請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23845完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃慶璨 | |
| dc.contributor.author | Chi-Jui Lee | en |
| dc.contributor.author | 李啟睿 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:11:40Z | - |
| dc.date.copyright | 2006-08-10 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
| dc.identifier.citation | 1. Biely, P. (1985). Microbial xylanolytic systems. Trends. Biotechnol. 3, 286.
2. Cereghino, J. L. & Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24, 45-66. 3. Chen, C.-C., Wu, P.-H., Huang, C.-T. et al. (2004). A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase. Enzyme Microb Tech 35, 315-320. 4. Couderc, R. & Baratti, J. (1980). Oxidation of methanol by the yeast Pichia pastoris: purification and properties of alcohol oxidase. Agric Biol Chem 44, 2279-2289. 5. Cregg, J. M., Madden, K. R., Barringer, K. J. et al. (1989). Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9, 1316-1323. 6. Damaso, M. C., Almeida, M. S., Kurtenbach, E. et al. (2003). Optimized expression of a thermostable xylanase from Thermomyces lanuginosus in Pichia pastoris. Appl Environ Microbiol 69, 6064-6072. 7. Durand, R., Rascle, C. & Fevre, M. (1996). Molecular characterization of xyn3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis. Curr Genet 30, 531-540. 8. Durand, R., Rascle, C. & Fevre, M. (1999). Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti. Appl Microbiol Biotechnol 52, 208-214. 9. Faber, N. K., Haima, P., Harder, W. et al. (1994). Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet 25, 305-310. 10. Gellissen, G. (2000). Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54, 741-750. 11. Gellissen, G. & Hollenberg, C. P. (1997). Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis -- a review. Gene 190, 87-97. 12. Gellissen, G., Hollenberg, C. P. & Janowicz, Z. A. (1994). Gene expression in methylotrophic yeasts. In Gene expression in recombinant microorganisms. (Smith, A., Ed), pp. 195-240. Marcel Dekker, Inc., New York. 13. Gellissen, G., Janowicz, Z. A., Weydemann, U. et al. (1992). High-level expression of foreign genes in Hansenula polymorpha. Biotechnol Adv 10, 179-189. 14. Gellissen, G., Kunze, G., Gaillardin, C. et al. (2005). New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - A comparison. FEMS Yeast Res 5, 1079-1096. 15. Georis, J., Giannotta, F., Lamotte-Brasseur, J. et al. (1999). Sequence, overproduction and purification of the family 11 endo-beta-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38. Gene 237, 123-133. 16. Gilbert, H. J., Hazlewood, G. P., Laurie, J. I. et al. (1992). Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin. Mol Microbiol 6, 2065-2072. 17. Guo, M., Lu, F., Du, L. et al. (2005). Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica. Appl Microbiol Biotechnol 1-5. 18. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95-98. 19. Harford, N., Cabezon, T., Colau, B. et al. (1987). Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen. Postgrad Med J 63 Suppl 2, 65-70. 20. Hiep, T. T., Kulikov, V. N., Noskov, V. N. et al. (1993). The 5-aminoimidazole ribonucleotide-carboxylase structural gene of the methylotrophic yeast Pichia methanolica: cloning, sequencing and homology analysis. Yeast 9, 1251-1258. 21. Hiep, T. T., Noskov, V. N. & Pavlov, Y. I. (1993). Transformation in the methylotrophic yeast Pichia methanolica utilizing homologous ADE1 and heterologous Saccharomyces cerevisiae ADE2 and LEU2 genes as genetic markers. Yeast 9, 1189-1197. 22. Huang, Y. H. (2005). Cloning and expression of anaerobic fungus Neocallimastix frontalis xylanase genes and characterization of the recombinant proteins. National Taiwan University, Taipei. 23. Huang, Y. H., Huang, C. T. & Hseu, R. S. (2005). Effects of dockerin domains on Neocallimastix frontalis xylanases. FEMS Microbiol Lett 243, 455-460. 24. Jenzelewski, V. (2002). Fermentation and primary product recovery. In Hansenula polymorpha: Biology and Applications. (Gellissen, Ed), pp. 156-174. Wiley-VCH Verlag GmbH, Weinheim. 25. Kulkarni, P. S., Raut, S. K., Patki, P. S. et al. (2006). Immunogenicity of a new, low-cost recombinant hepatitis B vaccine derived from Hansenula polymorpha in adults. Vaccine 24, 3457-3460. 26. Kurtzman, C. P. (1984). Synonomy of the yeast genera Hansenula and Pichia demonstrated through comparisons of deoxyribonucleic acid relatedness. Antonie Van Leeuwenhoek 50, 209-217. 