臺灣博碩士論文加值系統

本研究利用含有質體pET29a之重組大腸桿菌E.coli BL21 以不同誘導條件過量表現xyn45基因以分泌內切型木聚糖水解酶 (endo-1,4-β -xylanase; Xyn45) 到胞外。實驗上利用不同培養基 (LB、PM medium)、誘導劑種類 (IPTG、Lactose)和濃度高低，在搖瓶中比較Xyn45在胞內及胞外的蛋白質量及活性，並以饋料高密度發酵進行大量表達。在培養基的比較上， Xyn45在PM medium分泌至胞外的活性比起LB高出1.31倍。在誘導劑的比較上，低濃度IPTG誘導產生的Xyn45活性為Lactose的5.69倍，在誘導蛋白活性上IPTG皆優於Lactose。在誘導濃度的比較上，胞外Xyn45於PM以低濃度0.05 mM IPTG誘導比起高濃度1 mM，活性提升了5.96倍。在胞內及胞外的Xyn45活性比較上，以高濃度IPTG誘導時，胞外的Xyn45活性與胞內活性相近；而以低濃度IPTG誘導時，胞外的Xyn45活性比胞內活性高出4.4倍。推測以低濃度緩和的方式誘導，可提升Xyn45摺疊及生成融合蛋白的正確性，進而提升活性。在搖瓶小量培養時，最佳誘導條件為：PM中0.05 mM IPTG誘導，可得胞外最佳活性8.2 ± 2.9 U/ml及81.7 ± 14.9 U/mg，而胞外Xyn45占總分泌蛋白質之20.8%。大量培養則進行兩組不同誘導濃度之高密度發酵：第一組在PM中以IPTG 0.05 mM/OD600 (final conc. = 1.77 mM) 誘導，可得胞外Xyn45活性為11.7 U/ml (胞外與胞內活性相近)，OD600測得值為47.8，胞外Xyn45占總分泌蛋白質之37.4 %；第二組在PM中以 IPTG 0.05 mM (final conc. ) 誘導，可得胞外Xyn45活性為18.4 U/ml (胞外比胞內活性高出4.38倍)OD600測得值為62.67，而胞外Xyn45占總分泌蛋白質之37 %。以鹼液萃取麥麩所得之半纖維素，其中arabinose與xylose的比值為0.52，所含的木醣及阿拉伯醣總合約為16~17%，由麥麩原料所能萃取出的xylan比率為90.2%。

Extracellular endo-1,4-β-xylanase encoded by a gene xyn45 from Bacillus halodurans was over-expressed in recombinant Escherichia coli BL21 (DE3) using plasmid pET29a as the vector. The influence of culture medium (LB or PM medium) and concentration of inducer (IPTG or lactose in different concentration levels) on the production of this extracellular endoxylanase was studied. Using low concentration of IPTG as the inducer, the total activity of endoxylanase was higher in PM medium than that in LB medium. As the cultivation was in PM medium, the use of IPTG led to 5.7 times of enzymatic activity compared with the use of lactose as the inducer. Also, the use of low concentration (0.05 mM) of IPTG resulted in much higher endoxylanase activity in extracellular medium than the use of high concentration (1 mM) of IPTG. In PM medium containing 0.05 mM IPTG for induction, an activity of 8.2 ± 2.9 U/ml in the medium with a specific activity of 81.7 ± 14.9 U/mg was obtained. Furthermore, about 20.8% of over-expressed endoxylanase was found to be extracellular protein. The low concentration of inducer also resulted in a higher ratio of extracellular to intracellular activities. The recombinant E. coli was also cultivated to high-cell-density in fed-batch fermentation in PM medium and IPTG was used to induce the synthesis of extracellular endoxylanase. For large scale fermentation, PM medium containing IPTG with 0.05 mM/OD600 (final concentration = 1.77 mM) was used. As results, activity of extracellular Xyn45 was 11.7 U/ml (similar to activity of intracellular), OD600 = 47.8, and the yield of Xyn45 in total extracellular proteins is 37.4%. In the second group, PM medium containing IPTG 0.05 mM (final concentration) was used, the activity of extracellular Xyn45 was 18.4 U/ml (4.38 times higher than intracellular), OD600 = 62.67, and the yield of Xyn45 in total extracellular proteins is 37%.

