臺灣博碩士論文加值系統

酵母菌醛類去氫酶4（YOR374w）是一種位在粒線體內的去氫酶，需要鉀離子的活化，以及NAD+或NADP+當輔酶，在乙醇的代謝途徑扮演很重要的角色，尤其是以乙醇當碳源的環境下。為了了解醛類去氫酶對於其他不同代謝途徑是否有影響，經由選殖出正確ALD4基因，再利用大腸桿菌大量的表現出ald4p，透過管柱層析純化之後，對許多不同的受質做活性分析。在酵母菌的泛酸生合成途徑中，3-aminopropanal氧化成beta-alanine是重要的一個反應，而glycine betaine是一種滲透壓保護劑，對於細菌在高滲透壓環境下的生存很重要，是由choline氧化成betaine aldehyde再氧化生成glycine betaine，我們主要想了解ald4p對這兩種醛類3-aminopropanal和glycine betaine aldehyde是否有活性，希望能加以應用和建構出泛酸和glycine betaine更完整的代謝途徑。另外在蛋白質表現的過程中，我們利用lactose來誘導表現，比較以lactose誘導與IPTG誘導之最適條件，結果不論在單位菌數蛋白質的表現量和活性都是差不多的，但是lactose可獲得更多的菌數。在考量成本下，lactose會比IPTG佳。

The identity of the gene encoding the mitochondrial K(+)-activated acetaldehyde dehydrogenase (K(+)-ACDH) of Saccharomyces cerevisiae has been confirmed. The gene is situated on the right arm of chromosome XV, bears the systematic name YOR374w and the deduced product shows significant homology to other members of the S. cerevisiae aldehyde dehydrogenase (ALDH) family. K(+)-ACDH is required for growth on ethanol. In order to understand aldehyde dehydrogenase as to other metabolic pathways, we cloned ALD4 gene and used E.coli for over-expression and followed by purification. We made active analysis to various different substrate. In pantothenic acid biosynthesis, 3-aminopropanal oxidized to beta-alanine is an important reaction. Glycine betaine is an osmoprotectant which is important to a variety of organisms under the environment of high osmolarity . Mainly we want to understand whether ald4p has activities to these aldehydes, 3-aminopropanal and glycine betaine aldehyde in order to build a more comprehensive metabolic pathway for pantothenic acid and glycine betaine. In addition , we made use of lactose to induce ald4p and compared to the effect of IPTG. In all four host strains tested, both inducers are similar in expression of protein amount and activity based on the same unit amount of host cells. However, higher number of host cells is achieved by growth on lactose. To conside the cost-effect, lactose is a better inducer than IPTG.

目錄
壹. 第一章-緒論……………………………………………………….1
貳. 第二章-實驗步驟及材料………………………………………….6
第一節-實驗材料
（一）：儀器
（二）：藥品…………………………….…………………..8
第二節-ALD4基因選殖………………………………………11
（一）：啤酒酵母菌genomic DNA 分離
（二）：以聚合酶連鎖反應大量複製ALD4基因片段…..12
（三）：DNA瓊脂膠電泳分析（Agarose gel
electrophoresis）......................................................13
（四）：PCR產物純化..........................................................14
（五）：限制酶切割片段分析
（Restriction Enzyme Digest）.................................15
（六）：PCR產物片段補A..................................................16
（七）：選殖（TA clone）
1.勝任細胞製備（Competent Cell）
2.Ligation........................................................................17
3.轉型（Transformation）............................................ 18
（八）：篩選............... ...........................................................19
1.小量質體DNA製備
2.以限制酶處理確認plasmid.........................................21
3.定序ALD4片段..........................................................22
第三節-蛋白質表現....................................................................23
（一）：表現載體pET43之複製..........................................24
（二）：Ligation.....................................................................26
（三）：Transformation..........................................................28
1. transformation
2.以PCR方法確認plasmid...........................................29
3.以限制酶處理確認plasmid........................................30
（四）：轉形至表現載體.......................................................31
（五）：蛋白質表現條件.......................................................32
1.生長曲線
2.超音波破碎細胞..........................................................33
（六）：表現的蛋白質活性初測
（七）：蛋白質電泳分析......................................................34
第四節-蛋白質純化...................................................................37
（一）：疏水性膠體純化蛋白質
1.管柱裝填
2.蛋白質純化前準備.....................................................38
3.疏水性膠體純化蛋白質.............................................39
4.管柱膠體的清潔.........................................................40
（二）：離子交換膠體純化蛋白質
1.管柱裝填....................................................................41
2.蛋白質純化前準備
3.離子交換膠體純化蛋白質........................................42
4.管柱膠體的清潔........................................................43
（三）：親合性膠體純化蛋白質
1.管柱裝填....................................................................44
2.親合性膠體純化蛋白質
3.管柱膠體的清潔
（四）：蛋白質活性及濃度測定.........................................45
1.蛋白質活性測定
2.蛋白質濃度定量........................................................46
第五節-受質動力學分析............................................................47
（一）：Propionaldehyde活性分析
（二）：betaine aldehyde活性分析
（三）：γ-aminobutyraldehyde活性分析........................48
（四）：3-aminoproionaldehyde活性分析


參. 第三章-結果與討論........................................................................49
第一節- ALD4基因片段選殖
第二節- 蛋白質表現.................................................................53
第三節- 蛋白質純化.................................................................74
第四節- 動力學分析.................................................................83
肆. 附錄
一、TA clone NO.3定序後blast2比對結果…………..…….87
二、TA clone NO.3定序後blast2比對結果………..……….89
伍. 參考資料.........................................................................................91

