α-澱粉酶家族擁有三項共同特徵：在α-糖甘鍵上作用、擁有包含催化端的花籃狀蛋白結構、基因上有4個高度保留區分別和形成催化端與穩定花籃狀蛋白構形有關。本實驗利用基因轉殖方法，嘗試轉殖α-澱粉酶基因片段，透過隨機水解細菌染色體、轉殖入宿主細胞，製做Exiguobacterium sp.的基因圖譜，並以活性染色方式篩選菌株。成功轉殖的片段再經過修飾後轉殖入表達載體，配和表達宿主經過1 mM IPTG誘導後，透過蛋白質膠體染色可看出酵素明顯活性增加。
    原菌酵素活性測試為48.82 U/mg，酵素最適pH為6，最適反應溫度為37℃。

The α-amylase family has three common features：Act on α-glycosidic bonds and hydrolyze the bond to α-anomeric oligosacchrides or forming α-1,4 or α-1,6 bond、they all have the (β/α)8 or TIM barrel protein structure
contain the catalytic site、all of them have four highly conserved regions in their primary sequence which contain the amino acids that form the catalytic site and TIM barrel.
    DNA library was made by using partial digestion to digest Exiguobacterium sp. chromosome.Then clone into host cell selected by activity staining. Modified the sequence and clone again into pET expression vector system. Add 1 mM IPTG for induce a large amount of protein. Primary supernatant of Exiguobacterium sp. was concentrate and test by DNSA. The special activity was 48.82 U/mg. While the condense supernatant has the optimal condition on pH 6 and 37℃.

封面內頁
口試委員審議通過簽名表
授權書
致謝
中文摘要
英文摘要
目錄
圖表目錄

第一章	緒論
第一節	前言	1
第二節	澱粉結構與特性	1
第三節	澱粉酶酵素分類	3
第四節	產生麥芽寡糖澱粉酶的微生物	7
		
第二章	實驗材料與設備
第一節	實驗材料	8
第二節	實驗設備	9
第三節	培養基配置	11
		
第三章	實驗方法
第一節	篩選、分離菌株	16
第二節	鑑定分離的菌株	
	3.2.1		利用PCR方法鑑定菌株 16S rDNA	16
	3.2.2		DNA 洋菜電泳	19
	3.2.3		純化DNA	20
	3.2.4		DNA定序和比對	21
第三節	澱粉酶酵素活性測試	
	3.3.1		選殖株菌落檢測法	22
	3.3.2		SDS 聚丙烯醯胺膠體電泳 (SDS-PAGE)	23
	3.3.3		Zymogram	25
	3.3.4		醣解酵素活性測定法	26
第四節	澱粉酶基因轉殖	
	3.4.1		細菌染色體DNA的純化	29
	3.4.2		限制酶隨機切割細菌染色體和染色體純化	31
	3.4.3		限制酶切割載體、去磷酸化和載體純化	32
	3.4.4		DNA 黏合法	34
	3.4.5		勝任細胞製備	35
	3.4.6		電穿孔法與轉殖	37
	3.4.7		轉殖效率計算	38
	3.4.8		複製盤和活性測試	39
	3.4.9		基因圖譜、定序和比對	40
第五節	轉殖澱粉酶基因的表達	
	3.5.1		引子設計和PCR程式	41
	3.5.2		限制酶切割轉殖基因片段和表達載體的純化	42
	3.5.3		表達寄主選殖和製備	44
	3.5.4		轉殖基因片段黏合、轉殖和酵素活性測試	45
第六節	轉殖澱粉酶的純化	
	3.6.1		誘導和活性測試	46
		
第四章	結果與討論
第一節	16S rDNA鑑定分離的菌株	47
第二節	澱粉酶酵素活性篩選與推估蛋白質大小	47
第三節	澱粉酶基因轉殖與活性篩選	48
第四節	澱粉酶還原醣活性測試	51
第五節	誘導轉殖澱粉酶菌株與純化活性測試比較	52
第六節	澱粉酶保守區蛋白序列與結構比對	52
		
