Abstract

Highly sensitive thermostable biosensors for aldehydes are required in a series of applications related to solvents, cosmetics, perfume, wine, food and pharmaceutical industries. While extremophilic microorganisms are important source for stable catalysts for biotechnologies, psychrophilic bacterial strains represent a valuable source for extremozymes catalysing low temperature reactions. In view of constructing a stable, cold-active biosensor, with a wide-range substrate sensitivity to aliphatic and aromatic aldehydes, we obtained and characterized the recombinant aldehyde-dehydrogenase from Flavobacterium PL02 (F-ALDH), a psychrophilic bacterium isolated from Antarctic sea water. Structural analysis showed high amino acid sequence identity (89%) with the cold active Flavobacterium frigidimaris ALDH and 3D modelling indicated a similar structure with Staphylococcus aureus ALDH. The gene coding for F-ALDH was cloned in pHAT2 and expressed in E. coli at 25 ◦C as soluble protein. The enzyme was purified by Ni-affinity chromatography. Recombinant F-ALDH is a NAD+-dependent enzyme using a large variety of substrates, with high activity when using 4-isopropilbenzaldehyde and isovaleraldehyde. In comparison to currently used ALDH-based biosensors from mesophiles, this extremozyme is stable at both low and high temperatures (4–60 ◦C), representing a promising catalyst for ALDH-based biosensors.