Gene cloning, expression and characterization of aryl-alcohol dehydrogenase from Taiwanofungus camphorata

• Main Authors: Che-Chi Chang, 張哲綺
• Other Authors: Chi-Tsai Lin
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/53190940287284029269

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 100 === 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.