Details

Autor(en) / Beteiligte

• Kochhar, Sunil
• Chuard, Nathalie
• Hottinger, Herbert

Titel

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

Ist Teil von

• Biochemical and biophysical research communications, 1992-06, Vol.185 (2), p.705-712

Ort / Verlag

San Diego, CA: Elsevier Inc

Erscheinungsjahr

1992

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• 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, K m and k cat for pyruvate as well as for other 2-oxo acids and the subunit structure were identical to the wild-type enzyme.