探討雙功能與三功能纖維水解酵素之受質特異性

Substrate specificities of the bi-functional and tri-functional cellulase/xylanase/mannanase enzymes

陳浩銘(Hao-Ming Chen)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202103150

摘要

纖維素和半纖維素是地球上含量豐富的多醣類，透過發酵與酵素作用能夠被轉換成綠能使用。在我們先前的研究中，我們發現了兩種不同功能的酵素，一是來自 Thermotoga maritima 中的雙功能酵素TmCel5A，能夠分解纖維素與聚甘露糖，另一種則是來自Clostridium thermocellum 的CtCel5T，具有分解纖維素、聚木糖、聚甘露糖三種不同糖類的活性。根據這兩酵素的結構比較，我們發現在TmCel5A中210號的色胺酸與CtCel5T中360號的麩胺酸兩者在空間上與受質形成氫鍵作用的位置相似。且210號的色胺酸位於TmCel5A中的Tmloop上，而360號的麩胺酸位在CtCel5T的T2-loop上。於本研究中，我們透過將TmCel5A的210位點突變成麩胺酸，CtCel5T的360位點突變成色胺酸，藉此確認兩者對受質的活性是否也會因兩個關鍵位點的交換而有變化。結果顯示，E360W的突變種失去了催化水解聚木糖的能力但保留了部分的水解聚甘露糖的能力，由此可知麩胺酸對水解聚木糖的活性是很重要的。另一方面，W210E的突變種失去了原本能夠催化水解多糖的能力，由此可知色胺酸的存在對其來說是必要的。而我們也利用的電腦模型與分子對接技術，來顯示突變後受質與酵素間鍵結的變化來試圖觀察為何這些酵素會因此失去活性，事實上是否如此還需其後的實驗證實，本研究提供了交換的方式來加以驗證這兩種胺基酸在各酵素中的需求，相信這也能為日後開發酵素有所助益。

關鍵字

纖維酵素 ； 糖苷水解酶第5屬 ； 蛋白質改造工程 ； 分子對接分析

並列摘要

Cellulose and hemicellulose are abundant polysaccharides which can be converted to green energy by enzyme processing into monosaccharides and fermentation. Our previous studies showed the structures and functions of a bi-functional cellulase/mannanase from Thermotoga maritima, TmCel5A, and a tri-functional cellulase/xylanase/mannase from Clostridium thermocellum, CtCel5T. Based on these studies, a loop (Tmloop) from TmCel5A and a loop (T2-loop) from CtCel5T occupy similar positions, using a Tryptophan (Trp210) and a Glutamate (Glu360) for making H-bonding interactions with the ligands, respectively. In this study, we prepared a W210E mutant TmCel5A and a E360W mutant CtCel5T to study whether their substrate specificities can be manipulated to confirm these key residues. The results showed that E360W partially retained mannanase activity while losing all the xylanase activity, indicating E360 is important for xylanase activity. On the other hand, W210E caused loss of all three activities, suggesting it is an essential residue. According to the computer model and docking analysis, the changed interactions between ligand and key residues might explain the functional alternation of mutant enzymes. To sum up, our findings provide structure and activity information in GH5 enzymes useful for engineering multi-functional enzymes for biofuel production.

並列關鍵字

Cellulase ； GH5 ； protein engineering ； flexible loop ； docking analysis

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