臺灣博碩士論文加值系統

Fibrobacter succinogenes 1,3-1,4-β-D-葡聚醣水解酶（Fsβ-glucanase, E.C. 3.2.1.73）主要能特異性水解大麥、小麥與燕麥等穀類植物中的β-D-葡聚糖（β-D-glucan）或地衣聚醣（lichenan）中與β-1,3鍵結相鄰後的β-1,4鍵結，而形成三至五個寡醣組成的最終產物。由截短型Fibrobacter succinogenes 1,3-1,4-β-D-葡聚醣水解酶突變種Y42L晶體結構得知鈣和Tris離子從養晶溶液進入蛋白質共同成為複合物晶體，由酵素動力學實驗得知鈣和Tris離子分別是葡聚醣水解酶的非競爭型與競爭型的抑制劑。突變種結構中有三個鈣離子、二個醋酸根離子及二個Tris離子。除了一個鈣離子與原生種同位置外，另多兩個鈣離子分別位於靠近表面胺基酸Phe152及Glu154和活性區域通道入口處與Asp202有鍵結。其中一個Tris離子的結合位置正好是在催化活性區域-1位置，並與主要催化水解醣苷鍵的胺基酸Glu56及Glu60有氫鍵結合。另一個Tris離子與Phe193及Gly195有氫鍵結合，並與醋酸根離子對同一胺基酸（Phe193）在主鏈和支鏈上互相影響。二個醋酸根離子與鄰近苯環及支鏈之交互作用，可能有助於穩定結構，但不影響催化功能。

Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase, E.C.3.2.1.73) specifically hydrolyze the β-1,4 bonds when β-1,3 linkages are located prior to the β-1,4 bonds in β-D-glucan or lichenan. The final hydrolyzed products are tri- and penta- saccharides. Three calcium ions and two tris molecules are found in the truncated Fsβ-glucanase mutant Y42L structure. The first calcium ion is located at the same position as that of wild type. The second Ca2+ ion was found near the residues Phe152 and Glu154 on the protein’s surface, and the third one near the active site residue Asp202. Moreover, a tris molecule interacts with the catalytic residues Glu56 and Glu60 at subunit -1 of substrate. Based on the kinetic data, it is shown that the third Ca2+ ion and tris molecule are non-competitive and competitive inhibitors for the enzyme, respectively.

目錄

摘　要 i
ABSTRACT ii
誌謝 iii
目錄 iv
表目錄 vi
圖目錄 vii
簡寫表 viii
第一章 緒論 1
1.1 前言 1
1.2 1,3-1,4-β-D-葡聚醣結構 2
1.3 1,3-1,4-β-D-葡聚醣水解酶 3
1.4 蛋白質養晶及三維結構測定 5
1.5 Lineweaver-Burk雙倒數作圖法 6
第二章 實驗方法 8
2.1 蛋白質表現、純化與SDS-PAGE 8
2.2 蛋白質養晶方法 10
2.3 X-ray晶體數據收集 10
2.4 酵素活性 11
第三章 結果 13
3.1 突變種Y42L蛋白質純化 13
3.2 最適反應pH值 13
3.3 酸鹼與熱穩定性 14
3.4 突變種Y42L晶體結構 15
3.5 突變種Y42L中配體之鍵結位置及對其活性影響 23
3.5.1 鈣離子之鍵結位置與抑制劑作用 23
3.5.2 Tris之鍵結位置與抑制劑作用 27
3.5.3 醋酸根之鍵結位置與活性曲線圖 31
第四章 討論 34
4.1 突變種Y42L催化活性 34
4.2 第三個鈣離子對酵素活性的影響 35
第五章 結論 37
參考文獻 38

表目錄

表2. 1 蛋白質SDS-PAGE配方 10
表3. 1 突變種Y42L之X-ray數據分析與晶體結構統計數據 17
表3. 2 β-1,3-1,4-纖維三醣相鄰胺基酸之交互作用及鍵長 21
表3. 3 Tyr 42、Glu 47及β-1,3-1,4-纖維三醣之交互作用及突變前後鍵長22
表3. 4 突變種Y42L與鈣離子配位的元素及鍵長 25
表3. 5 突變種Y42L與Tris分子產生交互作用的元素及鍵長 29
表3. 6 突變種Y42L與醋酸根離子產生交互作用的元素及鍵長 33
表4. 1 葡聚醣水解酶（Glucanase）及突變種之酵素動力學數據 34

