Park Yong-Cheol, San Ka-Yiu, Bennett George N
Department of Biochemistry and Cell Biology, Rice University, 6100 Main St., Houston, TX 77005, USA.
Appl Microbiol Biotechnol. 2007 Aug;76(2):349-56. doi: 10.1007/s00253-007-0998-5. Epub 2007 May 22.
Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD(+)-binding motif and showed 54-75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 +/- 9 mU/mg using ethanol and NAD(+) as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3-C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD(+) preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.
乙醇脱氢酶（ADH）是醇类生产和利用中的关键酶。一些乙醇脱氢酶还催化由醇类和醛类形成羧酸酯。克隆了粗糙脉孢菌FGSC2489的ADH1和ADH3基因，并在重组大肠杆菌中表达，以研究它们的乙醇脱氢和羧酸酯形成能力。氨基酸序列的同源性分析和序列比对表明，粗糙脉孢菌的ADH1和ADH3含有一个锌结合共有序列和一个NAD(+)结合基序，与真菌ADH具有54%-75%的同一性。粗糙脉孢菌ADH1在大肠杆菌中表达，分别以乙醇和NAD(+)作为底物和辅因子时，比活性为289±9 mU/mg。对ADH3的表达和活性进行的相应实验得到的比活性为4 mU/mg。粗糙脉孢菌ADH1优先选择含有C3-C8碳的伯醇，而非仲醇，如2-丙醇和2-丁醇。粗糙脉孢菌ADH1具有5.3 mU/mg的特定羧酸酯形成活性，在18小时内积累了0.4 mM的乙酸乙酯。各种直链醇和醛的底物特异性表明，短链长度的醇和醛是羧酸酯生产的良好底物。粗糙脉孢菌ADH1是一种主要使用辅因子NAD(+)的伯醇脱氢酶，并且对短链醇和醛具有羧酸酯形成活性。
