(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
- 42 -
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
Mutation sites Oligo sequence(5’→3’)
I140X、F141X GTC CGG GTC NNK NNK AAG GAC TTT GAA ACC TCC AAC TGG ACC TTT GAC F163X TGG CAC CGC NNK TAC TGG CAC CAG
CCC GAC CTC AAC TGG GAC
F163X-rev CCA GTA MNN GCG GTG CCA GTA GTA GGC CTT GGC CAC GGG GTC
N244X-rev CCA CAT MNN GGC CTC GGC GAG GAG GAT CTT CCC GGG GCC
紅色標示代表突變的核苷酸，N會隨機突變成A、T、C、G；K會隨 機突變成G或T；M會隨機突變成A或C。
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表三. PDB 資料庫中 TtTS 的胺基酸序列搜尋結果
PDB
code identity similarity
Mycobacterium smegmatistrehalose synthase 3ZO9 59% 0.48 Mycobacterium tuberculosis
trehalose synthase 4LXF 58% 0.48 Deinococcus radiodurans
trehalose synthase 4TVU 57% 0.47 Geobacillus HTA-462
alpha-glucosidase 2ZE0 42% 0.41 Halothermothrix orenii
alpha-amylase 1WZA 39% 0.40 Bacillus subtilis isomaltase 4M56 38% 0.40 Pseudomonas mesoacidophila
MX-45 trehalulose synthase 1ZJA 37% 0.39 Protaminobacter rubrum
isomaltulose synthase 3GBD 34% 0.38 上述胺基酸序列覆蓋率皆高於 200 個胺基酸以上，前三名為海藻糖 合成酶，序列一致性與相似度最高，而後是水解酶，再來是同樣具 有異構化作用的蔗糖異構酶。此處所指 similarity 是經由 swiss model 網站的 BLOSUM62 substitution matrix 數學軟體換算標準化所得數 值，最大值為 1 (http://swissmodel.expasy.org/docs/help)
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表四. 距離麥芽糖5Å 以內胺基酸與麥芽糖作用力整理
-1 Subsite
residues Interaction
+1 Subsite
residues Interaction
D60 C4* I140
Y63 Stacking F141
H103 C6* F163
Q167 C6* A200
R197 Y203
D199
Nucleophile N244E242
Proton donor E309 C6*H307 C3* E313 C1*
D308
Transition stabilizer R387 C3*粗體字表示催化作用之胺基酸；*表示胺基酸與麥芽糖幾號碳上的 -OH形成氫鍵，空格欄位表示從pymol軟體顯示並沒有明顯的作用力
- 54 - Deinococcus radiodurans
trehalose synthase 4TVU 0.49 55% 0.39 Mycobacterium smegmatis
trehalose synthase 3ZO9 0.39 53% 1.58 Halomonas sp. H11
α-glucosidase 3WY2 0.38 38% 1.75 Mycobacterium tuberculosis
trehalose synthase 4LXF 0.36 53% 1.86 Bacillus subtilis
oligo-1,6-glucosidase 4M56 0.36 39% 1.82 Halothermothrix orenii
α-amylase 1WZA 0.36 37% 1.89 Pseudomonas
mesoacidophila MX-45 trehalulose synthase
1ZJA 0.36 34% 1.95
Protaminobacter rubrum
isomaltulose synthase 3GBD 0.36 33% 1.99 Klebsiella sp. Lx3
isomaltulose synthase 1M53 0.36 32% 1.98
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表六. 三級結構比對+1 subsite胺基酸
Enzyme +1 Subsite residue
Trehalose synthaseT. thermophilus
I140 F141 F163 A200 Y203 N244 E309 E313
T. aquaticusI138 F139 F161 A198 Y201 N242 E307 E311
Pimelobacter spI151 F152 F174 A211 Y214 N254 E328 E333
D. radioduransI150 F151 F173 A210 Y213 N253 E320 E324
M. smegmatisI171 F172 F194 A231 Y234 N274 E343 E347
M. tuberculosisI198 F199 F221 A258 Y261 N301 E370 E374
Sucrose isomeraseP.
mesoacidophila MX-45
F144 F145 F164 T201 T204 F256 N328 P329
Protaminobacte
r rubrum F143 F144 F163 T200 T203 F255 N328 P329 Klebsiella sp.
Lx3 F143 F144 F163 T200 T203 F255 N328 P329
Hydrolase
Halomonas sp.
