Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Abstract

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 inBombyx 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. coliexpression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coliBL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombi-nant 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. © 2011, Sociedade Brasileira de Genética.