Lignocellulosic residues from bioethanol production: a novel source of biopolymers for laccase immobilization.

RSC Advances, 13(20) , 13463–13471 (doi.org/10.1039/d3ra01520c )

Vázquez, V., Giorgi, V., Bonfiglio, F., Menéndez, P., Gioia, L., Ovsejevi, K.

The full utilization of the main components in the lignocellulosic biomass is the major goal from a biorefinery point of view, giving not only environmental benefits but also making the process economically viable. In this sense the solid residue obtained in bioethanol production after steam explosion pretreatment, enzymatic hydrolysis, and fermentation of the lignocellulosic biomass, was studied for further valorization. Two different residues were analyzed, one generated by the production of cellulosic ethanol from an energy crop such as switchgrass (Panicum virgatum) and the other, from wood (Eucalyptus globulus). The chemical composition of these by-products showed that they were mainly composed of lignin with a total content range from 70 to 83% (w/w) and small amounts of cellulose and hemicellulose. The present work was focused on devising a new alternative for processing these materials, based on the ability of the ionic liquids (IL) to dissolve lignocellulosic biomass. The resulting mixture of biopolymers and IL constituted the raw material for developing new insoluble biocatalysts. Active hydrogels based on fungal laccase from Dichostereum sordulentum 1488 were attained. A multifactorial analysis of the main variables involved in the immobilization process enabled a more direct approach to improving hydrogel-bound activity. These hydrogels achieved a 97% reduction in the concentration of the estrogen ethinylestradiol, an emerging contaminant of particular concern due to its endocrine activity. The novel biocatalysts based on fungal laccase entrapped on a matrix made from a by-product of second-generation bioethanol production presents great potential for performing heterogeneous catalysis offering extra value to the ethanol biorefinery

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Production of antioxidant whey hydrolysate using proteolytic extracts of Araujia sericifera var. hortorum latex

Biocatalysis and Agricultural Biotechnology, 44, 102453 (doi.org/10.1016/j.bcab.2022.102453)

Barros, M., Villadóniga, C., Cantera, A.M.B.

Proteins in whey from casein production were hydrolyzed with proteolytic extracts from the latex of Araujia sericifera var. hortorum. Hydrolysate fractions that were partially purified by RP-HPLC and evaluated using the ORAC method resulted in an antioxidant capacity of 112.4 ± 4.2 Trolox equivalents (μM). LC–MS/MS analysis of peptide fractions showed that peptides derived from β-lactoglobulin could be responsible for antioxidant activity. The sum of these results along with the fact that a controlled and reproducible production process was used, involving the use of natural resources and reaction conditions without excessive costs at an industrial scale, makes a promising contribution to improving antioxidant dairy products.

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Prospection of indigenous yeasts from Uruguayan Tannat vineyards for oenological applications

International Microbiology 25(4):733-744 ( doi: 10.1007/s10123-022-00257-6 )

Morera. G., de Ovalle, S., González-Pombo, P.

Prospection of yeasts from oenological environments can provide knowledge of new native strains that are capable of fermenting must and positively influence the composition and sensory characteristics of the wine. This work addressed the biotechnological characterization of indigenous yeasts of Tannat must, an emblematic and widespread vineyard of Uruguay. Fifty-three yeast isolates were morphologically characterized and further identified by amplification and sequencing of ITS and D1-D2 regions, grouping into a total of fifteen species. One isolate of each species was randomly chosen and evaluated for its technological traits. In presence of ethanol (6 to 16% v/v) and sulfur dioxide (40 mg/L), native Saccharomyces cerevisiae 3FS presented the best growth rates and minor lag phase. Regarding non-Saccharomyces strains, Starmerella bacillaris 3MS stood out for its behavior in vinification conditions, more closely related to S. cerevisiae strains. Saccharomyces cerevisiae 3FS, Starmerella bacillaris 3MS, and Saturnispora diversa 1FS conducted a successful fermentation process reaching a final ethanol concentration ≥ 10% v/v and presenting a killer and resistant phenotype, suggesting that they could be used as pure starter cultures, as well as in mixed culture fermentations. This preliminary screening and oenological characterization of indigenous Saccharomyces cerevisiae and non-Saccharomyces yeasts might be a useful tool to identify some strains as potential candidates for wine vinification.

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Genome sequencing and oenologically relevant traits of the Uruguayan native yeast Issatchenkia terricola

OENO One, 56(3), 103–119 (doi.org/10.20870/oeno-one.2022.56.3.5581 )

Dourron, J., de Ovalle, S., González-Pombo, P., Villarino, A., Ramón, A., Costábile, A.

Issatchenkia terricola 0621 is a non-Saccharomyces yeast strain isolated from Tannat grapes from Uruguayan vineyards; it stands out for its ability to produce high levels of β-glucosidase activity, which contributes to the aromatic complexity of wines. To delve into the potential oenological applications of this strain, its high-quality genome was obtained and explored, allowing the main central carbon and nitrogen metabolic pathways to be reconstructed. I. terricola is able to utilise glycerol as the sole carbon source in a way that has not previously been described for yeasts. The genes of the fermentome and those involved in stress resistance during winemaking were also identified, and differences were found when compared to S. cerevisiae, which may explain why I. terricola is unable to complete fermentation. The pathways responsible for natural aroma synthesis were also reconstructed, and the production of aromatic acids, alcohols, esters, acetates and lactones was verified experimentally. Finally, sequences encoding for β-glucosidases, in addition to the previously characterised one, were identified in the genome. The work presented here lays the groundwork for experimental research focused on the dissection of the metabolism of a native non-Saccharomyces strain and its application for oenological and biotechnological purposes.