中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅹ
表目錄 XIII
第一章 緒論 1
1.1 大腸桿菌 1
1.2 以大腸桿菌表達外源蛋白 1
1.3 木聚醣水解酶 3
1.3.1 木聚醣水解酶的作用機制 3
1.3.2 木聚醣水解酶的生產 4
1.4 木質纖維素 7
1.4.1 木質素 8
1.4.2 纖維素 9
1.4.3 半纖維素 9
1.4.4 麥麩及其組成 10
1.5 異質木聚醣 11
1.5.1異質木聚醣的結構 11
1.5.2異質木聚醣的萃取 13
1.6 實驗目的 13
第二章 實驗藥品 14
2.1 菌種來源 15
2.2 重組菌培養及誘導 15
2.3 高壓破菌 15
2.4 蛋白質定量 16
2.5 活性測定 16
2.6 SDS-PAGE 16
2.7 高密度發酵 17
2.8 木聚醣的萃取 17
2.9 組成分析 18
2.10 Scanning Electron Microscope (SEM) 18
2.11 烷基木醣苷的生產 18
第三章 實驗儀器與設備 19
3.1 重組菌培養相關 19
3.2 高壓破菌 20
3.3 蛋白質定量 20
3.4 活性測定 20
3.5 SDS-PAGE 20
3.6 高密度發酵 20
3.7 鹼處理法萃取木聚醣 20
3.8 組成分析 21
3.9 Scanning Electron Microscope (SEM) 21
3.10 Thin layer chromatography (TLC) 21
第四章 實驗步驟與方法 22
4.1 實驗設計 22
4.2 重組菌於搖瓶培養及誘導 23
4.2.1 塗盤培養 23
4.2.2 前培養 23
4.2.3 放大50 ml 23
4.2.4 誘導培養 24
4.3 高壓破菌 27
4.4 蛋白質定量 29
4.5 活性測定 30
4.6 SDS-PAGE 33
4.7高密度發酵 36
4.8木聚醣的萃取 37
4.8.1 去除澱粉 37
4.8.2 加酸軟化纏繞結構 38
4.8.3鹼處理法 38
4.8.2 酒精沉降 38
4.9 組成分析 39
4.9.1 澱粉含量測定 42
4.10 Scanning Electron Microscope (SEM) 43
4.11 烷基木醣苷的生產 44
4.11.1 Thin Layer Chromatography (TLC) 44
第五章 實驗結果與討論 46
5.1 搖瓶培養誘導條件比較 46
5.1.1 生長曲線 46
5.1.2 以SDS-PAGE觀測蛋白質表現 48
5.1.3蛋白質定量 50
5.1.4 活性比較 51
5.2 高密度發酵 54
5.2.1 生長曲線 54
5.2.2 以SDS-PAGE觀測蛋白質表現 56
5.2.3蛋白質定量及活性比較 58
5.3 木聚醣的萃取 61
5.3.1 麥麩組成 61
5.3.2 萃取所得之木聚醣組成 62
5.3.3 以SEM觀察麥麩及萃木聚糖外觀 64
5.4烷基木醣苷 66
第六章 結論與建議 67
6.1 結論 67
6.2 建議 69
參考文獻 70
附錄A 搖瓶及高密度SDS-PAGE重複實驗 79
附錄B Xyn45序列資料 80
附錄C HPLC及DNS檢量線 81
附錄D 搖瓶培養各組活性整理表 85
附錄E TLC重複實驗 86

