Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9.
A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9). It was identified to be a new strain of Bacillus licheniformis. The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18. The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31%. The enzyme was found to be composed of eight subunits identical in relative molecular mass (43,000). The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B. stearothermophilus enzyme [Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Oshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056-9062], although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH. However, they were markedly distinct from each other in halophilicity; the B. licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.
Nagata
S
S
Department of Bioresources Science, Kochi University, Japan.
Bakthavatsalam
S
S
Galkin
A G
AG
Asada
H
H
Sakai
S
S
Esaki
N
N
Soda
K
K
Ohshima
T
T
Nagasaki
S
S
Misono
H
H
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002712:Chlorides; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase