Filogenia molecular das enzimas isocitrato liase e malato sintase e sua evolução em Viridiplanta

The plant metabolism consists of a complex network of physical and chemical events resulting in photosynthesis, respiration, synthesis and degradation of organic compounds. This is only possible due to the different kinds of responses to many environmental variations that a plant could be subject through evolution, leading also to conquering new surroundings. The glyoxylate cycle is a metabolic pathway found in glyoxysomes plant, which has unique role in the seedling establishment. Considered as a variation of the citric acid cycle, it uses an acetyl coenzyme A molecule, derived from lipids beta-oxidation to synthesize compounds which are used in carbohydrate synthesis. The Malate synthase (MLS) and Isocitrate lyase (ICL) enzyme of this cycle are unique and essential in regulating the biosynthesis of carbohydrates. Because of the absence of decarboxylation steps as rate-limiting steps, detailed studies of molecular phylogeny and evolution of these proteins enables the elucidation of the effects of this route presence in the evolutionary processes involved in their distribution across the genome from different plant species. Therefore, the aim of this study was to establish a relationship between the molecular evolution of the characteristics of enzymes from the glyoxylate cycle (isocitrate lyase and malate synthase) and their molecular phylogeny, among green plants (Viridiplantae). For this, amino acid and nucleotide sequences were used, from online repositories as UniProt and Genbank. Sequences were aligned and then subjected to an analysis of the best-fit substitution models. The phylogeny was rebuilt by distance methods (neighbor-joining) and discrete methods (maximum likelihood, maximum parsimony and Bayesian analysis). The identification of structural patterns in the evolution of the enzymes was made through homology modeling and structure prediction from protein sequences. Based on comparative analyzes of in silico models and from the results of phylogenetic inferences, both enzymes show significant structure conservation and their topologies in agreement with two processes of selection and specialization of the genes. Thus, confirming the relevance of new studies to elucidate the plant metabolism from an evolutionary perspective

Bioinformatics analysis of the Pedobacter sp. NL19 genome

The last decades of the 20th century defined the genetic engineering advent, climaxing in the development of techniques, such as PCR and Sanger sequencing. This, permitted the appearance of new techniques to sequencing whole genomes, identified as next-generation sequencing. One of the many applications of these techniques is the in silico search for new secondary metabolites, synthesized by microorganisms exhibiting antimicrobial properties. The peptide antibiotics compounds can be classified in two classes, according to their biosynthesis, in ribosomal or nonribosomal peptides. Lanthipeptides are the most studied ribosomal peptides and are characterized by the presence of lanthionine and methylanthionine that result from posttranslational modifications. Lanthipeptides are divided in four classes, depending on their biosynthetic machinery. In class I, a LanB enzyme dehydrate serine and threonine residues in the C-terminus precursor peptide. Then, these residues undergo a cyclization step performed by a LanC enzyme, forming the lanthionine rings. The cleavage and the transport of the peptide is achieved by the LanP and LanT enzymes, respectively. Although, in class II only one enzyme, LanM, is responsible for the dehydration and cyclization steps and also only one enzyme performs the cleavage and transport, LanT. Pedobacter sp. NL19 is a Gram-negative bacterium, isolated from sludge of an abandon uranium mine, in Viseu (Portugal). Antibacterial activity in vitro was detected against several Gram-positive and Gram-negative bacteria. Sequencing and in silico analysis of NL19 genome revealed the presence of 21 biosynthetic clusters for secondary metabolites, including nonribosomal and ribosomal peptides biosynthetic clusters. Four lanthipeptides clusters were predicted, comprising the precursor peptides, the modifying enzymes (LanB and LanC), and also a bifunctional LanT. This result revealed the hybrid nature of the clusters, comprising characteristics from two distinct classes, which are poorly described in literature. The phylogenetic analysis of their enzymes showed that they clustered within the bacteroidetes clade. Furthermore, hybrid gene clusters were also found in other species of this phylum, revealing that it is a common characteristic in this group. Finally, the analysis of NL19 colonies by MALDI-TOF MS allowed the identification of a 3180 Da mass that corresponds to the predicted mass of a lanthipeptide encoded in one of the clusters. However, this result is not fully conclusive and further experiments are needed to understand the full potential of the compounds encoded in this type of clusters. In conclusion, it was determined that NL19 strain has the potential to produce diverse secondary metabolites, including lanthipeptides that were not functionally characterized so far.

