title: Molecular cloning and expression of the Streptomyces coniferyl alcohol dehydrogenase gene in Escherichia coli.
abstract: Coniferyl alcohol dehydrogenase (CADH) is a key enzyme in catabolism of lignin-related aromatic compounds in bacteria. In Streptomyces sp. NL15-2K, CADH is a tetramer of identical subunits with an individual molecular mass of 39 kDa. This work describes the cloning and sequencing of the CADH gene from Streptomyces sp. NL15-2K, optimization of a protocol for high-level active CADH expression, and purification of recombinant CADH. A BLAST search and motif analyses of the predicted CADH amino acid sequence indicated the enzyme belongs to the medium-chain zinc-dependent alcohol dehydrogenase group. Cell density at heat-shock treatment, temperatures for heat shock and culture, duration of heat shock, concentration of isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer, and culture time after induction were adjusted for optimal CADH expression. Expression of active CADH under optimized conditions was approximately 4-fold higher than in the absence of heat shock. CADH purified from recombinant Escherichia coli was in the tetrameric form, as was natural CADH from NL15-2K.
authors.affiliation: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan. m-nishi@yasuda-u.ac.jp
authors.forename: Motohiro
authors.initials: M
authors.lastname: Nishimura
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D008031:Lignin / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D008031:Lignin; D000429:Alcohol Oxidoreductases; C019931:coniferyl alcohol dehydrogenase