第五章	總結與未來展望                        65
第六章	參考文獻                              67

【1】	Dong-Heon Lee, Kye-Heon Oh and Hyung-Yeel Kahng (2009)   Molecular analysis of antioxidant genes in the extremohalophile marine bacterium Exiguobacterium sp. CNU020  Biotechnol Lett. Aug ; 31 (8):1245-51
【2】	Tan L, Qu YY, Zhou JT, Li A, Gou M. (2009) Identification and characteristics of a novel salt-tolerant Exiguobacterium sp. for azo dyes decolorization Appl Biochem Biotechnol. Dec;159(3):728-38
【3】	Okeke BC (2008) Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1 J Ind Microbiol Biotechnol. Dec;35(12):1571-9.
【4】	Debora F Rodrigues, Natalia Ivanova, Zhili He, Marianne Huebner, Jizhong Zhou and James M Tiedje (2008) Architecture of thermal adaptation in an Exiguobacterium sibiricum strain isolated from 3 million year old permafrost: A genome and transcriptome approach  BMC Genomics , 9:547doi:10.1186 /1471-2164-9-547
【5】	胡江, 代先祝, 李順鵬 (2005)阿特拉津及其降解菌的使用對土壤微生物群落的影響 應用生態學報, 8月, 第16卷, 第8期
【6】	Jun T. Park, Li-Ping Yu and James E. Rollings’ (1988)Substrate Structural Effects on Enzymatic Depolymerization of Amylose, Amylopectin, and Glycogen Department of Chemical Engineering Worcester Polytechnic Institute Worcester, Massachusetts 01 609
【7】	FRENCH, D. (1972). Fine structure of starch and its relationship to the organization of starch granules. J. Jpn. Soc. Starch Sci. 19:8.
【8】	Marc J.E.C. van der Maarel, Bart van der Veen,
Joost C.M. Uitdehaag, Hans Leemhuis, L. Dijkhuizen (2002) Properties and applications of starch-converting enzymes of the α-amylase family Journal of Biotechnology 94 137–155
【9】	N. S. Reddy, Annapoorna Nimmagadda and K. R. S. Sambasiva Rao (2003) An overview of the microbial α-amylase family African Journal of Biotechnology Vol. 2 (12), pp. 645-648, December 
【10】	Zhou JH, Baba T, Takano T, Kobayashi S, Arai Y. (1989) Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila. FEBS Lett. Sep 11;255(1):37-41.
【11】	Shida O, Takano T, Takagi H, Kadowaki K, Kobayashi S. (1992) Cloning and nucleotide sequence of the maltopentaose-forming amylase gene from Pseudomonas sp. KO-8940. Biosci Biotechnol Biochem. Jan;56(1):76-80.
【12】	Momma M. (2000) Cloning and sequencing of the maltohexaose-producing amylase gene of Klebsiella pneumoniae. Biosci Biotechnol Biochem. Feb;64(2):428-31.
【13】	Yoshiyuki Takasaki (1982) Production of Maltohexaose by a-Amylase from Bacillus circulans G-6 Agric. Biol Chem., 46 (6), 1539-1547
【14】	Ryuta Kanai, Keiko Haga, Toshihiko Akiba, Kunio Yamane and Kazuaki Harata (2006) Role of Trp140 at subsite −6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707 Protein Sci. March; 15(3): 468–477
【15】	Koichi Ogawa, Osamu Uejima, Teruo Nakakuki,Taichi Usui and Keiji Kainuma (1990) Enzymatic Synthesis of p-Nitrophenyl α-Maltoheptaoside by Transglycosylation of Maltohexaose-forming Amylase Agric. Biol. Chem., 54 (3), 581-586
【16】	Andrew D. Jamieson, Kenneth M. Pruitt and Robert C. Caldwell (1969) An Improved Amylase Assay J DENT RES. 48: 483
【17】	S. E. Chuang, A. L. Chen and C. C. Chao (1995) Growth of E. coli at low temperature dramatically increases the transformation frequency by electroporation Nucleic Acids Res. May 11; 23(9): 1641.
【18】	Keiji Kainuma, Katsuo Wako, Shoichi Kobayashi, Akiko Nogami and Shigeo Suzuki (1975) Purification and some properties of a novel maltohexaose-producing exo-amylase from Aerobacter aerogenes  Biochimica et Biophysica Acta, 410  333--346
【19】	Koichi Ogawa, Osamu Uejima, Teruo Nakakuki,Taichi Usui and Keiji Kainuma (1990) Enzymatic Synthesis of p-Nitrophenyl α-Maltoheptaoside by Transglycosylation of Maltohexaose-forming Amylase Agric. Biol. Chem., 54 (3), 581-586
【20】	Atsushi Narumi, Harumi Kaga, Yutaka Miura, Issei Otsuka, Toshifumi Satoh, Noriaki Kaneko and Toyoji Kakuchi (2006) Polystyrene Microgel Amphiphiles with Maltohexaose. Synthesis, Characterization, and Potential Applications Biomacromolecules, 7, 1496-1501
【21】	Kazukiyo Kobayashi, Hiroshi Sumitomo, Akira Kobayashi, Toshihiro Akaike (1988) Oligosaccharide-Carrying Styrene-Type Macromers. Polymerization and Specific Interactions Between the Polymers and Liver Cells Journal of Macromolecular Science, Part A, Volume 25, Issue 5 - 7 , pages 655 – 667
【22】	Megumi Miyazaki, Pham Van Hung, Tomoko Maeda and Naofumi Morita (2006) Recent advances in application of modified starches for breadmaking Trends in Food Science & Technology 17  591e599
【23】	Blanco A, Díaz P, Martínez J, Vidal T, Torres AL, Pastor FI. (1998) Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw Appl Microbiol Biotechnol. Jul;50(1):48-54.
【24】	Chun-Han Ko, Wen-Luen Chen, Chung-Hong Tsai, Wann-Neng Jane, Chia-Chen Liu and Jenn Tu (2007) Paenibacillus campinasensis BL11: A wood material-utilizing bacterial strain isolated from black liquor Bioresource Technology 98 2727–2733