27. Ledeboer, A. M., Edens, L., Maat, J. et al. (1985). Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13, 3063-3082. 28. Lee, J. D. & Komagata, K. (1980). Taxonomic study of methanol-assimilating yeasts. J. Gen. Appl. Microbiol. 26, 133-158. 29. Macauley-Patrick, S., Fazenda, M. L., McNeil, B. et al. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249-270. 30. Mayer, A. F., Hellmuth, K., Schlieker, H. et al. (1999). An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnol Bioeng 63, 373-381. 31. Melmer, G. (2005). Biopharmaceuticals and the industrial environment. In Production of Recombinant Proteins -Novel microbial and eukaryotic expression systems. (Gellissen, G., Ed), pp. 361-383. Wiley-VCH, Weinheim. 32. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426-428. 33. Muller, S., Sandal, T., Kamp-Hansen, P. et al. (1998). Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 14, 1267-1283. 34. Raschke, W. C., Neiditch, B. R., Hendricks, M. et al. (1996). Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris. Gene 177, 163-167. 35. Raymond, C. K., Bukowski, T., Holderman, S. D. et al. (1998). Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase. Yeast 14, 11-23. 36. Rodriguez, L., Narciandi, R. E., Roca, H. et al. (1996). Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris. Yeast 12, 815-822. 37. Romanos, M. A., Scorer, C. A. & Clare, J. J. (1992). Foreign gene expression in yeast: a review. Yeast 8, 423-488. 38. Sakai, Y. & Tani, Y. (1992). Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2. Gene 114, 67-73. 39. Smith, A. (1994). Gene expression in recombinant microorganisms. Marcel Dekker, Inc., New York. 40. Sohn, J. H., Choi, E. S., Kang, H. A. et al. (1999). A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1. Appl Microbiol Biotechnol 51, 800-807. 41. Song, H., Li, Y., Fang, W. et al. (2003). Development of a set of expression vectors in Hansenula polymorpha. Biotechnol Lett 25, 1999-2006. 42. Subramaniyan, S. & Prema, P. (2002). Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22, 33-64. 43. Tsai, C. T. (2003). Comparison of the Piromyces sp. T2 xylanase production by methylotrophic yeast Pichia pastoris and Pichia methanolica. National Taiwan University, Taipei. 44. van Dijk, R., Faber, K. N., Kiel, J. A. et al. (2000). The methylotrophic yeast Hansenula polymorpha: a versatile cell factory. Enzyme Microb Technol 26, 793-800. 45. Wang, H., Lu, F., Sun, Y. et al. (2004). Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica. Biotechnol Lett 26, 1569-1573. 46. Wegner, G. H. (1990). Emerging applications of the methylotrophic yeasts. FEMS Microbiol Rev 7, 279-283. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23845 | - |
| dc.description.abstract | 嗜甲醇酵母菌係指一群可以甲醇為唯一碳源生長的酵母菌，包括Candida，Hansenula，Pichia及Torulopsis等菌屬，目前主要以H. polymorpha、P. pastoris和P. methanolica廣泛地應用於各種蛋白質的大量生產，如工業酵素及醫藥用蛋白質等。然而，由於菌種特性使然，P. pastoris以甲醇誘導前常須先置換培養基才能提高蛋白質產量，而P. methanolica雖然不須置換培養基，但所生產的胞外蛋白質因過於複雜而不利於純化回收。本研究旨在利用另一種嗜甲醇酵母菌，H. polymorpha之野生型菌株及P. pastoris之商品化載體經置換啟動子後，搭配抗生素耐受度之篩選策略以建立操作方便及篩選快速的H. poymorpha表現系統，並以厭氣真菌Neocallimastix frontalis之木聚醣酶為目標蛋白質，藉以評估此系統在表現異源蛋白質時，能否排除上述其他酵母菌系統所遭遇的問題。結果顯示，帶有木聚醣酶基因的轉形株不僅可直接添加甲醇誘導表現，其胞外蛋白質亦以木聚醣酶為主，其中以MOX為啟動子者，於搖瓶及醱酵槽培養之木聚醣酶最高活性分別為615.37±45.56及2236.10±8.43 U/ml；而以FMD為啟動子之表現活性則為170.32±16.51及447.61±18.31 U/ml。 | zh_TW |
| dc.description.abstract | Among yeasts, the genera of Candida, Hansenula, Pichia and Torulopsis are capable of growing on methanol as the sole carbon source, hence are named as methylotrophic yeasts. Many applications of H. polymorpha, P. pastoris and P. methanolica to produce recombinant proteins such as industrial enzymes and therapeutic proteins have been described. However, the replacement of medium to enhance expression level before methanol induction and the complicated extracellular proteins have limited the extensive applications of P. pastoris and P. methanolica, respectively. In this study, the wild type strain and the commercial vectors replaced by two promoters, accompanied by a selection strategy of antibiotic tolerance were conducted to develop an easily and rapidly manipulated H. polymorpha expression system. Moreover, the target protein, Neocallimastix frontalis xylanase, was exploited to evaluate that this system overcomes the problems described above or not. As the results, recombinant xylanase was released after methanol induction without replacement of medium and occupied the vast majority of extracellular proteins. The highest expression levels of xylanase in flask cultivation and fermentation were 615.37±45.56 and 2236.10±8.43 U/ml of MOXZα-xyn11B’ 1B2-17; 170.32±16.51 and 447.61±18.31 U/ml of FMDZα- xyn11B’ 1B15, respectively. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:11:40Z (GMT). No. of bitstreams: 1 ntu-95-R93b47411-1.pdf: 2100738 bytes, checksum: ea25542a70f8dbca542f2b335727b8a9 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | FIGURE CONTENTS III
