NAD(+)-dependent glycerol (Gro) dehydrogenase (GroDHase) catalyzes the conversion of Gro into dihydroxyacetone (DHA), the first step for fermentative Gro metabolism in Escherichia coli. In this work, we cloned the gldA gene that codes for the E. coli GroDHase and homologously expressed, purified, and kinetically characterized the recombinant protein. To achieve this, the enzyme was over-produced using Gro supplemented growth medium and lactose as the inducer. The enzyme was highly purified using either pseudo-affinity chromatography or a simple heat-shock treatment, which is potentially valuable for industrial production of GroDHase. We detected efficient oxidation of Gro derived from biodiesel production to DHA by gas chromatography. The results presented in this work support recombinant GroDHase production in a biorefinery setting as a relevant tool for converting Gro into DHA for future biotechnological applications. (C) 2013 Elsevier Ltd. All rights reserved.