Abstract

Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 degrees C for 18 days. F-ALDH used both NAD(+) and NADP(+) and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory K-M and k(cat) values and the highest catalytic efficiency (0.50 mu M-1 s(-1)) for isovaleraldehyde. The enzyme was active in the 4-60 degrees C-temperature interval, with an optimal pH of 9.5, and a preference for NAD(+)-dependent reactions. Arrhenius plots of both NAD(P)(+)-dependent reactions indicated conformational changes occurring at 30 degrees C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.