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Characterization and Heterologous Gene Expression of a Novel Esterase from Lactobacillus casei CL96. Young J. Choi, 2004.A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp . The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (PmxaF), and alcohol oxidase (AOX1) promoters, respectively . The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558 . E . coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S · tag peptide . The recombinant L . casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose . The optimum pH and temperature of the purified enzyme were 7.0 and 37°C, respectively . Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C8), with Km and kcat values of 14 ± 1.08 µM and 1,245 ± 42.3 S–1, respectively . Flagella and Motility in Actinobacillus pleuropneumoniae. Erasmo Negrete-Abascal, 2003.Actinobacillus pleuropneumoniae has been considered nonmotile and nonflagellate . In this work, it is demonstrated that A . pleuropneumoniae produces flagella composed of a 65-kDa protein with an N-terminal amino acid sequence that shows 100% identity with those of Escherichia coli, Salmonella, and Shigella flagellins . The DNA sequence obtained through PCR of the fliC gene in A . pleuropneumoniae showed considerable identity (93%) in its 5' and 3' ends with the DNA sequences of corresponding genes in E . coli, Salmonella enterica, and Shigella spp . The motility of A . pleuropneumoniae was observed in tryptic soy or brain heart infusion soft agar media, and it is influenced by temperature . Flagella and motility may be involved in the survival and pathogenesis of A . pleuropneumoniae in pigs . 5-Keto-D-Gluconate Production Is Catalyzed by a Quinoprotein Glycerol Dehydrogenase, Major Polyol Dehydrogenase, in Gluconobacter Species. Kazunobu Matsushita, 2003.Acetic acid bacteria, especially Gluconobacter species, have been known to catalyze the extensive oxidation of sugar alcohols (polyols) such as D-mannitol, glycerol, D-sorbitol, and so on . Gluconobacter species also oxidize sugars and sugar acids and uniquely accumulate two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, in the culture medium by the oxidation of D-gluconate . However, there are still many controversies regarding their enzyme systems, especially on D-sorbitol and also D-gluconate oxidations . Recently, pyrroloquinoline quinone-dependent quinoprotein D-arabitol dehydrogenase and D-sorbitol dehydrogenase have been purified from G . suboxydans, both of which have similar and broad substrate specificity towards several different polyols . In this study, both quinoproteins were shown to be identical based on their immuno-cross-reactivity and also on gene disruption and were suggested to be the same as the previously isolated glycerol dehydrogenase (EC 1.1.99.22) . Thus, glycerol dehydrogenase is the major polyol dehydrogenase involved in the oxidation of almost all sugar alcohols in Gluconobacter sp . In addition, the so-called quinoprotein glycerol dehydrogenase was also uniquely shown to oxidize D-gluconate, which was completely different from flavoprotein D-gluconate dehydrogenase (EC 1.1.99.3), which is involved in the production of 2-keto-D-gluconate . The gene disruption experiment and the reconstitution system of the purified enzyme in this study clearly showed that the production of 5-keto-D-gluconate in G . suboxydans is solely dependent on the quinoprotein glycerol dehydrogenase . Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5'-Exonuclease, and Hybridization Probe Assays. D. T. Newby, 2003.Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence . A number of different approaches can be used to generate the fluorescence signal . Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus . The three assays utilized the same amplification primers to produce an identical amplicon . This amplicon spans a region of the B . abortus genome that includes portions of the alkB gene and the IS711 insertion element . All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg) . However, the greatest specificity was achieved with the hybridization probe assay .
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