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Cyclopeptides Natural Products as Herbicides and Inhibitors of Cyanobacteria: Synthesis of Versicotides E and F

Chemistry Select, 7 (27) e202201956 (doi.org/10.1002/slct.202201956 )

Posada, L., Rey, L., Villalba, J., Sol Colombo, S., Aubriot,L., Badagian, N., Brena, B., Serra, G.

The first total synthesis of cyclopeptides versicotide E and F, natural products produced by marine fungus Aspergillus versicolor LZD-14-1, was achieved in good yield by solid phase peptide synthesis (SPPS) of their linear precursors and solution phase cyclization. All the versicotides A−F were evaluated as herbicides and inhibitors of cyanobacterial growth. Versicotides A, B, D, E and F showed a significant inhibition of Rye grass seed's radicle growth at a concentration of 67 μg/mL. Versicotides A, B and D also inhibited seed germination and leaf development. On the other hand, Versicotides D and F caused a relevant reduction on Microcystis aeruginosa population when cultures on exponential growth were incubated with 40 μg/mL solutions of these compounds. Evaluation of the concentration of microcystins after these treatments showed that versicotide D inhibited the production of microcystins in a comparable extent to the positive control, colistine. These results indicate versicotides, with versicotides D and F as top hits, could be considered as lead candidates in the development of bioherbicides able to mitigate the environmental impact that the evolution of agriculture has had on water quality.

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Immobilized peptide-N-glycosidase F onto magnetic nanoparticles: A biotechnological tool for protein deglycosylation under native conditions

Biotechnology and Applied Biochemistry, 69 (1) 209 - 220 ( doi.org/10.1002/bab.2099)

Bidondo, L., Festari, F., Freire, T., Giacomini, C.

The elucidation of glycans biological function is essential to understand their role in biological processes, both normal and pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their interference in subsequent experiments. Here, we describe the immobilization of peptide-N-glycosidase F (PNGase F) onto silica magnetic nanoparticles with immobilization yields of 86% and activity yields of 12%. Immobilized PNGase F showed higher thermal stability than its soluble counterpart, and could be reused for at least seven deglycosylation cycles. It was efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease in specific lectin recognition of the glycoproteins (40%–80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.

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Trypanosoma cruzi-derived molecules induce anti-tumour protection by favouring both innate and adaptive immune responses

International Journal of Molecular Sciences, 23, p.15032 (doi.org/10.3390/ijms232315032)

Freire, T., Landeira, M., Giacomini, C., Festari, M.F. , Pittini, A., Cardozo, V. , Brosque ,A. , Monin, L. , Da Costa, V. , Faral-Tello, P., Robello, C. , Osinaga, E.

Lung cancer remains the leading cause of cancer mortality worldwide. Thus, the development of strategies against this type of cancer is of high value. Parasite infections can correlate with lower cancer incidence in humans and their use as vaccines has been recently explored in preclinical models. In this study, we investigated whether immunisations with a Trypanosoma cruzi lysate from epimastigotes protect from lung tumour growth in mice. We also explore the role of parasite glycans in the induction of the protective immune response. A pre-clinical murine cancer model using the lung tumour cell line LL/2 was used to evaluate the anti-tumour potential, both in preventive and therapeutic settings, of a T. cruzi epimastigote-derived protein lysate. Immunisation with the parasite lysate prevents tumour growth and induces both humoral and cellular anti-tumour immune responses to LL-2 cancer cells. The induced immunity and tumour protection were associated with the activation of natural killer (NK) cells, the production of interferon-γ (IFN-γ) and tumour cell cytotoxicity. We also show that mannose residues in the T. cruzi lysate induce Toll-like receptor (TLR) signalling. The evaluated T. cruzi lysate possesses anti-tumour properties likely by activating innate and adaptive immunity in a process where carbohydrates seem to be essential.

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Influence of β-glucosidases from native yeast on the aroma of Muscat and Tannat wines

Food Chemistry, 346, 128899 (doi.org/10.1016/j.foodchem.2020.128899)

De Ovalle, S., Brena, B.M., Gonzàlez Pombo, P.

It is now well established that β-glucosidases (BGLs) from non-Saccharomyces yeasts are key enzymes that hydrolyze grape-derived aroma precursors enhancing the flavour of wines. This work reports on the specificity for wine glycosides and the impact on wine aroma, of three native yeast β-glucosidases. Volatile compounds were analyzed by gas-chromatography and mass spectroscopy (GC–MS) and wine aroma was studied by sensory analysis. Issatchenkia terricola β-glucosidase stood out from the other β-glucosidases studied. The I. terricola BGL showed remarkable specificity for norisoprenoid aglycones such as: 3-oxo-7, 8-dihydro-alpha-ionol, 3-oxo-α-ionol, vomifoliol. This different specificity was perceived in the sensory tests. The judges described pleasant fruity, sweet, honey and raisin notes in both Tannat and Muscat wines treated with I. terricola BGL. These results are particularly remarkable for Tannat wines, since there are few reports concerning the application of β-glucosidases to enhance its aroma of Tannat, and none with BGLs from native yeasts.

