Biochemical Characterization of a Functional Recombinant Aryl-Alcohol Dehydrogenase from Taiwanofungus Camphorata

Chuian-Fu Ken ； Che-Chi Chang ； Lisa Wen ； Jenq-Kuen Huang ； Chi-Tsai Lin

《Botanical Studies》 55卷2月 (2014/02) Pp. 22-31

https://doi.org/10.1186/1999-3110-55-14

並列摘要

Background: Aryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds.Results: One TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (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 subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, 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 band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied. Conclusions: The coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.

並列關鍵字

Taiwanofungus camphorata ； Aryl-alcohol dehydrogenase ； Three-dimension structural model (3-D structural model) ； Veratraldehyde ； 3, 4-Dimethoxybenzyl alcohol ； 2, 4-Dimethoxybenzyl alcohol