臺灣博碩士論文加值系統

於前期研究中，本團隊由台灣水牛(Bubalus bubalis)瘤胃真菌群中選殖出一聚木醣酶(xylanase；EC3.2.1.8)基因，命名為xynR8，並發現該酵素之催化活性較文獻中其它聚木醣酶高出約120倍以上，因此於本研究中將xynR8選殖於pET21c載體中，並於E.coli BL21(DE3)中大量表達，再將所得重組酵素以親和管柱層析法等純化後，進行生化特性的探討與動力學分析。結果顯示重組型XynR8之Km為11.1mg/ml，kcat為389432.4 sec-1，其比活性較一般市售聚木醣酶高出約200倍以上，且酸鹼耐受範圍較廣(可耐pH 3~11)。為增加此酵素在工業上的應用價值，且探討其結構與功能的關係，本研究嘗試以定位突變改造此酵素，結果發現突變株XynR8(N41D較野生株有更好的酸鹼穩定度，而突變株XynR8(N85D)、XynR8(I165K) 、XynR8(I165R)則完全失去活性，經Deep View立體結構分析發現XynR8之立體結構與一般family 11之聚木醣酶相較，多了一個α-helix，是否因此使XynR8之催化活性較高，需進一步探討。此外，N41位於loop區內，此兩loop區對XynR8酸鹼耐受性之影響值得進一步研究。

Previously, a xylanase (EC 3.2.1.8) gene was subcloned from rumen fungi of Bubalus bubalis, named xynR8. The catalytic activity of XynR8 was found to be relatively estimated tobe at least 120-fold higher than other previously published xylanases. Thus, in this study, the gene of xynR8 was subcloned into pET21c vector and expressed in E.coli BL21(DE3). The resulting protein was purified by methods including affinity chromatography and characterized for its biochemical and kinetic properties. Km and kcat of the enzyme were determined to be 11.1 mg/ml and 389432.4 sec-1 respectively. The specific activity of the enzyme was estimated tobe at least 200-fold higher than those of commercially available xylanases. In addition, XynR8 appeared to have a broader range of pH tolerance (pH3~11). To increase its value in industrial application and to study its structure-function relationship, site-directed mutagenesis was conducted. Mutant XynR8(N41D) and XynR8(N58D) showed a better pH stability than the wild-type enzyme, whereas mutants XynR8(N85D), XynR8(I165K) and XynR8(I165R) showed no catalytic activities. Structure modeling by Deep view showed that the three-dimensional structure of XynR8 contained one additional α-helix than the general structure of family 11 xylanases. Whether this structural variation confers the high catalytic activity of XynR8 needs further investigation. In addition, N41 and N58 both were predicted to locate within two different loops the influence of these loop on the pH stability of XynR8 may be further explored.

目錄
中文摘要………………………………………………………………..Ⅰ
Abstract…………………………………...……………………………..Ⅱ
誌謝……………………………………………………………………..Ⅳ
目錄……………………………………………………………………..Ⅴ
圖表目錄……………………………………………………………… Ⅵ
第1章 前言……………………………………………...………………1
第2章 文獻回顧………………………………………………...………2
2.1聚木醣簡介…………………………………………….….……2
2.1.1聚木醣的結構(The structure of xylan)…………….……......2
2.1.2聚木醣酶的種類……………………..…………….………..6
2.2聚木醣酶(β-1,4-xylanase)之分類.………….……………..........7
2.3聚木醣酶的水解機制……………….…………………………10
2.4聚木醣酶之工業應用……………………………...…….…….12
2.4.1造紙工業之應用…………………..…………………..........12
2.4.2食品工業之應用………………………….………………...13
2.4.3飼料添加之應用……………………...………………….....13
2.5具特殊生化特性酵素之研究…………………….……………14
2.5.1耐熱性酵素………..………………..…………………........14
2.5.2耐酸鹼酵素……………………….………..…………….…16
2.6定位突變的技術………………...……………….……….........17
2.7研究目的與原理……………………………………………….18
第3章 材料與方法…………………………………………………….19
3.1實驗材料……………………………………………………….19
3.1.1菌株…………………………………………………………19
3.1.2載體……………………………….………………………..19
