The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius (original) (raw)

1994, European Journal of Biochemistry

Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IF0 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4°C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KC1. It retained about 90% of activity on incubation at 70°C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, Lleucine, NADH, 2-oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 11 8 mM, respectively, from initial-velocity analyses. The enzyme showed p r o 3 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 (Mr 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. Leucine dehydrogenase and valine dehydrogenase are NAD-dependent dehydrogenases which catalyze the reversible deamination of branched-chain L-amino acids to their 0x0 analogs. Leucine dehydrogenase widely occurs in endospore-forming bacteria such as bacilli [l, 21 and clostridia [3], and a non-sporing bacterium, Corynebacterium pseudodiphtheriticum [l, 41. The enzyme was purified from several microorganisms such as Bacillus sphaericus [l], and Bacillus cereus [ 5 ] , and also thermophiles, Bacillus stearothermophilus [6], Clostridium themzoaceticum [3], Bacillus caldolyticus [7] for their characterization and application. We have shown that the B. stearothermophilus enzyme is more useful for the industrial synthesis of aliphatic L-amino acids, and the analyses of L-branched-chain amino acids, their 0x0 analogs and the activity of serum leucine aminopeptidase owing to the higher stability [8, 91. The thermostable enzyme gene was cloned into Escherichia coli and the high thermostability facilitated the large-scale purification of the enzyme from E.