跳到主要內容

臺灣博碩士論文加值系統

(3.236.23.193) 您好!臺灣時間:2021/07/24 13:56
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:楊勝智
研究生(外文):Sheng-ChihYang
論文名稱:Daldinia caldariorum 263 (D263)之纖維分解酵素蛋白的大量表現及特性分析
論文名稱(外文):Expression and characterization of cellulases from Daldinia caldariorum 263 (D263)
指導教授:余淑美余淑美引用關係
指導教授(外文):Su-May Yu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:熱帶植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:53
中文關鍵詞:生質能源纖維素纖維分解酶Pichia pastoris
外文關鍵詞:biofuelcellulosecellulasePichia pastoris
相關次數:
  • 被引用被引用:0
  • 點閱點閱:124
  • 評分評分:
  • 下載下載:11
  • 收藏至我的研究室書目清單書目收藏:0
在以木質纖維素來生產生質能源的過程中,利用酵素將纖維素水解為葡萄糖是其中的一個重要步驟。能夠將纖維素水解的酵素主要分成三類,包括了內切型纖維分解酶(EC 3.2.1.4)、外切型纖維分解酶(EC 3.2.1.91)以及葡萄糖苷酶(EC 3.2.1.21)。內切型纖維分解酶主要用來水解纖維素的β-1,4糖苷鍵。而外切型纖維分解酶可以從纖維素長鏈的還原端或非還原端去分解纖維素,將其分解為較小的組合單位(以纖維二糖為單位的四糖、二糖)。葡萄糖苷酶則可以將纖維二糖水解為更小分子的葡萄糖。在本篇研究中,我們從台灣原生真菌(Daldinia caldariorum)的酵素中分別將其內切型分解酶(CelC)以及外切型分解酶(CelB)分離出來,並加以定性。先利用搖瓶的方式生產並收集其酵素;而我們的目標酵素,CelC,在SDS-PAGE中可以看到其分子量約為34 kDa,並在zymogram中看到其對carboxymethyl cellulose (CMC)具有相當高的酵素活性;而另一個酵素,CelB,在SDS-PAGE中可以看到其分子量約為43kDa,並在zymogram中看到其對4-methylumbelliferyl-β-D-cellobioside (MUC)具有相當高的酵素活性。我們利用蛋白質N端定序以及LC-MS/MS(液相層析質譜儀)的結果去設計degenerate primers(退化性引子),利用設計的引子將目標酵素CelC及CelB的結構基因放大後加以定序,得到其cDNA的序列。接著分別建構帶有CelC 跟CelB cDNA序列的載體(pGAPZαC),並將其轉入P. pastoris的表現系統中去做蛋白的表現,而表現出來的重組蛋白再接著做活性的分析。在本篇研究中,重組蛋白CelC跟CelB被成功的表現到Pichia蛋白表現系統中,且對其特性也有相當的研究,未來可進一步的使用在提升生質能源生產的效率上。
The hydrolysis of cellulose to glucose is one of major steps for production of biofuels from lignocellulose. Cellulases that hydrolyze cellulose are classified into three types, including endo-1,4-β-D-glucanases (EC 3.2.1.4), exo-1,4-β-D-glucanases (EC 3.2.1.91) and 1,4-β-D-glucosidases (EC 3.2.1.21). Endo-1,4-β-D-glucanases cleave the 1,4-β-D-glycosidic linkages of cellulose into small fragments. Exo-1,4-β-D-glucanases attack the reducing or nonreducing ends of cellulose chain to produce the constitutive unit of sugars (tetrasaccharides or disaccharide such as cellobiose). 1,4-β-D-glucosidases hydrolyze cellobiose into two molecules of glucose. In this study, I have identified an endo-1,4-β-D-glucanases (designed as CelC) and an exo-1,4-β-D-glucan cellobiohydrolase (designated as CelB) from a Taiwan indigenous fungus Daldinia caldariorum and determined their biochemical properties. These enzymes were produced and collected by shake-flask incubation. CelC displayed a molecular mass of approximately 34 kDa on SDS-PAGE and exhibited high enzymatic activity in zymogram assays using carboxymethyl cellulose (CMC) as the substrate. CelB displayed a molecular mass of approximately 43 kDa on SDS-PAGE and exhibited high enzymatic activity in zymogram assays using 4-methylumbelliferyl-β-D-cellobioside (MUC) as the substrate. In order to clone their cDNA sequences, degenerate primers were designed, based on the results of N-terminal sequencing and LC-MS/MS sequencing. Vectors containing the CelC or CelB cDNA were then constructed for expression in the P. pastoris expression system. Cellulase activities of recombinant enzymes were determined. In this study, recombinant CelC and CelB were successfully expressed in Pichia expression system. Characteristics of recombinant CelC and CelB proteins were well studied and could be used to improve the efficiency of cellulose hydrolysis.
