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研究生:林瑽羿
研究生(外文):Tsung-Yi Lin
論文名稱:探討海棲熱袍菌Cel5A 的生化鑑定及定點突變
論文名稱(外文):Biochemical characterization and site-directedmutagenesis of Cel5A from Thermotoga maritima
指導教授:梁博煌
口試委員:黃慶璨何孟樵
口試日期:2017-06-08
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:2017
畢業學年度:105
語文別:中文
論文頁數:46
中文關鍵詞:X光結晶學受質辨識機制酵素生物工程三功能酵素生物燃料
外文關鍵詞:X-ray crystallographysubstrate recognition mechanismenzymebioengineeringtri-functional enzymebiofuel
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植物生物質中的纖維素和半纖維素,經由水解酶處理後,可用於生產生物燃料。在我們先前的研究發現熱纖維梭菌的纖維素酶/木聚醣酶雙功能酵素CtCel5E和極端嗜熱海棲熱袍菌的纖維素酶/甘露聚醣酶雙功能酵素TmCel5A,兩者有高度的序列同源性,但卻擁有不同的雙功能活性。其中一部分序列具有顯著差異,稱之為flexible loop,當把CtCel5E的flexible loop置換為TmCel5A的序列後,得到一具有三功能酵素,稱為CtCel5E-Tmloop,具有纖維素酶/木聚醣酶/甘露聚醣酶活性。
於本論文中,經由表現、純化和定性在大腸桿菌中表現的重組蛋白TmCel5A,發現不只具有原本報告中提到的纖維素酶/甘露聚醣酶雙功能活性,亦同時具有木聚醣酶的活性。並藉由定點突變確認位於flexible loop區域的三個胺基酸H205、 W210、Y198,將三者突變為丙氨酸(alanine)後會顯著減少活性,說明三者對於催化作用的重要性,其中尤以H205和W210對甘露聚醣酶影響更為劇烈。
此外,藉由保留性的Tmloop結構比對發現4個纖維素酶,並表現熱纖維梭菌的纖維素酶CtCelC,發現不同於原本報告提到的單一活性,而同樣是一個三功能酵素具有纖維素酶/木聚醣酶/甘露聚醣酶的活性,說明了flexible loop此序列對多功能活性是必要的。
Cellulose and hemicelluloses from plant biomass can be used to produce biofuel after enzymatic process. We have engineered Clostridium thermocellum Cel5E (CtCel5E), a bifunctional cellulase/xylanase, into a tri-functional cellulase/xylanase/mannanase, named CtCel5E-Tmloop, by replacing its flexible loop region with that in Thermotoga maritima Cel5A (TmCel5A), a homologous bifunctional cellulase/mannanase, suggesting that the Tmloop might be essential for multi-functionality.
In this thesis, the recombinant TmCel5A expressed in E. coli was purified and characterized. Surprisingly, TmCel5A also showed low xylanase activity, in addition to the known cellulase and mannanase activities. By site-directed mutagenesis, H205A, W210A, and Y198A in the loop region showed significantly reduced activities, indicating their importance in catalysis. In particular, H205 and W210 are essential for mannanase activity. Moreover, by searching the conserved Tmloop structures in other cellulases, we identified Clostridium thermocellum CelC (CtCelC) also with tri-functional cellulase/xylanase/mannanase activity.
CONTENTS
中文摘要 iii
ABSTRACT iv
ABBREVIATIONS v
INTRODUTION 1
1.1 Alternative energy source 1
1.2 The composition of lignocellulosic biomass 1
1.3 Glycoside hydrolases 3
1.3.1 Cellulases 4
1.3.2 Hemicellulase 4
1.4 TmCel5A, a hyperthermophilic endoglucanase from Thermotoga maritima 5
1.5 Specific aim 6
MATERIALS AND METHODS 8
2.1 Materials 8
2.2 DNA Source and Bacterial Growth Condition. 8
2.3 Protein sequence alignment and structural analysis 9
2.4 Constructions of recombinant proteins 9
2.5 Site-directed Mutagenesis. 10
2.6 Expression and Purification of Recombinant Proteins. 11
2.7 Enzyme Activity Assays. 12
2.8 End-product Determination. 13
RESULTS 14
3.1 Sequence comparison of CtCel5E and TmCel5A 14
3.2 Expression and characterization of recombinant TmCel5A. 15
3.3 Site-directed mutagenesis of active-site residues in TmCel5A based on the structural comparison with CtCel5E. 16
3.4 Tmloop structure is important for mannanase activity. 19
DISCUSSION 22
TABPLES 25
REFERENCE 39
REFERENCE
1.Demain, A.L., M. Newcomb, and J.D. Wu, Cellulase, clostridia, and ethanol. Microbiology and molecular biology reviews, 2005. 69(1): p. 124-154.
