Genetics and Molecular Biology
Print version ISSN 1415-4757
WANG, Nan et al. Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori. Genet. Mol. Biol. [online]. 2011, vol.34, n.2, pp. 240-243. ISSN 1415-4757. http://dx.doi.org/10.1590/S1415-47572011000200013.
Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH.
Keywords : 5'-RACE PCRADH; enzymatic activity; recombinant protein.