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Title: 牛樟芝芳基醇脫氫酶之基因選殖、表現及酵素特性分析
Gene cloning, expression and characterization of aryl-alcohol dehydrogenase from Taiwanofungus camphorata
Authors: Che-Chi Chang
Contributors: NTOU:Institute of Bioscience and Biotechnology
Keywords: 牛樟芝;芳基醇脫氫酶
aryl-alcohol dehydrogenase;Taiwanofungus camphorata;AAD
Date: 2012
Issue Date: 2013-10-07T02:44:32Z
Abstract: 芳基醇脫氫酶 ( aryl-alcohol dehydrogenase;AAD ) 參與酪胺酸、苯丙胺酸代謝,以及芳香化合物的降解。本實驗由牛樟芝( Taiwanofungus camphorata ) 選殖出芳基乙醇脫氫酶 cDNA,全長1299個鹼基對,轉譯區有1047個鹼基對,可轉譯出348個胺基酸,預測分子量大小為39.1 kDa。為分析牛樟芝芳基乙醇脫氫酶 ( TcAAD ),將其轉譯區重組到表現載體 pYEX-S1,並轉形至 Saccharomyces cerevisiae 表現,後以鎳離子親合管柱純化。經純化的 TcAAD 進行12%十二烷基硫酸鈉聚丙烯酰胺凝膠電泳,可見明顯色帶,以藜蘆醛 ( veratraldehyde ) 作為基質,分析 TcAAD 還原基質之酵素動力學及其特性,得KM為0.25 mM 。於58℃下,半衰期為4.2 min,而熱失活常數 ( kd ) 則是7.42 x 10-2 min-1 。 TcAAD 置於pH 6-10的環境處理下,仍具50%以上活性,而以pH 4處理後,僅存12%的活性。又以苯甲醇 ( benzyl alcohol )、3, 4-二甲氧基苯甲醇( 3,4-dimethoxybenzyl alcohol ) 、2, 4-二甲氧基苯甲醇 ( 2,4-dimethoxybenzyl alcohol ) 、4-羥甲基苯甲酸作為基質 ( 4-(hydroxymethyl)benzoic acid ) ,分析 TcAAD 氧化基質之活性,當中以4-羥甲基苯甲酸活性最佳。
Aryl-alcohol dehydrogenase (AAD) play important roles in redox system via NADPH as a reductant. A TcAAD cDNA (1299 bp, HQ453361) encoding a putative AAD was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels (Kv1) subunit beta-2 ( PDB code 3EAU ). To characterize the TcAAD protein, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a single band at molecular mass of approximately 39.1 kDa on 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited AAD activity via veratraldehyde assay. The Michaelis constant (KM) value for veratraldehyde was 0.25 mM. The enzyme’s half-life of deactivation at 58C was 4.2 min, and its thermal inactivation rate constant kd was 7.4 x 10-2 min-1. The enzyme was most active at pH 6. The enzyme’s preferred substrate is 4-(hydroxymethyl)benzoic acid. It can also use other benzylcompounds as substrates including benzyl alcohol, 3,4-dimethoxybenzyl alcohol, 2,4-dimethoxybenzyl alcohol and veratraldehyde.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0019936009
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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