2.Wyman, C.E., Biomass ethanol: technical progress, opportunities, and commercial challenges. Annual Review of Energy and the Environment, 1999. 24(1): p. 189-226.
3.Lee, H., S. Hamid, and S. Zain, Conversion of lignocellulosic biomass to nanocellulose: structure and chemical process. The Scientific World Journal, 2014. 2014.
4.Fujita, Y., et al., Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Applied and environmental microbiology, 2004. 70(2): p. 1207-1212.
5.Kumar, P., et al., Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Industrial & engineering chemistry research, 2009. 48(8): p. 3713-3729.
6.Sannigrahi, P., Y. Pu, and A. Ragauskas, Cellulosic biorefineries—unleashing lignin opportunities. Current Opinion in Environmental Sustainability, 2010. 2(5): p. 383-393.
7.Pérez, J., et al., Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. International Microbiology, 2002. 5(2): p. 53-63.
8.Gírio, F.M., et al., Hemicelluloses for fuel ethanol: a review. Bioresource technology, 2010. 101(13): p. 4775-4800.
9.Ebringerová, A. Structural diversity and application potential of hemicelluloses. in Macromolecular Symposia. 2005. Wiley Online Library.
10.Campbell, M.M. and R.R. Sederoff, Variation in Lignin Content and Composition (Mechanisms of Control and Implications for the Genetic Improvement of Plants). Plant physiology, 1996. 110(1): p. 3.
11.Rubin, E.M., Genomics of cellulosic biofuels. Nature, 2008. 454(7206): p. 841.
12.Várnai, A., et al., Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood. Bioresource technology, 2011. 102(19): p. 9096-9104.
13.Cantarel, B.L., et al., The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic acids research, 2009. 37(suppl 1): p. D233-D238.
14.Wilson, D.B., Cellulases and biofuels. Current opinion in biotechnology, 2009. 20(3): p. 295-299.
15.Aspeborg, H., et al., Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC evolutionary biology, 2012. 12(1): p. 186.
16.Chen, Z., et al., Tracing determinants of dual substrate specificity in glycoside hydrolase family 5. Journal of Biological Chemistry, 2012. 287(30): p. 25335-25343.
17.Bayer, E.A., et al., Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 1998. 8(5): p. 548-557.
18.Henrissat, B. and A. Bairoch, New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 1993. 293(3): p. 781-788.
19.Zhang, X.Z. and Y.H.P. Zhang, Cellulases: characteristics, sources, production, and applications. Bioprocessing Technologies in Biorefinery for Sustainable Production of Fuels, Chemicals, and Polymers, 2013. 1: p. 131-146.
20.Shallom, D. and Y. Shoham, Microbial hemicellulases. Current opinion in microbiology, 2003. 6(3): p. 219-228.
21.Dodd, D. and I.K. Cann, Enzymatic deconstruction of xylan for biofuel production. GCB Bioenergy, 2009. 1(1): p. 2-17.
22.McCleary, B.V., β-D-Mannanase. Methods in Enzymology, 1988. 160: p. 596-610.
23.Ademark, P., et al., Softwood hemicellulose-degrading enzymes from Aspergillus niger: purification and properties of a β-mannanase. Journal of Biotechnology, 1998. 63(3): p. 199-210.
24.Pereira, J.H., et al., Biochemical characterization and crystal structure of endoglucanase Cel5A from the hyperthermophilic Thermotoga maritima. Journal of structural biology, 2010. 172(3): p. 372-379.
25.Wu, T.-H., et al., Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2011. 1814(12): p. 1832-1840.
26.Chhabra, S.R., et al., Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan-and mannan-based polysaccharides. Applied and environmental microbiology, 2002. 68(2): p. 545-554.
27.Shie, H.-Y., Structural analysis and functional improvement of plant polysaccharides degrading enzyme from Clostridium thermocellum. (Master''s Thesis) Retrieved from http://tulips.ntu.edu.tw, 2014.
28.Lynd, L.R., C.E. Wyman, and T.U. Gerngross, Biocommodity engineering. Biotechnology progress, 1999. 15(5): p. 777-793.
29.Chang, J.-J., et al., PGASO: A synthetic biology tool for engineering a cellulolytic yeast. Biotechnology for biofuels, 2012. 5(1): p. 53.
30.Lee, H.-L., et al., Construction and characterization of different fusion proteins between cellulases and β-glucosidase to improve glucose production and thermostability. Bioresource technology, 2011. 102(4): p. 3973-3976.
31.Lee, H.-L., et al., Mutations in the substrate entrance region of β-glucosidase from Trichoderma reesei improve enzyme activity and thermostability. Protein Engineering Design and Selection, 2012: p. gzs073.
32.Wang, H.M., et al., Parallel gene cloning and protein production in multiple expression systems. Biotechnology progress, 2009. 25(6): p. 1582-1586.
33.Sievers, F., et al., Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Molecular systems biology, 2011. 7(1): p. 539.
34.Gouet, P., E. Courcelle, and D.I. Stuart, ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics, 1999. 15(4): p. 305-308.
35.DeLano, W.L., Pymol: An open-source molecular graphics tool. CCP4 Newsletter On Protein Crystallography, 2002. 40: p. 82-92.
36.Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 1976. 72(1-2): p. 248-254.
37.Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry, 1959. 31(3): p. 426-428.
38.Zhang, Z., et al., Thin layer chromatography for the analysis of glycosaminoglycan oligosaccharides. Analytical biochemistry, 2007. 371(1): p. 118.
39.Henrissat, B., et al., Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proceedings of the National Academy of Sciences, 1995. 92(15): p. 7090-7094.
40.Zheng, B., et al., Crystal structure of hyperthermophilic endo-beta-1,4-glucanase: implications for catalytic mechanism and thermostability. J Biol Chem, 2012. 287(11): p. 8336-46.
41.Zheng, B., et al., Crystallization and preliminary crystallographic analysis of thermophilic cellulase from Fervidobacterium nodosum Rt17-B1. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 2009. 65(3): p. 219-222.
42.Oyama, T., et al., Mutational and structural analyses of caldanaerobius polysaccharolyticus Man5b reveal novel active site residues for family 5 glycoside hydrolases. PloS one, 2013. 8(11): p. e80448.
43.Chhabra, S.R. and R.M. Kelly, Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM). FEBS letters, 2002. 531(2): p. 375-380.
44.Benson, D.A., et al., GenBank. Nucleic acids research, 2013. 41(D1): p. D36-D42.
45.Bernstein, F.C., et al., The protein data bank. European Journal of Biochemistry, 1977. 80(2): p. 319-324.
46.Yuan, S.-F., et al., Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum. Journal of Biological Chemistry, 2015. 290(9): p. 5739-5748.
47.Vlasenko, E., et al., Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases. Bioresource Technology, 2010. 101(7): p. 2405-2411.
48.Sakon, J., et al., Crystal Structure of Thermostable Family 5 Endocellulase E1 from Acidothermus cellulolyticus in Complex with Cellotetraose†. Biochemistry, 1996. 35(33): p. 10648-10660.
49.Hilge, M., et al., High-resolution native and complex structures of thermostable β-mannanase from Thermomonospora fusca–substrate specificity in glycosyl hydrolase family 5. Structure, 1998. 6(11): p. 1433-1444.
50.Leggio, L.L. and S. Larsen, The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5. FEBS letters, 2002. 523(1): p. 103-108.
51.Schagerlöf, U., et al., Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites. Biomacromolecules, 2007. 8(8): p. 2358-2365.
52.GILKES, N.R., et al., Structural and functional relationships in two families of β‐1, 4‐glycanases. The FEBS Journal, 1991. 202(2): p. 367-377.
53.Notenboom, V., et al., Exploring the Cellulose/Xylan Specificity of the β-1, 4-Glycanase Cex from Cellulomonas fimi through Crystallography and Mutation†. Biochemistry, 1998. 37(14): p. 4751-4758.
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