RESUMO
A purified thermostable gellan lyase, produced by a thermophilic bacterium, Geobacillus stearothermophilus 98, was characterized in relation to its physicochemical properties. The gellan lyase was established to have a molecular weight of 216 kDa, defined by capillary gel electrophoresis. Amino acid analysis revealed high quantities of Lys, His, Ala, Val, Ile, Glx, and Pro residues. The circular dichroism revealed 45% beta-structure and practically lack of a-spiral domains. Kinetic studies showed high affinity of the enzyme to gellan as a substrate (Km = 0.21 microM). The thermal denaturation investigated by cicular dichroism showed a highly cooperative transition with a midpoint (Tm) at about 75 degrees C. A single product was identified after enzyme action on gellan. Large exothermic aggregation near Tm was observed by differential scanning calorimetry. Two types of gellan lyase crystals were reproducibly isolated.
Assuntos
Bacillus/enzimologia , Geobacillus stearothermophilus/enzimologia , Polissacarídeo-Liases/química , Aminoácidos/análise , Cromatografia em Camada Fina , Dicroísmo Circular , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Cinética , Polissacarídeo-Liases/isolamento & purificação , Polissacarídeo-Liases/metabolismo , TermodinâmicaRESUMO
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Assuntos
Biodiversidade , Lactobacillus , Fermentação , Suplementos Nutricionais/análise , Probióticos/análiseRESUMO
Fimbrial extracts from porcine enterotoxigenic Escherichia coli (ETEC) strains carrying F6 (987P) intestinal colonization factor antigen were obtained using the thermal shock method. The extracts were analyzed by SDSPAGE and immunoblotting using different fimbriae-specific antisera. Two major protein bands with molecular masses of 17.5 and 21.9 kDa were detected. The 21.9-kDa band was identified as the major subunit of F6 fimbrial antigen in strains of serogroups O9 and O141. The 17.5-kDa band was associated with porcine strains of serogroups O9 and O20 (AU)
Los extractos de fimbrias obtenidos usando el método de choque térmico a partir de cepas de origen porcino de Escherichia coli (ETEC) enterotoxigénica con el antígeno de colonización intestinal F6 (987P) fueron analizados por SDS-PAGE e immunotransferencia usando diferentes antisueros específicos frente a antígenos de fimbrias. Se detectaron dos bandas principales de proteínas de 17,5 kDa y 21,9 kDa según la cepa ensayada. La banda de 21,9 kDa fue identificada como la subunidad estructural principal del antígeno de fimbria F6 y se observó en las cepas de los serogrupos O9 yO141. La banda de 17,5 kDa se asoció a las cepas porcinas de los serogrupos O9 y O20 (AU)
Assuntos
Animais , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Fímbrias Bacterianas/microbiologia , Suínos/microbiologia , Diarreia/microbiologiaRESUMO
Fimbrial extracts from porcine enterotoxigenic Escherichia coli (ETEC) strains carrying F6 (987P) intestinal colonization factor antigen wereobtained using the thermal shock method. The extracts were analyzed by SDSPAGE and immunoblotting using different fimbriae-specific antisera. Two major protein bands with molecular masses of 17.5 and 21.9 kDa were detected. The 21.9-kDa band was identified as the major subunit of F6 fimbrial antigen in strains of serogroups O9 and O141. The 17.5-kDa band was associated with porcine strains of serogroups O9 and O20.
