臺灣博碩士論文加值系統

中文摘要
學 號：M9318019
論文題目：以Lactobacillus reuteri表現聚木醣酶基因xynR8
與其特性探討 總頁數：61
學校名稱：國立屏東科技大學 所別：生物科技研究所
畢業時間及摘要別：九十四學年度第二學期碩士學位論文摘要
研究生：林秀樺 指導教授：陳又嘉
論文摘要內容：
內切型聚木醣酶水解聚木醣後，能產生不同長度的短鏈木寡醣；這些木寡醣可作為益生素，能夠選擇性地促進益生菌的生長。若能將聚木醣酶基因選殖於益生菌中表達並添加於飼料中，可取代飼料中酵素的添加，使其水解產物供益生菌作為碳源。因此本研究將聚木醣酶xyn R8基因選殖於pNZ3004載體中並在益生菌-Lactobacillus reuteri Pg4中表達。重組酵素XYN R8以疏水性交互作用與離子交換層析法純化後，進行生化特性探討。結果顯示，基因重組的L. reuteri會有兩個性質具有聚木醣酶活性的產物分別是XYN R8-B與Xyn R8-A，以SDS-PAGE及活性染色分析發現其分子量分別為32kDa和34kDa，經LC-MS/MS分析顯示XYN R8-A為XYN R8未將訊號胜肽去除的產物；生化特性方面，除XYN R8-A的熱穩定性比XYN R8-B高外，其他特性均為相似，造成XYN R8-A的熱穩定性提高的原因，可能是XYN R8-A在N端訊號胜肽的疏水性殘基形成loop和β-strand，而使XNY R8-A結構更具穩定。
關鍵字：益生菌、益生素、聚木醣酶、木寡醣

Abstract
Student ID：M9318019
Title of Thesis：Experession and characterization of xylanase gene (xynR8) by Lactobacillus reuteri
Total Page：61
Name of Institute：National Pingtung University of Science Technoogy
Name of Department：Graduate Institute of Biotechnology
Graduate Date：June,2006 Degree Conferred：Master
Name of Student：Shiou-Hua Lin Adviser：Yo-Chia Chen
The Contents of Abstract in This Thesis：
Endoxylanases catalyze the reaction of xylan hydrolysis to produce short chain xylooligosaccharides(XOs) of various lengths, and these XOs can be used as prebiotics that are able to selectively stimulates the growth of probiotics. The cloned xylanase gene enables probiotics to express xylanase and hydrolyze xylan in the gut of livestocks without the addition of enzymes in the feed. These hydrolyzed products can be used by probiotics as a carbon source. In this study, a xylanase gene (xyn R8) was subcloned into the pNZ3004 vector and transformed into Lactobacillus reuteri Pg4. The expressed enzymes were purified by hydrophobic interaction chromatography and ion exchange chromatography for the determination of biochemical characteristics. There were two fractions, XYN R8-B and XYN R8-A, with xylanolytic activity were obtained after purification. According to the results of SDS-PAGE and zymogram, their molecular masses were estimated to be 34 and 32 kDa, respectively. XYN R8-A and XYN R8-B were further
identified by LC-MS/MS and revealed that XYN R8-A was XYN R8-B with uncutted signal peptide. Most of biochemical characteristics are similar between XYN R8-A and XYN R8-B except thermostability. XYN R8-A was able to tolorence higher temperature than XYN R8-B because the hydrophobic residus of signal peptide form extra loop　and　β- strand in N-terminal and stabilize the structure of XYN R8-A.
