Abstract

The gene encoding a thermophilic group 3 alcohol dehydrogenase from Thermotoga lettingae TMO was cloned and over-expressed in Escherichia coli. The full-length DNA sequence of TlADH was 1086-bp encoding a polypeptide of 361 amino acids. Comparative and bioinformatic analysis revealed that TlADH showed high similarity to group 3 ADHs from thermophilic and mesophilic alcohol dehydrogenases, but relatively highest similarity to T. maritime 1, 3-propanediol dehydrogenase. The optimal pH-values of butanol oxidation and butylaldehyde reduction of TlADH were 11.9 and 6.0, respectively. Kinetic parameters of the enzymes showed TlADH preferred NADP⁺ to NAD⁺ as a cofactor. TlADH can catalyze a range of primary alcohols oxidation, while it was inactive towards branched-chain alcohols and cyclitol. TlADH also can reduce aldehydes to corresponding alcohols, including aliphatic and aromatic aldehydes. It showed higher activity of aldehyde reduction than that of alcohol oxidation that may be relative to reactive aldehyde detoxification in cell metabolism. In the case of external ions addition we found a 5.23-fold increase in reaction activity by the adding of 1 mM MnCl₂. T. lettinga ADH was cloned and overproduced in a mesophilic heterologous expression system, and the recombinant enzyme was characterized. It will be helpful to understand more about the physiological role of group 3 alcohol dehydrogenase.

Key words: Group 3 ADH, bioinformatic analysis, recombinant expression, thermophilic,catalytic properties.