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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐駿森(Chun-Hua Hsu) | |
| dc.contributor.author | Chun-Jung Lin | en |
| dc.contributor.author | 林君蓉 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:01:07Z | - |
| dc.date.copyright | 2017-08-01 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-07-24 | |
| dc.identifier.citation | Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221.
Adiguzel, A., Nadaroglu, H., and Adiguzel, G. (2015). Purification and characterization of -mannanase from Bacillus pumilus (M27) and its applications in some fruit juices. J Food Sci Tech Mys 52, 5292-5298. Afonine, P.V., Grosse-Kunstleve, R.W., Echols, N., Headd, J.J., Moriarty, N.W., Mustyakimov, M., Terwilliger, T.C., Urzhumtsev, A., Zwart, P.H., and Adams, P.D. (2012). Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68, 352-367. Ardevol, A., and Rovira, C. (2015a). Reaction Mechanisms in Carbohydrate-Active Enzymes: Glycoside Hydrolases and Glycosyltransferases. Insights from ab Initio Quantum Mechanics/Molecular Mechanics Dynamic Simulations. J Am Chem Soc 137, 7528-7547. Ardevol, A., and Rovira, C. (2015b). Reaction Mechanisms in Carbohydrate-Active Enzymes: Glycoside Hydrolases and Glycosyltransferases. Insights from ab Initio Quantum Mechanics/Molecular Mechanics Dynamic Simulations. J Am Chem Soc 137, 7528-7547. Bissaro, B., Monsan, P., Faure, R., and O'Donohue, M.J. (2015a). Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases. Biochem J 467, 17-35. Bissaro, B., Monsan, P., Faure, R., and O'Donohue, M.J. (2015b). Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases. Biochem J 467, 17-35. Bourgault, R., Oakley, A.J., Bewley, J.D., and Wilce, M.C.J. (2005). Three-dimensional structure of (1,4)-beta-D-mannan mannanohydrolase from tomato fruit. Protein Sci 14, 1233-1241. Caffall, K.H., and Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohyd Res 344, 1879-1900. Chen, X., Cao, Y., Ding, Y., Lu, W., and Li, D. (2007). Cloning, functional expression and characterization of Aspergillus sulphureus beta-mannanase in Pichia pastoris. J Biotechnol 128, 452-461. Couturier, M., Roussel, A., Rosengren, A., Leone, P., Stalbrand, H., and Berrin, J.G. (2013). Structural and biochemical analyses of glycoside hydrolase families 5 and 26 beta-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis. J Biol Chem 288, 14624-14635. Daskiran, M., Teeter, R.G., Fodge, D., and Hsiao, H.Y. (2004). An evaluation of endo-beta-D-mannanase (Hemicell) effects on broiler performance and energy use in diets varying in beta-mannan content. Poult Sci 83, 662-668. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., et al. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36, W465-469. Dong, Y.H., Li, J.F., Hu, D., Yin, X., Wang, C.J., Tang, S.H., and Wu, M.C. (2016). Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii beta-mannanase, AuMan5A, to improve its enzymatic properties by rational design. Appl Microbiol Biotechnol 100, 3989-3998. dos Santos, C.R., Paiva, J.H., Meza, A.N., Cota, J., Alvarez, T.M., Ruller, R., Prade, R.A., Squina, F.M., and Murakami, M.T. (2012). Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-beta-D-mannanase. J Struct Biol 177, 469-476. Ducros, V.M., Zechel, D.L., Murshudov, G.N., Gilbert, H.J., Szabo, L., Stoll, D., Withers, S.G., and Davies, G.J. (2002). Substrate distortion by a beta-mannanase: snapshots of the Michaelis and covalent-intermediate complexes suggest a B(2,5) conformation for the transition state. Angew Chem Int Ed Engl 41, 2824-2827. Duffaud, G.D., McCutchen, C.M., Leduc, P., Parker, K.N., and Kelly, R.M. (1997). Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068. Appl Environ Microbiol 63, 169-177. Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132. Finch-Savage, W.E., and Leubner-Metzger, G. (2006). Seed dormancy and the control of germination. New Phytol 171, 501-523. Goncalves, A.M., Silva, C.S., Madeira, T.I., Coelho, R., de Sanctis, D., San Romao, M.V., and Bento, I. (2012). Endo-beta-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity. Acta Crystallogr D Biol Crystallogr 68, 1468-1478. Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280 ( Pt 2), 309-316. Henrissat, B., and Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293 ( Pt 3), 781-788. Holm, L., and Rosenstrom, P. (2010). Dali server: conservation mapping in 3D. Nucleic Acids Res 38, W545-549. Hoshikawa, K., Endo, S., Mizuniwa, S., Makabe, S., Takahashi, H., and Nakamura, I. (2012). Transgenic tobacco plants expressing endo-beta-mannanase gene from deep-sea Bacillus sp JAMB-602 strain confer enhanced resistance against fungal pathogen (Fusarium oxysporum). Plant Biotechnol Rep 6, 243-250. Huang, J.W., Chen, C.C., Huang, C.H., Huang, T.Y., Wu, T.H., Cheng, Y.S., Ko, T.P., Lin, C.Y., Liu, J.R., and Guo, R.T. (2014). Improving the specific activity of beta-mannanase from Aspergillus niger BK01 by structure-based rational design. Biochim Biophys Acta 1844, 663-669. Iglesias-Fernandez, R., Rodriguez-Gacio, M.C., Barrero-Sicilia, C., Carbonero, P., and Matilla, A. (2011). Three endo-beta-mannanase genes expressed in the micropylar endosperm and in the radicle influence germination of Arabidopsis thaliana seeds. Planta 233, 25-36. Kolpak, F.J., and Blackwell, J. (1976). Determination of the structure of cellulose II. Macromolecules 9, 273-278. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and Thornton, J.M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8, 477-486. Laskowski, R.A., and Swindells, M.B. (2011). LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 51, 2778-2786. Lassmann, T., Frings, O., and Sonnhammer, E.L. (2009). Kalign2: high-performance multiple alignment of protein and nucleotide sequences allowing external features. Nucleic Acids Res 37, 858-865. Lee, C.M., Lee, Y.S., Seo, S.H., Yoon, S.H., Kim, S.J., Hahn, B.S., Sim, J.S., and Koo, B.S. (2014). Screening and Characterization of a Novel Cellulase Gene from the Gut Microflora of Hermetia illucens Using Metagenomic Library. J Microbiol Biotechn 24, 1196-1206. Li, J.F., Zhao, S.G., Tang, C.D., Wang, J.Q., and Wu, M.C. (2012). Cloning and functional expression of an acidophilic beta-mannanase gene (Anman5A) from Aspergillus niger LW-1 in Pichia pastoris. J Agric Food Chem 60, 765-773. Lin, J.Y., Pantalone, V.R., Li, G.L., and Chen, F. (2011). Molecular Cloning and Biochemical Characterization of an Endo-beta-mannanase Gene from Soybean for Soybean Meal Improvement. J Agr Food Chem 59, 4622-4628. Linkies, A., and Leubner-Metzger, G. (2012). Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination. Plant Cell Rep 31, 253-270. McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. (2007). Phaser crystallographic software. J Appl Crystallogr 40, 658-674. Miller, G.L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31, 426-428. Moreira, L.R., and Filho, E.X. (2008). An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biotechnol 79, 165-178. Nonogaki, H., Gee, O.H., and Bradford, K.J. (2000). A germination-specific endo-beta-mannanase gene is expressed in the micropylar endosperm cap of tomato seeds. Plant Physiol 123, 1235-1246. Petersen, T.N., Brunak, S., von Heijne, G., and Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8, 785-786. Puchart, V., Vrsanska, M., Svoboda, P., Pohl, J., Ogel, Z.B., and Biely, P. (2004). Purification and characterization of two forms of endo-beta-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Biochim Biophys Acta 1674, 239-250. Rayle, D.L., and Cleland, R.E. (1992). The Acid Growth Theory of auxin-induced cell elongation is alive and well. Plant Physiol 99, 1271-1274. Robert, X., and Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42, W320-324. Rodriguez-Gacio, M.D.C., Iglesias-Fernandez, R., Carbonero, P., and Matilla, A.J. (2012). Softening-up mannan-rich cell walls. J Exp Bot 63, 3975-3988. Rossmann, M.G. (1990). The molecular replacement method. Acta Crystallogr A 46 ( Pt 2), 73-82. Rytioja, J., Hilden, K., Yuzon, J., Hatakka, A., de Vries, R.P., and Makela, M.R. (2014). Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes. Microbiol Mol Biol R 78, 614-649. Sabini, E., Schubert, H., Murshudov, G., Wilson, K.S., Siika-Aho, M., and Penttila, M. (2000). The three-dimensional structure of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5. Acta Crystallogr D Biol Crystallogr 56, 3-13. Shastak, Y., Ader, P., Feuerstein, D., Ruehle, R., and Matuschek, M. (2015). beta-Mannan and mannanase in poultry nutrition. World Poultry Sci J 71, 161-173. Tailford, L.E., Ducros, V.M., Flint, J.E., Roberts, S.M., Morland, C., Zechel, D.L., Smith, N., Bjornvad, M.E., Borchert, T.V., Wilson, K.S., et al. (2009). Understanding how diverse beta-mannanases recognize heterogeneous substrates. Biochemistry 48, 7009-7018. van Zyl, W.H., Rose, S.H., Trollope, K., and Gorgens, J.F. (2010). Fungal beta-mannanases: Mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45, 1203-1213. Vincent, F., Gloster, T.M., Macdonald, J., Morland, C., Stick, R.V., Dias, F.M., Prates, J.A., Fontes, C.M., Gilbert, H.J., and Davies, G.J. (2004). Common inhibition of both beta-glucosidases and beta-mannosidases by isofagomine lactam reflects different conformational itineraries for pyranoside hydrolysis. Chembiochem 5, 1596-1599. Vogel, J. (2008). Unique aspects of the grass cell wall. Curr Opin Plant Biol 11, 301-307. von Freiesleben, P., Spodsberg, N., Blicher, T.H., Anderson, L., Jorgensen, H., Stalbrand, H., Meyer, A.S., and Krogh, K.B. (2016). An Aspergillus nidulans GH26 endo-beta-mannanase with a novel degradation pattern on highly substituted galactomannans. Enzyme Microb