表目錄
表1：PCR反應溶液…………………………………………………….13
表2：Restriction Enzyme Digest.............................................................15
表3：PCR產物補A反應液組成……………………………………..16
表4：TA liogation反應試劑…………………………………………...18
表5：EcoRI確認plasmid反應試劑……………………………….….21
表6：NdeI限制酶作反應試劑…………………………………………21
表7： SpeI切出需要的ALD4和pET43片段反應溶液…………….26
表8： NdeI切出需要的ALD4和pET43片段反應溶液
表9：ligation反應溶液…………………………………………………28
表10：以PCR以確認ligation的反應溶液…………………………...29
表11：以NdeI和SpeI限制酶進行確認反應液………………………30
表12：Ald4p活性初測溶液…………………………………….…..…33
表13：12.5 ％SDS-PAGE分離凝膠組成…………………….………34
表14：3.7 ％ SDS-PAGE焦集凝膠組成…………………………….35
表15：活性偵測溶液組成……………………………………………..45
表16：各種條件之活性（單位mM/min）……………………….….…..73
表17：疏水性膠體純化的鹽類梯度表…………………………….…..75
表18：蛋白質純化結果表……………………………….…………….82
表19：各受質不同濃度下的活性……………………….…………….83

[1].Wayne D. Tessier, Philip G. Meaden, Francis M. Dickinson, Melvin Midgley（1998）Identification and disruption of the gene encoding the K(+)-activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae. FEMS Microbiology Letters 164,29-34
[2].White WH, Skatrud PL, Xue Z, Toyn JH.（2003）Specialization of function among aldehyde dehydrogenases: the ALD2 and ALD3 genes are required for beta-alanine biosynthesis in Saccharomyces cerevisiae. Genetics Society of America 2003 Jan;163(1):69-77.
[3].Falkenberg P and Strøm AR (1990) Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli. Biochim Biophys Acta. 1990 1034(3):253-9.
[4].Mori N et al. (1980) Agric. Biol. Chem. 44: 3015-3016.
[5].Boch J et al. (1997) Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine. Arch Microbiol.168(4):282-9.
[6].Russell R and Scopes RK (1994) Use of hydrophobic chromatography for purification of the membrane-located choline dehydrogenase from a Pseudomonas strain. Bioseparation 4(4):279-84.
[7].ROTHSCHILD HA and BARRON ES (1954) The oxidation of betaine aldehyde by betaine aldehyde dehydrogenase. J Biol Chem. 209(2):511-23.
[8].Chern MK and Pietruszko R (1995) Human aldehyde dehydrogenase E3 isozyme is a betaine aldehyde dehydrogenase. Biochem Biophys Res Commun. 213(2):561-8.
[9].Kurys G et al. (1989)Human aldehyde dehydrogenase. Purification and characterization of a third isozyme with low Km for gamma-aminobutyraldehyde. J Biol Chem. 264(8):4715-21.
[10].Vaz FM et al. (2000) Molecular and biochemical characterization of rat γ-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis. J Biol Chem. 275(10):7390-4.
[11].Dragolovich J and Pierce HK (1994) J. Exp. Zool. 270:417-425.
[12].Perrino LA, Pierce SK. (2000) Betaine aldehyde dehydrogenase kinetics partially account for oyster population differences in glycine betaine synthesis. J Exp Zool. 286(3):238-49.
[13].Weigel P et al. (1986) Plant Physiol. 82:753-759.
[14].Weretilnyk EA and Hanson AD (1988) Betaine aldehyde dehydrogenase polymorphism in spinach: genetic and biochemical characterization. Biochem Genet. 26(1-2):143-51.
[15].Pan SM et al. (1981) Plant Physiol. 67:1105-1108.
[16].Trossat C et al. (1997). Transgenically Expressed Betaine Aldehyde Dehydrogenase Efficiently Catalyzes Oxidation of Dimethylsulfoniopropionaldehyde and ω-Aminoaldehydes. Plant Physiol. 113(4): 1457-1461.
[17].http://www.windrug.com/pic/10/11/18/17/11/003.htm
[18].http://bbs.fh21.com.cn/php2/detail.php?table=drug&id=86
[19].http://biocyc.org/
[20].Barak AJ (1993) Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration. Alcohol Clin Exp Res. 17(3):552-5.
[21].Gasch AP et al. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 11(12): 4241-57.
[22].http://db.yeastgenome.org/cgi-bin/search/textSearch?query=choline&type=gp; Dec.25, 2004.
[23].Robert S.Donovan,Campbell W. Robinson, and Bernard R. Glick（2000）Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter. Can J Microbiol. 46(6):532-41
[24].Rose A. Monteiro, Emanuel M. Souza, M. Geoffery Yates, Fảbio O. Pedrosa, and Leda S. Chubatsu.（2000）Use of lactose to induce expression of soluble NifA protein domains of Herbaspirillum seropedicae in Escherichia coli. Can J Microbiol. 46(11):1087-90
[25].XINPING WANG, CRAIG J. MANN, YINLIN BAI, LI NI, AND HENRY WEINER.（1998）Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae. JOURNAL OF BACTERIOLOGY,p. 822–830
[26].Gary L.PETERSON.（1983）Determination of Total Protein.METHODS IN ENZYMOLOGY.91(12):95-119
[27].http://juang.bst.ntu.edu.tw/index.htm