一、中文文獻

朱運平， 江正强， 李里特， 李道義, 沈颖桀，烷基木糖苷的酶法合成及其纯化，過程工程學報 (2004)第四卷，第六期，572-576。

陳文恆，郭家倫，黃文松，王嘉寶，纖維酒精技術之發展，農業生技產業季刊 (2007) ，第九期，62-69。

王萍，呂姍姍，鹼液提取小麥麩皮木聚糖的研究，食品研究與開發(2006)，7-10。

鄭仲賢，以蛋白質體學方法研究基因重組之大腸桿菌表現雙標誌融合蛋白質及提升蛋白質溶解度，國立中正大學化學工程研究所博士論文 (2010)。

李育錚，大量生產木聚醣水解酶重組蛋白以將廢棄農作物轉化為木寡醣，國立中正大學分子生物研究所碩士論文(2012)。

許紹彥，於大腸桿菌中利用σ32因子與目標蛋白質共表達來改善酵素蛋白質的可溶性及活性，國立中正大學化學工程研究所碩士論文(2012)。
李慧玲，鹼處理稻草桿之酵素水解與發酵，國立中正大學化學工程研究所碩士論文(2008)。

施沂佑，小麥麩皮中arabinoxylan的鹼萃取方法探討及其理化性質研究，國立台灣大學農業化學研究所碩士論文(2003)。

曾詩喬，神經鞘磷脂水解酶的表現、純化及定性，國立成功大學生物科技研究所碩士論文(2002)

二、英文文獻

Babu, K.R., Seaminathan, S., Marten, S., Khanna, N., Rinas, U., Production of interferon-_ in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins. Applied Microbiology and Biotechnology(2000) 53, 655–660.

Bacic, A., and Stone, B.A., Isolation and ultrastructure of aleurone cell components from wheat and barley. Aust J Plant Physiol (1981a) 8: 453-474.

Bacic, A., and Stone, B.A., Chemistry and organization of aleurone cell wall components from wheat and barley. Aust J Plant Physiol (1981b) 8: 475-495.

Balakrishnan, H., Srinivasan, M.C., Rele, M.V. Extracellular protease activities in relation to xylanase secretion in an alkalophilic Bacillus sp. Biotechnology Letters (1997).

Bataillon, M., Mathaly, P., Nunes Cardinali, A. P., Duchiron, F. Extraction and purification of arabinoxylan from destarched wheat bran in a pilot scale. Industrial Crops and Products (1998) 8: 37–43.

Beg, Q., Kapoor, M., Mahajan, L., and Hoondal, G. Microbial xylanases and their industrial applications: a review. Applied Microbiology and Biotechnology (2001) 56: 326-338.

Brillouet, J.M. and Joseleau, J.P. Investigationof the structure of a heteroxylan from the outer pericarp (beeswing bran) of wheat kemel. Carbohydrate Research (1987) 159: 109-126.

Choi, J. H., Jeong, K..J., Kim, S.C., Lee, S.Y., Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence. Applied Microbiology and Biotechnology (2000) 53: 640–645.

Cinthia A. A. S., Maria, P. F. L. and Gustavo, G. F., Biotransformation of Pequi and Guavira Fruit Wastes via Solid State Bioprocess Using Pleurotus Sajor-Caju. International Journal of Bioscience, Biochemistry and Bioinformatics (2013) Vol. 3, No. 2,

Drouet, P., Zhang, M. and Legoy, M. D., Enzymatic Synthesis of Alkyl p-D-Xylosides by Transxylosylation and Reverse Hydrolysis. Biotechnology and Bioengineering (1994) 43: p.p. 1075-1080.

DuPont, M.S. and Selvendran, P.R. Hemicellulosic polymers from the cell walls of beeswing wheat bran: Part 1, polymers solubilised by alksli at 2°. Carbohydrate Research (1987) 163: 99-113.

