Bioquímica e Biologia Molecular
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11837
Navegar
Item Characterization of a heat-resistant extracellular protease from Pseudomonas fluorescens 07A shows that low temperature treatments are more effective in deactivating its proteolytic activity(Journal of Dairy Science, 2016-10) Alves, Maura P.; Salgado, Rafael L.; Eller, Monique R.; Vidigal, Pedro Marcus P.; Carvalho, Antonio Fernandes deThis work discusses the biological and biochemical characterization of an extracellular protease produced by Pseudomonas fluorescens. The enzyme has a molecular weight of 49.486 kDa and hydrolyzes gelatin, casein, and azocasein, but not BSA. Its maximum activity is found at 37°C and pH 7.5, but it retained almost 70% activity at pH 10.0. It was shown to be a metalloprotease inhibited by Cu2+, Ni2+, Zn2+, Hg2+, Fe2+, and Mg2+, but induced by Mn2+. After incubation at 100°C for 5 min, the enzyme presented over 40% activity, but only 14 to 30% when submitted to milder heat treatments. This behavior may cause significant problems under conditions commonly used for the processing and storage of milk and dairy products, particularly UHT milk. A specific peptide sequenced by mass spectrometer analysis allowed the identification of gene that encodes this extracellular protease in the genome of Pseudomonas fluorescens 07A strain. The enzyme has 477 AA and highly conserved Ca2+- and Zn2+-binding domains, indicating that Ca2+, the main ion in milk, is also a cofactor. This work contributes to the understanding of the biochemical aspects of enzyme activity and associates them with its sequence and structure. These findings are essential for the full understanding and control of these enzymes and the technological problems they cause in the dairy industry.Item Genotypic and phenotypic characterization of Staphylococcus aureus causing persistent and nonpersistent subclinical bovine intramammary infections during lactation or the dry period(Journal of Dairy Science, 2015-01) Klein, R. C.; Veh, K. A.; Ster, C.; Keefe, G.; Lacasse, P.; Scholl, D.; Roy, J. P.; Haine, D.; Dufour, S.; Talbot, v; Ribon, A. O. B.; Malouin, F.Staphylococcus aureus is a significant pathogen frequently causing persistent intramammary infections (IMI) in dairy cows. We compared some genotypic and phenotypic characteristics of 285 strains collected from quarter milk samples from cows with persistent and nonpersistent subclinical IMI across Canada. Variable number of tandem repeats typing was used to infer the persistence of the same S. aureus strain in 3 consecutive quarter milk samples collected at intervals of 3 wk during lactation or before and after dry-off. All first isolates of the series were used as the representative strains from persistent IMI and were compared with nonpersistent strains for the presence of genes seg, sen, sec, and tst as well as by spa typing. Biofilm production in vitro and hld-RNAIII expression levels were also quantified. The gene seg was associated with a reduction in the likelihood of the bacteria to cause a persistent IMI during lactation. Strains persisting through the dry period produced significantly more biofilm in vitro than strains that do not persist after calving. Also, we showed that strains expressing more hld were more likely to be non- persistent during either lactation or through the dry period. Three spa types were predominant (t529, t267, and a novel type: t13401). In the strains studied, the spa type t529 was the most frequent, and 97.0% of the strains of this spa type carried both sen and seg. Strains from the spa type t529 were less likely to cause a persis- tent IMI in the dry period. Most (86.7%) of the strains of the novel spa type (t13401) were negative for seg, sen, or both and produced significantly more biofilm in vitro than t529 and t267. The present study expanded our current knowledge on the genotypic and phenotypic traits of S. aureus strains recovered from persistent and nonpersistent IMI in Canada.