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Structural insights in galectin-1-glycan recognition: Relevance of the glycosidic linkage and the N-acetylation pattern of sugar moieties

Bioorganic & Medicinal Chemistry, 44, 11630 ( doi.org/10.1016/j.bmc.2021.116309 )

Porciúncula, C., Cagnoni, A.J., Fontana, C., Mariño, K., Saenz Méndez, P., Giacomini, C., Irazoqui, G.

Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for β-galactosides such as N-acetyllactosamine (β-d-Galp-(1 → 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for β-(1 → 6) galactosides, including β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was evaluated, and their performance was compared to that of β-(1 → 4) and β-(1 → 3) galactosides. To this end, the trisaccharide β-d-Galp-(1 → 6)-β-d-GlcpNAc-(1 → 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing β-(1 → 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving β-(1 → 4) and β-(1 → 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔGbindcalc) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔGbindexp), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.

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Rapid freshwater discharge on the coastal ocean as a mean of long distance spreading of an unprecedented toxic cyanobacteria bloom

Science of the Total Environment, 754, 142362 (doi.org/10.1016/j.scitotenv.2020.142362)

Kruk, C., Martìnez A., De la Escalera, G., Trinchin, R., Manta, G., Segura, A., Piccini, C., Brena, B.M., Yannicelli, B., Fabiano, G., Calliari, D.

Cyanobacterial toxic blooms are a worldwide problem. The Río de la Plata (RdlP) basin makes up about one fourth of South America areal surface, second only to the Amazonian. Intensive agro-industrial land use and the construction of dams have led to generalized eutrophication of main tributaries and increased the intensity and duration of cyanobacteria blooms. Here we analyse the evolution of an exceptional bloom at the low RdlP basin and Atlantic coast during the summer of 2019. A large array of biological, genetic, meteorological, oceanographic and satellite data is combined to discuss the driving mechanisms. The bloom covered the whole stripe of the RdlP estuary and the Uruguayan Atlantic coasts (around 500 km) for approximately 4 months. It was caused by the Microcystis aeruginosa complex (MAC), which produces hepatotoxins (microcystin). Extreme precipitation in the upstream regions of Uruguay and Negro rivers' basins caused high water flows and discharges. The evolution of meteorological and oceanographic conditions as well as the similarity of organisms' traits in the affected area suggest that the bloom originated in eutrophic reservoirs at the lower RdlP basin, Salto Grande in the Uruguay river, and Negro river reservoirs. High temperatures and weak Eastern winds prompted the rapid dispersion of the bloom over the freshwater plume along the RdlP northern and Atlantic coasts. The long-distance rapid drift allowed active MAC organisms to inoculate freshwater bodies from the Atlantic basin, impacting environments relevant for biodiversity conservation. Climate projections for the RdlP basin suggest an increase in precipitation and river water flux, which, in conjunction with agriculture intensification and dams' construction, might turn this extraordinary event into an ordinary situation.

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Microcystin ELISA in water and animal serum for an integrated environmental monitoring strategy

International Journal of Environmental Analytical Chemistry, 1-13 (doi.org/10.1080/03067319.2021.1881073)

Brena, B.M., Font, E., Pírez. M., Badagian, N., Cardozo, E., Perez, A., Bonilla, S.

Free-range livestock can be exposed to toxic cyanobacteria by drinking water from eutrophic natural sources. This study reports on the analysis of livestock drinking water sources from ranches in an eutrophic subtropical basin of South America (33ºS, 57°W), where frequent Microcystis spp. blooms and occasionally dense scums were found. To minimise monitoring costs, microcystins (MCs) were determined in serum and water with an in-house developed enzyme linked immunosorbent assay (ELISA). The water results were compared with those of liquid chromatography – tandem mass spectrometry (LC-MS/MS) for the most common congeners (MC-LR, MC-RR; MC-YR, MC-LA). Both methods were strongly correlated along a broad range of concentrations. The concentrations of MCs in the livestock drinking water were up to 3,700 µg/L. This is about 1,000 times higher than the Australian and New Zealand livestock MCs guidance values (cattle = 4.2; sheep = 3.9 µg/L) as well as the California-EPA sub-chronic action level (3 µg/L) and implies a high risk of poisoning. The animals exposed to MCs concentrations 70–100 times higher than these guidelines showed no significant alterations in liver enzymes. Nevertheless, serum concentrations of up to 0.11 µg/L MCs were recorded by ELISA. These results reveal the potential of MCs-ELISA in serum as a sensitive, minimally invasive method for risk assessment in integrated environmental monitoring strategies.

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Las floraciones de cianobacterias tóxicas comprometen el uso del agua del Río Negro, Uruguay

INNOTEC, e577., 2021. (doi.org/10.26461/22.08)

Bonilla, S., Aubriot, L., Haakonsson, S., Illarze, M., Díaz Isasa, I., Brena, B.M.

