RESUMO

Defects in the crystalline structure of quartz facilitate the connection with the alkali hydroxides, since under a high alkalinity condition (e.g., in concrete), the Si-O bonds of quartz are easily broken. This study set out to investigate the influence of the deformation structures of quartz on its susceptibility to the alkali⁻silica reaction. A granite, a protomylonite, and a mylonite were selected for this study. Using optical microscopy, the quartz grains contained in these rocks were quantified and their texture characterized. The quartz samples extracted from the rocks were analyzed by magnetic nuclear resonance, to evaluate their potential for dissolving silica as well as changes in their atomic scale before and after the reaction with alkali hydroxides. These analyses were compared with the results of the accelerated mortar bar test. The study showed that the quartz with intense undulatory extinction and deformation bands denotes the most favorable condition to the development of the alkali⁻silica reaction. However, on an atomic scale, the slightly deformed grains were highly prone to react. Thus, in a high alkalinity condition, over a long period of time, any quartz tends to develop the alkali⁻silica reaction, regardless of the deformation degree of the grain.

RESUMO

Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. ß-glucosidases (EC 3.2.1.21) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 ß-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-ß-d-glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-ß-d-xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 Šresolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/ß sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.

Assuntos

Bifidobacterium adolescentis/enzimologia , Probióticos , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato

RESUMO

ß-glucosidases are glycoside hydrolases able to cleave small and soluble substrates, thus producing monosaccharides. These enzymes are distributed among families GH1, GH2, GH3, GH5, GH9, GH30 and GH116, with GH1 and GH3 being the most relevant families with characterized enzymes to date. A recent transcriptomic analysis of the fungus Trichoderma harzianum, known for its increased ß-glucosidase activity as compared to Trichoderma reesei, revealed two enzymes from family GH1 with high expression levels. Here we report the cloning, recombinant expression, purification and crystallization of these enzymes, ThBgl1 and ThBgl2. A close inspection of the enzymatic activity of these enzymes surprisingly revealed a marked difference between them despite the sequence similarity (53%). ThBgl1 has an increased tendency to catalyze transglycosylation reaction while ThBgl2 acts more as a hydrolyzing enzyme. Detailed comparison of their crystal structures and the analysis of the molecular dynamics simulations reveal the presence of an asparagine residue N186 in ThBgl2, which is replaced by the phenylalanine F180 in ThBgl1. This single amino acid substitution seems to be sufficient to create a polar environment that culminates with an increased availability of water molecules in ThBgl2 as compared to ThBgl1, thus conferring stronger hydrolyzing character to the former enzyme.

Assuntos

Trichoderma/enzimologia , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Glicosilação , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Trichoderma/metabolismo , beta-Glucosidase/isolamento & purificação

RESUMO

Here the statistics concerning X-ray data processing and structure refinement are given, together with the substrate preference analysis for ThBgl1 and ThBgl2. Finally, the analysis of the influence of temperature and pH on the activities of both enzymes are shown.