Technol 83, 68-77. Wang, D., Kim do, H., Seo, N., Yun, E.J., An, H.J., Kim, J.H., and Kim, K.H. (2016). A Novel Glycoside Hydrolase Family 5 beta-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40T and Its Transglycosylase Activity. Appl Environ Microbiol 82, 4340-4349. Wang, J., Zeng, D., Liu, G., Wang, S., and Yu, S. (2014a). Truncation of a mannanase from Trichoderma harzianum improves its enzymatic properties and expression efficiency in Trichoderma reesei. J Ind Microbiol Biotechnol 41, 125-133. Wang, Y., Azhar, S., Gandini, R., Divne, C., Ezcurra, I., and Aspeborg, H. (2015). Biochemical characterization of the novel endo-beta-mannanase AtMan5-2 from Arabidopsis thaliana. Plant Sci 241, 151-163. Wang, Y., Vilaplana, F., Brumer, H., and Aspeborg, H. (2014b). Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana. Planta 239, 653-665. Wilkins, M.R., Gasteiger, E., Bairoch, A., Sanchez, J.C., Williams, K.L., Appel, R.D., and Hochstrasser, D.F. (1999). Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112, 531-552. Xia, W., Lu, H., Xia, M., Cui, Y., Bai, Y., Qian, L., Shi, P., Luo, H., and Yao, B. (2016). A Novel Glycoside Hydrolase Family 113 Endo-beta-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family. Appl Environ Microbiol 82, 2718-2727. Xu, X., Zhang, Y., Meng, Q., Meng, K., Zhang, W., Zhou, X., Luo, H., Chen, R., Yang, P., and Yao, B. (2013). Overexpression of a fungal beta-mannanase from Bispora sp. MEY-1 in maize seeds and enzyme characterization. PLoS One 8, e56146. Yamabhai, M., Sak-Ubol, S., Srila, W., and Haltrich, D. (2016). Mannan biotechnology: from biofuels to health. Crit Rev Biotechnol 36, 32-42. Yan, D., Duermeyer, L., Leoveanu, C., and Nambara, E. (2014). The functions of the endosperm during seed germination. Plant Cell Physiol 55, 1521-1533. Yeoman, C.J., Han, Y., Dodd, D., Schroeder, C.M., Mackie, R.I., and Cann, I.K. (2010). Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol 70, 1-55. Zhou, P., Liu, Y., Yan, Q., Chen, Z., Qin, Z., and Jiang, Z. (2014). Structural insights into the substrate specificity and transglycosylation activity of a fungal glycoside hydrolase family 5 beta-mannosidase. Acta Crystallogr D Biol Crystallogr 70, 2970-2982. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20738 | - |
| dc.description.abstract | 內切-1,4-β-甘露聚醣酶 (endo-1,4-β-mannanase, β-mannanase, EC. 3.2.1.78) 在植物發芽和生長調控上扮演重要的角色，此酵素主要功能為水解β-1,4甘露聚醣使植物細胞壁軟化，以利於種子胚軸之突出與胚乳的消耗，然而目前對於植物來源β-mannanase 之結構及功能研究甚少。而本論文選擇大豆之β-mannanase作為研究對象，原因除了大豆是全球重要經濟作物外，在食品、飼料和工業應用上亦有很大的潛力。經基因體探勘，大豆中有21個β-mannanase基因，其中GmMAN19-1於親緣關係樹上較有其獨特性，因此以此基因為首要目標。 即時PCR結果顯示GmMAN19-1 基因於發芽7天後之子葉組織中表現，而經純化後的GmMAN19-1重組蛋白為一嗜酸性酵素且在pH 4.6有最大活性，且對直鏈型多醣有較好之水解能力。為了獲得更詳細的資訊，我們利用蛋白質結晶學解出GmMAN19-1以及其與受質五醣之複合體結構，意外發現此複合體結構包含兩種五醣的結合模式，分別呈現出糖苷水解酶受質 (subsites：-3,-2,-1,+1,+2) 和轉糖酵素受質 (subsite：-5,-4,-3,-2,-1) 的結合狀態。此外，GmMAN19-1與其他真菌來源之β-mannanase的結構比較顯示，GmMAN19-1於結構上多出了兩個延伸的loop，造成了較狹窄的活性位裂口。以解出的複合體結構為基礎，為嘗試提高GmMAN19-1對支鏈性甘露聚醣的選擇性，進行循理設計將GmMAN19-1突變。在我們所構築的五個突變株中，以Q267W最具潛力，因其對於支鏈型甘露聚醣的關華豆膠相對於野生株有50% 的比活性提升，而Q267W突變株，還有Y264W突變株及其複合物等蛋白質結構，也被進行結構解析並探討。整體而言，我們的研究結果提供GmMAN19-1受質專一性與轉醣基能力的結構觀點，並顯示了植物型β-mannanase和真菌來源之β-mannanase於結構上的差異。且特別是，Q267W對於支鏈型受質的水解有相當的潛力，提供了未來對於GmMAN19-1進行酵素工程以及大豆分子育種的依據。 | zh_TW |