Las floraciones de cianobacterias tóxicas generan impactos negativos a nivel ambiental, económico, y en la salud humana y animal. Se realizó un análisis de datos históricos (1989-2020, n = 423) y un experimento de enriquecimiento de nutrientes para estudiar las cianobacterias del Río Negro, principal río interno del país. En base a indicadores cuantitativos (biovolumen, abundancia de células de cianobacterias y observación visual), se definieron cuatro niveles de peligrosidad de exposición a cianobacterias tóxicas. Las cianobacterias más frecuentes (Microcystis sp. y Dolichospermum sp.) son productoras potenciales de diversas toxinas, incluyendo algunas que no han sido analizadas aún en Uruguay. Se advierte un deterioro ambiental creciente desde el año 2000, pautado por el aumento de la biomasa de cianobacterias y las concentraciones de toxinas (microcistinas). Los nutrientes en el agua indican eutrofización avanzada, asociada al incremento del área agrícola de la cuenca. Los resultados experimentales demostraron el papel clave de los nutrientes y el tiempo de residencia en el favorecimiento de estos organismos. Las floraciones tóxicas de cianobacterias amenazan seriamente los múltiples servicios ecosistémicos que brinda el río, siendo indispensable la instrumentación de planes de monitoreo de cianobacterias y medidas de manejo para controlar la eutrofización a largo plazo.

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A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens

Biochemistry and Biophysics Reports, 26, 10094 (doi.org/10.1016/j.bbrep.2021.100940)

Bidondo, L., Landeira, M., Festari, F., Freire, T., Giacomini, C.

Background: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation; Methods: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.; Results: Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.; Conclusions: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes.; General significance: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.

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Semillas criollas de maíz de Uruguay y contaminación con transgenes

Editores: Viviane Camejo Pereira e Fábio Dal Soglio. Universidad Federal do Rio Grande do Sul. Serie Ensino, Aprendizagem e Technologias, 2020. ISBN 978-65-5725-007-5. pp 135-159.

Galeano, P., Beltrán,M., Machado, S., Fossatti, M., González, T., Arleo, M., Porta, B., Vidal, R., Martinez, C., Franco Fraguas,L., Galván, G.

Capítulo V en el Libro “A conservação das sementes crioulas: uma visão interdisciplinar da agrobiodiversidade".

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Nuevas metodologías para el análisis de microcistinas en peces. Estudio de Astraloheros facetus expuestos in vitro.

INNOTEC (2020) v.: 20, 10 - 29. ( link al artículo )

Badagian, N., Letarmendia, M., Pirez, M., Carnevia, D., Brena, B.M.

La alta incidencia de floraciones de cianobacterias productoras de microcistinas en el país y la región representa un riesgo muy elevado para humanos y animales. A fin de estudiar el impacto y la presencia de las microcistinas (MCs) en ani-males, es importante disponer de métodos simples de bajo costo. Como primera aproximación a estos objetivos en peces, se estudiaron Astraloheros facetus (Cas-tañetas) expuestas a una floración de Microcystis spp (MCs 60 y 600 μgMCs/L) en un bioensayo subcrónico (18 días). Si bien no hubo mortalidad, la histopato-logía mostró infiltración grasa en el hígado, más relevante en los peces expuestos a la mayor concentración. Para analizar MCs en pescados se optimizaron dos mé-todos inmunoquímicos sensibles basados en un anticuerpo recombinante de llama (nanobody) de alta especificidad: ELISA y MALDI-TOF cuantitativo, utilizando partículas magnéticas funcionalizadas. Los métodos fueron recientemente desa-rrollados localmente. La excelente correlación ELISA/MALDI-TOF (rSpearman = 0,988, p< 10-7) resalta el potencial de este ELISA como herramienta simple y costo-efectiva para minimizar las muestras a analizar por métodos de referencia. Las concentraciones de MCs en las Castañetas fueron relevantes, acordes con bioensayos en otras especies y peces de la naturaleza. Esto destaca la importancia de analizar MCs en pescado para consumo.

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Characterization of the cellulase-secretome produced by the Antarctic bacterium Flavobacterium sp. AUG42

Microbiological Research, vol 223-225, 13-21 (doi.org/10.1016/j.micres.2019.03.009)

Herrera,L.M., Braña, V., Franco Fraguas, L., Castro-Sowinski, S.

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.

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Enzymatic synthesis of non-natural trisaccharides and galactosides; Insights of their interaction with galectins as a function of their structure

Carbohydrate Research, v.: 472 p.:1 - 15 (doi.org/10.1016/j.carres.2018.10.011 )

Porciúncula Gonzalez, C. Cagnoni, A.J., Mariño, K.V., Fontana, C., Saenz Mendez, P., Irazoqui, G., Giacomini, C.