Purificação, caracterização e atividade bioinseticida de um inibidor de tripsina de sementes de Crotalaria pallida

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulphate fractionation, affinity chromatography on immobilized Trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named ITC, had Mr of 32.5 kDa by SDS-PAGE and was composed by two subunits with 27.7 and 5.6 kDa linked by disulphide bridges, a typical characteristic of Kunitz-Inhibitor family. ITC was stable until 50°C, and at 100°C its residual activity was of about 60%. Also, ITC was stable at pHs 2 to 12. The inhibition of trypsin by ITC was non-competitive, with a Ki of 8,8 x 10-7M. ITC inhibits weakly other serine proteinases such as chymotrypsin and elastase. The inhibition of papain (44% of inhibition), a cysteine proteinase was an indicative of the bi-functionality of ITC. In vitro assays against digestive proteinases from several Lepdoptera, Diptera and Coleoptera pests were made. ITC inhibited in 100% digestive enzymes of Ceratitis capitata (fruit fly), Spodoptera frugiperda and Alabama argillacea, the last one being a cotton pest. It also inhibited in 74.4% Callosobruchus maculatus (bean weevil) digestive enzymes, a Coleoptera pest. ITC, when added in artificial diet models, affected weakly the development of C. capitata larvae and it had a WD50 of 2.65% to C. maculatus larvae

Influência dos fatores abióticos na composição química dos óleos essenciais.

Os óleos essenciais constituem um dos mais importantes grupos de matérias primas para as indústrias de alimentos, farmacêutica, perfumaria e afins. São constituídos por uma mistura complexa de diversas classes de substâncias, dentre elas os fenilpropanóides, mono e sesquiterpenos, pertencentes ao metabolismo secundários das plantas. O metabolismo secundário por sua vez pode ser influenciado, dentre outros, por fatores genéticos, climáticos (temperatura, intensidade de luz, efeito sazonal, etc.) e edáficos. Observa-se que, grande parte das espécies popularmente utilizadas, encontra-se próxima ao estado silvestre, mantendo forte interação com o ambiente. Informações sobre o efeito de condições ambientais no metabolismo secundário de plantas provêm principalmente de esforços da pesquisa para maximizar a produção de constituintes ativos de espécies medicinais e aromáticas. Como aplicação prática, avanços no sentido de compreender a influência dos fatores ambientais na regulação de biossíntese de metabólitos secundários, podem contribuir para um aumento na produção de compostos de interesse nestas espécies. Neste artigo será abordada a ação de alguns fatores abióticos no rendimento e composição de óleos essenciais, com base nos dados da literatura, sem a pretensão de esgotar este extenso tema.

Avaliação de métodos de deteção de genótipos de cianobactérias produtoras de cianotoxinas em amostras ambientais

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Estudo da associação dos genes GGR, GGPPS2 e GGPPS6 em Arabidopsis thaliana L. (Brassicaceae) a compostos alelopáticos

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Effect of post-emergent herbicides application on carotenoid concentration in green corn.

Considering the influence of herbicides in the metabolism of the carotenoids in corn, the objective of the present study was to evaluate the effect of herbicides and genotype on carotenoids concentration. The green corn hybrids BRS 1030 and P30F53 were subjected to a post-emergent herbicides application at 20 and 30 days after emergence. Carotenoids were extracted from corn grains and analyzed to quantify ?- and ?-carotene, lutein, zeaxanthin, ?-cryptoxanthin, total carotenoids (TC), and total of vitamin A carotenoids precursors (proVA). The application of foramsulfuron + iodosulfuron-methyl-sodium (40 + 2.6 g ha-1), nicosulfuron (20 g ha-1), mesotrione (120 g ha-1) and tembotrione (80 g ha-1 and 100 g ha-1) promoted higher concentration of carotenoids in fresh green corn. Lutein, zeaxanthin, ?-cryptoxanthin, ?-carotene, ?-carotene, proVA carotenoids, and TC concentration increased after foramsulfuron + iodosulfuron-methyl-sodium in late application (V5 to V6), nicosulfuron in both applications, mesotrione applied post-initial (V3 to V4), tembotrione (100 g ha-1) in both applications and tembotrione (80 g ha-1) in late post-application, at least for one hybrid. The content of carotenoids in the green corn kernels differed between ?BRS 1030? and ?P30F53?. Our results suggest a possibility of significant increase of carotenoids in green corn kernels through the handling of corn production with post-emergent herbicides.