Navegando por Autor "Salgado, Rafael L."
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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 Identification of an emergent porcine circovirus-2 in vaccinated pigs from a brazilian farm during a postweaning multisystemic wasting syndrome outbreak(Genome Announcements, 2014-03-20) Salgado, Rafael L.; Vidigal, Pedro M. P.; Souza, Luiz F. L. de; Onofre, Thiago S.; Gonzaga, Natália F.; Eller, Monique R.; Bressan, Gustavo C.; Fietto, Juliana L. R.; Almeida, Márcia R.; Silva Júnior, AbelardoThree porcine circovirus-2 strains were isolated from pigs on a Brazilian farm during an outbreak, indicating a vaccine failure. They present identical genomic sequences, with high identities to other isolates that were also related to vaccination failures, supporting the recent theory about an antigen drift being associated with vaccine failures throughout the world.Item Temperature modulates the production and activity of a metalloprotease from Pseudomonas fluorescens 07A in milk(Journal of Dairy Science, 2018-02) Alves, Maura P.; Salgado, Rafael L.; Eller, Monique R.; Dias, Roberto Sousa; Paula, Sérgio Oliveira de; Carvalho, Antonio Fernandes deThis work evaluated the expression and activity of a metalloprotease released by Pseudomonas fluorescens 07A in milk. Low relative expression of the protease by the strain was observed after incubation for 12 h at 25°C while the strain was in the logarithmic growth phase. After 24 h, protease production significantly increased and remained constant for up to 48 h, a time range during which the strain remained in the stationary phase. Conversely, at refrigeration temperatures, at 12 h the strain was still in the lag phase and expressed the protease at higher levels than when the logarithmic phase was reached. Casein fractions were highly degraded by P. fluorescens 07A, the purified protease, and the bacterial pellet on d 7 of incubation at 25°C and to a lesser extent at 10°C for the sample incubated with the bacterium. Heat treatment at 90°C for 5 min completely inactivated the proteolytic activity of the purified protease and the bacterial pellet. This work contributes to the knowledge about the conditions of milk storage that influence the production and activity of this extracellular metalloprotease. The results demonstrate the need to find alternative strategies to control the synthesis and activity of proteolytic enzymes in the dairy industry to ensure the quality of processed products.