Galectins are a family of carbohydrate-recognizing proteins that by interacting with specific glycoepitopes can mediate important biological processes, including immune cell homeostasis and activation of tolerogenic circuits. Among the different members of this family, Galectin 1 and 3 have shown pro-tumorigenic effects, being overexpressed in numerous neoplasic diseases, proving to be relevant in tumor immune escape, tumor progression and resistance to drug-induced apoptosis. Thus, generation of specific glycosides that could inhibit their pro-tumorigenic ability by blocking their carbohydrate recognition domain is one of the current major challenges in the field. Considering that galectin-ligand binding strength is closely related to the ligand structure, analysis of this relationship provides valuable information for rational design of high-affinity ligands that could work as effective galectin inhibitors. Taking profit of the ability of glycosidases to catalyze transglycosylation reactions we achieved the enzymatic synthesis of β-d-Galp-(1 → 6)-β-d-Galp-(1 → 4)-d-Glcp (2), a mixture of β-d-Galp-(1 → 6)-β-d-Glcp-(1 → 4)-d-Glcp (5) and β-d-Galp-(1 → 3)-β-d-Glcp-(1 → 4)-d-Glcp (6), and finally benzyl β-d-galactopyranoside (9), with reaction yields between 16 and 27%. All the galactosides were purified, and characterized using 1H and 13C nuclear magnetic resonance spectroscopy. Docking results performed between the synthesized compounds and human Galectin 1 (hGal-1) and human Galectin 3 (hGal-3) showed that the replacement of a glucose moiety linked to the terminal galactose with a galactose moiety, decreases the affinity for these galectins. Moreover, regarding the interglycosidic bond the most favorable β-Gal linkage seems to be β(1 → 4) followed by β(1 → 3) and β(1 → 6) for hGal-1, and β(1 → 4) followed by β(1 → 6) and β(1 → 3) for hGal-3. These results were in accordance with the IC50 values obtained with in vitro solid phase inhibition assays. Therefore, docking results obtained in this work proved to be a very good approximation for predicting binding affinity of novel galactosides.

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Pseudozyma sp. isolation from Eucalyptus leaves and its hydrolytic activity over xylan

Biocatalysis and Agricultural Biotechnology, v. 21, p.10128 ( DOI: 10.1016/j.bcab.2019.101282 )

Botto, E., Gioia, L., Menéndez, P., Rodríguez, P.

Eucalyptus leaves were investigated as a source for the isolation of xylanase producing microorganisms. A total of 37 isolates were obtained after a series of enrichment steps. Seven of the isolates were xylanase positive in an agar screening experiment and were further analyzed in liquid media with beechwood xylan as carbon source. A yeast identified as Pseudozyma sp. showed the highest xylanase activity in tested conditions. Afterwards, different lignocellulosic residues were studied as substrates for xylanase production by this strain and the best results were obtained with corncob. Yeast's xylanase with a molecular weight of 19.9 kDa showed the maximum activity at pH 4.8 and 50 °C. Thermostability was observed at 30 °C with a 60% activity retention after 10 days. By the hydrolytic activity of the enzyme was characterized as an endoxylanase, similar as the ones found in family GH 10, from the products obtained by beechwood xylan hydrolysis.

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Survery of b-galactosidases properties: applications to transgalactosylation process

En: Beta Galactosidase Properties, Structure and Functions, 65-115, 2019, ed. Eloy Kras, Nova Science Publishers, Inc , New York,ISSN/ISBN: 978-1-53615-605-8

Porciúncula Gonzalez, C., Giacomini, C., Irazoqui, G.,

b-galactosidases (b-D-galactohydrolase, EC are enzymes that hydrolyzes terminal b 1-4 galactosides. They belong to the GH 1, GH 2, GH 35 and GH 42 of the GH-A superfamily of glycoside hydrolases. They are widely distributed in nature and can be find in animals, plants and several microorganisms (yeast, fungi and bacteria). In nature, they function as hydrolases. In plants, they remove terminal b 1-4 galactose from polymers containing galactose, in animals and microorganisms they catalyzes the hydrolysis of lactose in galactose and glucose. The most studied b-galactosidases are those from microorganism, such as Escherichia coli, Aspergillus oryzae, Bacillus circulans, Streptococcus thermophilus, Kluyveromyces lactis. This allows a collection of b-galactosidases with different properties (as optimum pH and temperature, thermal stability, stability against pH, denaturing agents, etc.), which offer a great versatility of conditions to use these enzymes for different applications. One of the first biotechnological application of these enzymes was for lactose hydrolysis in order to produce lactose reduced milk and dairy products for consumption of lactose intolerant people. Besides, a reduced lactose content in dairy product avoids lactose crystallization improving their organoleptic properties. Nevertheless, under adequate conditions they are able to catalyze the synthesis of oligosaccharides and galactosides. Two methods have been used for this purpose, reverse synthesis and transglycosylation. The first one involves the inversion of the hydrolysis reaction starting from a mixture of monosaccharides and nucleophilic hydroxylated molecules. This method requires high concentration of reactants as well as the presence of co-solvents in order to reduce water activity. On the other hand transglycosylation mechanisms is kinetically controlled and catalyzes the transfer of a galactosyl moiety from a galactosyl donor (lactose, ortho-nitrophenyl-b-D-galactopyranosyde, lactulose) to an hydroxylated nucleophile. Aliphatic alcohols, hydroxylated aminoacids such as serine and threonine, polyols, flavonoids, and monosaccharides among others have proved to be good glycosyl acceptors. The use of the transgalactosylation mechanism is an excellent alternative to the complex chemical synthesis as it allows the synthesis of b-galactosides and b-galactooligosaccharides in a single step preserving the anomeric center configuration. Even though glycosidases are stereospecific they are not always regiospecific and so the generation of isomers mixtures could take place, depending on the structure of the acceptor molecule as well as on the source of the b-galactosidase. Regarding synthetic applications, they have been used for enzymatic synthesis of: galactooligosaccharides with prebiotic properties as food additives; galactooligosaccharides as building blocks for synthesis of branched oligosaccharides; galactosides with potential activity as galectin inhibitors or antitumor agent; alkyl galactosides as non-ionic surfactants. b-galactosidases have also been used for galactosylation of drugs in order to improve their hydrophilicity and bioavailability. In this chapter we will make an update regarding b-galactosidases classification, mechanisms, characterization of their properties and applications.

