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研究生:葉婷娟
研究生(外文):Ting-Juan Ye
論文名稱:探討台灣本土嗜熱菌Meiothermus taiwanensis WR220中endo-β-1,4-glucanase之生化特性
論文名稱(外文):Basic properties and characterizations of endo-β-1,4-glucanase from Meiothermus taiwanensis WR220
指導教授:吳世雄吳世雄引用關係
指導教授(外文):Shih-Hsiung Wu
口試委員:梁博煌花國鋒黃人則
口試委員(外文):Po-Huang LiangKuo-Feng HuaJen-Tse Huang
口試日期:2014-07-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:85
中文關鍵詞:Meiothermus taiwanensisGlucanaseVP-ITC熱穩定性酵素動力學
外文關鍵詞:Meiothermus TaiwanensisGlucanaseVP-ITCthermal stabilitykinetics
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在自然界中,細菌及真菌或是一些昆蟲的體內常含有纖維素水解酵素(例如:內切型纖維素分解酵素,endo-β-1,4-glucanase),這些水解酵素可以幫助分解植物細胞壁,以提供生物體內的碳來源。在工業應用上,纖維素分解酵素可用於再生能源的生產或是用於造紙業,也常做為家畜的飼料或酒類發酵的添加物。經由對Meiothermus taiwanensis WR220的全基因體解序,我們發現一個很少存在於Thermus species,註解為內切型纖維素分解酵素的酵素(Mta-glucanase)。從sequence alignment的結果中發現了一段低保留度的未知區域(269-379),其在已知的纖維素分解酵素中表現出極低同源性。因此,為了闡明這個未知區域的功能,我們利用基因轉殖技術做出了GlucanaseWT與GlucanaseΔ269-379這兩個重組蛋白,經由圓二色光譜儀(CD)的分析,我們發現雖然GlucanaseWT與GlucanaseΔ269-379有相似的二級結構,但GlucanaseWT中含有比GlucanaseΔ269-379略高的β-sheet比例;而經由熱穩定性實驗與活性實驗,我們發現GlucanaseWT不論在熱穩定性或是受質水解活性都比GlucanaseΔ269-379來的好。為了更加深入探討他們對β-1,4-glucan(例如:CMC)的酵素機制,我們比較了兩個蛋白的酵素動力學參數,根據兩個蛋白的Km與kcat,我們認為這段未知區域的主要功能可能和受質的結合力有關,藉由homology modeling結果,我們發現這段區域可能是可以與carbohydrate結合的lectin-like構型。經由恆溫滴定微卡計(VP-ITC)實驗結果,我們驗證了這段未知區域可能是與醣受質(例如:CMC)結合的區域。綜合以上的實驗結果,GlucanaseWT中的269-379這段未知區域不但可以增加蛋白的熱穩定性與受質水解活性,也可以增加蛋白與受質的結合力,這些特性都顯示Mta-Glucanase在日後工業應用上具有更好的發展潛力。

In nature, bacteria, fungi and insects often utilize some cellulases (e.g., endo-β-1,4-glucanase), to acquire their carbon sources from hydrolyzing plants’ cell walls. Industrially, these enzymes are often applied to renewable energy production or paper industry, and can also be used as additives for animal feeds and alcohol fermentation. From whole genome sequence of Meiothermus taiwanensis WR220, we found a protein annotated as endo-β-1,4-glucanase (Mta-Glucanase), which is less commonly observed in Thermus species. Interestingly, the result from sequence alignments, an uncommon region (269-379) was identified, sharing no homology to any other known glucanases. Therefore, in order to elucidate the function of that extra region, we constructed both wild type (GlucanaseWT) and deletion mutant (Glucanase269-379). Based on circular dichroism, although they shared similar CD spectrum, GlucanaseWT contained more percentage of beta-sheet than that of Glucanase269-379. Not only the thermal stability but also the activity of GlucanaseWT is better than those of the mutant. To gain more insight into the mechanism underlying β-1,4-glucan (e.g., CMC), we compared the basic enzyme kinetic parameters of them. According to their Km and kcat, we thought the function of that extra region seems to play a role in binding to the substrate. By homology modeling, we found that region might be similar to the domain in lectin-like proteins responsible for carbohydrate-binding. Together with the results from isothermal titration calorimetry (VP-ITC), we demonstrated the extra region as a sugar-binding domain essential for CMC binding. Taken together, the extra region of GlucanaseWT plays important roles in not only thermal stability but also the affinity to the substrate. These findings are of vital importance in future industrial applications.