圖目錄

圖1. 1 不同來源之β-葡聚醣酶之三維結構差異性 1
圖1. 2 1,3-1,4-β-D-葡聚醣之結構、構型及分子間氫鍵示意圖 3
圖1. 3 1,3-1,4-β-D-葡聚醣水解酶的反應機制 4
圖1. 4 1,3-1,4-β-D-葡聚醣水解酶之水解產物 4
圖1. 5 Lineweaver-Burk雙倒數圖 6
圖1. 6 抑制劑Lineweaver-Burk雙倒數圖 7
圖2. 1 實驗流程圖 8
圖3. 1 突變種Y42L親和性層析結果及電泳分析膠片 13
圖3. 2 突變種Y42L之最適反應pH值曲線圖 14
圖3. 3 突變種Y42L之酸鹼穩定性曲線圖 15
圖3. 4 突變種Y42L之熱穩定曲線圖 15
圖3. 5 突變種Y42L的結構及晶體單元中的蛋白質排列位置圖。 16
圖3. 6 突變種Y42L與原生種（wild-type）的結構重疊圖 18
圖3. 7 突變種Y42L之電子密度圖 19
圖3. 8 突變種Y42L與β-1,3-1,4-纖維三醣產生氫鍵鍵結的胺基酸 19
圖3. 9 突變種Y42L與β-1,3-1,4-纖維三醣存在著凡德瓦爾作用力的胺基酸20
圖3. 10 突變種Y42L突變前後與β-1,3-1,4-纖維三醣之交互作用 22
圖3. 11 突變前後之分子表面圖 23
圖3. 12 配體相對位置之整體結構圖 23
圖3. 13 鈣離子鍵結關係及電子密度圖 24
圖3. 14 鈣離子對突變種Y42L之抑制曲線圖 26
圖3. 15 鈣離子對突變種Y42L之動力學抑制圖 27
圖3. 16 Tris離子鍵結關係及電子密度圖 28
圖3. 17 Tris對突變種Y42L之抑制曲線圖 30
圖3. 18 Tris對突變種Y42L之動力學抑制圖 31
圖3. 19 醋酸根離子鍵結關係及電子密度圖 32
圖3. 20 醋酸根離子對突變種Y42L之活性曲線圖 33
圖4. 1 第三個鈣離子位於活性區域裂縫入口處附近 36