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Oriented Functionalization of magnetic beads with in vivo biotinylated nanobodies for rapid MALDI-TOF MS ultrasensitive quantitation of microcystins in biological samples

Analytical Chemistry, 91, 15, 9925–9931 (doi/10.1021/acs.analchem.9b01596)

Pírez, M., Brena, B.M., González-Sapienza, G.

Here we present a new analytical method where immunoconcentration of the analyte is coupled to quantitative matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis allowing in minutes the identification and highly sensitive quantitation of microcystins (MCs) as model targets. The key element is a site-specific in vivo biotinylated nanobody of broad cross-reactivity with microcystins. The single biotin moiety at the C-terminus and the small size of the nanobody (15 kDa) enable its oriented and tightly packed immobilization on magnetic beads, providing a highly efficient capture of the toxin. The binding capacity of the bioadsorbent is partially loaded with an easily synthesized internal standard for MS quantitation. After capture, the beads are directly dispensed on the MALDI-TOF MS target enabling the identification and sensitive quantitation of the microcystin (MC) congeners. Since salts and contaminants are removed during the concentration step, no cleanup or other sample treatments are needed. The method was validated with a large number of water and serum samples with excellent precision and recovery at quantitation limits of 0.025 μg/L of MC.

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Assessing multimetric trophic state variability during an ENSO event in a large estuary (Rio de la Plata, South America).

Regional Studies in Marine Sciences, v 28, 100565 ( doi.org/10.1016/j.rsma.2019.100565)

Brugnoli, E., Muniz, P., Venturini, N., Brena, B.M., Rodríguez, A., Garcia Rodriguez, F.

During the “El Niño”- Southern Oscillation (ENSO) event 2009–2010, trophic state uni (TSI’s) and multimetric (TRIX’s) indexes were determined in the north coast of the Río de la Plata (RdlP). The relationships of such indexes with the hydrological, physical and chemical parameters were explored to identify climatic variability or anthropogenic effects on the eutrophication process. Eleven oceanographic sampling campaigns at 25 stations were conducted along the north coast of RdlP from 2009 to 2011. Temperature, salinity and oxygen saturation, water transparency (Secchi depth), chlorophyll (Chl a) content and total nutrient concentrations (total nitrogen and phosphorous) were determined. Trophic indexes (TSI Chl a, TSI TP, TRIX NP-Voll. and TRIX NP-Mvdeo.) were quantified and the RdlP flow was measured. RdlP flow showed significant differences between ENSO phases, with higher values during “El Niño” (EN), negative and significant correlations with salinity, Secchi depth and positive with total nutrients. The trophic status indexes showed spatial and temporal variability and associations with flow, physical and chemical parameters. Trophic state indexes showed spatial and temporal heterogeneity and were strongly associated to environmental conditions (hydrological and seasonal oscillations) during the study period but also to the anthropogenic factors affecting the north coast of RdlP. The system has been classified as mesotrophic to hypertrophic, depending on the selected indicator. According to these results, we suggest the use of a multimetric trophic state indicator, i.e., the TRIX index, as explains better the causes and effects of the eutrophication processes. To implement the TRIX index, (TRIX NP-Mvdeo.) we suggest researchers to consider a large number of local records (Chl a, nutrients, oxygen) at seasonal scales. This index is adapted to the environmental conditions of the study zone and complemented with other community structure indexes, and should be used for eutrophication assessment programs in the north coast of RdlP.

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Combined Danio rerio embryo morbidity, mortality and photomotor response assay: A tool for developmental risk assessment from chronic cyanoHAB exposure.

Science of the Total Environment, v.: 697, 134210 (doi.org/10.1016/j.scitotenv.2019.134210)

Roegner, A., Truong, L., Weirich, C., Pirez, M., Brena, B.M.,Miller, T.M., Tanguay, R.

