建立醣甘水解酵素之生物資訊分析平台---以LPHase, MTSase,及MTHase為應用模型---子計畫四:藉由合理化設計以改善麥芽寡糖甘海藻糖生成甘及麥芽寡糖甘海藻糖水解甘的基質特異性(III)

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Project title

Code/計畫編號

NSC99-2627-B019-001

Project Coordinator/計畫主持人

Tsuei-Yun Fang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=2149918

Year

2010

Start date/計畫起

01-08-2010

Expected Completion/計畫迄

31-07-2011

Bugetid/研究經費

600千元

ResearchField/研究領域

生物技術(理)

麥芽寡糖?海藻糖生成?(maltooligotrehalose synthas, MTSase)能催化分子內的轉糖?反應 (transglycosylation)，將麥芽寡糖 (maltooligosaccharide)的還原性末端-1,4 糖?鍵結轉變為-1,1 糖?鍵結，產生麥芽寡糖?海藻糖(maltooligotrehalose)。麥芽寡糖?海藻糖水解? (maltooligotrehalose trehalohydrolase, MTHase)可水解麥芽寡糖?海藻糖分子上鄰近 α-1,1 鍵結之 α-1,4鍵結而產生海藻糖。然而此二酵素皆具有水解麥芽寡糖產生葡萄糖的副反應，而造成海藻糖產量下降。所建構之多重突變型 S. solfataricus ATCC35092 MTHase 基因，於 E. coli 表現酵素，經純化後，F332Y/K358E/K362R、F332Y/K358E、K358E/K362R 等多重突變型MTHases 之酵素動力學分析結果顯示，原生型與突變型F332Y/K358E/K362R、F332Y/K358E、F332Y/K362R、K358E/K362R等 MTHases 水解海藻糖?麥芽寡糖之催化效率 (kcat/KM) 非常近似。然而海藻糖產量試驗顯示，原生型與 F332Y/K358E/K362R、F332Y/K358E、F332Y/K362R、K358E/K362R 等突變型酵素之海藻糖產量皆無顯著差異。由於氫鍵對於酵素催化機制扮演非常重要的角色，因此本實驗藉由清大所模擬’S. solfataricus KM1 MTHase之胺基酸殘基與基質麥芽三糖?海藻糖可能產生之氫鍵’ 的結果，選出五個與基質可能產生氫鍵之胺基酸殘基進行定位突變轉變成alanine。所建構之突變型 S. solfataricus ATCC35092 MTHase 基因，於 E. coli 表現與純化後，Y155A、Y155F、D156A、H195A、R447A 與 E450A等 MTHases 水解海藻糖?麥芽寡糖之催化效率分別為原生型的0.63, 3.04, 26, 0.28, 15及79%。而Y155A, Y155F與H195A MTHases具有顯著降低的催化效率與顯著增加的過渡狀態能量，顯示 Y155及H195 兩殘基與基質間具有氫鍵。利用電腦模擬 MTSase 與麥芽五糖的結合方式，期望能找到與基質結合相關的胺基酸殘基。綜合電腦模擬的結果發現，K390、D394、E517、D596及R600等胺基酸殘基皆位於 subsite -2到 -4之間，可與麥芽五糖產生氫鍵，所以將D394、D596及R600於Sulfolobus solfataricus ATCC 35092 中相對的胺基酸殘基D411、D610及R614 突變成Alanine，以確認其與基質之氫鍵是否確實存在。酵素動力學分析結果顯示D411A、D610A及R614A具有顯著降低的催化效率與顯著增加的過渡狀態能量，顯示 D411、D610及R614三殘基與基質間具有氫鍵。針對可能影響 MTSase 基質結合部位上基質結合相關的胺基酸設計突變點，期望藉由增加基質與基質結合部位之氫鍵，評估 MTSase 轉糖?活性及水解活性之影響。P402S MTSase 之催化效率為原生型之 201.52 % ，具有顯著降低的過渡狀態能量，故推測 P402S MTSase 可能增加與基質麥芽五糖間形成一個氫鍵。最後，以 isoamylase、 MTSases 與 MTHase ，於 75 ℃ 、36 小時反應下之最高產量，原生型 MTSase 海藻糖產量為 81.47%， P402S MTSase 海藻糖產量為 86.87% 。英文摘要Maltooligosyltrehalose synthase (MTSase) catalyzes an intramolecular transglycosylation to produce maltooligosyltrehalose by converting the -1,4-glucosidic linkage at the reducing end of maltooligosaccharide to an -1,1-glucosidic linkage. Maltooligosyltrehalose can be further hydrolyzed by maltooligosyltrehalose trehalohydrolase (MTHase) to produce trehalose. The wild-type, F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, and K358E/K362R MTHases had similar catalytic efficiencies (kcat/KM) on G3T and G5. The results of trehalose production indicated that the final trehalose yields of all MTHases had no significant difference.Five residues of MTHase, which were proposed to form hydrogen bond with substrate maltotriosyltrehalose by NTHU using computer simulation, were chose to mutate to alanine. Mutations at residues Y155, D156, H195, R447, and E450 were constructed to identify the simulation-suggested H-bonds. The significantly lowered catalytic efficiencies and significant increases in transition-state energy of the mutant Y155A, Y155F, and H195A MTHases, indicating that residues Y155 and H195 form H-bonds with the substrate.The simulation results suggest that residues D411, D610 and R614 of MTSase form H-bonds with the substrate. In order to confirm the existence of H-bonds, mutagenesis was used to demolish the proposed H-bonds by altering the residues to alanine. The significantly lowered catalytic efficiencies and significant increases in transition-state energy of the mutant enzymes, indicating that those residues form H-bonds with the substrate. In order to study the effect of an additional H-bond, mutant MTSases were constructed. The catalytic efficiency of P402S MTSase was 201% of that of wild-type MTSase. The significant decrease in transition-state energy of the P402S MTSase, suggesting that P402S MTSase has an additional hydrogen bond between substrate and enzyme.

Keyword(s)

海藻糖
麥芽寡糖?海藻糖生成酶
麥芽寡糖?海藻糖水解酶
氫鍵trehalose
突變
maltooligosyltrehalose synthase;
trehalohydrolase
maltooligosyltrehalose
hydrogen bond
mutation

DSpace CRIS

建立醣甘水解酵素之生物資訊分析平台---以LPHase, MTSase,及MTHase為應用模型---子計畫四:藉由合理化設計以改善麥芽寡糖甘海藻糖生成甘及麥芽寡糖甘海藻糖水解甘的基質特異性(III)

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