TABLE CONTENTS IV 摘要 V ABSTRACT VI 1. INTRODUCTION 1 1.1. Gene expression system 1 1.2. Expression systems based on methylotrophic yeasts 3 1.2.1 Pichia pastoris 4 1.2.2 Pichia methanolica 5 1.3. Hansenula polymorpha as the versatile cell factory 7 1.4. Neocallimastix frontalis xylanase Xyn11B’ 9 1.5. Aim of this study 11 2. MATERIALS AND METHODS 13 2.1 Strains and cultivation 13 2.1.1 Hansenula polymorpha NCYC495 13 2.1.2 Escherichia coli Top 10 13 2.2 Fermentation 14 2.2.1 Batch fermentation 14 2.2.2 High cell density fermentation 14 2.3 Vector construction 15 2.3.1 Cloning of MOX and FMD promoters 15 2.3.2 Construction of pMOXZα-xyn11B’ and pFMDZα-xyn11B’ 17 2.4 Transformation procedures of H. polymorpha 19 2.4.1 Preparation of H. polymorpha competent cells 19 2.4.2 Electroporation procedure 19 2.5 Selection of H. polymorpha transformants 20 2.6 Enzyme assay 20 2.7 Western blot 21 2.8 Southern hybridization 22 3. RESULTS AND DISCUSSION 23 3.1 Cloning of MOX and FMD promoters in H. polymorpha 23 3.2 Construction of pMOXZα-xyn11B’ and pFMDZα-xyn11B’ 27 3.3 Selection of H. polymorpha transformants 29 3.3.1. Zeocin tolerance plate assay 29 3.3.2. Xylanase activity assay in tube cultures 31 3.3.3. Analysis of transformants by Southern hybridization 33 3.4 Expression of recombinant Xyn11B’ in flasks 35 3.4.1. Xylanase activity induced by methanol 36 3.4.2. SDS-PAGE and western blot 39 3.4.3. Xylanase activity induced by different induction modes 42 3.5 Expression of recombinant Xyn11B’ in fermentors 44 3.5.1. Batch fermentation 44 3.5.2. High cell density fermentation 47 4. CONCLUSIONS 51 5. FUTURE WORKS 52 REFERENCES 53 APPENDIX 58 1. Recipe of fermentation basal salts medium in this study 58 Figure contents Figure 1. Methanol utilization pathway in methylotrophic yeasts. 6 Figure 2. The schematic framework of this study. 12 Figure 3. The expression vectors used in this study. 18 Figure 4. Agarose gel electrophoresis of gradient PCR with MOXp and FMDp primer sets. 24 Figure 5. Alignment of two MOXp clones, MOX-7 and MOX-9, with AY550079 (1~1508 bp), MOX. 25 Figure 6. Alignment of two FMDp clones, FMD-1 and FMD-2, with AY550077 (1~624 bp), FMD. 26 Figure 7. Agarose gel electrophoresis of gradient PCR with Xyn-F and Xyn-R primers. 28 Figure 8. Zeocin tolerance plate assay of H. polymorpha transformed with pFMDZα-xyn11B’ (partial transformants). 30 Figure 9. Xylanase activity in tube cultivation. 32 Figure 10. Southern analysis of transformants genomic DNA. 34 Figure 11. Xylanase activity induced by methanol in flasks. 38 Figure 12. SDS-PAGE analysis of recombinant Xyn11B’. 40 Figure 13. Western blot analysis of recombinant Xyn11B’. 41 Figure 14. Xylanase activity induced by different induction modes. 43 Figure 15. Recombinant Xyn11B’ production in batch fermentation. 46 Figure 16. Recombinant Xyn11B’ production in high cell density fermentation. 48 Figure 17. Effects of cell density on xylanase production in fermentation. 50 Table contents Table 1. Primers and PCR conditions in this study. 16 Table 2. Xylanase production in flasks 35 Table 3. Xylanase production in fermentors 49 | |
| dc.language.iso | en | |
| dc.title | 利用漢遜酵母MOX及FMD啟動子異源表現瘤胃真菌Neocallimastix frontalis之木聚醣酶 | zh_TW |
| dc.title | Heterologous expression of Neocallimastix frontalis xylanase in Hansenula polymorpha by MOX and FMD promoters | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊盛行,陳浩仁,陳又嘉,許瑞祥 | |
| dc.subject.keyword | 漢遜酵母,瘤胃真菌,木聚醣酶, | zh_TW |
| dc.subject.keyword | Hansenula polymorpha,Neocallimastix frontalis,xylanase,MOX,FMD, | en |
| dc.relation.page | 58 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-23 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-95-1.pdf 未授權公開取用 | 2.05 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。