Key words：probiotic, prebiotic, xylanase, xylooligosaccharides

目錄
中文摘要 Ⅰ
英文摘要 Ⅱ
致謝 Ⅳ
目錄 Ⅴ
圖表目錄 Ⅷ
第一章前言 1
第二章文獻回顧 2
2.1 益生菌 2
2.1.1 益生菌之定義 2
2.1.2 益生菌與腸道菌相 2
2.2益生素 3
2.2.1 木寡醣的結構與特性 3
2.2.1 木寡醣的生理功能與應用 4
2.3 聚木醣與聚木醣酶 5
2.3.1 聚木醣的結構 5
2.3.2 聚木醣的降解 6
2.3.3 聚木醣酶的來源與種類 8
2.3.4 β-1,4-聚木醣酶分類 9
2.3.5 聚木醣酶的應用 13
2.4乳酸菌 15
2.4.1乳酸菌的醣類代謝 15
2.4.2以糖類誘導的表現系統 17
2.4.3 Lactobacillus reuteri 18
2.5研究目的 18
第三章材料與方法 19
3.1實驗架構 19
3.2聚木醣酶基因轉殖至L. reuteri Pg4中表現 20
3.2.1 實驗菌株與pNZR8建構 20
3.2.2 E. coli 培養 20
3.2.3 E. coli質體之抽取 20
3.2.4 L. reuteri Pg4勝任細胞製作 21
3.2.5 pNZR8轉形至L. reuteri Pg4表達 21
3.2.6 L. reuteri R8轉型株之篩選 21
3.2.7基因定序 22
3.2.8菌種保存 22
3.3. 聚木醣酶純化 22
3.3.1 L. reuteri R8培養 22
3.3.2 疏水性交互作用層析 22
3.3.3 離子交換層析 23
3.3.4 聚木醣酶活性測定 23
3.3.5 蛋白質測定 24
3.3.6 蛋白質鑑定 24
3.4. 聚木醣酶生化特性分析 24
3.4.1 聚木醣酶分子量分析 24
3.4.2 最適反應酸鹼值和酸鹼值穩定性分析 26
3.4.3 最適反應溫度與耐熱性分析 26
3.4.4 水解產物分析 27
3.4.5 聚木醣酶酵素動力學分析 27
第四章 結果與討論 29
4.1確認pNZ R8轉形至L. reuteri Pg4中與XYN R8生產 29
4.1.1 剛果紅染色確認 29
4.1.2 序列之確認 30
4.1.3 XYN R8生產 30
4.2 聚木醣酶純化 32
4.2.1疏水性交互作用層析 32
4.2.2離子交換層析 33
4.2.3聚木醣酶分子量分析 36
4.2.4 XYN R8-A與XYN R8-B 身分鑑定 38
4.2.4 XYN R8-A與XYN R8-B立體結構模擬 39
4.3 聚木醣酶生化特性分析 40
4.3.1水解產物分析 40
4.3.2聚木醣酶最適反應酸鹼值 42
4.3.3聚木醣酶酸鹼穩定性 44
4.3.4聚木醣酶最適反應溫度 46
4.3.5聚木醣酶熱穩定性 48
4.3.6聚木醣酶酵素動力學參數 50
第五章結論 53
參考文獻 54
作者簡介 61



























圖表目錄
表1. 不同微生物聚木醣酶之特性 10
表2. 水解聚木醣之酵素所屬醣苷水解家族的類別 11
表3. 聚木醣酶的應用 14
表4. XYN R8 聚木醣酶純化結果 35
表5. XYN R8聚木醣酶動力學參數 50
表6. XYN R8-A和XYN R8-B生化特性 52
圖1. 木寡醣的應用 5
圖2. 聚木醣的化學結構 7
圖3. 來自微生物的聚木醣酶可截切聚木醣結構中不同取代基的位置 8
圖4. 乳酸菌糖類發酵代謝途徑 16
圖5. 實驗架構 19
圖6. 含有pNZR8的L. reuteri Pg4轉形株分解聚木醣所產生的透明環 29
圖7. xynR8基因序列與推譯胺基酸序列 31
圖8. L. reuteri R8聚木醣酶疏水性交互作用層析 32
圖9. L. reuteri R8聚木醣酶離子交換作用層析 33
圖10. L. reuteri R8聚木醣酶純化後SDS-PAGE與活性染色電泳分析 37
圖11. XYN R8-A與XYN R8-B身分鑑定 38
圖12. XYN R8-A和XYN R8-B 立體結構模擬 39
圖13. 重組酵素XYN R8-A與XYN R8-B 水解產物分析 41
圖14. 重組酵素XYN R8-A和XYN R8-B最適反應酸鹼值 43
圖15. 重組酵素XYN R8-A與XYN R8-B酸鹼穩定性 45
圖16. 重組酵素XYN R8-A和XYN R8-B最適反應溫度 47
圖17. 重組酵素XYN R8-A和XYN R8-B聚木醣酶熱穩定性 49
圖18. XYN R8-A與XYN-R8 B之Lineweaver-Burk reciprocal double plot 51

參考文獻
許育如 (2001) 篩選腸道來源之乳酸桿菌為益生菌之探討。碩士論文。國立中興大學。
黃崇真 (2004) 腸道微生物與益生菌。食品工業月刊。36(3):4-15。
張珍田 (2001) 木寡醣之特性與生理功能。食品工業月刊。 33(6):11-18。
楊潔彬 (1996) 乳酸菌：生物學基礎及應用。第一版。北京市：中國輕工業。98-137。
Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondal (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol. 56:326–338.
