臺灣博碩士論文加值系統

第一部分:醣&;#33527;水解&;#37238;家族5（GH5）擁有超過3000個來自原核生物和真核生物的酵素，包括纖維水解&;#37238;、纖維二糖水解&;#37238;、聚殼醣水解&;#37238;、聚甘露醣水解&;#37238;和聚木醣水解&;#37238;，有著種類繁多的特殊性。CtCel5E和TmCel5A，皆是GH5成員，兩者有著高度的序列同源性，卻具有不同的雙功能活性，分別是纖維水解&;#37238;/聚木醣水解&;#37238;和纖維水解&;#37238;/聚甘露醣水解&;#37238;。以SCHEMA分析兩個蛋白質的一級結構，找到一段有顯著差異的序列。並根據先前的結晶結構，此段有差異的序列在CtCel5E之中，沒有得到足夠的電子雲密度，故稱之為flexible loop。將CtCel5E的flexible loop序列置換為TmCel5A的序列，得到突變種CtCel5E，是一個三功能酵素，同時擁有纖維水解&;#37238;/聚木醣水解&;#37238;/聚甘露醣水解&;#37238;。在活性改良中，發現除了flexible loop序列置換，再加上第267號苯丙胺酸(Phe)突變為丙胺酸(Ala)，會獲得活性更好的三功能酵素。再將三功能酵素結合三種二糖水解&;#37238;，即可降解混合的人工受質成為三種單醣。再者，我們利用單定點突變實驗去找出哪些胺基酸對於聚甘露醣水解&;#37238;的活性是必要的。我們的研究提供如何將雙功能&;#37238;改造成三功能&;#37238;的理論基礎，有助於酵素的改造工程，並希望對未來生質能源工業應用有助益。
第二部分:纖維水解&;#37238;家族主要負責纖維素的分解，分成三大類別1.纖維內切水解&;#37238;，任意的從纖維素長醣鏈中切出纖維二醣。2.纖維外切水解&;#37238;，從還原端或非還原端開始將纖維長醣鏈依序切出纖維二醣。3.纖維二醣水解&;#37238;，將纖維二醣切成兩個葡萄醣單醣。而葡萄醣即可進一步發酵成酒精。生質能工業積極尋找新類型的纖維水解&;#37238;或是改造現有的水解&;#37238;，進而提高水解&;#37238;分解效率，希望能利於未來的發展。於是在一個有機體內結合兩種纖維水解&;#37238;和纖維二醣水&;#37238;是一個很好的生產葡萄糖的策略。李曉苓博士等成功地將纖維內切水解&;#37238;(CtCD)和纖維二醣水解&;#37238;(CcBG)融合成一個蛋白質(CtCD-CcBG)，這樣的融合蛋白提高了纖維素長醣鍊轉換成葡萄醣單醣的效率，並且增加其耐熱性。在本篇論文中，我們利用一系列的蛋白質結構分析方法去探討融合蛋白高轉換率的原因。從分析型超速離心實驗(AUC)中得知融合蛋白會是一個多聚體(oligomer)並且有個能對抗蛋白水解&;#37238;的核心結構，再利用cross-link 實驗和LC-MS/MS 分析找到融合蛋白相互作用的地方，再配合上結晶結構的資料，建立了融合蛋白的模擬結構。因此，我們知道到融合蛋白有兩個較接近的活性位，能夠高效率的將纖維長醣鍊轉換成葡萄醣單醣。希望利用這些結果，可以進一步改造出更好的融合蛋白質，有著受質接力的通道，更進一步提升葡萄糖生成的效率，利於生質能源未來的發展。

Part1:The glycoside hydrolase 5 (GH5) family contains more than 3000 prokaryotic and eukaryotic enzymes with a large variety of specificities, including endoglucanses, cellobiohydrolases, chitosanases, mannanases and xylanases. Two GH5 enzymes, CtCel5E and TmCel5A, possess different bi-functional activities, cellulase/xylanase and cellulase/mannanase, respectively, although they share sequence homology. The amino acid sequences of these two enzymes are aligned based on SCHEMA and a block was found significantly different in sequence. As revealed by the two protein crystal structures, a flexible loop (without visible electron density) in this block exists in CtCel5A, but not in TmCel5E. The mutant CtCel5E with replacement of this block by the corresponding one in TmCel5A became a tri-functional enzyme with all three cellulase/xylanase/mannanase activities. Through optimization, the best engineered tri-functional enzyme was found to contain loop region replacement plus a F267A mutation. The tri-functional enzyme in combination with three disaccharide-degrading enzymes allowed the complete degradation of mixed artificial substrate into monosaccharides. Moreover, several other amino acids were mutated to test their roles in determining the substrate specificities. Our study provides rationale for engineering a bi-functional enzyme into a tri-functional enzyme, which could be potentially useful for biomass degradation for biofuel production.