3.1.3用於定序之引子…………………….………………….…..19
3.1.4定位點突變(site-directed mutagenesis)之引子………........20
3.1.5電泳分子量標記( molecular weight markers )……..……...20
3.1.6抗體………………....…………………………….…….…..20
3.1.7大腸桿菌培養基…………………………………………....20
3.1.8緩衝溶液及試劑……………………..…………...……….21
3.1.8.1 DNA分析用試劑………………...………….………..21
3.1.8.2 5 X TBE buffer (用於DNA電泳)…….….……...……21
3.1.8.3 DNS試劑(用於木聚醣酶活性測試)……….………...22
3.1.8.4 蛋白質純化用試劑………..……..…………………....22
3.1.8.5 蛋白質電泳分析之膠體……………....…………..…..24
3.1.8.6 蛋白質電泳膠體固定染色液……………..…...….…..25
3.1.8.7 蛋白質電泳膠體脫色液………………….…………...25
3.1.8.8 薄層層析(thin-layer chromatography)展開液…..…....25
3.1.8.9 薄層層析呈色液……………………………...……....25
3.1.8.10 其它化學藥品分別是……………..………….………26
3.1.8.11 主要儀器及設備………………………….….….……26
3.2實驗方法……………………………………………………….27
3.2.1 xynR8之次選殖(subcloning)………………………………27
3.2.1.1xynR8-pGEX 5X-1質體之小量純化…………….……..27
3.2.1.2 xynR8-pGEX 5X-1質體之電泳分析……….……….…28
3.2.1.3 xynR8基因片段之純化.………………………………..28
3.2.2 質體(pET-21C)之製備…………………..…………...…....29
3.2.3 xynR8/Bam HⅠ/Not Ⅰ基因片斷與pET-21c/ Bam HⅠ/Not Ⅰ 片段之接合反應(ligation)….…………...…....29
3.2.4 E.coli BL21熱休克勝任細胞(compent cells for transformation by heat shock)之製備..................................30
3.2.5 XynR8-pET-21C質體轉型至E.coli BL21……………..…..30
3.2.6 XynR8以IPTG誘導表現之最適時間點測試………...…....30
3.2.7 以聚丙烯醯胺凝膠電泳( Sodium dodecyl sulfate polyacrylamide gel electrophoresis；SDS-PAGE)………..31
3.2.8 XynR8木聚醣酶表現位置之鑑定……………..………..…31
3.2.9 西方墨點法……………………………..…………….…...32
3.2.10 xynR8之定位突變………………………..…………….…32
3.2.11 野生型XynR8及各突變株之大量表達……………...…..33
3.2.12 以陽離子管柱(CM-Sepharose)純化XynR8......................33
3.2.13 以Ni-NTA管柱純化XynR8…………………...…….…...34
3.2.14 純化後XynR8之透析…..……………….....………….….34
3.2.15 XynR8活性染色分析(zymogram)………………..…...….35
3.2.16 XynR8酵素之最適反應溫度測試…..................................35
3.2.17 XynR8酵素之最適反應pH測試…………………....…….35
3.2.18 XynR8酵素之熱穩定性測試…..........................…………35
3.2.19 XynR8酵素之pH穩定性測試………………………….…36
3.2.20 XynR8酵素之受質專一性分析……………..……………36
3.2.21 XynR8酵素之水解產物分析…………………..…………37
3.2.22 XynR8之酵素動力學分析…..………………..………….37
第4章 結果…………………………………………………………….38
4.1選殖xynR8基因於pET21c載體並轉殖於E. coli BL21 (DE3)……………………………………………………….…38
4.2 XynR8聚木醣酶表達之最適時間點測試及表現位置之 鑑定…………………………………………………...………38
4.3 XynR8之定位突變………………..….……………………...39
4.4 XynR8之大量表達與純化…………………...………………40
4.5 XynR8活性染色分析………………………………...………41
4.6 XynR8酵素之最適反應溫度………………………………..41
4.7 XynR8酵素之最適反應pH試驗……………………………42
4.8 XynR8酵素之熱穩定性試驗………………………...……...42
4.9 XynR8酵素之pH穩定性試驗…………………………...…...42
4.10 XynR8酵素之受質專一性分析…………………….………43
4.11 XynR8酵素之水解產物分析……………………...……….43
4.12酵素之動力學分析…………………………….…………...44
第5章討論…………………………………………………………...…71
第6章 結論…………………………………………………………….74
參考文獻…………………………………………..……………………75
附錄……………………………………………………………………..82
作者簡介………………………………………………………………..84






圖表目錄
圖1. 聚木醣的組成結構……………………….…..…………………3
圖2. 硬木聚木醣之結構……………………………………...………4
圖3. 軟木聚木醣之結構……………………………………………...5
圖4. 各endo-1,4-β-xylanase家族之立體結構………………………9
圖5. 聚木醣酶催化聚木醣水解之機制圖………………………….11
圖6. PVX耐熱性聚木醣酶酵素之立體結構圖…...……………….15
圖7. 野生型XynR8之表達量隨IPTG誘導時間之不同而變化…45
圖8. XynR8表現位置之鑑定…………………………………..…..46
圖9. XynR8與PVX胺基酸序列比對…………………….……….47
圖10. XynR8與BCX胺基酸序列比對……………………………..48
