ABSTRACT
El género Pseudomonas es una fuente importante de proteasas; sin embargo, su uso está restringido en la industria alimentaria. El clonaje permite aprovechar la capacidad catalítica de estas enzimas mediante su producción en microorganismos inocuos. Por otro lado, las leguminosas son fuentes ricas en proteínas, a partir de las cuales se pueden obtener compuestos con valor agregado mediante procesos de hidrólisis enzimática. En este estudio, se produjo y caracterizó una proteasa recombinante (PT4) alcalina y termoestable de Pseudomonas aeruginosa M211, para la obtención de hidrolizados proteicos de leguminosas. Para ello, el gen de la proteasa se clonó en el vector pJET1.2/blunt utilizando E. coli DHalfa como hospedero. El análisis de la secuencia nucleotídica parcial de la proteasa indicó un 99 % de similitud con Peptidasas de la Familia M4 de Pseudomonas aeruginosa. La enzima recombinante presentó un peso molecular de 80 kDa, demostró ser activa y estable en condiciones alcalinas y termófilas con un pH y temperatura óptimos de 8 y 60 °C, respectivamente, y fue inhibida por EDTA. Además, hidrolizó proteínas de semillas de Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis, obteniéndose fracciones peptídicas menores a 40 kDa. Esta proteasa recombinante se podría utilizar en la elaboración de hidrolizados proteicos funcionales a partir proteínas de distintas fuentes y residuos agroalimentarios.
The genus Pseudomonas is an important source of proteases; however, in the food industry the use of this bacterium is restricted. Cloning allows for the use of the proteolytic activity of Pseudomonas proteases through their production in innocuous microorganisms. Leguminous are protein-rich sources from which value-added compounds can be obtained through enzymatic hydrolysis. In this study, an alkaline and thermostable recombinant protease (PT4) from Pseudomonas aeruginosa M211 was cloned and characterized in order to obtain protein hydrolysates from leguminous. Therefore, protease gene was cloned into the pJET1.2 / blunt vector using E. coli DHalpha as a host. Analysis of protease partial nucleotide sequence showed 99% homology with Peptidases M4 Family from Pseudomonas aeruginosa. The molecular weight of the recombinant enzyme was 80 kDa, it was active and stable under alkaline and thermophilic conditions, presented an optimum pH and temperature of 8 and 60 °C, respectively, and was inhibited by EDTA. In addition, it hydrolysed Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis proteins, obtaining peptide fractions less than 40 kDa. This recombinant protease could be used in the elaboration of functional hydrolysates using protein from different sources and agricultural waste.
Subject(s)
Peptide Hydrolases/metabolism , Protein Hydrolysates/metabolism , Pseudomonas aeruginosa/enzymology , Recombinant Proteins/metabolism , Peptide Hydrolases/genetics , Temperature , Enzyme Stability , Cloning, Molecular , Hydrogen-Ion Concentration , FabaceaeABSTRACT
It is well known that the risk of development of gastric cancer (GC) in Helicobacter pylori-infected patients depends on several factors. Thus, the aim of this study was to investigate the effect of proinflammatory cytokine gene polymorphisms for IL-1β, IL-1RN and TNF-α on the development of GC in a Brazilian population. A total of 202 biopsies obtained from Brazilian patients with chronic gastritis and GC were included in the study. Infection with H. pylori cagA+ was determined by the polymerase chain reaction (PCR) as previously described. IL-1β, IL-1RN and TNF-α polymorphism genotyping was performed by restriction fragment length polymorphism PCR. Associations between gene polymorphisms, clinical diseases and virulence markers were evaluated using either the χ² test or the Fisher exact test. Our results demonstrated that the IL-1β -511 C/C and IL-1β -511 C/T alleles were associated with chronic gastritis in H. pylori-positive patients (P = 0.04 and P = 0.05, respectively) and the IL-1β -511 C/C genotype was associated with GC (P = 0.03). The frequency of IL-1RN alleles from patients with chronic gastritis and GC indicated that there was no difference between the genotypes of the groups studied. Similar results were found for TNF-α -308 gene polymorphisms. Our results indicate that the IL-1β -511 C/C and C/T gene polymorphisms are associated with chronic gastritis and GC development in H. pylori-infected individuals.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Gastritis/genetics , Helicobacter pylori , Helicobacter Infections/genetics , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin-1beta/genetics , Stomach Neoplasms/genetics , Tumor Necrosis Factor-alpha/genetics , Alleles , Brazil , Chronic Disease , DNA, Bacterial/analysis , Genetic Predisposition to Disease , Genotype , Gastritis/immunology , Gastritis/microbiology , Helicobacter Infections/immunology , Polymorphism, Genetic , Polymorphism, Restriction Fragment Length , Stomach Neoplasms/immunology , Stomach Neoplasms/microbiologyABSTRACT
Our goal was to identify herbivory patterns from two insect guilds associated with Cnidoscolus quercifolius in a tropical deciduous forest in northeastern Brazil. We sampled four different habitats: (1) forest edge, (2) mesic (near to the perennial water source), (3) forest interior and (4) rupestrian fields. Habitat edge had lower leaf damage than rupestrian, mesic and forest interior habitats. Nevertheless, abundance of galls at the edge habitat was higher than at mesic, forest interior and/or rupestrian habitats. There was no difference in gall mortality by natural enemies among the four habitats sampled, demonstrating the absence of any influence of top-down controls related to abundance of galls. Trophic relationships were not related to the patterns of distribution among habitats of two insect herbivorous guilds associated with C. quercifolius. Our results demonstrated that environmental heterogeneity of dry forests can significantly alter important ecological interactions and experimental studies are needed to better understand the mechanisms responsible for differences in herbivory among habitats.
Nosso objetivo foi identificar os padrões de herbivoria de duas guildas de insetos herbívoros associadas à Cnidoscolus quercifolius, em uma floresta estacional seca do nordeste do Brasil. Foram amostrados quatro diferentes habitats: (1) borda florestal; (2) mésico (próximo a uma fonte de água perene); (3) interior florestal, e (4) rupestre. O habitat borda apresentou uma quantidade de dano foliar inferior aos habitats rupestre, mésico e interior florestal. Diferentemente, a abundância de galhas no habitat borda foi superior aos habitats mésico, interior florestal e rupestre. Não houve diferença na mortalidade de galhas por inimigos naturais entre os quatro habitats, o que revela ausência de influência de controles top-down relacionados à abundância de galhas. Relações tróficas não parecem influenciar os padrões de distribuição entre habitats das duas guildas de insetos herbívoros associadas à C. quercifolius. Nossos resultados demonstram que a heterogeneidade ambiental de florestas secas pode alterar significativamente importantes interações ecológicas e que trabalhos experimentais são necessários para uma melhor compreensão dos mecanismos responsáveis pelas diferenças nas taxas de herbivoria entre habitats.