參考文獻

1.Roubroeks, J.P., Andersson, R., and Aman, P. (2000). Structural features of (1→3),(1→4)-β-D-glucan and arabinoxylan fractions isolated from rye bran. Carbohydrate Polymers 42, 3-11.
2.Tosh, S.M., Wood, P.J., Wang, Q., and Weisz, J. (2004). Structural characteristics and rheological properties of partially hydrolyzed oat β-glucan: the effects of molecular weight and hydrolysis method. Carbohydrate Polymers 55, 425-436.
3.Papageorgiou, M., Lakhdara, N., Lazaridou, A., Biliaderis, C.G., and Izydorczyk, M.S. (2005). Water extractable (1→3,1→4)-β-D-glucans from barley and oats: An intervarietal study on their structural features and rheological behaviour. Journal of Cereal Science 42, 213-224.
4.Collins, T., Gerday, C., and Feller, G. (2005). Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews 29, 3-23.
5.Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal 280(Pt 2), 309-316.
6.Henrissat, B., and Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal 293(Pt 3), 781-788.
7.Vasur, J., Kawai, R., Andersson, E., Igarashi, K., Sandgren, M., Samejima, M., and Stahlberg, J. (2009). X-ray crystal structures ofPhanerochaete chrysosporiumLaminarinase 16A in complex with products from lichenin and laminarin hydrolysis. FEBS Journal 276, 3858-3869.
8.Bohm, N., and Kulicke, W.-M. (1999). Rheological studies of barley (1→3)(1→4)-β-glucan in concentrated solution: mechanistic and kinetic investigation of the gel formation. Carbohydrate Research 315, 302-311.
9.Tosh, S.M., Brummer, Y., Wood, P.J., Wang, Q., and Weisz, J. (2004). Evaluation of structure in the formation of gels by structurally diverse (1→3)(1→4)-β-D-glucans from four cereal and one lichen species. Carbohydrate Polymers 57, 249-259.
10.Vincken, J.-P., Schols, H.A., Oomen, R.J.F.J., McCann, M.C., Ulvskov, P., Voragen, A.G.J., and Visser, R.G.F. (2003). If Homogalacturonan Were a Side Chain of Rhamnogalacturonan I. Implications for Cell Wall Architecture. Plant Physiology 132, 1781-1789.
11.Agoub, A.A., Giannouli, P., and Morris, E.R. (2009). Gelation of high methoxy pectin by acidification with D-glucono-δ-lactone (GDL) at room temperature. Carbohydrate Polymers 75, 269-281.
12.Doublier, J.-L., and Wood, P.J. (1995). Rheological properties of aqueous solutions of (1 → 3)(1 → 4)-β-D-glucan from oats (Avena sativa L.). Cereal Chemistry 72, 335–340.
13.Woodward, J.R., Phillips, D.R., and Fincher, G.B. (1983). Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure. Carbohydrate Polymers 3, 207-225.
14.Cui, W., Wood, P.J., Blackwell, B., and Nikiforuk, J. (2000). Physicochemical properties and structural characterization by two-dimensional NMR spectroscopy of wheat β-D-glucan—comparison with other cereal β-D-glucans. Carbohydrate Polymers 41, 249-258.
15.Tvaroska, I., Ogawa, K., Deslandes, Y., and Marchessault, R.H. (1983). Crystalline conformation and structure of lichenan and barley β-glucan. Canadian Journal of Chemistry 61, 1608-1616.
16.Jensen, M.S., Bach Knudsen, K.E., Inborr, J., and Jakobsen, K. (1998). Effect of β-glucanase supplementation on pancreatic enzyme activity and nutrient digestibility in piglets fed diets based on hulled and hulless barley varieties. Animal Feed Science and Technology 72, 329-345.
17.Humbert-Goffard, A., Saucier, C., Moine-Ledoux, V., Canal-Llauberes, R.-M., Dubourdieu, D., and Glories, Y. (2004). An assay for glucanase activity in wine. Enzyme and Microbial Technology 34, 537-543.
18.Garry, B.P., Fogarty, M., Curran, T.P., and O''Doherty, J.V. (2007). Effect of cereal type and exogenous enzyme supplementation in pig diets on odour and ammonia emissions. Livestock Science 109, 212-215.
19.Leek, A.B.G., Callan, J.J., Reilly, P., Beattie, V.E., and O’Doherty, J.V. (2007). Apparent component digestibility and manure ammonia emission in finishing pigs fed diets based on barley, maize or wheat prepared without or with exogenous non-starch polysaccharide enzymes. Animal Feed Science and Technology 135, 86-99.
20.Koolman, J., and Roehm, K.H. (2008). Color atlas of biochemistry, 2nd edition Edition.
21.Trinci, A.P.J., Davies, D.R., Gull, K., Lawrence, M.I., Bonde Nielsen, B., Rickers, A., and Theodorou, M.K. (1994). Anaerobic fungi in herbivorous animals. Mycological Research 98, 129-152.
22.Teather, R.M., and Erfle, J.D. (1990). DNA sequence of a Fibrobacter succinogenes mixed-linkage β-glucanase (1,3-1,4-β-D-glucan 4-glucanohydrolase) gene. Journal of Bacteriology 172(7), 3837-3841.
