Identification Of A Novel Aldehyde Dehydrogenase Gene From The Metagenomic Library Of Arctic Deep-sea Sediments

Aldehyde dehydrogenase(ALDH) is distributed widely in eukaryotic and prokaryotic organisms and could reduce damage of aldehydes and ketones to human body by catalyzing the degrade of them, followed by excretion from human body. However, over50%of the Asian people lack this enzyme, causing serious diseases. Therefore, study on acetaldehyde dehydrogenase is of great importance. Isolation and characterization of acetaldehyde dehydrogenases from unique environments is gaining more attention, due to the possibility that these extreme environments could contribute to unique physiological features to these enzymes.The present study focused on the characterization of acetaldehyde dehydrogenase gene from arctic deep-ocean sediments metagenomic library.Firstly, we screened one positive clone from this library, and found it contains an entire open reading frame(1158bp, named as ccs24-5-a) encoding386amino acids with a molecular weight of41.7281kDa.(an accession number in Gene bank:JF737989). Blast analysis revealed that its encoding product is composed of a critical domain conserved in acetaldehyde dehydrogenase from other sources. Based on phylogenetic analysis, it was proposed to be isolated from Jannaschia or Loktanella because it has a closer distance to two strains in these two genera (Jannaschia sp. CCS1and Loktanella vestfoldensis SKA53). Comparison of this protein with six homologous supported some conservative sites on all of them, except that the N-terminal of ccs24-5-a. is shorter than others. We also observed that a bacteria regulatory protein-encoding gene is located in the upstream of t ccs24-5as well an unknown function protein gene in the downstream of it.Next, we performed amplified the full-length ccs24-5-aand cloned it onto a commonly-used expression vector pET-28-a and established a prokaryotic expression system in E.coli BL21(DE3). But, we found it could express mainly in the form of inclusion body, even after we tried to optimize the induction conditions. Then, we attempted to co-express molecular chaperon with ccs24-5-a by introducing pKJE7and pTF16respectively into this system. Finally, we could obtained soluble ccs24-5-a protein under optimized conditions (1mmol/L L-arabinose,0.25mmol/L IPTG,28℃)Subsequently, we established an enzymatic assay system using purified ccs24-5-a and NAD+as the substrate, and found that it showed a high activity (30U per mL) at the optimal reaction temperature (35℃) and the optimal pH (7.5).