| dc.description.abstract | Endo-1,4-β-mannanase (β-mannanase, EC. 3.2.1.78) is a hydrloase that catalyzes cleavage of β-1-4 bonds in the mannan polymer. This enzyme family is involved in soften of the mannan-rich cell walls and consumption of endosperm, which is benifical to radicle protrusion upon seed germination. However, there is limited information about the structural and functional relationship of plant-type β-mannanase. In this study, plant-type β-mannanases from soybean (Glycine max) were studied, since soybean is not only a globally important commercial crops, but also a potential material for use in food, feed or industrial applications. Using genome mining, we find out that there are 21 types of β-mannanase gene in the genome of soybean, and GmMAN19-1 was selected as primiary target due to its unique position on phylogentic tree. RT-PCR data showed GmMAN19-1 was expressed only in the cotyledons tissue after 7-day germination. Purified recombinant GmMAN19-1 was acidophilic with a pH optimum of 4.6, and exhibited a higher activity to linear polysaccharides. For detailed information, crystal sturctures of GmMAN19-1 in apo form and in complex with mannopentose were determined. Intriguingly, the complex structure existed two distinct binding modes of mannopentaose, presented as the substrates for glycohydrolase (subsides -3, -2, -1, +1, +2) and transglycohydrolase (subsides -5, -4, -3, -2, -1), respectively. In addition, structural comparison of GmMAN19-1 with other β-mannanases from fungus reveals that GmMAN19-1 has two extended loops, producing a narrower active site cleft. Based on the solved structure of GmMAN19-1/pentaose complex, rational design was conducted to engineer GmMAN19-1 in an attempt to alter the substrate selectivity toward branched mannans. Among the 5 mutants we constructed, the most promising Q267W showed a 50% increase in specific activity toward the branched-mannan guar gum by comparison with the wild-type enzyme. GmMAN19-1-Q267W, GmMAN19-1-Y264W and its complex were also structurally characterized. Taken together, our findings provide structural insights into the substrate specificity and transglycosylation activity of GmMAN19-1 and demonstrate the structural differences between plant-type and fungal β-mannanase. In particular, Q267W mutant shows potential to hydrolysis branched substrate, which providing a basis for further enzymatic engineering of GmMAN19-1 and molecular breeding of soybean. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:01:07Z (GMT). No. of bitstreams: 1 ntu-106-R04623011-1.pdf: 4102001 bytes, checksum: a9f5ae607b1edeec012569fe27738994 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 壹、前言 1
1.1 植物細胞壁 1 1.1.1 細胞壁組成成分 2 1.2甘露聚醣 4 1.2.1直鏈型甘露聚醣 (linear mannans) 5 1.2.2半乳甘露聚醣 (galactomannans) 5 1.2.3葡甘露聚醣 (glucomannans) 6 1.2.4半乳葡甘露聚醣 (galactoglucomannan) 7 1.3醣苷水解酶 (Glycoside hydrolase families, GH) 的定義與分類 7 1.4 β-mannanase (甘露聚醣酶) 8 1.4.1 β-mannanase於植物體之功能 9 1.4.2 β-mannanase來源與功能介紹 11 1.4.3目前β-mannanase之結構研究 13 1.4.4 β-mannanase 產業上的應用 14 1.5 研究目的 14 貳、材料與方法 15 2.1實驗材料 15 2.1.1.植物樣本來源 15 2.2實驗方法 15 2.2.1.