圖11. 野生型XynR8與突變株N85D，I165R及I165K以IPTG 誘導後產物之比較………………………………………...….49
圖12. 一般family 11之聚木醣酶立體結構圖………………..…….50
圖13. 野生型XynR8以Deep View軟體分析之立體結構………...50
圖14. XynR8以phosphate buffer pH 7.8經CM-sepharose管柱純 化之結果…………..…………………………….…………….51

圖15. XynR8以phosphate buffer pH 7.8經Ni-NTA管柱純化之 結果……………………………………………………………52
圖16. XynR8以50 mM citric acid buffer , pH6.5經CM- sepha- rose管柱層析的結果分析………………...………………….53
圖17. XynR8以50 mM citric acid buffer , pH6.5經Ni-NTA管柱 層析的結果…………..………………………………….…….54
圖18. XynR8經CM-sepharose管柱及Ni-NTA管柱純化後的產 物以SDS-PAGE分析…………...…..……..…………….…....55
圖19. 突變株N85D、I165R及I165K之活性染色分析……..….…57
圖20. 野生型XynR8之活性染色分析……………….……………..58
圖21. XynR8最適反應溫度分析…………….……….……………..59
圖22. XynR8之最適反應pH分析…………….……………………60
圖23. XynR8之熱穩定性試驗……………….……………………...61
圖24. XynR8之pH穩定性試驗……………….…………..………..62
圖25. XynR8之pH 11穩定性試驗…………..………………….…..63
圖26. XynR8酵素之水解產物分析………………….…………..….65
圖27. XynR8之動力學分析…………….………………………..….67
表1 endo-1,4-β-xylanase之醣苷水解酶家族...……………………...8
表2 於E.coli中表達來自不同微生物之xylanase基因…….……...18
表3 野生型XynR8的純化結果…………………………………….56
表4 XynR8受質專一性分析………….…………………………….64
表5 XynR8之酵素動力學分析參數..................................................68
表6 源自於細菌、真菌及酵母菌之聚木醣酶之生化特性….……..69
附圖1 pET21c(+)載體之圖譜………..………………………………82
附圖2 pET21c(+) multiple cloning site之序列………………………83

參考文獻
林順富(2001) 生物化學。偉明圖書有限公司，Harcourt Asia Pte Ltd. 合作出版，104-173。
蔡芷芸(2006) Streptomyces thermonitrificans NTU-88聚木醣醣酶之純化與生化特性探討，40-79。
Alberto, R. A., P. Sanchez, J. Calvete, M. Raida, J. M. F. Abalos, and R. Santamaria (1997) Analysis of xysA, a gene from Streptomyces halstedii JM8 That Encodes a 45-kilodalton modular xylanase, Xys1. American Society for Microbiology. 63:2983-2988.
Andrews, S. R., E. J. Taylor, G. Pell, F. Vincent, V. M. A. Ducros, G. J. Davies, J. H. Lakey, and H. J. Gilbert (2004) The use of forced protein evolution to investigate and improve stability of family 10 xylanases. The Journal of Bioidgical Chemistry. 279:54369-54379.
Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnol prog. 15:147-157.
Bastawde, K. B. (1992) Xylan structure, microbial xylanases, and their- mode of action. World J Microbiol Biotechnol. 8:353–368.
Bedford, M.R., and H. L. Classen (1992) The influence of dietary xylanase on intestinal viscosity and molecular weight distribution of carbohydrates in rye-fed broiler chick. pp 361-370. In: Visser J, Beldman G, vanSomeren MAK, Voragen AGJ (eds) Xylans and xylanases, Elsevier, Amsterdam.
Beg, Q. K., B. Bhushan, M. Kapoor, and G. S. Hoondal (2001) Microbial xylanases and their industrial applications: a review. Appl Microbial Biotechnol. 56:326-338.


Biely, P. (1985) Microbial xylanolytic systems. Trends Biotechnol Lett. 3:286-290.
Bourne, Y., and B. Henrissat (2001) Glycoside hydrolases and glycosyltransferases: families and functional modules. Current Opinion in Structural Biology.11:593-600.
Carmona, E. C., M. R. Brochetto-Braga, A. A. Pizzirani-Kleiner, and J. A. Jorge (1998) Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor. FEMS Microbiol Lett. 166:311-315.