摘要 i
Abstract ii
Acknowledgements iv
Table of contents v
List of tables vii
List of figures viii
Chapter 1: Introduction 1
1.1 Biofuel, one of the renewable energies 1
1.2 Composition of lignocellulosic biomass 2
1.2.1 Cellulose. 2
1.2.2 Hemicellulose 3
1.2.3 Lignin 3
1.3 Pretreatment 4
1.4 Cellulase 4
1.4.1 Endo-glucanase (EC 3.2.1.4) 4
1.4.2 Exo-glucanase (EC 3.2.1.91) 5
1.4.3 Beta-glucosidase (EC 3.2.1.21) 5
1.4.4 Discovery of cellulases 5
1.4.5 Glycoside hydrolase (GH) family classification 6
1.5 Introduction of Daldinia caldariorum 263 6
1.6 Expression systems overview 7
1.7 Thesis objectives 8
Chapter 2: Material and method 9
2.1 Cultivation condition and crude enzyme activities detection 9
2.2 Enzyme activities detection by zymograms 10
2.3 SDS-PAGE and protein sequencing 11
2.4 Cloning of cellulase genes 11
2.5 Bioinformatics analysis 12
2.6 Cloning cellulase genes into Pichia expression system 13
2.7 Purification 15
2.8 Characterization 15
2.8.1 Optimum pH and temperature 15
2.8.2 pH and temperature stability 16
2.8.3 Effect of various ions on enzymatic activity 16
Chapter 3: Results 17
3.1 D263 has promising cellulase activities 17
3.2 CelC 17
3.2.1 CelC, a gene encoding endo-glucanase that belongs to GH family 5 17
3.2.2 Expression and characterization of recombinant CelC 18
3.3 CelB 19
3.3.1 CelB, a gene encoding exo-glucanase that belongs to GH family 6 19
3.3.2 Expression and characterization of recombinant CelB 20
Chapter 4: Discussion 21
Chapter 5: Summary 23
Chapter 6: Tables and figures 24
References 49
Appendices 51
Bitzer, J., Laessoe, T., Fournier, J., Kummer, V., Decock, C., Tichy, H. V., . . . Stadler, M. (2008). Affinities of Phylacia and the daldinoid Xylariaceae, inferred from chemotypes of cultures and ribosomal DNA sequences. [Research Support, Non-U.S. Gov't]. Mycol Res, 112(Pt 2), 251-270. doi: 10.1016/j.mycres.2007.07.004
Bollok, M., Resina, D., Valero, F., & Ferrer, P. (2009). Recent Patents on the Pichia Pastoris Expression System: Expanding the Toolbox for Recombinant Protein Production. Recent Patents on Biotechnology, 3, 192-201.
Dragone, G., Fernandes, B., Vicente, A. A., & Teixeira, J. A. (2010). Third generation biofuels from microalgae. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, 1355 - 1366.
