Molecular cloning and expression of the complete DNA sequence encoding NAD+-dependent acetaldehyde dehydrogenase fromAcinetobacter sp. strain HBS-2

摘要

The aldehyde dehydrogenase (ALDH) gene fromAcinetobacter sp. strain HBS-2 was cloned and characterized. The ORF of the gene was a 1467-bp sequence that encoded a 489-amino-acid protein. Strain HBS-2 carries a newALDH gene that differs from the knownALDH genes inAcinetobacter, and the product coded is a member of the NAD+-dependent ALDH family with 99% identity to several knownAcinetobacter baumannii orthologs. The determined sequence exhibited 56, 59 and 39% identity with human mitochondrial ALDH2, and ALDH fromPseudomonas aeruginosa andEscherichia coli, respectively. TheALDH gene was cloned into the pET28a(+) expression vector and expressed inE. coli BL21 cells under the control of the IPTG-inducible promoter T7. Two-thirds of the recombinant enzyme was available in soluble form, and its molecular mass was estimated at 57 kDa by SDS-PAGE. After purification on an Ni-sepharose column, the recombinant protein was found to have a specific activity of 60.6 U/mg protein. TheKm values of the enzyme were 15.18 μM for acetaldehyde, 2.12 μM for formaldehyde, and 0.49 μM for propionaldehyde. These results indicate that ALDH from strain HBS-2 preferentially reacts with propionaldehyde. However,Vmax of the enzyme for acetaldehyde was determined to be the highest. The recombinant enzyme exhibited novel biochemical characteristics. Its activity was significantly elevated in the presence of Mn2+. These findings are different from those reported for ALDHs from other microorganisms.