摘要 I
Abstract II
目錄 III
表目錄 VII
圖目錄 VI
第一章 緒論 1
1-1 能源議題 1
1-1.1 能源短缺與替代能源開發 1
1-1.2 生質能(Bio-mass) 2
1-2 纖維素(cellulose) 3
1-2.1 植物細胞壁 3
1-2.2 纖維素結構 4
1-3 纖維素水解酵素(cellulase) 6
1-3.1 纖維素水解酵素之分類 7
1-3.2 纖維素水解酵素之應用 8
1-4 嗜熱性細菌 10
1-4.1 嗜熱性細菌Thermus/Meiothermus群的基本介紹 11
1-4.2 Meiothermus屬 11
1-4.3 嗜熱菌屬的應用 12
1-5 研究動機與實驗目的 14
第二章 實驗儀器與材料 15
2-1 實驗材料 15
2-1.1 染色體DNA萃取(chromosome DNA extraction) 15
2-1.2 質體(plasmid DNA)製備 15
2-1.3 細菌培養 16
2-1.4 SDS- PAGE 16
2-1.5 西方印漬術(Western blot) 17
2-1.6 ThermoFluor Assay (Tm值) 17
2-1.7 DNSA assay (3,5-dinitrosalicylic acid assay) 18
2-1.8 各種緩衝溶液配方 18
2-1.9 Isothermal Titration Calorimetry (ITC) 20
2-1.10 其他 21
2-2 實驗儀器 22
2-2.1 聚合脢連鎖反應(Polymerase Chain Reaction,PCR) 22
2-2.2 細菌培養 22
2-2.3 蛋白質分析 22
2-2.4 SDS- PAGE和Western blot 23
2-2.5 其他 23
第三章 實驗方法 24
3-1 染色體DNA萃取 24
3.1.1 嗜熱菌培養基配置 24
3.1.2 染色體DNA萃取 24
3-2 質體製備 25
3-2.1 聚合酶連鎖反應 ( Polymerase chain reaction,PCR ) 25
3-2.2 從電泳凝膠中回收DNA ( Gel extraction ) 27
3-2.3 大腸桿菌培養基製備 27
3-2.4 載體純化 27
3-2.5 限制酶反應 28
3-2.6 接合反應 ( Ligation ) 29
3-2.7 大腸桿菌的轉形作用 ( transformation ) 29
3-2.8 菌落PCR (colony PCR) 29
3-2.9 質體純化與定序 30
3-3 蛋白質表現與純化 31
3-3.1 小量表現蛋白質 31
3-3.2 大量表現蛋白質 31
3-3.3 破菌與純化 31
3-3.4 蛋白質濃度鑑定 32
3-3.5 蛋白質電泳分析 (SDS-PAGE) 32
3-3.6 西方印漬術(Western blot) 33
3-4 蛋白二級結構分析 33
3-5 蛋白Tm值測定 35
3-6 蛋白活性分析 35
3-7 Kinetic參數的計算 36
3-8 恆溫滴定微卡計(Isothermal Titration Calorimetry, ITC)實驗 37
3-8.1 突變蛋白活性位置(active site) 37
3-8.2 ITC實驗 39
第四章 實驗結果 41
4-1 生物資訊學(BLAST)與序列分析(Alignment) 41
4-2 演化樹(phylogenic tree) 43
4-3 蛋白結構預測(homology modeling) 44
4-4 Signal peptide分析 45
4-5 大量表現與純化蛋白 46
4-6 蛋白二級結構分析 47
4-7 熱穩定性比較 49
4-8 活性測試 50
第五章 總結與討論 53
5-1 Glucanase269-379對GlucanaseWT其酵素結合與活性影響之探討 53
5-1.1 Glucanase269-379結構預測 53
5-1.2 Enzyme Kinetics 54
5-1.3 Isothermal Titration Calorimetry (ITC) 55
5-1.4 由Homology modeling探討Glucanase269-379對酵素活性之影響 62
5-2 GlucanaseWT與commercial Glucanase活性比較 65
5-3 總結 67
第六章 參考文獻 68
第七章 附件 73


1.Ragauskas, A.J., et al., The path forward for biofuels and biomaterials. Science, 2006. 311(5760): p. 484-9.
2.Demain, A.L., M. Newcomb, and J.H. Wu, Cellulase, clostridia, and ethanol. Microbiol Mol Biol Rev, 2005. 69(1): p. 124-54.
3.Lynd, L.R., et al., Fuel ethanol from cellulosic biomass. Science, 1991. 251(4999): p. 1318-23.
4.Sun, Y. and J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol, 2002. 83(1): p. 1-11.
5.Yang, Y.L., et al., Structural elucidation of phosphoglycolipids from strains of the bacterial thermophiles Thermus and Meiothermus. J Lipid Res, 2006. 47(8): p. 1823-32.
6.Subramaniyan, S. and P. Prema, Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol, 2002. 22(1): p. 33-64.