Biely, P., M. Vrsanska, M. Tenkanen, and D. Kluepfel (1997) Endo-beta-1,4-xylanase families: differences in catalytic properties. J. Biotechnol. 57: 151–166.
Bray, M. R., and A. J. Clarke (1992) Action pattern of xylooligosaccharide hydrolysis by Schizophyllum commune xylanase A. Eur. J. Biochem. 204: 191–196.
Eleonora, C. C., M. R. Brochetto-Braga, A. A. Pizzirani-Kleniner, and J. A. Jorge (1998) Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor. FEMS Micro. Biol. Lett. 166:311-315.
Collins, T., C. Gerday, and G. Feller (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 1:3-23.
Christakopoulos, P., P. Katapodis, E. Kalogeris , D. Kekos, B. J. Macris,
H. Stamatis, and H. Skaltsa (2003) Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases. J. Biol. Macromol. 31:171-175.
Collins, T., M. A. Meuwis, I. Stals, M. Claeyssens., G. Feller, and C. Gerday (2002) A novel family 8 xylanase: functional and physico-chemical characterization. J. Biol. Chem. 277: 35133 -35139.
de Man, J. C., M. Rogosa, and M. E. Sharpe (1960) A medium for the cultivation of Lactobacilli. J. Appl. Bacteriol. 23:130-135.
de Vos, W. M. (1999) Gene expression systems for lactic acid bacteria. Curr. Opin. Microbiol. 2:289-295.
Decelle, B., A. Tsang, and R. K. Storms (2004) Cloning, functional expression and characterization of three Phanerochaete chrysosporium endo-1,4-beta-xylanases. Curr. Genet. 3:166-175.
Ebringerovfi, A., and Z. Hromfidkov (1997) The effect of ultrasound on the structure and properties of the watersoluble corn hull heteroxylan. Ultrason Sonochem. 4:305-309.
Frederick, M. M., C. Kiang, J. R. Frederick, and P. J. Reilly (1985) Purification and characterization of endo-xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points. Biotechnol Bioeng. 27:525–532.
Fuller, R. (1989) Probiotics in man and animals. J. Appl. Bacteriol. 66:365-378.
Fioramonti, J., V. Theodorou, and L. Bueno (2003) Probiotics: what are they? What are theireffects on gut physiology? Best Practice & Research Clinical Gastroenterology.17:711-724.
Gosalbes, M. J., J. A. Perez-Gonzalez, R. Gonzalez, and A. Navarro (1991) Two beta-glycanase genes are clustered in Bacillus polymyxa: molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)- (1,4)-glucanase. J. Bacteriol. 173:7705–7710.
Garrote, G., H. Dominguez, and J. C. Parajo (2002) Autohydrolysis of corncob: study of non-isothermal operation for xylooligosaccharide production. J. food Eng .52:211–218.
Gupta, N., R. M. Vohra, and G. S. Hoondal (1992) A thermostable extracellular xylanase from alkalophilic Bacillus sp. NG-27. Biotechnol Lett. 14:1045–1046.
