Cloning and overexpression of the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene in Escherichia coli:purification and characterization of the recombinant enzyme.

Nestlé Research Centre, Lausanne, Switzerland.

The Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene was amplified by the polymerase chain reaction and cloned into an Escherichia coli expression plasmid pKK223.3. Attempts to clone the full-length chromosomal DNA encoding D-lactate dehydrogenase from a partial Sau3AI lambda phage library or an enriched clone bank in E. coli were unsuccessful. The recombinant plasmid pKBULDH containing the amplified gene overexpressed D-lactate dehydrogenase (greater than 30% of total soluble protein) following induction of the tac promotor with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by two chromatographic steps with 76% recovery of enzyme activity. All the properties of the recombinant protein, e.g., optimum pH and temperature, Km and k(cat) for pyruvate as well as for other 2-oxo acids and the subunit structure were identical to the wild-type enzyme.

PMID: 1610363 [PubMed - indexed for MEDLINE]