The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53,269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80 degrees C; ammonia-dependent reaction, 90 degrees C).

Methyl parathion hydrolase (MPH) from a methyl parathion-degrading Burkholderia cepacia indigenous to Thailand was purified to apparent homogeneity by three steps of column chromatography using Resource S, Sephadex G100, and Octyl Sepharose 4FF columns. Its molecular mass was determined to be 35 kDa, and the pI to be 8.5. The recombinant plasmid pGT1, containing the MPH-encoding gene, mpdB, cloned into pGEX-4T-2 was over-expressed in Escherichia coli as GST-MPH fusion protein. The recombinant MPH was purified to homogeneity by a single step, using GSTPrep FF affinity column, with the molecular mass identical to that of the native enzyme. The purified enzyme had the specific activity of about 1,600 unit mg(-1) protein and the yield of about 75%, a 39-fold increase in recovery compared to that of the native enzyme. The optimal temperature and pH were 25°C and 9.0, respectively. The MPH was stable, with its activity unchanged for 48 h at 4°C, and reduced to 50% after 5 h and to 45% after 48 h at 25°C. The enzyme activity remained 80-90% after 8-15 h at pH 6-7. Cd(2+), Co(2+), and Zn(2+) ions at the concentration of 1 mM enhanced the activity; while sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and ethylenediaminetetraacetate (EDTA) reduced it. The enzyme also showed cross reactivity with other insecticides within the organophosphate group, and the kinetic parameters for individual substrates were investigated. Since MPH from B. cepacia has wide potential applications in detoxification and detection of organophosphate compounds, this study provides important basis for its future use.

N-Terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) lacking 5'-3' exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR). In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase, delta Tth polymerase gave readable sequencing patterns which extend up to more than 500 bases from the primer site on cycle sequencing and automated sequencing. The delta Tth polymerase was used for the standard and mutagenic PCR, and net amplification of the DNA and the mutations accumulated during PCR were analyzed. Under mutagenic PCR, the mutation rates were 7.0 x 10(-4) (Tth) and 8.3 x 10(-4) (delta Tth) per nucleotide per cycle of amplification, which were 4-9 times higher than the rates under standard PCR.

No abstract

The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric form (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60°C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.

5-Aminolevulinic acid (ALA) is an intermediate in the biosynthesis of tetrapyrroles. Its current production is expensive. We have developed a low-cost medium for Propionibacterium acidipropionici to produce extracellular ALA. When grown at 35 °C on a medium containing 3 % (w/v) food-grade sodium lactate supplemented with 18 g glycine/l, 4.05 g succinate/l, 1.8 g glucose/l, pH 7, it produced ALA up to 7.7 g/l over 6 days. Plant-growth promoting activity assays showed that the ALA was biologically active.

The glutamate dehydrogenase from Pyrococcus sp. KOD1 (Pk-GDH) was purified to homogeneity and its enzymatic characteristics for glutamate production were analyzed. Pk-GDH exhibited glutamate producing activity from glutamine, which was found in some higher eukaryotic GDHs. However, the k t /K m values for ammonia and glutamine were 15U10 Q min 3I mM and 0.4U10 Q min 3I mM, respectively, indicating that the enzyme utilizes ammonia predominantly for glutamate production. Kinetic parameters of Pk-GDH were compared with those of Pk-GltA, which was previously identified as a glutamate synthase homologue. The turnover number for glutamine of Pk-GltA (4.1U10 Q min 3I mM) was 10-fold higher than that of Pk-GDH (0.4U10 Q min 3I mM). Therefore, Pk-GDH is less efficient than Pk-GltA for glutamate production. Pk-GDH utilized both NADPH and NADH as a cofactor, Pk-GltA utilized only NADPH. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

No abstract