Duku, M. H., Gu, S., & Hagan, E. B. (2011). A comprehensive review of biomass resources and biofuels potential in Ghana. Renewable and Sustainable Energy Reviews, 15(1), 404-415. doi: 10.1016/j.rser.2010.09.033
Filpponen, I. (2009). The Synthetic Strategies for Unique Properties in Cellulose Nanocrystal Materials.
Jeffries, T. W. (1994). Biodegradation of lignin and hemicelluloses. Biochemistry of Microbial Degradation, 233 - 277.
Katahira, S., Mizuike, A., Fukuda, H., & Kondo, A. (2006). Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain. [Research Support, Non-U.S. Gov't]. Appl Microbiol Biotechnol, 72(6), 1136-1143. doi: 10.1007/s00253-006-0402-x
Kuhad, R. C., Gupta, R., & Singh, A. (2011). Microbial cellulases and their industrial applications. Enzyme Res, 2011, 280696. doi: 10.4061/2011/280696
Kumar, P., Barrett, D. M., Delwiche, M. J., & Stroeve, P. (2009). Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research, 48(8), 3713-3729. doi: 10.1021/ie801542g
Larson, E. D. (2008). Biofuel production technologies: status, prospects and implications for trade and development. Paper presented at the United Nations Conference on Trade and Development.
Leonard, L. M. (2006). Melzer’s, Lugol’s or Iodine for Identifi cation of White-spored Agaricales? McIlvainea, 16, 43 - 51.
Levi, M. A. (2009). Energy and agriculture: The future of biofuels. Paper presented at the 2020 EUROPEAN AGRICULTURE: CHALLENGES &POLICIES.
50
Mandels, M., & Reese, E. T. (1956). Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. Journal of Bacteriology, 73, 269-278.
Mellitzer, A., Weis, R., Glieder, A., & Flicker, K. (2012). Expression of lignocellulolytic enzymes in Pichia pastoris. Microb Cell Fact, 11(1), 61. doi: 10.1186/1475-2859-11-61
Pirog, R. (2005). World Oil Demand and its Effect on Oil Prices CRS Report for Congress (Vol. RL32530).
Ryu, D. D. Y., & Mandels, M. (1980). Cellulase: biosynthesis and application. Enzyme and Microbial Technology, 2, 91 - 102.
Taherzadeh, M. J., & Karimi, K. (2008). Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int J Mol Sci, 9(9), 1621-1651. doi: 10.3390/ijms9091621
Undersander, D., McCaslin, M., Sheaffer, C., Whalen, D., Miller, D., Putnam, D., & Orloff, S. (2009). LOW LIGNIN ALFALFA: REDEFINING THE YIELD/QUALITY TRADEFOFF. Paper presented at the Western Alfalfa & Forage Conference.
Vicente Díaz martínez, M. C. M. (2010). Expression systems for recombinant proteins (II). Producing organisms. Biotech Spain.
Whalley, A. J. S., & Edwards, R. L. (1997). The Xylariaceae: A Case Study in Biological and Chemical Diversity. Paper presented at the Biodiversity and Bioresources: Conservation and Utilization.
Zhang, A. L., Luo, J. X., Zhang, T. Y., Pan, Y. W., Tan, Y. H., Fu, C. Y., & Tu, F. Z. (2009). Recent advances on the GAP promoter derived expression system of Pichia pastoris. [Research Support, Non-U.S. Gov't]. Mol Biol Rep, 36(6), 1611-1619. doi: 10.1007/s11033-008-9359-4Bitzer, J., Laessoe, T., Fournier, J., Kummer, V., Decock, C., Tichy, H. V., . . . Stadler, M. (2008). Affinities of Phylacia and the daldinoid Xylariaceae, inferred from chemotypes of cultures and ribosomal DNA sequences. [Research Support, Non-U.S. Gov't]. Mycol Res, 112(Pt 2), 251-270. doi: 10.1016/j.mycres.2007.07.004
Bollok, M., Resina, D., Valero, F., & Ferrer, P. (2009). Recent Patents on the Pichia Pastoris Expression System: Expanding the Toolbox for Recombinant Protein Production. Recent Patents on Biotechnology, 3, 192-201.
