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(II)

View Statistics Email Alert RSS Feed

Information

Details

Project title

Code/計畫編號

NSC98-2627-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=1896633

Year

2009

Start date/計畫起

01-08-2009

Expected Completion/計畫迄

31-07-2010

Bugetid/研究經費

1558千元

ResearchField/研究領域

食品科技(工)

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。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“Sulfolobus solfataricus KM1 MTHase之胺基酸殘基與基質麥芽三糖苷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。我們利用先前所發表的“巨引子引導及免接合酶之PCR定位突變法”(megaprimed and ligase-free PCR-based site-directed mutagenesis method)，得到突變型 MTHase 之基因，並於 E. coli 表現酵素，先經小量培養並收集菌體，經lysis buffer 作用獲得粗酵素液，初步之活性分析結果與 wild type 相比之下，Y155A 及 H195A MTHases活性幾乎完全喪失，表示 Y155 及 H195 與基質間極可能有氫鍵存在。R447A、D156A 和 E450A 活性分別下降了 61%、54% 和 16%。這些突變是否造成氫鍵的喪失，則須待獲得純化酵素後，再進行動力學分析以確認之。英文摘要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. Hydrogen bond plays an important role in the mechanisms of enzymatic catalysis. Five residues, which were proposed to form hydrogen bond with substrate maltotriosyltrehalose by National Tsing Hua University using computer simulation, were chose to mutate to alanine by side-directed mutagenesis. Our previous reported “A novel megaprimed and ligase-free PCR-based site-directed mutagenesis method” was used to construct mutated genes. Then the mutated genes were transferred to E.coli to express the mutated MTHases. The cells expressed the mutated MTHases were grown, collected, and lysed to obtain crude enzyme. The preliminary activity assays shown that Y155A and H195A MTHases almost completely lost their activities, and R447A, D156A, and E450A MTHases have decreased 61, 54, and 16% activities, respectively. The completely lost of activity by mutation indicated the possible presence of hydrogen bonds between substrate and residues Y155 and H195. The mutated MTHases will be purified and the enzyme kinetics will then be carried out to evaluate the status of hydrogen bonds.

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(II)

Details

Description