Gomez de Segure, B., and M. Fevre (1993) Purification and characterization of two 1,4-beta-xylan endohydrolases from the rumen fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 11:3654-3660.
Gibson, G. R., and M. B. Roberfroid (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr. 125: 1401–1412.
Hespell, R. B., and T. R. Whitehead (1990) Physiology and genetics of xylan degradation by gastrointestinal tract bacteria. J. Dairy. Sci .73:3013-3022.
Hurlbert, J. C., and J. F. Preston (2001) Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi. J. Bacteriol. 6:2096-2100.
Huis in’t veld, J. H. J., R. Havenaar, and P. Matrau (1994) Establishing a specific basis for probiotic R&D. TIBTECH J. 12:6-8.
Ito, K., H. Ogasawara, T. Sugimoto, and T. Ishikawa (1992) Purification and properties of acid stable xylanases from Aspergillus kawachii. Biosci. Biotechnol Biochem. 56:547–550.
Isolauri, E., S. Salminen, and A. C. Ouwehand (2004) Probiotics. Best Practice & Research Clinical Gastroenterology. 18:299-313.
Jeffries, T. W. (1996) Biochemistry and genetics of microbial xylanases. Curr. Opin. Biotechnol. 7:337–342
Jaskari, J., P. Kontula, A. Siitonen, H. Jousimies-Somer, T. Mattila-Sandholm, and K. Poutanen (1998) Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains. Appl. Microbiol Biotechnol. 49:175-181.
Kleywegt, G. J., J. Y. Zou, C. Divne, G. J. Davies, I. Sinning, J. Stahlberg, T. Reinikainen, M. Srisodsuk, T. T. Teeri, and T. A. Jones (1997) The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 Å resolution, and a comparison with related enzymes. J. Mol. Biol. 272: 383–397.
Kulkarni, N., A. Shendye, and M. Rao (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev.4:411-456.
Kelly, C. T., M. R. O’Mahony, and W. M. Fogarty (1989) Extracellular xylanolytic enzymes of Paecilomyces varioti. Biotechnol Lett1. 1:885–890.
Khasin, A., I. Alchanati, and Y. Shoham (1993) Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl. Environ Microbiol. 59:1725–1730.
Khanna, S., and P. Gauri (1993) Regulation, purification and properties of xylanase from Cellulomonas fimi. Enzyme Microb. Technol. 15:990–995.
Kobayashi, T., M. Okazaki, S. Fujikawa, and K. Koga (1991) Effect of xylooligosaccharide on feces of men. Nipp. Nogei. Kai. 65:1651-1653.
Liu, J. R., B. Yu, 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. FEMS Microbiol Lett. 15:233-241.
Lin, J., L. Ndlovu, M. S. Singh, and B. Pillay (1999) Purification and biochemical characteristics of β-D-xylanase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. Biotechnol Appl. Biochem. 30:73–79.
Larson, S. B., J. Day, A. P. Barba de la Rosa, N. T. Keen, and A. McPherson (2003) First crystallographic structure of a xylanase from glycoside hydrolase family 5: implications for catalysis. Biochemistry. 42:8411–8422.
Lopez, C., A. Blanco, and F. I. J. Pastor (1998) Xylanase production by a new alkali-tolerant isolate of Bacillus. Biotechnol Lett. 20:243–246.
Matte, A., and C. W. Forsberg (1992) Purification characterization and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85. Appl. Environ Microbiol.1:157-168.
Manning, T. S., and G. R. Glenn (2004) Prebiotic. Best Practice & Research Clinical Gastroenterology. 18:287-298.
Melchiorsen, C. R., K. V. Jokumsen, J. Villadsen, H. Israelsen, and J. Arnau (2002) The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis. Appl. Microbiol Biotechnol. 58:338-344.
Neeta, K., C. Jyoti, and R. Mala(1995) Characterization of the recombinant xylanases in Escherichia coli from an alkaliphilic thermophilic Bacillus sp. NCIM 59. Enzyme Microb. Technol. 11:972-976.