Fausch, H., Kundig, W., and Neukom, H. Ferulic acid as a component of a glycoprotein from wheat flour. Nature (1963) 199-287.

Gavit, P., Better, M., Production of antifungal recombinant peptides in Escherichia coli. Journal of Biotechnology(2000) 79, 127–136.

Giudicianni, P., Cardone, G., R. Ragucci, Cellulose, hemicellulose and lignin slow steam pyrolysis: Thermal decomposition of biomass components mixtures. Journal of Analytical and Applied Pyrolysis volume 100, March 2013, Pages 213–222

Gruppen, H., Hamer, R.J. and Voragen, A.G.J. Water unextractable cell wall material from wheat flour 2. Fractionation of alkali extractable polymers and comparison with water extractable arbinisexylans. J. Cereal Science (1992) 16: 53-67.

Hromádková, Z., Ebringerová, A. Structural features of a rye-bran arabinoxylan with a low degree of branching. Carbohydrate Research Volume 163, Issue 1, (1987), Pages 73-79

Iiyama, K., Lam, T.B.T., Stone, B.A., Covalent cross-links in the cell wall. Plant Physiol., 104 (1994), pp. 315-320
Kadi, N., Belloy, L., Chalier, P. and Crouzet, J. C. Enzymatic Synthesis of Aroma Compound Xylosides Using Transfer Reaction by Trichoderma longibrachiatum Xylanase. J. Agric. Food Chem. (2002), 50, 5552-5557.

Khandeparker, R.., and Numan, M.T. Bifunctional xylanases and their potential use in biotechnology. J Ind Microbiology & Biotechnology (2008) 35: 635-644.

Lee, S.Y., High cell density cultivation of Escherichia coli. Trends in Biotechnology (1996) 14, 98–105.

Li, Y., Chen, C.X., von Specht, B., and Hahn, H.P., Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli. Gene (2002) 25: 437–447.

Lin, Y. S., Tseng, M.J. and Lee, W.C., Production of xylooligosaccharides using immobilized endo-xylanase of Bacillus halodurans. Process Biochemistry 46: (2011) 2117–2121.

Liu, Y. C., Liao, L. C. and Wu, W. T., Cultivation of Recombinant Echerichia coli to Achive High Cell Density with High Level of Penicillin G Acylase Activity. Proc. Natl. Sci. ROC(B) (2000) 24:156-160.

MacDougall, A.J., Selvendran, R.R., Chemistry, architecture, and composition of dietary fibre from plant cell walls. Handbook of Dietary Fibre, Marcel Dekker, New York (2001) 281–319
Maes, C. and Delcour, J. A., Structural Charaterisation of Water-extractable and Water-unextractable Arabinoxylans in Wheat Bran. Journal of Cereal Science 35: (2002) 315-326.

Makrides, S.C., Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Review (1996) 60: 512–538.

Mamo, G., Hatti-Kual, R., and Mattiasson, B., A thermostable alkaline active endo-β-1,4-xylanase from Bacillus halodurans S7: purification and characterization. Enzyme Microb. Technol., (2006) 39: 1492-1498.

Mamo, G., Kasture, S., Faryar, R., Hashim, S., Hatti-Kaul, R., Surfactants from xylan: Production of n-octyl xylosides using a highly thermostable xylanase from Thermotoga neapolitana. Process Biochemistry (2010) 45: 700–705.

Manisseri, C., Gudipati, M. Bioactive xylo-oligosaccharides from wheat bran soluble polysaccharides. Food Science and Technology (2010) 43:421–430.

Maria, I.R., Aneli, M.B., Ana, F.D.V., Asae, S.E., XYLANASE PRODUCTION BY TRICHODERMA HARZIANUM RIFAI BY SOLID STATE FERMENTATION ON SUGARCANE BAGASSE. Brazilian Journal of Microbiology (2002) 33: 67-72

Miller, G.L., Blum, R., Glennon, W.E., and Burton, A.L., Measurement of carboxymethylcellulasr activity. Analytical Biochemistry (1960) 1: 127-132.