23.Chen, H., Li, X.-L., and Ljungdahl, L.G. (1997). Sequencing of a 1,3-1,4-β-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin. Journal of Bacteriol 179(19), 6028-6034.
24.Tsai, L.-C., Shyur, L.-F., Cheng, Y.-S., and Lee, S.-H. (2005). Crystal Structure of Truncated Fibrobacter succinogenes 1,3-1,4-β-D-Glucanase in Complex with β-1,3-1,4-Cellotriose. Journal of Molecular Biology 354, 642-651.
25.Tsai, L.-C., Shyur, L.-F., Lee, S.-H., Lin, S.-S., and Yuan, H.S. (2003). Crystal Structure of a Natural Circularly Permuted Jellyroll Protein: 1,3-1,4-β-D-Glucanase from Fibrobacter succinogenes. Journal of Molecular Biology 330, 607-620.
26.Chen, J.-L., Tsai, L.-C., Wen, T.-N., Tang, J.-B., Yuan, H.S., and Shyur, L.-F. (2001). Directed Mutagenesis of Specific Active Site Residues on Fibrobacter succinogenes 1,3-1,4-β-D-Glucanase Significantly Affects Catalysis and Enzyme Structural Stability. Journal of Biological Chemistry 276, 17895-17901.
27.Sulzenbacher, G., Driguez, H., Henrissat, B., Schulein, M., and Davies, G.J. (1996). Structure of the Fusarium oxysporum Endoglucanase I with a Nonhydrolyzable Substrate Analogue: Substrate Distortion Gives Rise to the Preferred Axial Orientation for the Leaving Group. Biochemistry 35, 15280-15287.
28.Davies, G.J., Mackenzie, L., Varrot, A., Dauter, M., Brzozowski, A.M., Schulein, M., and Withers, S.G. (1998). Snapshots along an Enzymatic Reaction Coordinate: Analysis of a Retaining β-Glycoside Hydrolase. Biochemistry 37, 11707-11713.
29.Planas, A. (2000). Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1543, 361-382.
30.Malet, C., and Planas, A. (1998). From β-glucanase to β-glucansynthase: glycosyl transfer to α-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile. FEBS Letters 440, 208-212.
31.Erfle, J.D., Teather, R.M., Wood, P.J., and Irvin, J.E. (1988). Purification and properties of a 1,3-1,4-β-D-glucanase (lichenase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli. Biochemical journal 255(3), 833-841.
32.Kumagai, Y., and Ojima, T. (2009). Enzymatic properties and the primary structure of a β-1,3-glucanase from the digestive fluid of the Pacific abalone Haliotis discus hannai. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 154, 113-120.
33.Kawai, R., Igarashi, K., Yoshida, M., Kitaoka, M., and Samejima, M. (2005). Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium. Applied Microbiology and Biotechnology 71, 898-906.
34.Cui, S.W., and Wang, Q. (2009). Cell wall polysaccharides in cereals: chemical structures and functional properties. Structural Chemistry 20, 291-297.
35.Chen, J.-H., Tsai, L.-C., Huang, H.-C., and Shyur, L.-F. (2010). Structural and catalytic roles of amino acid residues located at substrate-binding pocket in Fibrobacter succinogenes 1,3-1,4-β-D-glucanase. Proteins: Structure, Function, and Bioinformatics 78, 2820-2830.
36.Lin, Y.-S., Tsai, L.-C., Lee, S.-H., Yuan, H.S., and Shyur, L.-F. (2009). Structural and catalytic roles of residues located in β13 strand and the following β-turn loop in Fibrobacter succinogenes 1,3-1,4-β-D-glucanase. Biochimica et Biophysica Acta (BBA) - General Subjects 1790, 231-239.
37.Nelson, D.L., and Cox, M.M. (2005). Principles of Biochemistry, fourth edition Edition.
38.Margui, E., Queralt, I., and Hidalgo, M. (2009). Application of X-ray fluorescence spectrometry to determination and quantitation of metals in vegetal material. Trends in Analytical Chemistry 28, 362-372.
39.Miller, G.L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry 31, 426-428.
40.Cortes, A., Cascante, M., Cardenas, M.L., and Cornish-Bowden, A. (2001). Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition : new ways of analysing data. Biochemical journal 357(Pt 1), 263-268.
41.Tsai, L.-C., Hsiao, C.-H., Liu, W.-Y., Yin, L.-M., and Shyur, L.-F. (2011). Structural basis for the inhibition of 1,3-1,4-β-D-glucanase by noncompetitive calcium ion and competitive Tris inhibitors. Biochemical and Biophysical Research Communications 407, 593-598.
42.Walti, M., Roulin, S., and Feller, U. (2002). Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves. Plant Physiology and Biochemistry 40, 363-371.
43.Zhang, X.-y., Ruan, H., Mu, L., He, G.-q., Tang, X.-j., and Chen, Q.-h. (2006). Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution. Journal of Zhejiang University Science A 7, 1948-1955.
44.Price, A.C., Zhang, Y.-M., Rock, C.O., and White, S.W. (2004). Cofactor-Induced Conformational Rearrangements Establish a Catalytically Competent Active Site and a Proton Relay Conduit in FabG. Structure 12, 417-428.