Freshwater harmful algal blooms produce a broad array of bioactive compounds, with variable polarity. Acute exposure to cyanotoxins can impact the liver, nervous system, gastrointestinal tract, skin, and immune function. Increasing evidence suggests chronic effects from low-level exposures of cyanotoxins and other associated bioactive metabolites of cyanobacterial origin. These sundry compounds persist in drinking and recreational waters and challenge resource managers in detection and removal. A systematic approach to assess the developmental toxicity of cyanobacterial metabolite standards was employed utilizing a robust and high throughput developmental Danio rerio embryo platform that incorporated a neurobehavioral endpoint, photomotor response. Subsequently, we applied the platform to cyanobacterial bloom surface water samples taken from temperate recreational beaches and tropical lake subsistence drinking water sources as a model approach. Dechorionated Danio rerio embryos were statically immersed beginning at four to six hours post fertilization at environmentally relevant concentrations, and then assessed at 24 h and 5 days for morbidity, morphological changes, and photomotor response. At least one assessed endpoint deviated significantly for exposed embryos for 22 out of 25 metabolites examined. Notably, the alkaloid lyngbyatoxin–a resulted in profound, dose-dependent morbidity and mortality beginning at 5 μg/L. In addition, hydrophobic components of extracts from beach monitoring resulted in potent morbidity and mortality despite only trace cyanotoxins detected. The hydrophilic extracts with several order of magnitude higher concentrations of microcystins resulted in no morbidity or mortality. Developmental photomotor response was consistently altered in environmental bloom samples, independent of the presence or concentration of toxins detected in extracts. While limited with respect to more polar compounds, this novel screening approach complements specific fingerprinting of acutely toxic metabolites with robust assessment of developmental toxicity, critical for chronic exposure scenarios.

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Biodegradation of acid dyes by an immobilized laccase: an ecotoxicological approach

Environmental Science: water research & technology, 2018, 4 (12) , 2125 – 2135. (DOI: 10.1039/C8EW00595H )

Gioia,L., Ovsejevi, K. , Manta,C. , Menéndez, P.

Synthetic dyes in watercourses resulting from industrial effluent discharges cause serious ecological impacts, besides carcinogenic and mutagenic effects on human health. Thus, it is important to develop effective methods to remove the dyes from industrial wastewaters, and also to carry out adequate toxicity studies to establish their safety. Azo dyes are the main class of industrial dyes and important environmental contaminants. We have examined the decolorization of two azo dyes (Acid Red 88 and Acid Black 172) by a native Trametes villosa laccase immobilized on thiolsulfinate-agarose as well as the effect of different redox mediators in the reactions. The method was effective for the decolorization of both dyes. The immobilization method did not affect the capacity of the biocatalyst for dye degradation. Therefore, the insoluble enzyme removed 97% of the color of AR88 and 92% of AB172 in 24 hours at 22 °C in the presence of the selected redox mediators, vanillin (0.1 mM) and violuric acid (1.0 mM), respectively. In addition, the immobilized enzyme kept 78% of its initial capacity for decolorization of AR88 after three cycles of use. The ecotoxicological evaluation of the solutions showed a great variation depending on the biological systems used. In the phytotoxicity test, the decolorization products were not toxic to plants, whereas in Daphnia magna and Microtox® bioassays an acute residual toxicity was found. The last outcome shows the importance of using a battery of bioassays to determine the remaining ecotoxicity of the treated effluents before their discharge into the aquatic environment.

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Thiol-cyclodextrin: a new agent for controlling the catalytic activity of polyphenol oxidase from red delicious apple.

Journal of Food Science & Technology, 3(2)1-11, 2018. (DOI: 10.25177/JFST.3.2.2 )

Peralta-Altier, G., Manta, C., Ovsejevi, K.

Polyphenol oxidase (PPO, EC is the main enzyme responsible for enzymatic browning, a natural process which produces deterioration of fruits and vegetables. One alternative to prevent this undesirable process is to inhibit the catalytic activity of PPO by encapsulating enzyme’s substrates in cyclodextrins (CD). In this article the effect of a Thiol-CD on PPO from Red Delicious apple was studied, demonstrating that this compound is a powerful tool for controlling oxidative processes in food. Thiol-CD could encapsulate the polyphenols, natural substrates of the enzyme, by means of the cyclodextrin hydrophobic internal cavity. Simultaneously, through the thiol group, it could inactivate the PPO by reducing the copper ions from the active site of the enzyme. Moreover, thiol moieties could decrease the browning by reducing the quinones generated by oxidative processes. The isolation and purification of PPO from apple was performed in order to study those effects, different polyphenols, chlorogenic acid (CA) and 4-methylcatechol (4-MC), were assayed as enzymatic substrates. Both β -CD and Thiol-CD exhibited better enzyme inhibition value for CA than for 4-MC. Moreover, Thiol-CD showed an extraordinary performance compared with β-CD. When CA 10 mM was used, 5 mM β-CD gave 11 % PPO inhibition, meanwhile nearly 100 times less concentration of Thiol-CD (45 μM) gave 100 % of enzyme inhibition.

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Immobilization of b-galactosidase and a-mannosidase onto magnetic nanoparticles: A strategy for increasing the potentiality of valuable glycomic tools for glycosylation analysis and biological role determination of glycoconjugates

Enzyme and Microbial Technology, v. 117 p.45 - 55 (DOI: 10.1016/j.enzmictec.2018.05.012)

Rodriguez, E., Francia, K., Brossard, N., García Vallejo, J.J., Kalay, H., van Kooyk, Y., Freire, T., Giacomini, C.

Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae β-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70-90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40-50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.

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Production and characterization of a beta-glucosidase from Issatchenkia terricola and its use for hydrolysis of aromatic precursors in Cabernet Sauvignon wine LWT-Food

Science and Technology v. 87, 515 – 522 (doi.org/10.1016/j.lwt.2017.09.026)

De Ovalle, S., Cavello, I., Brena, B.M., Caballito, S., González-Pombo, P.