H11 L143 D144 F161 G198 H201 W235 Q304 N305 B. subtilis
I141 F142 F161 V198 S201 N252 Q328 P329
H. oreniiI143 F144 F163 T200 F203 G270 V331 V332
分成三大群比較+1-subsite胺基酸差異，第一群為TS，第二大群為蔗 糖異構酶，第三大群為水解酶，其中，相同胺基酸以粗體表示
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表七. 定位飽和突變胺基酸定序 (A)
Compared to WT Mutants Sequencing Mutation
Mutants with similar conversion rate
Wild type
Mutants with very little conversion rate
Wild type
Compared to WT Mutants Sequencing Mutation
Mutants with similar conversion rate
Mutants with very little conversion rate
Wild type
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(C)
Compared to WT Mutants Sequencing Mutation
Mutants with similar conversion rate
Mutants with very little conversion rate
Wild type
Compared to WT Mutants Sequencing Mutation
Mutants with similar conversion rate
Mutants with very little conversion rate
Wild type
- 58 -
- 59 -
表九. 突變株異構化能力統整
- 60 -
(A)
(B)
- 61 -
(C)
圖一. TtTS 最適溫度 65℃轉化率分析 (A) 以 125mM 麥芽糖為受質；
(B) 以 125mM 海藻糖為受質；
(C) 以 50mM 蔗糖為受質。
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(A)
(B)
- 63 -
(C)
圖二. TtTS 低溫 30℃轉化率分析 (A) 以 125mM 麥芽糖為受質；
(B) 以 125mM 海藻糖為受質；
(C) 以 50mM 蔗糖為受質。
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(C)
V (μmol/min)
substrate concentration (mM)
(D)
V (μmol/min)
substrate concentration (mM)
(E)
V (μmol/min)
substrate concentration (mM)
(B)
substrate concentration (mM)
V (μmol/min)
(A)
substrate concentration (mM)
V (μmol/min)
(F)
V (μmol/min)
substrate concentration (mM)
- 65 -
圖三. TtTS 酵素動力學
(A) 65℃反應不同濃度麥芽糖；(B) 65℃反應不同濃度海藻糖；(C) 65
℃反應不同濃度麥芽糖；(D) 30℃反應不同濃度海藻糖；(E) 65℃反 應不同濃度蔗糖；(F) 30℃反應不同濃度蔗糖；(G) 65℃反應 15mM 葡萄糖與不同濃度麥芽糖；(H)統整 65℃與 30℃反應麥芽糖、海藻 糖、蔗糖的 KM值，以及生成產物的 Vmax、Kcat、Kcat over KM值。
(I ) 0.57μg TtTS 於 65℃反應麥芽糖，以及額外添加 15mM 葡萄糖之 麥芽糖的 KM值，以及生成產物的 Vmax、Kcat、Kcat over KM值
(G)
V (μmol/min)
substrate concentration (mM)
(H)
(I)
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(A)
(B)
圖四. 以 α-麥芽糖為主反應的轉化率分析
海藻糖生成量隨時間變化圖 (A)長時間，海藻糖最終產量於三個處 理組中並無差異 (B)一小時內，要產生 35μg 海藻糖，α-amylase 處理 30 分鐘組別較處理 5 分鐘組別所需反應時間快了一倍
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註 : 分析條件為 ACN / MQ = 75 : 25
圖五. GI-coupled TtTS 之反應
以 HPLC 分析反應後果糖（fructose, F）、葡萄糖(glucose, G)、麥芽 糖(maltose, M)、海藻糖(trehalose, T)隨著不同條件 GI-coupled TtTS 的時間變化，圖中數值代表濃度(mM)，紅色代表最大海藻糖濃度。
當加入 100mg GI 與 TtTS 一起反應 0.5 小時，TtTS 偏向水解葡萄糖 且使 GI 達到葡萄糖與果糖之平衡，使產生海藻糖濃度剩下 4.9mM。
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(A)
註 : 分析條件為 ACN / MQ = 85 : 15
GI-0h GI-1h GI-2h
G
M T
F G
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(B)
圖六. 在不時間加入 GI-coupled TtTS 之反應
(A)HPLC 圖譜 (B) 面積量化結果，實心方格代表海藻糖轉化，空心 方格代表葡萄糖轉化，箭頭代表加入 GI 時間，加入 GI 量為 20mg。
(A)HPLC 圖譜 (B) 面積量化結果，實心方格代表海藻糖轉化，空心 方格代表葡萄糖轉化，箭頭代表加入 GI 時間，加入 GI 量為 20mg。