Cheng, H.-L., L.-C. Tsai, S.-S. Lin, H.-S. Yuan, N.-S. Yang, S.-H. Lee, and L.-F. Shyur (2002)Mutagenesis of Trp54 and Trp203 residues on Fibrobacter succinogenes 1,3-1,4--D-glucanase significantly affects catalytic activities of the enzyme. Biochemistry 41: 8759-8766.
Christov, L. P., and B. A. Prior (1993) Esterases of xylan-degrading microorganisms: production, properties, and significance. Enzyme Microbiol. Technol. 15:460-475.
Cleemput, G., M. Hessing, M. Van Oort, M. Deconynck, and J. A. Delcour (1997) Purification and Characterization of a [beta]-D- Xylosidase and an endo-xylanase from Wheat Flour. Plant Physiol. 113:377-386.
Collins, T., C. Gerday, and G. Feller (2005) Xylanases, xylanase families and extremophilic xylanase. FEMS Microbiol Rev. 29:3-23.
Courtin, C.W., G. G. Gelders, and J. A. Delcour (2001) Use of two endoxylanases with diVerent substrate selectivity for understanding arabinoxylan functionality in wheat Xour breadmaking. Journal of Cereal Chemistry 78:564-571.
Fushinobu, S., K. Ito, M. Konno , T. Wakagi, and H. Matsuzawa (1998) Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Protein Eng. 11:1121-1128.
Gebler, J., N. R. Gilkes, M. Claeyssens, D. B. Wilson, P. Beguin, W. W. Wakarchuk, D. G. Kilburn, J. R. C. Miller, R. A. Warren, and S. G. Withers (1992) Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases. The Journal of Biological Chemistry. 267:12559-12561.
Gilkes, N. R., B. Henrissat, D. G. Kilburn, R. C. Miller, and R. A. Warren (1991) Domains in microbial beta-1,4-glycanases: sequence conservation, function, and enzyme families. Microbiological Reviews. 55:303-315.
Gomes, J., I. I. gomes, K. Terler, N. Gubala, G. Ditzelmuller, and W. Steiner (2000) Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus. Enzyme Microb Technol. 27:414-422.
Gruber, K., G. Klintschar, M. Hayn, A. Schlacher, W. Steiner, and C. Kratky (1998) Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies. Biochemistry. 37:13475-13485.
Hakulinen, N., O. Turunen, J. Janis, M. Leisola, and J. Rouvinen (2003) Three-dimensional structures of thermophilic beta-1,4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa. Comparison of twelve xylanases in relation to their thermal stability. European Journal of Biochemistry. 270: 1399-1412.
Hazlewood, G. P., and H. J. Gilbert (1993) Molecular biology of hemicellulases. P.103. In: Coughlan, M. P. and G. P. Hazlewood (eds), Hemicelluloses and Hemicellulases. Portland Press, London.
Henrissat, B., M. Claeyssens, P. Tomme, L. Lemesle, and J. P. Mornon (1989) Cellulase families revealed by hydrophobic cluster analysis. Gene. 81:83-95.
Henrissat, B., and P. M. Coutinho (2001) Classification of glycoside hydrolases and glycosyltransferases from hyperthermophiles. Methods Enzymol. 330:183-201.
Jeffries, T. W. (1996) Biochemistry and genetics of microbial xylanase. Current Opinion in Biotechnology. 7:337-342.
Jiang, Z., X. Li, S.Yang, L. Li, and S.Tan (2005) Improvement of the bread making quality of wheat Xour by the hyperthermophilic xylanase from Thermotoga maritima. Food Research International. 38:37-43.
Jimenez, L., C. Martinez, I. Perez, and F. Lopez (1997) Biobleaching procedures for pulp from agricultural residues using Phanerochaete chrysosporium and enzymes. Process Biochem. 32(4):297-304.
Joshi, M. D., G. Sidhu, I. Pot, G. D. Brayer, S. G. Withers, and L. P. McIntosh (2000) Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J. Mol. Biol. 299:255-279.
Kuhad, R. C., and A. Singh (1993) Lignocellulosic biotechnology: current and future prospects. Crit Rev Biotechnol. 13:151-172.
Kulkarni, N., A. Shendye, and M. Rao (1999) Molecular and biotech- nological aspects of xylanases. FEMS Microbiology reviews 23:411-456.
Kumar, P.R., S. Eswaramoorthy, P. J. Vithayathil, and M. A. Viswamitra (2000) The tertiary structure at 1.59 Å resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti bainier. Journal of Molecular Biology. 295:581-593.
