CLONING AND OVEREXPRESSION OF THE LACTOBACILLUS-BULGARICUS NAD+- DEPENDENT D-LACTATE DEHYDROGENASE GENE IN ESCHERICHIA-COLI - PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

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 Sau3Al λ phage library or an enriched clone bank in E. coli were unsuccessful. The recombinant plasmid pKBULDH containing the amplified gene overexpressed D-lactate dehydrogenase (>30 % of total soluble protein) following induction of the tac promotor with isopropyl-β-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 kcat for pyruvate as well as for other 2-oxo acids and the subunit structure were identical to the wild-type enzyme. © 1992.