Part2:Cellulase system is responsible for degrading cellulosic materials, involving three major groups of enzymes, endoglucanases, exoglucanases and β-glucosidases, which cleave at random the internal amorphous area of the cellulose polysaccharide chain, act in a processive manner on the reducing or nonreducing ends to release cellobiose as a major product, and then hydrolyze soluble cellobiose into glucose that can be further fermented to ethanol. For bioethanol industry, seeking novel types of cellulolytic enzymes or engineering the existing enzymes to improve their abilities are actively pursued1. Combining both cellulase and β-glucosidase in the same organism is a good strategy to produce glucose2-3. Dr. Hsiao-Lin Lee et al. fused cellulases from Clostridium thermocellum, a cellulosomal endoglucanase CtCD, with a β-glucosidase CcBG from Clostridium cellulovorans in a single polypeptide chain to efficiently convert cellulosic substrates into glucose without accumulation of cellobiose and improve the thermostability in comparison with the mixture of single CtCD andCcBG enzymes4. In this study, we found that CtCD-CcBG is an oligomer based on AUC analysis and has a resistance core structure against the protease cleavage. Using cross-link, LC-MS/MS and database search, we identified the interaction sites between CtCD andCcBG. Based on the crystal structures of CtCD and CcBg, we established the modeling structure of CtCD-CcBG to realize that two active sites in CtCD-CcBG are closer so that the fused enzyme has higher efficiency to cleave cellulose into glucose. With the knowledges, we could further design better fused protein of cellulase and β-glucosidase with enhanced substrate channeling to increase the yield of glucose for biomass industry.

PART(1)中文摘要………………………………………………………………. ……vi
Abstract…………………………………………………………………………......... vii
Abbreviationa………………………………………………………………………….viii
PART(2)中文摘要……………………………………………………………………...ix
Abstract…………………………………………………………………………………x

Part(1)
(1) Introduction………………………………………………………………………..1
1.1 The requirement for alternative energy source……………………………….1
1.2 Structure of lignocellulosic biomass…………………………………...........1
1.3 Glycoside hydrolases…………………………………………………………2
1.4 Overview of the conversion of biomass to biofuel…………………………..4
1.5 Specific aim of this study…………………………………………………….4
(2) Materials and methods……………………………………………………………..6
2.1 Reagents……………………………………………………………………....6
2.2 Protein sequence alignment and structural analysis…………………………..6
2.3 DNA source and bacterial strains……………………………………………..6
2.4 Cloning of three mutant CtCel5E…………………………………………….7
2.5 Site-directed mutagenesis…………………………………………………….8
2.6 Expression and purification of recombinant proteins………………………..9
2.7 Determination of enzyme activity……………………………………………10
2.8 Determination of enzyme kinetics……………………………………………11
2.9 End-product determination…………………………………………………..12
2.10 Pretreatment of rice straw…………………………………………………...13
2.11 Analysis of hydrolytic end product by GC-MASS…………………………..13
2.12 CtCel5E Tmloop+F267A structure model…………………………………...14
2.13 Cloning, expression and purification of three chimeric CtCel5E……………15
(3) Result……………………………………………………………………….………16
3.1 Sequence comparison of CtCel5E and TmCel5A by SCHEMA……………...16
3.2 Expression and characterization of three CtCel5E engineered enzymes……..16
3.3 Hydrolytic behavior of the engineered enzymes toward CMC, BW and LBG.18
3.4 Improve enzyme activity by F267A mutation………………………………...19
3.5 Hydrolytic behavior of the engineered enzymes in combination with
glucosidase, xylosidase, and mannosidease………………………………..20
3.6 Time courses and hydrolytic products of wild-type and mutant CtCel5E in
degradation of natural substrate…………………………………………………..22
3.7 CtCel5E Tmloop+F267A structure modeling by pymol………………………22
(4) Discussion…………………………………………………………………………..23
4.1 Improve enzyme thermostability……………………………………………...23
Tables…………………………………………………………………………………...24
Figures………………………………………………………………………………….28
Reference……………………………………………………………………………….46

Part(2)
(1)Introduction…………………………………………………………………………50
1.1 The hybrid CtCD-CcBG enzyme system……………………………………..50
1.2 Explore the quaternary structure of CtCD–CcBG…………………………….50
1.3 Identification of inter or intra -cross-links in CtCD-CcBG by LC-MS/MS.....51
1.4 Specific aim of this study……………………………………………………..52
(2) Materials and methods……………………………………………………………..53
2.1 Preparation of CtCD-CcBG…………………………………………………..53
2.2 AUC analysis………………………………………………………………….53
2.3 Native page……………………………………………………………………54
2.4 Limited proteolysis……………………………………………………………54
2.5 CtCD-CcBG cross-linking…………………………………………………….55
2.6 Sample preparation for MS analysis…………………………………………..56
2.7 Mass spectrometric analysis…………………………………………………..56
2.8 Database searching……………………………………………………………57
(4) Result……………………………………………………………………….………59
3.1 Native states of CtCD, CcBG, CtCD+CcBG mixture and CtCD-CcBG……...59
3.2 Limited proteolysis of native CtCD, CcBG, CtCD+CcBG mixture and CtCD-CcBG….59
3.3 The interaction between two protein components of CtCD-CcBG………..…60
3.4 CtCD-CcBG tetramer structure modeling…………………………………….62
(4) Discussion…………………………………………………………………………..64
4.1 Substrate channel…………………………………………………………...…64
Figures………………………………………………………………………………….65
Reference……………………………………………………………………………….83

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