大豆GmMAN19-1基因取得與表現測定 15 2.2.1.1大豆β-mannananse之基因體探勘 (genome mining) 與其親緣關係分析 15 2.2.1.2大豆 RNA 萃取與 cDNA 反轉錄 16 2.2.1.3 GmMAN19-1 和GmMAN11 之基因表現測定 17 2.2.2 GmMAN19-1和GmMAN11重組蛋白的製備 18 2.2.2.1目標基因放大與蛋白質表現載體之構築 18 2.2.2.2質體抽取與DNA定序比對 19 2.2.2.3蛋白質表現與純化 20 2.2.2.4 SDS-PAGE膠體電泳分析 21 2.2.2.5膠體過濾層析法 (Gel filtration chromatography) 22 2.2.2.6蛋白質濃縮 23 2.2.2.7蛋白質濃度測定 23 2.2.3大豆β-mannanase生化特性分析 23 2.2.3.1酵素活性試驗 23 2.2.3.1.1 β-mannanase之最適 pH 值與 pH 耐受性實驗 24 2.2.3.1.2 β-mannanase之最適溫度與溫度耐受性實驗 25 2.2.3.1.3 β-mannanase酵素動力學測定 25 2.2.3.2圓二色光譜 (Circular Dichroism, CD) 實驗 25 2.2.3.2.1熱變性曲線測定 26 2.2.3.2.2二級結構構型觀察 26 2.2.3.3示熱差掃描螢光法 (differential scanning fluorimetry) 蛋白質穩定性測試 27 2.2.3.4以薄層層析 (Thin layer chromatography, TLC) 檢測轉醣化活性 27 2.2.4 X-ray晶體繞射實驗法 28 2.2.4.1蛋白質結晶測試 29 2.2.4.2蛋白質晶體條件篩選 29 2.2.4.3蛋白質晶體形成條件微調 29 2.2.4.4 X-ray 晶體繞射數據收集及處理 30 2.2.4.5相位角決定方法與結構精修 31 2.2.4.6 GmMAN19-1晶體浸潤甘露五醣 31 2.2.4.7 Ramachandran Plot 32 2.2.5 物種間胺基酸序列與蛋白結構之比較 32 2.2.5.1 胺基酸序列比對 32 2.2.5.2 蛋白質結構比對 33 2.2.6 定點突變實驗 33 參、結果 34 3.1大豆β-mannanase基因體探勘與表現測定 34 3.2 GmMAN19-1與MBP-GmMAN11表現與純化 35 3.3生化特性分析 36 3.3.1 pH 對GmMAN19-1之影響 36 3.3.2 溫度對GmMAN19-1之影響 37 3.3.3 GmMAN19-1酵素動力學測定 37 3.4 GmMAN19-1的結構鑑定 38 3.4.1 GmMAN19-1 蛋白質晶體培養 38 3.4.2 以甘露五醣 (M5) 浸潤 GmMAN19-1 晶體 39 3.4.3 GmMAN19-1 X-ray 繞射數據分析與單位晶格判斷 39 3.4.4 GmMAN19-1/M5 X-ray繞射數據分析與單位晶格判斷 40 3.4.5 GmMAN19-1與GmMAN19-1/M5蛋白質晶體結構建立 40 3.4.6 GmMAN19-1 蛋白質構型 41 3.4.7 GmMAN19-1與M5之結合模式 41 3.4.8 GmMAN19-1的受質辨認機制 42 3.4.9支鏈取代影響活性原因 43 3.5 GmMAN19-1與其他β-mannanase酵素構型之比較 43 3.5.1 GmMAN19-1和 structural relatives 之整體比較 43 3.5.2 GmMAN19-1和結構相似之β-mannanase於正負結合位的差異 44 3.6定點突變之選擇與考量 45 3.6.1突變株之蛋白表現與純化 46 3.6.2各突變株之水解能力分析 46 3.6.3突變株熱變性曲線結果 47 3.6.4以示熱差掃描螢光法測定WT與突變株之熱穩定性 47 3.6.5突變株之酵素結構鑑定 48 3.6.5.1突變株GmMAN19-1-Q267W、GmMAN19-1-Y264W和GmMAN19-1-E186A蛋白質晶體培養 48 3.6.5.2以甘露五醣 (M5) 浸潤GmMAN19-1之突變株晶體 48 3.6.5.3突變株蛋白晶體繞射數據分析與單位晶格判斷 48 3.6.5.4突變株蛋白質晶體結構建立 49 3.6.5.5突變株與WT之結構差異 50 肆、討論 51 4.1 GmMAN19-1於植物體內的功能 51 4.2 GmMAN19-1與其他已知結構的比較 52 4.2.1 GmMAN19-1受質複合體結果突顯負結合位Y41、Y77和F354之特殊處 52 4.2.2 GmMAN19-1和真菌來源β-mannanase活性差異探討 53 4.2.3 GmMAN19-1之受質特異性與催化機制推測 54 4.3突變株與WT的比較 55 4.3.1GmMAN19-1-Q267W對水解作用和轉醣基化能力之影響 55 4.3.2 GmMAN19-1-Y264之高度保留性原因 56 4.4 突變株與WT的熱穩定性差異 57 4.4.1 以CD測定之結果探討 57 4.4.2示熱差掃描螢光法蛋白質穩定性測試 57 伍、結論 58 陸、圖表 59 柒、參考文獻 108 捌、附錄 114 | |
| dc.language.iso | zh-TW | |
| dc.subject | 轉醣基化 | zh_TW |
| dc.subject | 內切甘露聚醣? | zh_TW |
| dc.subject | 大豆 | zh_TW |
| dc.subject | 酵素學 | zh_TW |
| dc.subject | 蛋白質結晶學 | zh_TW |
| dc.subject | protein crystalization | en |
| dc.subject | transglycosylation | en |
| dc.subject | β-mannanase | en |
| dc.subject | soybean | en |
| dc.subject | enzymology | en |
| dc.title | "大豆內切-1,4-β-甘露聚醣酶之受質專一性與
轉醣基能力的結構觀點與探討" | zh_TW |
| dc.title | Structural insights into the substrate specificity and transglycosylation activity of an endo-1,4-β-mannanase from soybean (Glycine max) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立(Nei-Li Chan),方翠筠(Tsuei-Yun Fang) | |
| dc.subject.keyword | 內切甘露聚醣?,大豆,酵素學,蛋白質結晶學,轉醣基化, | zh_TW |
| dc.subject.keyword | β-mannanase,soybean,enzymology,protein crystalization,transglycosylation, | en |
| dc.relation.page | 124 | |
| dc.identifier.doi | 10.6342/NTU201701843 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2017-07-24 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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