7.Bayer, E.A., et al., The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu Rev Microbiol, 2004. 58: p. 521-54.
8.Szijarto, N., et al., Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces. J Biotechnol, 2008. 136(3-4): p. 140-7.
9.Lynd, L.R., et al., Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol, 2005. 16(5): p. 577-83.
10.Bhat, M.K., Cellulases and related enzymes in biotechnology. Biotechnol Adv, 2000. 18(5): p. 355-83.
11.Russell, J.B. and J.L. Rychlik, Factors that alter rumen microbial ecology. Science, 2001. 292(5519): p. 1119-22.
12.Krause, D.O., et al., Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics. FEMS Microbiol Rev, 2003. 27(5): p. 663-93.
13.Pace, N.R., A molecular view of microbial diversity and the biosphere. Science, 1997. 276(5313): p. 734-40.
14.Rondon, M.R., R.M. Goodman, and J. Handelsman, The Earth''s bounty: assessing and accessing soil microbial diversity. Trends Biotechnol, 1999. 17(10): p. 403-9.
15.Tringe, S.G., et al., Comparative metagenomics of microbial communities. Science, 2005. 308(5721): p. 554-7.
16.Ferrer, M., et al., Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ Microbiol, 2005. 7(12): p. 1996-2010.
17.Dodd, D. and I.K. Cann, Enzymatic deconstruction of xylan for biofuel production. Glob Change Biol Bioenergy, 2009. 1(1): p. 2-17.
18.Yeoman, C.J., et al., Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol, 2010. 70: p. 1-55.
19.Lynd, L.R., et al., Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev, 2002. 66(3): p. 506-77, table of contents.
20.Gong, X., et al., Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene. BMC Res Notes, 2012. 5: p. 566.
21.Wu, T.H., et al., Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose. Biochim Biophys Acta, 2011. 1814(12): p. 1832-40.
22.Kristjansson, J.K. and G.A. Alfredsson, Distribution of Thermus spp. in Icelandic Hot Springs and a Thermal Gradient. Appl Environ Microbiol, 1983. 45(6): p. 1785-9.
23.Brock, T.D. and H. Freeze, Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J Bacteriol, 1969. 98(1): p. 289-97.
24.O., S.K., Hyperthermophiles: isolation, callifications. Edited by K. Horikoshi and W.D. Grant. New York: Wiley-Liss, 1998. 322: p. 165-170.
25.Fujiwara, S., S. Okuyama, and T. Imanaka, The world of archaea: genome analysis, evolution and thermostable enzymes. Gene, 1996. 179(1): p. 165-70.
26.Hensel,et al,, Chemotaxonomic and molecular-genetic studies of the genus Thermus: enidence for a phylogenetic relationship of Thermus aquaticus and Thermus rubber to the genus Deiococcus. Int. J. Syst. Bacteriol., 1986. 36: p. 444.
27.Egorova, L.A. and L.G. Loginova, [Selection of a culture forming alkaline phosphatase from thermophilic bacteria in the genus Thermus]. Mikrobiologiia, 1984. 53(2): p. 242-5.
28.Tenreiro, S., M.F. Nobre, and M.S. da Costa, Thermus silvanus sp. nov. and Thermus chliarophilus sp. nov., two new species related to thermus ruber but with lower growth temperatures. Int J Syst Bacteriol, 1995. 45(4): p. 633-9.
29.Chung, A.P., et al., Meiothermus cerbereus sp. nov., a new slightly thermophilic species with high levels of 3-hydroxy fatty acids. Int J Syst Bacteriol, 1997. 47(4): p. 1225-30.
30.Chen, M.Y., et al., Meiothermus taiwanensis sp. nov., a novel filamentous, thermophilic species isolated in Taiwan. Int J Syst Evol Microbiol, 2002. 52(Pt 5): p. 1647-54.
31.Lamosa, P., et al., Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus. Appl Environ Microbiol, 2000. 66(5): p. 1974-9.
32.Shen, P.Y., et al., Fatty acid distribution in mesophilic and thermophilic strains of the genus Bacillus. J Bacteriol, 1970. 103(2): p. 479-81.
33.Ray, P.H., D.C. White, and T.D. Brock, Effect of growth temperature on the lipid composition of Thermus aquaticus. J Bacteriol, 1971. 108(1): p. 227-35.
34.Yang, F.L., et al., TLR-independent induction of human monocyte IL-1 by phosphoglycolipids from thermophilic bacteria. Glycoconj J, 2008. 25(5): p. 427-39.
35.Niehaus, F., et al., Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol, 1999. 51(6): p. 711-29.
