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 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.
Kochhar
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Nestlé Research Centre, Lausanne, Switzerland.
Chuard
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N
Hottinger
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H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D045022:Lactate Dehydrogenases; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase