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脢海藻糖生成脢及麥芽寡糖脢海藻糖水解脢的基質特異性(I)

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Project title

Code/計畫編號

NSC97-2627-B019-004

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=1674690

Year

2008

Start date/計畫起

01-08-2008

Expected Completion/計畫迄

31-07-2009

Bugetid/研究經費

1416千元

ResearchField/研究領域

生物技術(理)

麥芽寡糖苷海藻糖水解酶 (maltooligotrehalose trehalohydrolase, MTHase) 可水解海藻糖苷麥芽寡糖分子上鄰近 α-1,1 鍵結之 α-1,4 鍵結而產生海藻糖，但也能水解麥芽寡糖分子還原端之 α-1,4 鍵結，水解產生葡萄糖。本實驗將與基質次結合部位相關之胺基酸進行多重點突變，探討多點突變後對於 MTHase 活性與基質選擇性之影響。利用蛋白質工程技術得到突變型 MTHase 之基因，於 E. coli 表現酵素，經熱處理、陰離子交換樹脂層析得到高純度之各突變 MTHase。純化後，F332Y/K358E/K362R、F332Y/K358E、K358E/K362R 等MTHases 之比活性與原生型相近， F332Y/K362R 的比活性為原生型 MTHase 的 78.2% ，而 N384D、N384T、N384V 三突變酵素之比活性介於原生型之11.1% 至 16.3% 之間。經過 80℃ 保溫兩小時後，原生型與突變型酵素皆具有良好的熱穩定性，顯示圖變型酵素具有穩定的構形。而原生型與突變型 F332Y/K358E/K362R、F332Y/K358E、F332Y/K362R、K358E/K362R 與 N384D 等酵素之最適作用溫度皆為 85℃，突變型 N384T 和 N384V MTHases 的最適作用溫度降為 75℃。突變型與原生型酵素的最適作用 pH 值皆為 pH 5.0。經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、N384D、N384T 與 N384V 等 MTHases 水解海藻糖苷麥芽寡糖之催化效率 (kcat/KM)，分別為 147.2、155.2、140.8、113.1、128.5、29.1、21.5 和 21.7 S-1．mM-1；在 MTHase 水解麥芽寡糖的催化能力方面，則分別為 3.21、2.95、4.41、2.10、2.49、0.70、0.37 與 0.40 S-1．mM-1。接著以水解海藻糖苷麥芽寡糖產生海藻糖之催化效率較佳之突變型與原生型酵素進行海藻糖產量試驗，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。由此結果分析，經突變後之各酵素並無法提高海藻糖之最終產量。而產量試驗中的葡萄糖生成對於海藻糖生成最初反應速率的比值與海藻糖產量具有負相關性，以具有較低速率比值的 MTHase 具有相對較高海藻糖產量的趨勢。 Maltooligosyltrehalose trehalohydrolase (MTHase) mainly hydrolyzes the α-1, 4 linkage adjacent to the α-1, 1 bond of maltooligosyltrehalose to release trehalose, and it can also hydrolyze the α-1, 4 linkage at the reducing end of maltooligosaccharides to release glucose. In order to understand the effects of substrate-binding subsite residues on MTHase hydrolytic activities and substrate selectivity, site-directed mutagenesis was used to construct multiple mutations at the residues related to substrate-bindings in this study. The plasmids contained mutant treZ gene were obtained by PCR using mutation-designed primers. The wild-type and mutant plasmids were then transformed into Escherichia coli BL21(DE3)- CodonPlus to express wild-type and mutant MTHases, respectively. The specific activities of purified wild-type, mutant F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, K358E/K362R, N384D, N384T and N384V MTHases were 421.7 U/mg, 378.7 U/mg, 414.2 U/mg, 329.9 U/mg, 443.3 U/mg, 68.7 U/mg, 46.6 U/mg and 48.6 U/mg, respectively. All mutant MTHases showed good thermostabilities at 80℃ for 2 hours. These results indicated that the mutant enzymes were well folded and have the same structure as that of wild-type MTHase. Wild-type, F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, K358E/K362R, and N384D MTHases showed an optimal activity at 85℃, while N384T and N384V MTHases had that at 75℃. All the MTHases showed an optimal activity at pH 5.0. The kinetic studies indicates that wild-type, F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R, and K358E/K362R MTHases had similar catalytic efficiencies (kcat/KM) on G3T and G5, while N384D, N384T and N384V MTHases were smaller than wild-type MTHase. According to kinetic analysis, we chose F332Y/K358E/K362R, F332Y/K358E, F332Y/K362R and K358E/K362R MTHases to study the trehalose production. The initial rates of glucose formation to trehalose formation had inverse correlation with trehalose yields. Results indicated that the final trehalose yields of all MTHases had no significant difference.

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脢海藻糖生成脢及麥芽寡糖脢海藻糖水解脢的基質特異性(I)

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