請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5517
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 謝文陽 | |
dc.contributor.author | Shih-Shuan Cheng | en |
dc.contributor.author | 鄭世璿 | zh_TW |
dc.date.accessioned | 2021-05-15T18:01:23Z | - |
dc.date.available | 2014-09-03 | |
dc.date.available | 2021-05-15T18:01:23Z | - |
dc.date.copyright | 2014-09-03 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-21 | |
dc.identifier.citation | Altschul, S. F., W. Gish, W. Miller, E. W. Myers and D. J. Lipman (1990). 'Basic local alignment search tool.' Journal of molecular biology 215(3): 403-410
Balqis, Z. S. and A. Rosma (2011). 'Artocarpus integer leaf protease: Purification and characterisation.' Food chemistry 129(4): 1523. Bellamy, W. D. (1974). 'Biotechnology report: single cell proteins from cellulosic wastes.' Biotechnol bioeng 16(7): 869-880. Benson, D. A., M. Cavanaugh, K. Clark, I. Karsch-Mizrachi, D. J. Lipman, J. Ostell and E. W. Sayers (2013). 'GenBank.' Nucleic acids research 41(Database issue): D36-42. Bhat, M. K. (2000). 'Cellulases and related enzymes in biotechnology.' Biotechnol advance 18(5): 355-383. Cavaco, P. (1998). 'Mechanism of cellulase action in textile processes.' Carbohydrate polymer 37: 273-277. Cheng, H. R. and N. Jiang (2006). 'Extremely rapid extraction of DNA from bacteria and yeasts.' Biotechnology letters 28(1): 55-59. Douady, C. J. and C. L. Nesbo (2007). 'Reconstructing and interpreting evolutionary relationships.' Methods for general and molecular microbiology C. A. Reddy, T. J. Beveridge, J. A. Breznak et al., ASM Press. Efron, B. (1979). '1977 Rietz lecture - Bootstrap methods - Another look at the Jackknife.' Annals of statistics 7(1): 1-26. Fell, J. W., T. Boekhout, A. Fonseca, G. Scorzetti and A. Statzell-Tallman (2000). 'Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis.' International journal of systematic and evolutionary microbiology 50(pt3): 1351-1371. Hall, B. G. (2013). 'Building phylogenetic trees from molecular data with MEGA.' Molecular biology and evolution 30(5): 1229-1235. Jain, D., I. Pancha, S. K. Mishra, A. Shrivastav and S. Mishra (2012). 'Purification and characterization of haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: A potential additive for laundry detergents.' Bioresource technology 115: 228-236. Jukes, T. H. and C. R. Cantor (1969). 'Evolution of protein molecules.' Mammalian protein metabolism H. N. Munro and J. B. Allison, Academic Press. Laemmli, U. K. (1970). 'Cleavage of structural proteins during assembly of head of bacteriophage-T4.' Nature 227(5259): 680-685. Munn, C. B. (2011). Marine microbiology: ecology and applications (2 ed.: Garland Science). Page, R. D. M. and E. C. Holmes (1998). Molecular evolution: a phylogenetic approach Blackwell Science. Pearson, W. R. (2007). 'Characterization of bacterial genome sequences by similarity searching.' Methods for general and molecular microbiology. Ryu, D. D. Y. and M. Mandels (1980). 'Cellulase: biosynthesis and application.' Enzyme microbiology and technology 2: 92-102. Saitou, N. and M. Nei (1987). 'The neighbor-joining method: a new method for reconstructing phylogenetic trees.' Molecular biology and evolution 4(4): 406-425. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk (1985). 'Measurement of protein using bicinchoninic acid.' Analytical biochemistry 150(1): 76-85. Sun, T. T. (2004). 'Excessive trust in authorities and its influence on experimental design.' Nature reviews molecular cell Biology 5(7): 785-799. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar (2011). 'MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.' Molecular biology and evolution 28(10): 2731-2739. Tolan, J. S. and B. Foody (1999). 'Cellulase from submerged fermentation.' Advances in Biochemical Engineering: Biotechnology 65(41-67). Watson, J. D. (2008). Molecular biology of the gene 6th ed.:Pearson/Benjamin Cummings. Yan, B. Q., X. L. Chen, X. Y. Hou, H. L. He, B. C. Zhou and Y. Z. Zhang (2009). 'Molecular analysis of the gene encoding a cold-adapted halophilic subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp SM9913: Cloning, expression, characterization and function analysis of the C-terminal PPC domains.' Extremophiles 13(4): 725-733. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5517 | - |
dc.description.abstract | 本研究從新北市瑞芳的磅磅仔附近海水中分離出具有纖維素分解能力之細菌。經16S rRNA 定序後,建立分離株之親緣關係樹,結果顯示為弧菌屬 (Vibrio)。本研究對分離株所產生的纖維素分解酶進行分離、純化並探討該酵素的一般性質,以及評估工業上的應用。結果顯示:分離株使用250 mL具凹槽錐形瓶,添加0.5% CMC、pH 6、培養液50 mL,在30 °C 下,震盪培養72 小時後,纖維素分解酶的活性最佳。粗酵素液經由硫酸銨沉澱、透析、並以陰離子交換管柱層析 (DEAE-FF)分離出分子量約60 kDa、36 kDa 及 20 kDa三種纖維素分解酶。此纖維素分解酶在pH 6、50˚C時有最佳活性;在 pH 4~9 以及4~40 ˚C有良好的穩定性。本酵素受界面活性劑的影響,在1% SDS及 1% Tween 80 中,活性皆僅剩下約原本的25 % ;而1% 的 Tween 20使其活性降低約 55 %。另一方面,Ethylenediaminetetraacetic acid (EDTA) 和phenylmethanesulfonylfluride (PMSF) 等酵素抑制劑皆降低了本酵素活性 30 ~ 40 %,而2-Mercaptoethanol (β-ME) 幾乎不造成影響。在工業用途評估測試中,本酵素對微晶纖維素、石蓴、紙張和甘蔗渣皆具有分解能力,其中對微晶纖維素的分解能力最好,對石蓴和紙張次之,對甘蔗渣最差。 | zh_TW |
