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.
