酵母菌醛類去氫酶4之選殖、異源表現、純化及其動力學分析

Cloning, heterologous expression, purification, and kinetic analysis of aldehyde dehydrogenase 4 of Saccharomyces cerevisiae.

陳俊傑(Jyun-Jie Chen)

指導教授 ： 陳銘凱

淡江大學/理學院/生命科學研究所碩士班/碩士(2005年)

https://doi.org/10.6846/TKU.2005.00109

摘要

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。

關鍵字

酵母菌 ； 醛類去氫酶

並列摘要

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.

並列關鍵字

aldehyde dehydrogenase 4 ； Saccharomyces cerevisiae

參考文獻

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