The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius.
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.
Ohshima
T
T
Department of Chemistry, Kyoto University of Education, Fukakusa, Japan.
Nishida
N
N
Bakthavatsalam
S
S
Kataoka
K
K
Takada
H
H
Yoshimura
T
T
Esaki
N
N
Soda
K
K
D000190:Actinomyces / Q000201:enzymology* / Q000235:genetics*; D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050803:Leucine Dehydrogenase; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors
D011994:Recombinant Proteins; D009243:NAD; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase