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Lecture 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°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%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 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. [ABSTRACT FROM AUTHOR]

Alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile. Bacillus sphaericus DSM 461, b ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme has a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75°C for at least 60 min and with incubation in pH 5.5-9.5 at 75°C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20°C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate. 2-oxobutryrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM. [ABSTRACT FROM AUTHOR]

4-α-Glucanotransferase was purified from cells of Thermococcus litoralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optimal temperature for its activity was 90°C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligosaccharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive saccharides were produced. On incubation of amylose with the T. litoralis enzyme, glucoamylase-resistant but α-amylase—sensitive molecules were produced, but the amount of reducing sugar showed only slight increases. These results indicate that the T. litoralis enzyme catalyzes not only intermolecular transglycosylation to produce linear α-l,4-glucan, but also intramolecular transglycosylation to produce cyclic α-l,4-glucan (cycloamylose), similarly to potato 4-α-glucanotransferase (called disproportionating enzyme). The gene encoding the Z litoralis 4-α-glucanotransferase was cloned, sequenced and expressed in Escherichia coli. The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77 883 Da. The amino acid sequence of the T. titoralis enzyme showed high similarity with those of α-amylases of Pyrococcus furiosus, a hyperthermophilic archaeon, and Dictyoglomus thermophilum, an extremely thermophilic bacterium, but little similarity with those of other known 4-α-glucanotransferases. [ABSTRACT FROM AUTHOR]