Biochemical and structural characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family

[Abstract]: A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60 C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester. The tridimensional structures of EstD11 and the inactive mutant have been determined at atomic resolutions. Structural and bioinformatic analysis, confirm that EstD11 belongs to the family IV, the hormone-sensitive lipase (HSL) family, from the α/β-hydrolase superfamily. The canonical a/b hydrolase domain is completed by a cap domain, composed by two subdomains that can unmask of the active site to allow the substrate to enter. Eight crystallographic complexes were solved with different substrates and reaction products that allowed identification of the hot-spots in the active site underlying the specificity of the protein. Crystallization and/or incubation of EstD11 at high temperature provided unique information on cap dynamics and a first glimpse of enzymatic activity in vivo. Very interestingly, we have discovered a unique Met zipper lining the active site and the cap domains that could be essential in pivotal aspects as thermo-stability and substrate promiscuity in EstD11Ministerio de Ciencia e Innovación; BFU2017-90030-

Base de datos de abejas ibéricas

Las abejas son un grupo extremadamente diverso con más de 1000 especies descritas en la península ibérica. Además, son excelentes polinizadores y aportan numerosos servicios ecosistémicos fundamentales para la mayoría de ecosistemas terrestres. Debido a los diversos cambios ambientales inducidos por el ser humano, existen evidencias del declive de algunas de sus poblaciones para ciertas especies. Sin embargo, conocemos muy poco del estado de conservación de la mayoría de especies y de muchas de ellas ignoramos cuál es su distribución en la península ibérica. En este trabajo presentamos un esfuerzo colaborativo para crear una base de datos de ocurrencias de abejas que abarca la península ibérica e islas Baleares que permitirá resolver cuestiones como la distribución de las diferentes especies, preferencia de hábitat, fenología o tendencias históricas. En su versión actual, esta base de datos contiene un total de 87 684 registros de 923 especies recolectados entre 1830 y 2022, de los cuales un 87% presentan información georreferenciada. Para cada registro se incluye información relativa a la localidad de muestreo (89%), identificador y colector de la especie (64%), fecha de captura (54%) y planta donde se recolectó (20%). Creemos que esta base de datos es el punto de partida para conocer y conservar mejor la biodiversidad de abejas en la península ibérica e Islas Baleares. Se puede acceder a estos datos a través del siguiente enlace permanente: https://doi.org/10.5281/zenodo.6354502ABSTRACT: Bees are a diverse group with more than 1000 species known from the Iberian Peninsula. They have increasingly received special attention due to their important role as pollinators and providers of ecosystem services. In addition, various rapid human-induced environmental changes are leading to the decline of some of its populations. However, we know very little about the conservation status of most species and for many species, we hardly know their true distributions across the Iberian Peninsula. Here, we present a collaborative effort to collate and curate a database of Iberian bee occurrences to answer questions about their distribution, habitat preference, phenology, or historical trends. In total we have accumulated 87 684 records from the Iberian Peninsula and the Balearic Islands of 923 different species with 87% of georeferenced records collected between 1830 and 2022. In addition, each record has associated information such as the sampling location (89%), collector and person who identified the species (64%), date of the capture (54%) and plant species where the bees were captured (20%). We believe that this database is the starting point to better understand and conserve bee biodiversity in the Iberian Peninsula. It can be accessed at: https://doi.org/10.5281/zenodo.6354502Esta base de datos se ha realizado con la ayuda de los proyectos EUCLIPO (Fundação para a Ciência e a Tecnologia, LISBOA-01-0145-FEDER-028360/EUCLIPO) y SAFEGUARD (ref. 101003476 H2020 -SFS-2019-2).info:eu-repo/semantics/publishedVersio

Temperature- and pH-sensitive nanohydrogels of Poly(N-Isopropylacrylamide) for food packaging applications: modelling the swelling-collapse behaviour

