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An aldehyde dehydrogenase (AldH), from Klebsiella pneumoniae was cloned, expressed, and characterized for its properties in E. coli BL21(DE3). The enzyme effectively converts 3-hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, to 3-hydroxypropionic acid (3-HP), a commercially valuable platform chemical. It also exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. The enzyme showed preference towards NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes.
When several aldehydes tested, the specific activity of 27.7 U mg-1 protein was recorded for 3-HPA at pH 8.0 and 45°C. The halfsaturation constant (Km) and the specificity constant (kcat) for 3-HPA in the presence of NAD+ were 0.48 mM and 41.44ⅹ103 M-1 S-1, respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol, 2-mercaptoethanol, or semicarbazide hydrochloride. Reduction in the AldH activity was
noted in the presence of certain metal ions like Hg2+, Ag+, Cu2+, Fe2+ and Zn2+. This study demonstrated that AldH of K. pneumoniae was very specific towards NAD+ and highly active in converting 3-HPA to 3-HP.