Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. VII. Nucleotide sequence of the lactate dehydrogenase gene from the mesophilic bacterium Bacillus megaterium. Preparation and properties of a hybrid lactate dehydrogenase comprising moieties of the B. megaterium and B. stearothermophilus enzymes.
The lactate dehydrogenase (LDH) gene of a mesophilic bacterium, Bacillus megaterium (DSM 090), was cloned in E. coli HB 101 using a pEMBL vector and synthetic oligonucleotide probes. The gene was strongly expressed in the vector used if the orientation of the insert allowed the LDH promoter and the vector's lac promoter to direct transcription in the same direction. The gene and its 5' and 3' flanking regions have been sequenced. Codon usage patterns of LDH genes from mesophilic and thermophilic bacilli were compared and found to be characteristically different. A hybrid gene was constructed from fragments of the LDH genes from B. stearothermophilus (coding for aa 15-100) and B. megaterium (coding for aa 101-331). The hybrid LDH, named S100M, was more thermostable than B. megaterium LDH, less thermostabile than B. stearothermophilus LDH and unlike the two wildtype enzymes, it could not be activated by Fru-P2.
D001410:Bacillus megaterium / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis*; D004795:Enzyme Stability; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011489:Protein Denaturation
D004269:DNA, Bacterial; D007770:L-Lactate Dehydrogenase