Okazak, M., S. Fujikawa, and N. Mastsumoto (1990) Effect of xylooligosaccharide on the growth of Bifidobacteria. Bifidobacteria Microflora. 9:77-86.
Penttila, M., P. Lehtovaara, H. Nevalainen, R. Bhikhabhai, and J. Knowles (1986) Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene. 1692:253–263.
Parker, R. B. (1974) Probiotics, the other half of the antibiotic story. Anim. Nutr. Health. 29:4-8.
Peitsch, M. C. (1995) Protein modeling by E-mail. Bio/Technology. 13: 658-660.
Radionova, N. A., N. V. Dubovaya, E. V. Eneiskaya, L. I. Matrinovich., I. M. Gracheva, and A. M. Bezborodov (2000) Purification and characterization of endo-(14)-beta-xylanase from Geotrichum candidum 3C. Appl. Biochem. Microbiol. 36:460–465.
Shao, W., S. DeBlois, and J. Wiegel (1995) A high molecular weight, cell-associated xylanase isolated from exponentially growing Thermoanaerobacterium sp. strain JW/SL-YS485. Appl. Environ Microbiol. 61:937–94.
Siitonen, S., H. Vapaatalo, S. Salminen, A. Gordin, M. Saxelin, R. Wikberg, and A. Kirllola (1990) Effect of Lactobacillus GG yoghurt in prevention of antibiotic associated diarrhoea. Ann. Med. 22:57-59.
Tony, C., C. Gerday, and G. Feller (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 29:3–23.
Tenkanen, H., J. Puls, and K. Poutanen (1992) Two major xylanases of Trichoderma reesei. Enzyme Microb. Technol. 14:566–574.
Tateyama, I., K. Hashii, I. Johno, T. Iino, K. Hiraik, Y. Suwa, and Y. Kiso (2005) Effect of xylooligosaccharide intake on severe constipation in pregnant women. J. Nutr. Sci. Vitaminol. 51:445-448.
Talarico, T. L., I. A. Casas, T. C. Chung, and W. J Dobrogosz (1988) Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob Agents Chemother. 32:1854-1858.
van Rooijen, R. J., M. J. Gasson, and W. M. de Vos (1992) Characterization of the Lactococcus lactis lactose operon promoter : contribution of flanking sequences and LacR repressor to promoter activity. J. Bacteriol. 174:2273-2280.
van Petegem, F., T. Collins, M. A. Meuwis, C. Gerday, G. Feller, and J. van Beeumen (2002) Crystallization and preliminary X-ray analysis of a xylanase from the psychrophile Pseudoalteromonas haloplanktis. Acta Crystallogr. Biol. Crystallogr. 58: 1494–1496.
Vazquez, M. J., J. L. Alonso, H. Dominguez, and J. C. Parajo (2000) Xylooligosaccharides: manufacture and applications. Trends food Sci. Technol. 11:387–393.
Vrsanska, M., I. V. Gorbacheva, Z. Kratky, and P. Biely (1982) Reaction pathways of substrate degradation by an acidic endo- 1,4-beta-xylanase of Aspergillus niger. Biochim Biophys Acta. 704: 114–122.
Winterhalter, C., and W. Liebel (1995) Two extremely thermostablexylanases of the hyperthermophilic bacterium Thermotoga maritama MSB8. Appl. Environ Microbiol. 61:1810–1815.
Wicher, K. B., M. Abou-Hachem, S. Halldorsdottir, S. H. Thorbjarnadottir, G. Eggertsson, G. O. Hreggvidsson, E. N. Karlsson, and O. Holst (2001) Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus. Appl. Microbiol Biotechnol. 55:578-584.
Yang, H. M., B. Yao, H. Y. Luo, W. Z. Zhang, Y. R. Wang, T. Z. Yuan, Y. G. Bai, N. F. Wu, and Y. L. Fan (2005) Hydrophobic interaction between β-sheet B1 and B2 in xylanase XYNB influencing the enzyme thermostability. Chinese Journal of Biotechnology. 21:414-419.