Publications

• Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9.

  Nagata S., Bakthavatsalam S., Galkin A.G., Asada H., Sakai S., Esaki N., Soda K., Ohshima T., Nagasaki S., Misono H.

  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 pU ... >> More

  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. << Less

  Appl. Microbiol. Biotechnol. 44:432-438(1995) [PubMed] [EuropePMC]

• Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases.

  Nagata S., Tanizawa K., Esaki N., Sakamoto Y., Ohshima T., Tanaka H., Soda K.

  The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was fou ... >> More

  The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance. << Less

  Biochemistry 27:9056-9062(1988) [PubMed] [EuropePMC]

• Cloning, sequencing and overexpression of the leucine dehydrogenase gene from Bacillus cereus.

  Stoyan T., Recktenwald A., Kula M.R.

  The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70-80% identity with LeuDH's from the ... >> More

  The L-leucine dehydrogenase gene from Bacillus cereus (DSM 626) was cloned from a partial genomic library and sequenced. The open reading frame has 1101 bp and codes for a protein of 39.9 kDa. The deduced amino acid sequence of the LeuDH from B. cereus shares 70-80% identity with LeuDH's from the thermophilic strains B. stearothermophilus and Thermoactinomyces intermedius. The active protein was overexpressed in Escherichia coli to yield approximately 30% of the total soluble protein. << Less

  J. Biotechnol. 54:77-80(1997) [PubMed] [EuropePMC]

• The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius.

  Ohshima T., Nishida N., Bakthavatsalam S., Kataoka K., Takada H., Yoshimura T., Esaki N., Soda K.

  Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4 degrees C) for over two years. The enzyme was more thermostable at higher c ... >> More

  Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4 degrees C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KCl. It retained about 90% of activity on incubation at 70 degrees C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, L-leucine, NADH, 2-oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 118 mM, respectively, from initial-velocity analyses. The enzyme showed pro-S stereospecificity for hydrogen transfer of NADH in the reductive amination. The enzyme gene was cloned into Escherichia coli and its complete DNA sequence was determined. The leucine dehydrogenase gene (leudh) consists of a 1098-bp open reading frame and encodes 366 amino acid residues corresponding to a subunit (M(r) 40586) of the octameric enzyme. The amino acid sequence of the enzyme showed 80.7% similarity with that of the Bacillus stearothermophilus enzyme. The enzyme was overproduced in E. coli JM 109 having a recombinant plasmid, pULDH2, which was constructed from pUC18 and the leudh gene. The enzyme was purified from the cell extract to homogeneity in one day, with 78% recovery. << Less

  Eur. J. Biochem. 222:305-312(1994) [PubMed] [EuropePMC]