36.Ramaley, R.F. and J. Hixson, Isolation of a nonpigmented, thermophilic bacterium similar to Thermophilic bacterium similar to Thermus aquaticus. J Bacteriol, 1970. 103(2): p. 527-8.
37.Kaledin, A.S., A.G. Sliusarenko, and S.I. Gorodetskii, [Isolation and properties of DNA polymerase from extreme thermophylic bacteria Thermus aquaticus YT-1]. Biokhimiia, 1980. 45(4): p. 644-51.
38.Sambrook, J., and D. W. Russell., Molecular cloning:a laboratory manual. 2001.
39.Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970. 227(5259): p. 680-5.
40.Brahms, S. and J. Brahms, Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J Mol Biol, 1980. 138(2): p. 149-78.
41.Phillips, K. and A.H. de la Pena, The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Curr Protoc Mol Biol, 2011. Chapter 10: p. Unit10 28.
42.Pantoliano, M.W., et al., High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen, 2001. 6(6): p. 429-40.
43.Dodds, C., et al., Blood-sugar response of normal adults to dextrose, sucrose, and liquid glucose. Lancet, 1959. 1(7071): p. 485-8.
44.Danner, M., et al., Folding and assembly of phage P22 tailspike endorhamnosidase lacking the N-terminal, head-binding domain. Eur J Biochem, 1993. 215(3): p. 653-61.
45.Miller, G.L., Use of dinitrosalicylic acid reagent for determination of resucing sugar. Anal. Chem., 1959. 31: p. 426-428.
46.Sami, e.a., Biochemical characterization of endo-1, 4-β-D-glucanase activity of a green insect pest Aulacophora foveicollis (Lucas). Life Science Journal, 2008. 5(2): p. 30 – 36.
47.Lee, H.L., et al., Construction and characterization of different fusion proteins between cellulases and beta-glucosidase to improve glucose production and thermostability. Bioresour Technol, 2011. 102(4): p. 3973-6.
48.Zhang, Z., et al., Thin-layer chromatography for the analysis of glycosaminoglycan oligosaccharides. Anal Biochem, 2007. 371(1): p. 118-20.
49.Celestino, K.R., R.B. Cunha, and C.R. Felix, Characterization of a beta-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry. BMC Biochem, 2006. 7: p. 23.
50.Furtado, G.P., et al., A designed bifunctional laccase/beta-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse. Protein Eng Des Sel, 2013. 26(1): p. 15-23.
51.Liu, W.C., et al., Engineering of dual-functional hybrid glucanases. Protein Eng Des Sel, 2012. 25(11): p. 771-80.
52.Addington, T., et al., Re-engineering specificity in 1,3-1, 4-beta-glucanase to accept branched xyloglucan substrates. Proteins, 2011. 79(2): p. 365-75.
53.Johnson, P.E., et al., Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC. Biochemistry, 1998. 37(37): p. 12772-81.
54.Christopher D. Warner, G.C.-U., Substrate Binding by the Catalytic Domain and Carbohydrate Binding Module of Ruminococcus flavefaciens FD-1 Xyloglucanase/ Endoglucanase. nd. Eng. Chem. Res., 2013. 52 (1): p. 30–36.
55.Shih, Y.P., et al., High-throughput screening of soluble recombinant proteins. Protein Sci, 2002. 11(7): p. 1714-9.
56.Chiu, J., et al., Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Res, 2004. 32(21): p. e174.
57.Zhang, Y., I-TASSER: fully automated protein structure prediction in CASP8. Proteins, 2009. 77 Suppl 9: p. 100-13.
58.Roy, A., A. Kucukural, and Y. Zhang, I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc, 2010. 5(4): p. 725-38.
59.Borriss, R., K. Buettner, and P. Maentsaelae, Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases. Mol Gen Genet, 1990. 222(2-3): p. 278-83.
60.Akita, M., et al., Crystallization and preliminary X-ray study of alkaline mannanase from an alkaliphilic Bacillus isolate. Acta Crystallogr D Biol Crystallogr, 2004. 60(Pt 8): p. 1490-2.
61.Huang, W., et al., Crystal structure of Proteus vulgaris chondroitin sulfate ABC lyase I at 1.9A resolution. J Mol Biol, 2003. 328(3): p. 623-34.
62.Ilari, A., et al., Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition. FEBS J, 2009. 276(4): p. 1048-58.
63.Fujii, R., M. Kitaoka, and K. Hayashi, One-step random mutagenesis by error-prone rolling circle amplification. Nucleic Acids Res, 2004. 32(19): p. e145.
64.Milev, S., Isothermal titration calorimetry: Principles and experimental design. GE, 2013: http://bcmp.med.harvard.edu/sites/bcmp.med.harvard.edu/files/facilities/ITC200%20training_pdf.pdf.




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