I.重組酵母菌醛類去氫酶ALD4p之定性分析: 與其天然酵素之比較 II.啤酒酵母菌酒精去氫酶ADH4之過度表現、純化、與定性分析

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题名:	I.重組酵母菌醛類去氫酶ALD4p之定性分析: 與其天然酵素之比較 II.啤酒酵母菌酒精去氫酶ADH4之過度表現、純化、與定性分析
其它题名:	I. characterization of the purified recombinant ALD4p of saccharomyces cerevisiae: comparison with its natural counterpart. II. overexpression, purification, and characterization of ADH4 of saccharomyces cerevisiae
作者:	蔡宗佑;Tsai, Tzung-you
贡献者:	淡江大學生命科學研究所碩士班陳銘凱;Chern, Ming-kai
关键词:	啤酒酵母菌;酒精去氫酶;醛類去氫酶;Saccharomyces cerevisiae;alcohol dehydrogenase IV;aldehyde dehydrogenaseIV
日期:	2008
上传时间:	2010-01-11 02:26:22 (UTC+8)
摘要:	在第一部份，酵母菌醛類去氫酶4為一種存在於粒腺體內的去氫酶，會被K+離子所活化及需要 NAD+、NADP+ 當作輔酶，當乙醇為碳源的代謝途徑下，ALD4扮演了重要的角色。為了比較醛類去氫酶4和其天然酵素之定性分析，我們先將ALD4基因，利用大腸桿菌 BL21大量的表現 ald4p，並且透過管柱層析之後，測定對不同受質的活性，而 Ald4 天然酵素的部份，利用親和性膠體管柱純化後，比較重組的 ald4p與天然ald4p的活性與在蛋白質的二級與四級構型。第二部份，酵母菌酒精去氫酶4為存在於細胞質內的去氫酶，會被 Zn2+離子所調控，屬於 MDR家族之一。當 ADH1基因存在的條件之下 adh4p的表現會被抑制，所以我們先利用基因重組的方式將酵母菌基因體中 Adh1基因的部份破壞，再將已連接到 pVT101L的 ADH4基因送進已重組過的基因體之中，再誘導酵母菌 adh4p大量表現，透過管柱層析純化之後，檢測其對於受質膽鹼之動力學活性，並探討此活性與甜菜鹼（betaine）代謝之關係。 In Part I, aldehyde dehydrogenase 4 of Saccharomyces cerevisiae is a K+-activated mitochondrial enzyme using NAD+ and NADP+ as coenzymes. ALD4 plays a major role during growth on ethanol as carbon source. To compare the characteristics of the purified recombinant ald4p with tose of natural counterpart, we firstly used E.coli for overexpression of ald4p, and followed by purification. Then we made kinetic analysis with various kinds of substrate. In ald4p natural counterpart, we performed kinetic active analysis on the same substrates as for the purified recombinant ald4p after purification by affinity column. We also compared the secondary and tertiary structures between the recombinant ald4p and its natural counterpart. In Part II, yeast alcohol dehydrogenase IV exists in the cytosol, is a member of the MDR superfamily and activated by Zn2+. Since adh1 could repress the adh4 expression, we used the one-step gene disruption to knockout the adh1 gene in yeast genome. (BJ2168). We then transform the expression vector pVT101L which contains ADH4 ORF into the ADH1 strain, and over-express adh4p. Finally, we determined the enzyme activities with ethanol and choline after purification. The kinetic properties are discussed in relation to choline-betaine pathway.
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