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Biochemical characterization of a functional recombinant aryl‐alcohol dehydrogenase from Taiwanofungus camphorata (583.4)
Author(s) -
Huang JenqKuen,
Chang CheChi,
Ken ChuianFu,
Wen Lisa,
Lin ChiTsai
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.583.4
Subject(s) - recombinant dna , enzyme , complementary dna , biochemistry , chemistry , microbiology and biotechnology , dehydrogenase , molecular mass , alcohol dehydrogenase , nad+ kinase , biology , gene
A TcAAD cDNA encoding a putative aryl‐alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata (Tc). 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 K M , V max 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. Grant Funding Source : National Science Council of the Republic of China, Taiwan under grant 100‐2313‐B‐019‐003‐MY3