糖苷水解酶家族5（GH5）催化各種多醣的降解作用，在這個家族中包含了內切葡聚醣酶，甘露聚醣酶，木聚醣酶和基丁聚醣酶等催化不同醣類的酵素活性，這個特性使其成為在酵素工程中關鍵的發現來源和加工目標。然而，目前為止GH5酶仍然缺乏有力的結構 - 活性關係，這限制了我們來合理的研發活性更為優異的酵素。在本篇研究中，我們發現了一個來自Clostridium thermocellum的GH5酶，它顯示出具有催化分解纖維素，木聚醣和甘露聚醣的活性。為了闡明此酵素中何氨基酸對於何種活性的影響較為重要，我們利用了在此三功能酵素的活化位進行位點直接突變實驗來解決此問題。初步數據中顯示Glu193和Glu316為催化殘基，Tyr270對酶催化中的所有三種活性（纖維素酶/木聚醣酶/甘露聚醣酶）都是至關重要的。此外，Met277在甘露聚醣酶活性中起著至關重要的作用。另外，Asn351和Glu360在木聚醣酶的活性上扮演了關鍵的角色。而在環路置換實驗中，我們發現T2-loop上的Glu360與Tmloop上的Trp210在位置上重疊，導致結構尺度上的不協調現象，此發現也為木聚醣酶和甘露聚醣酶活性之間的此消彼長的現象提供了解釋。總體而言，這些研究結果提供了更多GH5酶在結構與活性的關係間的細節，且這些發現可能對於生物燃料工業應用中蛋白質工程的未來成功至關重要。

Glycoside Hydrolase Family 5 (GH5) enzymes catalyze various kinds of polysaccharide depolymerization, including endoglucanse, mannanase, xylanase and chitosanase, making them key discovery and engineering targets. However, there remains a lack of structure-activity relationships on GH5 enzymes that limit us to rationally develop better enzymes. Here we present a GH5 enzyme from Clostridium thermocellum, which shows activity against cellulose, xylan and mannan. In order to elucidate which amino acids are responsible for three substrate specificities, site-direct mutagenesis experiments were performed. The preliminary data revealed that Glu193 and Glu316 are catalytic residues and Tyr270 is critical for all three activities (cellulase/xylanase/mannanase) in enzyme catalysis. Moreover, Met277 plays a vital role in mannanase activity. In addition, Asn351 and Glu360 are crucial for xylanase activity. In loop replacement experiments, we found that Glu360 on T2-loop was overlapping with Trp210 on Tmloop, resulting in structural incoordination and providing an explanation for the antagonism between xylanase and mannanase activity. Overall, these findings provide more details in GH5 enzymes that could be essential to the future success of protein engineering in biofuel industrial applications.

中文摘要 1
ABSTRACT 2
ABBREVIATIONS 3
INTRODUCTION 4
1.1 The requirement for alternative energy source 4
1.2 Structure of lignocellulosic biomass 4
1.3 Glycoside hydrolases 5
1.4 Overview and difficulties in the development of biomass biofuel 7
1.5 Specific aim of this study 8
MATERIALS AND METHODS 10
2.1 Reagents 10
2.2 DNA source and bacterial strains 10
2.3 Constructions of Recombinant Proteins 10
2.4 Expression and Purification of Recombinant Proteins 11
2.5 Site-Directed Mutagenesis 12
2.6 Protein sequence alignment and structural analysis 13
2.7 CtCel5T-oligosaccharide complex structural modeling 13
2.8 Determination of Enzyme Activity 14
2.8.1 3,5-dinitrosalicylic acid (DNS) reagent preparation 14
2.8.2 Optimal pH and temperature for enzyme activity 14
2.8.3 Enzyme activity assays 15
2.8.4 Determination of enzyme kinetics 15
2.9 End-product determination 16
2.10 Loop replacement in CtCel5T and TmCel5A 17
RESULTS 18
3.1 Cloning, expression and characterization of CtCel5T 18
3.2 Analysis of hydrolytic end products 19
3.3 Structures of CtCel5T-substrate complexes by molecular modeling 20
3.4 Site-directed mutagenesis of active-site residues in CtCel5T 22
3.5 Sequence and structure comparison with CtCel5E and TmCel5A 24
3.6 T2-loop and Tm-loop are uncoordinated from a structural perspective 25
DISCUSSION 28
TABLES 32
FIGURES 40
REFERENCE 62

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