化麥芽糖 之突變株，能力與異構化蔗糖相當，而完全無法異構化或水解麥芽 糖之突變株，對於蔗糖同樣沒有反應。如多點飽和突變株 I140、
F140L、F163V、N244，海藻糖最大轉化率為 50.4%，海藻酮糖最大 轉化率 58.2%，與 WT 相當，而突變株 I140A 對麥芽糖與蔗糖皆無 反應，這樣的結果顯示 TtTS 具有相同活性中心，是透過類似的催化 方式異構化麥芽糖與蔗糖；其中，N244V 突變株異構化蔗糖生成產 物偏向異麥芽酮糖，從 WT 海藻酮糖與異麥芽酮糖組成比為 30 : 1 變為 1: 1.5，顯示 N244 位點可決定產物專一性(product specificity)。
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- 36 - 得最後製得麥芽糖晶體以 β-構型居多(Xiaoling, 1998)。雖然當麥芽糖
晶體溶於水中會發生自發性變旋作用產生 α-麥芽糖，但最終平衡時 結果來看(圖四、B)，只有一小時內 TtTS 反應速率有因為 α-amylase 處理較久而提升，再長時間的海藻糖產量不論任何處理組皆相近，
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三、GI-coupled TtTS 反應分析
加入 GI 至 TtTS 反應麥芽糖溶液中，可將水解副產物葡萄糖異構
- 38 - 實驗只使用單一引子進行 QuickChange 突變，即便後續有以 DpnI 處 理，但含有半甲基化(hemimethylated)之親代股 DNA 抵抗 DpnI 作用 能力較高，不易清除，且後續含有缺口(nick)之新合成 DNA 又較完 整親代股 DNA 難轉型至 E.coli，使得最後突變庫中包含高比例之親 代股基因(Vovis and Lacks, 1977)。這樣的現象可透過互補引子對的 設計，排除傳統 QuickChange 操作時無法以新合成股作為模板進行 放大的缺點，以提高基因合成效率、減少親代股基因相對數量(Liu and Naismith, 2008)。
而本實驗使用 NNK 進行多點飽和突變，所需篩選突變庫數量可 減少為 NNN 之一半(Patrick et al., 2003)，但為了避免前述所產生之現 象，需要進行更多的篩選(over sampling)，勢必會造成實驗負擔，且 NNK 仍有胺基酸冗贅現象(codon redundancy)，如 Arginine、Leucine 包含了三種密碼子組成，會因此增加特定胺基酸出現頻率。若使用 NDT/VHG/TGG (D,G = no C、V = no T、H = no G) 搭配，只需要 22
- 39 - 株異構化能力不到 WT 的一半(圖二十五)。根據 Arnold and Georgiou 的統計分析結果，失活突變株約占突變庫之 40%~50%為最適合操作 定向演化的突變率，其基因(平均為含一千個鹼基)約含有 1 至 2 個胺 基酸突變(Arnold and Georgiou, 2003)。越高突變率則可能因為引入終 止密碼子或不利於結構摺疊之胺基酸而提高失活突變株的比例(Guo 證 96 孔突變株之反應結果，可發現利用 Maltase-coupled TS assay 作 為篩選改善異構化能力之突變株方式，分析所得突變株轉化率須高
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20 倍體積培養後，分離 TtTS 蛋白再反應結果與高通量反應結果一 致，顯示此高通量系統大致上能反映酵素異構化程度，但顧及 Maltase-coupled TS assay 會有誤差存在，因此後續進行 I140X、N244X 以及多點飽和與隨機突變所得可能突變株，直接取反應結果(尚未進 定值，稱為溫度因素(temperature factor or B factor)，若 B factor 越高 代表此胺基酸於整體酵素結構中受溫度影響的搖擺程度越大，不利 於酵素整體結構的穩定(Yuan et al., 2003)，因此藉由突變高 B factor 胺基酸可能會使酵素耐熱性與穩定性上升，例如以 Bacillus subtilis 脂肪酶結構中高 B factor 胺基酸建立飽和突變庫，透過重複飽和突變
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(iterative saturation mutagenesis)，疊加具有改善耐熱性質之突變株，
最終篩選到耐熱性提升 490 倍之突變株(Reetz et al., 2006)；或是以 Serratia plymuthica AS9 麥芽糖異構酶結構中高 B factor 胺基酸建立 定位突變，找到熱穩定性提升 7.6 倍且催化效率增加 38.2%的突變株 (Duan et al., 2016)，雖然 B factor 存在於解析度較高之蛋白晶體時最 能反映胺基酸受溫度影響的狀態(Parthasarathy and Murthy, 2000)，但 透過軟體計算，亦能直接分析預測一級序列胺基酸之 B factor
(Schlessinger et al., 2006)。
TS 結構中控制受質以及產物進出如同閥門的 subdomain 7 與 選數量(Bosley and Ostermeier, 2005)，需要仰賴更高效率之篩選系 統。結合流式細胞儀與螢光激活訊號之超高通量篩選技術，可減少 所需樣本與反應試劑體積，且每小時可分析高達 107之突變株，非常 適合進行酵素定向演化之篩選工具(Agresti et al., 2010)，參考 Colin
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等人於 2015 發表的水解酶篩選方式，搭配本實驗可規畫初步流程為 附錄十三。將多點飽和、隨機或 B factor 突變策略所建立之突變基 因，轉型至 E. coli，蒐集轉型後細胞株搭配破菌液以及酵素反應受質 麥芽糖，通過油包水珠之生成器，因為油水不互融之特性可於體外 模擬細胞與外界環境的分隔狀態(in vitro compartmentalization)，生成 的每一個油包水珠中包含單一個突變株細胞，經過一段反應時間，
使細胞破菌後，釋出的酵素與受質反應，並利用雙層包覆方式“水- 油 - 水”(water in oil in water)，混合兩種油包水珠(紅色通道的酵素與綠 色通道之麥芽糖水解酶和 GOD/POD 檢測劑)，通入另一個水包油珠 生成器，經過穩定油包水珠之脂溶性溶劑(黃色通道)與去離子水(藍 色通道)形成水包油珠，靜置一段時間使麥芽糖水解酶將未轉化為海 藻糖之殘餘麥芽糖分解成葡萄糖後，通入分析器。由於替換 POD 受 質，從本實驗使用的 o-Dianisidine dihydrochloride 變為能激發出螢光 訊號之 Amplex RedR，當突變株含有較弱螢光訊號則代表異構化能力 提升，蒐集此種突變株後，定序其 DNA，可獲得改善性質之突變酵 素。綜合本研究之結果，可以提供從事 TS 之蛋白質工程的參考。
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表一. 定位突變與定位飽和突變引子設計 Mutation sites Oligo sequence(5’→3’)
F141Y GTC CGG GTC ATC TAT AAG GAC TCT AGA
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表二. 多點飽和突變引子設計
表二. 多點飽和突變引子設計