產黃纖維單胞桿菌(Cellulomonas flavigena NTOU1)幾丁質酶C.f NTOU1 Chi63分子，已由本研究室從其染色體DNA選殖出基因全長，並於大腸桿菌Rosetta(DE3)pLysS的表現系統中，表現出具有幾丁質酶活性的重組酵素。但所表現的蛋白質量少，且以His-Tag親和性層析純化重組蛋白質效果不佳，尚未完成C.f NTOU1 Chi63之酵素動力學研究。
本研究室多年來以大腸桿菌表現系統表現微生物AcD1ChiAG561幾丁質酶，且用6xHis-Tag親和性層析純化此重組幾丁質酶，均極為成功。故以AcD1ChiAG561為借鏡，全面改寫Cellulomonas flavigena NTOU1 Chi63核苷酸序列，使其更適合大腸桿菌表現系統以取得高純度之C.f NTOU1 Chi63重組幾丁質酶，來完成其酵素動力學的研究。
以AcD1ChiAG561幾丁質酶基因進行改寫之C.f NTOU1 Chi63重組幾丁質酶基因(C.f NTOU1 Chi63 ver.1)，其所表現的重組蛋白質量仍然偏少，且以親和性層析純化重組蛋白質的效果不如預期。因此再以大腸桿菌使用之最適化之基因密碼來改寫C.f NTOU1 Chi63重組幾丁質酶基因序列(C.f NTOU1 Chi63 ver.2)，可惜仍未有良好的蛋白質表現與純化效果，來完成此酵素動力學的研究。

A full-length chitinase gene encoded in the chromosome DNA from Cellulomonas flavigena NTOU1 has been cloned and its recombinant enzyme has been expressed in E.coli Rosetta(DE3)pLysS with chitinase activity in this laboratory. The kinetic study of this enzyme, C.f NTOU1 Chi63, has not been completed due to the low level of protein expression and its poor His-Tag affinity protein purification.
However, a chitinase gene cloned from another bacteria, AcD1ChiAG561, has been successfully expressed and His-Tag affinity purified with a stable enzyme activity during the past several years in this laboratory.
A modified version of C.f NTOU1 Chi63 gene (C.f NTOU1 Chi63 ver.1) was artificially synthesized based on the codon usage in AcD1chiAG561 and was expressed in the same E.coli expression system. Unfortunately, both its protein expression level and His-Tag affinity protein purification did not have a significant progress. Therefore, a third approach using the E.coli optimized codon usages for C.f NTOU1 Chi63 was chosen (C.f NTOU1 Chi63 ver.2) for this improvement purpose. Again, this trial has not got a significant improvement on the protein expression level and His-Tag affinity protein purification efficiency. The recombinant C.f NTOU1 Chi63 ver.2 remained to keep its enzyme activity.

Keywords：Cellulomonas flavigena, Chitinase, Artificial Engineering

中文摘要.......................................................................................... I
Abstract........................................................................................... II

第一章 前言...................................................................................... 1
1.1 幾丁質及幾丁聚醣........................................................................ 1
1.2 幾丁質水解酵素............................................................................ 2
1.3 Cellulomonas flavigena NTOU 1...................................................... 5
1.4 研究背景與目的............................................................................ 7

第二章 實驗材料與方法.................................................................. 19
2.1 實驗材料及藥品............................................................................ 19
2.2 C. flavigena NTOU 1幾丁質酶人工改造基因................................... 28
2.3 Cellulomonas flavigena NTOU 1 重組幾丁質酶基因表現與純化....... 34

第三章 實驗結果.............................................................................. 49
3.1 C. flavigena NTOU 1 Chi63幾丁質酶的人工改造與其蛋白質表現..... 49
3.2 C. flavigena NTOU 1 Chi63 及其人工改造的基因蛋白質表現的誘導條件...................................................................................................
50
3.3 C. flavigena NTOU1 chi63人工改造幾丁質酶的純化........................ 51
第四章 討論.................................................................................... 74
4.1 C. flavigena NTOU 1 Chi63幾丁質酶的人工改造與其蛋白質表現..... 74
4.2 C. flavigena NTOU 1 Chi63 及其人工改造的基因蛋白質表現的誘導條件...................................................................................................
75
4.3 C. flavigena NTOU1 chi63人工改造幾丁質酶的純化........................ 76

第五章 結論與未來展望................................................................ 77

第六章 參考文獻............................................................................ 79

許美芳，1995。Cellulomonas flavigena NTOU 1降解幾丁質與蝦殼及幾丁質酶純化之研究。國立台灣海洋大學水產食品科學研究所碩士論文。基隆。

王三郎。1996。水產資源利用學。高立圖書有限公司。台北市。

江晃榮。2000。幾丁質生技產品-在醫療、食品及環保上之應用。財團法人生物技術開發中心。

林姵妏。2003。Cellulomonas flavigena NTOU 1 幾丁質酶基因之選殖。
國立台灣海洋大學生物科技研究所碩士論文。基隆。

沈芳伶。2005。Cellulomonas flavigena NTOU 1 幾丁質酶基因之選殖II。國立台灣海洋大學生物科技研究所碩士論文。基隆。

葉哲誠。2007。Cellulomonas flavigena NTOU 1 幾丁質酶基因之選殖III。
國立台灣海洋大學生物科技研究所碩士論文。基隆。

鄭蓓淋。2014。產黃纖維單胞桿菌幾丁質酶基因的選殖與生化特性。
國立台灣海洋大學生物科技研究所碩士論文。基隆。

陳宏年。2015。產黃纖維單胞桿菌幾丁質酶C.f NTOU1 Chi63的結構及功能與其衍生分子的關係。國立台灣海洋大學生物科技研究所碩士論文。基隆。

吳宗勳。2017。產黃纖維單胞桿菌幾丁質酶C.f NTOU1 Chi50重組蛋白質新碳端標記對其親和性層析純化的影響。國立台灣海洋大學生物科技研究所碩士論文。基隆。

Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72：248.