Murphy, J. D., McCarthy, K. Ethanol production from energy crops and wastes for use as a transport fuel in Ireland. Applied Energy (2005) 148-166

Nakamura, T., Toshima, K., and Matsumura, S. One-step synthesis of n-octyl-D-xylotrioside, xylobioside and xyloside from xylan and n-octanol using acetone powder of Aureobasidium pullulans in supercritical fluids. Biotechnology Letters (2000)22: 1183–1189,

Naveena, B. J., Altaf, Md., Bhadrayya, K.., Madhavendra, S. S., Gopal Reddy, Direct fermentation of starch to l(+) lactic acid in SSF byLactobacillus amylophilus GV6 using wheat bran as support and substrate: medium optimization using RSM. Process Biochemistry (2005) 40, Issue 2, Pages 681–690

Ng, A. and Waldron, K. W., Effect of Steaming on Cell Wall Chemistry of Potatoes (Solanum tuberosum Cv. Bintje) in Relation to Firmness. J. Agric. Food Chem. (1997) 45: 3411-3418.

Ninawe, S., Kapoor, M., and Kuhad, R.C. Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32. Bioresource Technology (2008) 99: 1252-1258.

Ochs, M., Muzard, M., Plantier-Royon, R., Estrined, R. and Remond, C., Enzymatic synthesis of alkyl b-D-xylosides and oligoxylosides from xylans and from hydrothermally pretreated wheat bran. Green Chem.(2011) 13, 2380-2388.

Qiu, J., Swartz, J.R., Georgiou, G.,. Expression of active human tissue-type plasminogen activator in Escherichia coli. Applied and Environmental Microbiology (1998) 64: 4891–4896.

Qian, Z. G., Xia, X. X., Choi, J. H. & Lee, S. Y. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnol. Bioeng (2008) 101: 587–601.

Rinb, S.G. and Selvendran, R.R. Isolation and analysis of cell wall material from beeswing wheat bran. (Triricum aestivum) (1980) 19: 1730-1732.

Rye, C.S., and Withers, S.G. Glycosidase mechanisms. Current Opinion in Chemical Biology (2000) 4: 573-580.

Saha B.C. Bothast RJ Enzymology of xylan degradation. In: Imam SH, Greene RV, Zaidi BR (eds) Biopolymers: utilizing natures advanced materials. American Chemical Society, Washington, D.C. (1999), pp 167-194.

Saha B.C. Hemicellulose bioconversion. J. Ind. Microbiol. Biotechol., 30 (2003), pp. 279-291

Schooneveld-Bergmans, M.E.F. van Dijk, Y.M., Beldman, G., Voragen, A.G.J. Wheat bran glucuronoarabinixylans. Journal of Cereal Science. (1999) 29: Pages 49–61.

Sivakesava, S., Xu, Z.N., Chen, Y.H., Hackett, J., Huang, R.C., Lam, E., Kam, T.L., Siu, K.L., Wong, R.S.C., Wong, W.K.R., Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli. Process Biochemisty (1999) 34, 893–900.

Tseng, M. J., Yap, M. N., Ratanakhanokchaic, K., Kyuc, K. L., Chen, S. T., Purification and characterization of two cellulase free xylanases from an alkaliphilic Bacillus firmus. Enzyme and Microbial Technology (2002) Volume 30, Issue 5, 2, Pages 590–595.

Vázquez, M.J, Alonso, J.L, Domı́nguez, H., and Parajó, J.C. Xylooligosaccharides: manufacture and applications. Trends in Food Science & Technology (2000) 11: 387-393.

Yim, S.C., Jeong, K.J., Chang, H.N., Lee, S.Y., 2001. High level secretory production of human G-CSF by fed-batch culture of recombinant Escherichia coli. Bioprocess and Biosystems Engineering 24: 249–254