Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3) from the hyperthermophilic Archeon Pyrococcus furiosus was purified to homogeneity by chromatography on anion-exchange, molecular-exclusion and hydrophobic-interaction media. The purified native enzyme had an M(r) of 270,000 +/- 15,000 and was shown to be a hexamer with identical subunits of M(r) 46,000. The enzyme was exceptionally thermostable, having a half-life of 3.5 to more than 10 h at 100 degrees C, depending on the concentration of enzyme. The Km of the enzyme for ammonia was high (9.5 mM), indicating that the enzyme is probably active in the deaminating, catabolic direction. The coenzyme utilization of the enzyme resembled the equivalent enzymes from eukaryotes rather than eubacteria, since both NADH and NADPH were recognized with high affinity. The enzyme displayed a preference for NADP+ over NAD+ that was more pronounced at low assay temperatures (50-70 degrees C) compared with the optimal temperature for enzyme activity, 95 degrees C.     

Screening of yeast strains capable of hyperproducing amylolytic enzymes

Summary Fifteen strains of yeast, which produced an extracellular amylolytic enzymes, were isolated from nature. One of them produced more than 100 times the enzyme activity in comparison with the 14 strains and the extremely hyperproducing strain of yeast was identified asCandida sp. 347. Paper chromatograms of the amylolytic enzyme demonstrated activity of amyloglucosidase. The optimum pH for activity of the enzyme was 5.5–6.0 and optimum temperature was 60°C.     

A tripartite HIV-1 tat-env-rev fusion protein.

A 26 kd protein reactive with antiserum to the transactivator tat of the Human Immunodeficiency Virus Type 1 (HIV-1) has been detected in virus producing cells. The 26 kd protein is shown to be a tripartite fusion protein including coding sequences of the tat, envelope (env) and regulator of virion expression (rev) genes. Fusion of these coding sequences occurs by use of a previously undescribed exon within env. This 26 kd protein, designated tnv, has tat but no rev activity detectable with the assay used. The existence of other less abundant tat and rev related proteins in HIV-1 producing cells is also noted.     

Identification of the mpl gene encoding UDP-N-acetylmuramate: L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase in Escherichia coli and its role in recycling of cell wall peptidoglycan.

A gene, mpl, encoding UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelat e ligase was recognized by its amino acid sequence homology with murC as the open reading frame yjfG present at 96 min on the Escherichia coli map. The existence of such an enzymatic activity was predicted from studies indicating that reutilization of the intact tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate occurred and accounted for well over 30% of new cell wall synthesis. Murein tripeptide ligase activity could be demonstrated in crude extracts, and greatly increased activity was produced when the gene was cloned and expressed under control of the trc promoter. A null mutant totally lacked activity but was viable, showing that the enzyme is not essential for growth.     

Mapping of the entomocidal fragment of Spodoptera -specific Bacillus thuringiensis toxin CryIC

 Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI poteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins. Received: 15 April 1996/Accepted: 10 July 1996     

Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Transfer of conjugative plasmid pAMβ1 from Lactococcus lactis to mouse intestinal bacteria

Conjugal transfer of plasmid pAMβ1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 × 10⁻³ and with lower frequencies to other species. In vivo , using gastric intubation with the pAMβ1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis . However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 × 10⁻³, in mice whose anus was artificially closed after gastric intubation with pAMβ1-bearing Lactococcus lactis . These results demonstrate clearly that pAMβ1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.     

Charactererization of novel non-toxic Bacillus thuringiensis isoloates from Korea

J.Y. ROH, H.W. PARK, B.R. JIN, H.S. KIM, Y.M. YU AND S.K. KANG. 1996. Four Bacillus thuringiensis isolates from soil samples produced parasporal inclusions which were non-toxic to insects. The isolates were named B. thuringiensis NTB-1, NTB-2, NTB-3 and NTB-4. The parasporal inclusions were shown to be ovoid by phase contrast and scanning electron microscopy. The serotypes of the four isolates were determined by agglutination using 33 antisera; NTB-1 and NTB-4 seemed to be subsp. isruelensis ,and NTB-2 seemed to be subsp. pondzcheriensis . NTB-3 did not react with the 33 antisera. However, comparison of parasporal protein and plasmid DNA patterns of the four isolates with those of 15 known non-toxic B. thuringiensis strains demonstrated that the four isolates are novel.     

Synthesis of biodegradable emulsifier using lipase in anhydrous pyridine

Summary Synthesis of a bioemulsifier using a lipase from Pseudomonas sp. with fructose and vinyl laurate was carried out in anhydrous pyridine. The synthetic product was identified as laurylfructose with an emulsifying activity on various hydrocarbons, edible oils and petroleum oils. The compound reduced the surface tension of distilled water from 72 mN/m to 29 mN/m and the interfacial tension of water/n-hexadecane from 50 mN/m to 6 mN/m.