New enzymes isolated from the biodiversity of native wine ecosystems could contribute to increase the varietal character of regional wines. This study reports on the production and characterization of Issatchenkia terricola, beta-glucosidase and its potential to release red-wine aromatic compounds. The enzyme, a monomer of 48 kDa with an isoelectric point of 3.5 is tolerant to glucose and ethanol, properties compatible with enological use. Although fed-batch is usually the most suitable system for enzyme production in submerged culture, in this case the yield was practically the same as in batch culture. Enzyme productivity was increased 2-fold in synthetic medium with glucose with respect to the YPG and 3–8-fold with respect to other media assayed. After enzymatic treatment, GC-MS analysis of the released aglycones demonstrated significant increases in the concentration of phenols (83%) and norisoprenoids (65%). According to the judges of the sensory panel, the treatment resulted in a wine with dried fruits and raisins notes, as compared to the control, which was found more sweet and fruity. This, together with the lack of activity on anthocyanin glycosides, highlights the potential of this enzyme in enology, since its high selectivity allowed the development of aroma without compromising wine color.

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Effects of wind mixing in a stratified water column on toxic cyanobacteria and microcystin-LR distribution in a subtropical reservoir

Bulletin of Environmental Contamination and Toxicology, v.101 (5): 611-616 (doi.org/10.1007/s00128-018-2446-x)

González-Piana, M., Piccardo, A., Ferrer, C., Brena, B.M., Pírez, M., Fabian, D., Chalar, G.

We analyzed the effects of stratification changes due to wind on the vertical cyanobacteria distribution and microcystin-LR concentrations in a reservoir and assessed the implications for water management. Under stratified conditions, the highest microcystin concentrations (up to 4.16 µg/L) and toxic cyanobacteria biovolume occurred in the epilimnion (~ 1 m). The lowest microcystin concentrations were between 0.02 and 1.28 µg/L and occurred in the hypolimnion (~ 20 m). A cold front passage associated with high wind velocities induced water column mixing, promoting the redistribution of microcystin-LR and cyanobacteria throughout the water column and increasing their concentrations in deeper zones. Microcystin-LR concentration was positively correlated with cyanobacteria biovolume (r = 0.747) and chlorophyll a concentration (r = 0.798). Changes in thermal profile due to wind would imply a greater challenge for drinking water treatment plants, since high cyanobacterial and microcystin concentrations could reach deep-water intakes.

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Tesis de posgrado

Tesis de maestría

Biocatálisis aplicada a la síntesis de radiotrazadores: síntesis de S-adenosil metionina por metodologías biocatalíticas

Diego Umpiérrez. Tutoras: Gabriela Irazoqui, Sonia Rodríguez .

Finalizada en 2023.

Análisis de glicoproteínas de Trypanosoma cruzi como potenciales ligandos de receptores de tipo Toll de la inmunidad innata

Mercedes Landeira. Tutoras: Teresa Freire, Cecilia Giacomini, Florencia Festari.

Finalizada en 2021.

Link a la tesis

Explotación del genoma de Issatchenika terricola para identificación de glicosidasas con potencial aplicación en enología

Juliette Dourron. Tutoras: Paula Gonzalez Pombo, Ana Ramón.

Finalizada en 2020.

Link a la tesis

Celulasas sicrófilas, una innovación en la industria del bioetanol

Lorena Herrera Marrero. Tutoras: Laura Franco Fraguas, Susana Castro.

Finalizada en 2019.

Link a la tesis

Tesis de doctorado

Actividad Proteolítica del Látex de Plantas Indígenas Uruguayas. Obtención de Péptidos Antimicrobianos y/o Antioxidantes

Miriam Barros. Tutoras: Ana María Cantera, Carolina Villadóniga.

Finalizada en 2023.

Link a la tesis

Producción, caracterización bioquímica e inmovilización de lipasas microbianas y sus aplicaciones

Agustín Castilla. Tutoras: Gabriela Irazoqui, Sonia Rodríguez.

Finalizada en 2022.

Link a la tesis

β-glucosidasas de cepas nativas y su potencial en la liberación de aromas de vino. Desarrollo de nuevas estrategias para su uso en la industria enológica.

Stefanie de Ovalle. Tutoras: Paula Gonzalez Pombo, Beatriz Brena.

Finalizada en 2021.

Link a la tesis

Desarrollo y validación de métodos sencillos y rápidos para cianotoxinas en el monitoreo ambiental

Vania Macarena Pírez. Tutores: Beatriz Brena, Gualberto González-Sapienza.

Finalizada en 2019.

Link a la tesis

Diseño racional y síntesis enzimática de galactósidos con potencial actividad como inhibidores de galectinas

Cecilia Porciúncula. Tutoras: Cecilia Giacomini, Gabriela Irazoqui, Patricia Saenz Méndez.

Finalizada en 2019.

Link a la tesis

Purificación, caracterización funcional y estructural de cisteín proteinasas presentes en frutos maduros de Bromelia antíacantha. Evaluación de sus posibles aplicaciones.

Diego Vallés. Tutora: Ana María Cantera.

Finalizada en 2019.

Link a la tesis


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