Kunkel, T. A., (1985) Rapid and efficient site-specific mutagenesis without phenotype selection. Proceedings of the National Academy of Sciences of the United States of America. 82: 488-492.
La Grange, D. C., M. claeyssens, I. S. Pretorius, and W. H. Van Zyl (2000) Coexpression of the Bacillus Pumilus beta-xylosidase (xynB) gene with the Trichoderma resei beta xylanase 2(xyn2) gene in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 54:195-200.
Liu, J. R., Y. Bi, S. H. Lin, K. J. Cheng, and Y. C. Chen (2005) Direct cloning of a xylanase gene from the mixed genomic DNA of rumen fungi and its expression in intestinal Lactobacillus reuteri. Microbiology Letters. 251:233-241.
Lo Leggio, L., S. Kalogiannis, M. K. Bhat, and R. W. Pickersgill (1999) High resolution structure and sequence of T. aurantiacus xylanase I: implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-barrel architecture. Proteins. 36:295-306.
McCarter, J. D., and S. G.Withers (1994) Mechanisms of enzymatic glycoside hydrolysis. Current Opinion in Structural Biology. 4:885-892.
Messner, K., and E. Serbotnik (1994) Biopulping: An overview of developements in an environmentally safe paper making technolgy. FEMS Microbiology Reviews. 13:351-364.
Morales, P., A. Madrarro, A. Flors, J. M. Sendra, and J. A. P. Gonzalez (1995) Purification and characterization of a xylanase and arabinofuranosidase from Bacillus polymyxa. Enzyme Microb Technol. 17:424-429.
Niehaus, F., C. Bertoldo, M. Kahler, and G. Antranikian (1999) Extremophiles as a source of novel enzymes for industrial applications. Appl Microbiol Biotechnol. 51:711-729.
Polizeli, M. L., A.C.S. Rizzatti, R. Monti, H. F. Terenzi, J. A. Jorge, and D. S. Amorim (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol. 67:577-591.
Prade, R.A. (1995) Xylanases: from biology to biotechnology. Biotech- nology and genetic engineering Reviews. 13:100-131.
Querol, E., J. A. Perez-Pons, and A. Mozo-Villarias (1996) Analysis of protein conformational characteristics related to thermostability. Protein Eng. 9: 265-271.
Rye, C. S., and S. G. Withers (2000) Glycosidase mechanisms. Current Opinion Chemistry Biology. 4:573-580.
Shah, A. R., R. K. Shah, and Madamwar, D. (2005) Improvement of the quality of whole wheat bread by supplementation of xylanase from Aspergillus foetidus. Bioresource Technology.
Techapun, C., N. Poosaran, M. Watanabe, and K. Sasaki (2003) Thermostable and alkaline-tolerant microbial cellulase-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: a review. Process Biochem. 38:1327-1340.
Torronen, A., and J. Rouvinen (1997) Structural and functional properties of low molecular weight endo-1,4-β-xylanases. Journal of Biotechnology. 57:137-149.
Vanparidon P. A., Booman JCP, Selten GCM, Geerse C, Barug D, deBot PHM, Hemke G (1992) Application of fungal endoxylanase in poultry diets. pp 371-378. In: Visser J, Beldman G, vanSomeran MAK, Voragen, AGJ (eds) Xylans and xylanases. Elsevier, Amsterdam.
Vogt, G., S. Woell, and P. Argos (1997) Protein thermal stability, hydrogen bonds and ion pairs. Journal of Molecular Biology. 269:631-643.
Wong, K. K. Y., and J. N. Saddler(1993) Multiplicity of 1,4-xylanase in microorganisms, functions and applications. Microbiol Rev. 52 :305-317.
Wong, K.K.Y., L.U.L. Tan, and J.N. Saddler (1988) Multiplicity of beta-1,4-xylanases in microorganisms: functions and applications. Microbiological Reviews. 52:305-317.
Wong, K. K. Y., and J. N. Saddler (1992a) Applications of hemicellulases in the food, feed, and pulp and paper industries. pp. 127-143. In: Hemicellulose and Hemicellulases (Coughlen, P.P. and Hazlewood, G.P., Eds.) Portland Press, London.
Wong, K. K. Y., and J. N. Saddler (1992b) Trichoderma xylanase, their properties and purification. Crit Rev Biotechnol. 12:413-435.
Zechel, D. L., and S. G.Withers (2000) Glycosidase mechanisms: anatomy of a finely tuned catalyst. Accounts of Chemical Research. 33:11-18.