Chen, H.C., Ho, W.L., Moody, M.W., and Jiang, S.T. (1992) Modification of Cellulomonas flavigena NTOU 1 characteristics for the production of shrimp hydrolysates. Journal of food science：an official publication of the Institute of Food Technologists, 57：271-276.

Chernin, L.S., Fuente, L.D.L., Sobolev, V., Haran, S., Vorgias, C.E., Oppenheim, A.B., and Chet, I. (1997) Molecular cloning, structural analysis, and expression in Escherichia coil of chitinase gene from Enterobacter agglomerans. Appl. Environ. Microbiol., 63：834-839.


D’Aquila, R.T., L.J. Bechtel, J.A. Viteler, J.J. Eron, P. Gorczyca, and J.C. Kaplin (1991) Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic Acids Res. 19：3749.

Davies, G. and Herissat, B. (1995) Structure and mechanisms of glycosyl hydrolases. Struc., 3：853-859.

Dahiya, N., Tewari, R., and Hoondal, G.S. (2006). Biotechnological aspects of chitinolytic enzymes: a review. Applied Microbiology and Biotechnology 71, 773-782.

Frandberg, E., and Schnurer, J. (1998) Antifungal activity of chitinolytic bacterial isolated from airtight stored cereal grain. Can. J. Microbiol., 44：121-127.

Fujita, K., Shimomura, K., Yamamoto, K., Yamashita, T., and Suzuki, K. (2006). A chitinase structurally related to the glycoside hydrolase family 48 is indispensable for the hormonally induced diapause termination in a beetle. Biochemical and biophysical research communications 345, 502-507.

Gupta, R.G., Saxena, R.K., Chaturvedl, P., and Virdi, J.S. (1995) Chitinase production by Steptomyces viridificans: its potential in fungal cell wall lysis. J. App. Bacteriol., 78：378-383.

Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. J.Biochem.280：309-316.

Henrissat, B.and Bairoch, A. (1993).New families in the classification of glycosyl hydrolases, based on amino acid sequence similarities. J. Biochem.293：781-788.

Hsu, F.J. and Chen, H.C. (1987) A study on the processing of marine sauce. J. Fish. Soc. Taiwan, 14：90-103.

Jon D. R, Arthur F. M, Edward M. M, and Hart, P. J. (1998). Structural analysis shows five glycohydrolase families diverged from a common ancestor. J. Exp. Zool. 282:127-132.

Keddie, R.M. (1974) Cellulomonas in：Bergey’s Manual of Determinative Bacteriology. 8th ed. Buchanan, R.E., and Gibbons, N.E. eds. The Williams and Wilkins Company, Baltimore, MD., 629-631.

Kurita, K. (2001). Controlled functionalization of the polysaccharide chitin. Prog Polym Sci 26, 1921-1971.

Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

Mullis, K.B. (1991) The polymerase chain reaction in an anemic mode：How to avoid cold oligodeoxyribonuclear fusion. PCR Methods Appl., 1:1.

Perrakis, A., Tews, I., Wilson, K.S., and Vorgias, C.E. (1996b) Structural aspects on the catalytic mechanism of chitinases, hevamine, and chitobiases. “ Far away and yet so close ? ” In：Chitin Enzymology, vol. 2 (ed. By Muzzarelli, R.A.A.) , pp. 109-122. Atec Edizioni Press. Senigallia. Italy.

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In Molecular cloning. A laboratory manual. Cold Sping Harbor Laboratory Press, New York.

Sandford, P. A. (1989). Chitosan: commercial uses and potential applications. In: Chitin and Chitosan, Proceedings of the Fourth International Conference on Chitin and Chitosan (eds. by Skjak-Braek, G. et al.), pp.51-69. Elsevier, New York.

Sneath, P.H.A., Mair, N.S., Sharpe, M.E., and Holt, J.G. (1986) Cellulomonas in：Bergey’s Manual of Systematic Bacteriology. Edited by Stackebrandt, E. and Keddie, R.M. The Williams and Wilkins Company, USA. 2：1325-1329.

Synowiecki, J., and Al-Khateeb, N.A. (2003). Production, properties, and some new applications of chitin and its derivatives. Crit Rev Food Sci 43, 145-171.

Terwisscha van Scheltinga, A.C., Armand, S., Kalk, K.H., Isogai, A., Henrissat, B., and Dijkstra, B.W. (1995) Stereochemistry of chitin hydrolysis by a plant chitinase / lysozyme / and X-ray structure of a complex with allosamidin：Evidence for substract assisted catalysis. Biochem., 34：15619-15623.

Tomokazu Kawase, Akihiro Saito, Toshiya Sato, Ryo Kanai, Takeshi Fujii, Naoki Nikaidou, Kiyotaka Miyashita, and Takeshi Watanabe. (2004) Distribution and Phylogenetic Analysis of Family 19 Chitinases in Actinobacteria. Applied and Environmental Microbiology 70: 1135-1144.

Tronsmo A, Harman GE (1993). Detection and quantification of N-acetyl-beta-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal Biochem 208: 74–79.

Whelan, H.A. and Pembroke, J.T. (1989) The effect of protease on stability of cellulase and xylanase from Cellulomonas flavigena. Biotechnology Letters, 11：891-894.

Younes, I., Hajji, S., Frachet, V., Rinaudo, M., Jellouli, K., and Nasri, M. (2014). Chitin extraction from shrimp shell using enzymatic treatment. Antitumor, antioxidant and antimicrobial activities of chitosan. Int J Biol Macromol 69, 489-498.