dc.description.abstract | In this study, an isolate from sea water of Rueifang, Xinbei, had been tested for its cellulase activity. After the 16S rRNA was sequenced to establish phylogenetic tree of this isolate, which belonged to the genus Vibrio. Cellulase of this isolate was purified and characterized. Optimal cellulase yielding conditions for the culture were determined. For the best cellulase production, the culture was incubated at 30 °C with 50 mL LB broth (pH 6) in 250 mL flask and shaken for 72 hours (75 rpm). CMC (1%) was added as an cellulase inducer in LB broth. The cellulase was purified by ion exchange chromatography after ammonium sulfate precipitation, dialysis, and desalting. The molecular mass of the cellulases determined by SDS-PAGE was approximately 60 kDa, 36 kDa and 20 kDa. The cellulase showed optimum activity at pH 6 and 50 °C, and was stable at pH 4-9 and 4-40°C. When the cellulase was treated with various surfactants, 70 % activity was inhibited by 1% (g/mL) SDS and 1% Tween 80 , and had a lost of 55 percent under the effect of Tween 20. The cellulase was inactivated by 5 mM phenylmethanesulfonylfluride (PMSF) and 5 mM ethylenediaminetetraacetic acid (EDTA), but was not influenced by 2-Mercaptoethanol. Finally, in the industrial application tests, the cellulase showed better ability to decompose microcrystalline cellulose than to Ulva and paper, but showed weaker ability to break down sugar cane bagasse than to any others substrates. | en |
dc.description.provenance | Made available in DSpace on 2021-05-15T18:01:23Z (GMT). No. of bitstreams: 1 ntu-103-R99241208-1.pdf: 2929395 bytes, checksum: df89fa4f01d9e30cab8fdf7d97331f80 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄
中文摘要............................................................................................... VI Abstract...................................................................................….VII 第一章前言..................................................................................................1 1.1 纖維素 ............................................................................................................1 1.2 纖維素分解酶 ................................................................................................2 1.3 纖維素分解酶的應用 ................................................................................... 3 1.4 研究動機 ....................................................................................................... 3 第二章材料和方法........................................................................................4 2.1 菌株取得 ........................................................................................................4 2.2 實驗材料與設備 .............................................................................................4 2.3 分離株 16S rRNA 基因序列的取得並與基因資料庫比較 ..........................6 2.4 纖維素分解酶的活性測試 .....................................................................11 2.5 纖維素分解酶之培養條件測試 .....................................................................12 2.6 細菌大量培養及誘導酵素產生 .....................................................................13 2.7 粗纖維素分解酶的製備 .................................................................................13 2.8 硫酸銨沈澱和透析 .........................................................................................13 2.9 蛋白質定量 .....................................................................................................14 2.10 纖維素分解酶的純化 ...................................................................................14 2.11蛋白酶的電泳分析 ........................................................................................15 2.12 酵素最適反應溫度 .......................................................................................16 2.13 酵素之熱穩定性 ...........................................................................................16 2.14 酵素最適反應pH ..........................................................................................16 2.15 酵素在不同 pH 下之穩定性 ......................................................................16 2.16 蛋白抑制劑對酵素活性的影響 ..................................................................17 2.17 界面活性劑對酵素活性的影響 ...................................................................17 2.18 纖維素分解酶之應用測試 ...........................................................................17 第三章結果..................................................................................................18 3.1 分離株的親緣關係鑑定 .................................................................................18 3.2 纖維素分解酶初步測試 .................................................................................18 3.3 最佳培養條件測試 .........................................................................................18 3.4 不同條件下分離株生長情形的比較 .............................................................19 3.5 纖維素分解酶的純化 ..................................................................................19 3.6 纖維素分解酶的分子量分析 .........................................................................20 3.7 纖維素分解酶的特性探討 .............................................................................21 3.8 纖維素分解酶的應用測試 .............................................................................22 第四章討論..................................................................................................23 4.1 分離株的鑑定 .................................................................................................23 4.2 剛果紅測試 .....................................................................................................23 4.3 分離株的生長速率及纖維素分解酶的活性 .................................................23 4.4 纖維素分解酶的純化 .....................................................................................24 4.5 纖維素分解酶的特性 .....................................................................................25 4.6 纖維素分解酶的應用測試 ....................................................................26 第五章結論..................................................................................................28 參考文獻.................................................................................................29 表目錄 表1、纖維素分解酶純化表.................................................................................32 圖目錄 圖1、分離株 131 的親緣關係樹.........................................................................33 圖2、剛果紅染色法....................................................................................34 圖3、分離株 131 與其他菌株的剛果紅染色比較圖.....................................35 圖4、DNS還原糖標準曲線................................................................36 圖5、蛋白質濃度標準曲線 .....................................................................37 圖6、分離株在不同溫度培養下的酵素活性 ...........................................38 圖7、分離株在不同 pH 培養下的酵素活性................................................39 圖8、分離株在不同誘導物濃度培養下的酵素活性.......................................40 圖9、分離株在不同體積培養下的酵素活性...................................................41 圖10、分離株在不同溫度培養下的生長情形.....................................42 圖11、分離株在不同 pH 培養下的生長情形......................................43 圖12、分離株在不同誘導物濃度培養下的生長情形...............................44 圖13、硫酸銨沈澱各分劃的纖維酶活性.................................................45 圖14、硫酸銨沈澱各分劃的蛋白質濃度..............................................................46 圖15、硫酸銨沈澱各分劃的蛋白質總量.................................................47 圖16、硫酸銨沈澱各分劃的纖維酶比..................................................48 圖17、陰離子交換層析之管柱層析圖....................................................49 圖18、SDS-PAGE 及活性染色.............................................................................50 圖19、纖維素分解酶在不同溫度之活性.............................................51 圖20、纖維素分解酶在不同溫度之熱穩定性...........................................52 圖21、纖維素分解酶在不同 pH 之活性..................................................53 圖22、纖維素分解酶在不同 pH 之穩定性..........................................................54 圖23、蛋白抑制劑對纖維酶的影響............................................................55 圖24、界面活性劑對纖維酶的影響............................................................56 圖25、纖維酶對甘蔗、微晶纖維素、石蓴和影印紙的分解能力.................57 附錄目錄 附錄1、纖維素的基本結構.......................................................................58 附錄2、纖維素的結晶型和非結晶型區域........................................................59 附錄3、三種不同類型的纖維素分解酶作用方式.............................................60 附錄4、分離株的 16s rRNA 核苷酸序列.........................................................61 附錄5、硫酸銨百分飽和濃度表.................................................................62 附錄6、最佳產酶培養條件測試...................................................................63 附錄7、培養基與培養液成分......................................................................64 | |
dc.language.iso | zh-TW | |
dc.title | 弧菌屬分離株所產纖維素分解酶之純化與特性探討 | zh_TW |
dc.title | Purification and characterization of cellulase
from a Vibrio isolate | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳卓昇,簡文達,李宗徽,陳彥霖 | |
dc.subject.keyword | 纖維素,纖維素分解?,弧菌屬,酵素抑制劑,界面活性劑,陰離子交換層析, | zh_TW |
dc.subject.keyword | cellulose,cellulase,Vibrio,enzyme inhibitors,surfactants,anionic exchange chromatography, | en |
dc.relation.page | 64 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2014-08-22 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 海洋研究所 | zh_TW |
顯示於系所單位: | 海洋研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf | 2.86 MB | Adobe PDF | 檢視/開啟 |
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。