Dragone, G., Fernandes, B., Vicente, A. A., & Teixeira, J. A. (2010). Third generation biofuels from microalgae. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, 1355 - 1366.
Duku, M. H., Gu, S., & Hagan, E. B. (2011). A comprehensive review of biomass resources and biofuels potential in Ghana. Renewable and Sustainable Energy Reviews, 15(1), 404-415. doi: 10.1016/j.rser.2010.09.033
Filpponen, I. (2009). The Synthetic Strategies for Unique Properties in Cellulose Nanocrystal Materials.
Jeffries, T. W. (1994). Biodegradation of lignin and hemicelluloses. Biochemistry of Microbial Degradation, 233 - 277.
Katahira, S., Mizuike, A., Fukuda, H., & Kondo, A. (2006). Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain. [Research Support, Non-U.S. Gov't]. Appl Microbiol Biotechnol, 72(6), 1136-1143. doi: 10.1007/s00253-006-0402-x
Kuhad, R. C., Gupta, R., & Singh, A. (2011). Microbial cellulases and their industrial applications. Enzyme Res, 2011, 280696. doi: 10.4061/2011/280696
Kumar, P., Barrett, D. M., Delwiche, M. J., & Stroeve, P. (2009). Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research, 48(8), 3713-3729. doi: 10.1021/ie801542g
Larson, E. D. (2008). Biofuel production technologies: status, prospects and implications for trade and development. Paper presented at the United Nations Conference on Trade and Development.
Leonard, L. M. (2006). Melzer’s, Lugol’s or Iodine for Identifi cation of White-spored Agaricales? McIlvainea, 16, 43 - 51.
Levi, M. A. (2009). Energy and agriculture: The future of biofuels. Paper presented at the 2020 EUROPEAN AGRICULTURE: CHALLENGES &POLICIES.
50
Mandels, M., & Reese, E. T. (1956). Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. Journal of Bacteriology, 73, 269-278.
Mellitzer, A., Weis, R., Glieder, A., & Flicker, K. (2012). Expression of lignocellulolytic enzymes in Pichia pastoris. Microb Cell Fact, 11(1), 61. doi: 10.1186/1475-2859-11-61
Pirog, R. (2005). World Oil Demand and its Effect on Oil Prices CRS Report for Congress (Vol. RL32530).
Ryu, D. D. Y., & Mandels, M. (1980). Cellulase: biosynthesis and application. Enzyme and Microbial Technology, 2, 91 - 102.
Taherzadeh, M. J., & Karimi, K. (2008). Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int J Mol Sci, 9(9), 1621-1651. doi: 10.3390/ijms9091621
Undersander, D., McCaslin, M., Sheaffer, C., Whalen, D., Miller, D., Putnam, D., & Orloff, S. (2009). LOW LIGNIN ALFALFA: REDEFINING THE YIELD/QUALITY TRADEFOFF. Paper presented at the Western Alfalfa & Forage Conference.
Vicente Díaz martínez, M. C. M. (2010). Expression systems for recombinant proteins (II). Producing organisms. Biotech Spain.
Whalley, A. J. S., & Edwards, R. L. (1997). The Xylariaceae: A Case Study in Biological and Chemical Diversity. Paper presented at the Biodiversity and Bioresources: Conservation and Utilization.
Zhang, A. L., Luo, J. X., Zhang, T. Y., Pan, Y. W., Tan, Y. H., Fu, C. Y., & Tu, F. Z. (2009). Recent advances on the GAP promoter derived expression system of Pichia pastoris. [Research Support, Non-U.S. Gov't]. Mol Biol Rep, 36(6), 1611-1619. doi: 10.1007/s11033-008-9359-4
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