建構與特性分析具有提高功效與熱穩定性之多功能纖維素/半纖維素分解酶

Abstract

木質纖維素可被酵素水解並轉換成生質燃料。在實驗室先前的研究中，有一個從熱纖梭菌純化出來的雙功能水解酶(CtCel5E)，其具有的活性可分解兩種最主要組成植物細胞壁的多醣，分別為纖維素(為葡萄糖的多聚醣)和聚木醣(為木糖聚合而成的半纖維素主要構造)，使之最終分解為纖維二醣與木二醣。在之前碩士論文中，上述的雙功能酵素將與來自細菌或真菌的葡萄醣苷酶(CcBglA or TrBgl2 P172L)接合形成具cellulase, xylanase, 及β-glucosidase三種活性之融合酵素。在另一篇碩士論文中，CtCel5E中的一個flexible loop被另一來自T. maritima可分解纖維素及甘露醣的雙功能酵素TmCel5A置換而稱為CtCel5E_Tmloop，因此增加了甘露聚糖酶 (mannanase)活性。而在本篇論文發現，CtCel5E-CcBglA此融合酵素還增加了熱穩定性。CtCel5E還與另一個具木糖苷酶與葡萄糖苷酶活性的雙功能酵素接合，比起兩個雙功能酵素的混合反應，此融合酵素一樣擁有更好的活性與熱穩定性。此論文證明了一種有效與便捷的方法來合併不同酵素活性以利於生質原料的處理。為了更進一步達成統合生物加工過程(consolidated bioprocessing)，融合酵素被篩選並轉殖於嗜甲醇酵母菌內。我們的最終目標是利用生物工程來創造一個能同時使用葡萄糖與木糖的釀酒酵母發酵系統，來生產生質酒精。另外，為了增進三功能酵素(CtCel5E_Tmloop)的甘露聚糖酶活性，我們利用電腦軟體疊合一些甘露聚糖酶與CtCel5E_Tmloop的結構，並藉此結構分析來預測酵素工程的突變位置，接著使用點突變與DNA片段插入刪除實驗來驗證。

Lignocellulosic biomass can be enzymatically degraded and converted into biofuel. We have studied a bifunctional cellulase/xylanase from thermophilic C. thermocellum (CtCel5E), which can digest two major plant cell wall polysaccharides, cellulose (a polymer of glucose) and xylan (a major hemicellulose composed of xylose), into disaccharides cellobiose and xylobiose. In the previous thesis, the bifunctional enzyme was fused with a bacterial β-glucosidase from C. cellulovorans (CcBglA) or a fungal β-glucosidase from Trichoderma reesei (TrBgl2 P172L) to form tri-functional fusion enzymes. In another thesis, a flexible loop of CtCel5E was replaced with the corresponding loop in a bifunctional cellulase/mannanase from T. maritima, called TmCel5A, to form a tri-functional enzyme with additional mannanase activity. In this thesis, we found CtCel5E-CcBglA showed enhanced thermostability. Furthermore, CtCel5E was fused with a bifunctional β-glucosidase/β-xylosidase (RuBG3A) to degrade cellulose/hemicellulose into glucose and xylose also with better efficiency than the mixture of two parental enzymes and enhanced thermostability. This study demonstrates an efficient and convenient way of consolidating different enzyme activities for synergistic biomass processing. A further step, to achieve consolidated bioprocessing, suitable fusion enzymes were selected to be incorporated into Pichia pastoris . Our final goal is to engineer a glucose/xylose-utilizing Saccharomyces cerevisiae fermentative system, to produce bioethanol. In addition, to improve the mannanase activity of the tri-functional CtCel5E_Tmloop, superimposing some structure of β-mannanase with CtCel5E_Tmloop by computer software was achieved for analyzing the strategy of enzyme engineering. Enzyme engineering was performed by site-directed mutagenesis and sequence deletion/insertion experiments.