15 páginas, 5 tablas, 8 figuras.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Temperature-sensitive poly(N-isopropylacrylamide) (PNIPA) nanohydrogels were synthesized by nanoemulsion polymerization in water-in-oil systems. Several cross-linking degrees and the incorporation of acrylic acid as comonomer at different concentrations were tested to produce nanohydrogels with a wide range of properties. The physicochemical properties of PNIPA nanohydrogels, and their relationship with the swelling-collapse behaviour, were studied to evaluate the suitability of PNIPA nanoparticles as smart delivery systems (for active packaging). The swelling-collapse transition was analyzed by the change in the optical properties of PNIPA nanohydrogels using ultraviolet-visible spectroscopy. The thermodynamic parameters associated with the nanohydrogels collapse were calculated using a mathematical approach based on the van't Hoff analysis, assuming a two-state equilibrium (swollen to collapsed). A mathematical model is proposed to predict both the thermally induced collapse, and the collapse induced by the simultaneous action of two factors (temperature and pH, or temperature and organic solvent concentration). Finally, van't Hoff analysis was compared with differential scanning calorimetry. The results obtained allow us to solve the problem of determining the molecular weight of the structural repeating unit in cross-linked NIPA polymers, which, as we show, can be estimated from the ratio of the molar heat capacity (obtained from the van't Hoff analysis) to the specific heat capacity (obtained from calorimetric measurements).This work was funded by grant (MAT 2006-11662-CO3-CO2-C01/MAT 2010-21509-C03-01/EUI 2008-00115) from the “Ministerio de Educación y Ciencia” (Spain). Clara Fuciños was funded by a predoctoral scholarship from University of Vigo (Spain)Peer reviewe

Design of a procedure for obtaining a protein concentrate prepared from tuna cooking water

5 páginas, 3 tablas, 3 figuras.-- Póster presentado al FOODSIM 2010, June 24-26, 2010, CIMO Research Centre, Braganca, PortugalAnalytical ultrafiltration using three TFF cartridges differing on the cut-off (10, 30 and 100 kDa) were used to desalination and recovery of the collagenous fraction from the tuna cooking water. Best results were obtained with the 30 kDa membrane. The obtained concentrate had collagenous nature and provided functionality to be capable of gelling at temperatures close to the room temperatureN

Tuna processing wastewater and by-products as source of protein for muscle injection

1 póster presentado en el Total Food Conference 2014 Science and technology for the economic and sustainable exploitation of agri-food chain wastes and co-products, Norwich, Uk, 11-13 November (2014)Tuna processing wastewater (TPW) results from the tuna cooking process in canning industries. Their main soluble components are salt, solubilized sarcoplasmic and myofibrillar proteins and a higher proportion of gelatin. Because of this, have a high organic load and strong contaminant impact. The recuperation of a gelling fraction from this stream, by mean ultrafiltration technologies to use them as injection solution for protein fortification of fish preparations could constitute a alternative, which wasn’t enough tested until nowThis poster is presented as a part of the EU’s Seventh Framework Programme Project Biotechnologies to Valorise the regional food Biodiversity in Latin America BiValBi (PIRSES‐GA‐2013‐611493).N

One-step chromatographic method to purify -lactalbumin from whey for nanotube synthesis purposes

A one-step anion-exchange chromatography method (NaCl gradient elution on a DEAE Sepharose Fast Flow gel column) was developed to purify -lactalbumin (-LA) from whey protein isolate. -LA nearly 100% pure (based on the total protein content) was obtained with a yield of about 39%. Besides pure -LA, which was the main objective of this work, highly pure -lactoglobulin was also obtained with a yield of about 59%. The high purity of the obtained -LA samples allowed its use to synthesise protein nanotubes with excellent gelation properties for their use as food thickeners and bioactive carriers. The samples purity degree obtained (based on the total protein content) was critical in the formation of proper nanotubes instead of random aggregates, which produced opaque and weak gels, less useful for food applications.Clara Fuciños gratefully acknowledge her Postdoctoral grant (I2C 2014) from Consellería de Cultura, Educación e Ordenación Universitaria(XuntadeGalicia,Spain).Thisstudywassupportedbythe Portuguese Foundation for Science and Technology (FCT, Portugal) under the scope of the strategic funding of UID/BIO/04469/2013 unit and the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER027462).info:eu-repo/semantics/publishedVersio

Accumulation of copper and histopathological alterations in the oyster Crassostrea angulata

13 páginas, 6 figuras.[EN] Specimens of Crassostrea angulata were exposed to sublethal copper concentrations (200 and 600 µg L-1 Cu2+) during 2 to 30 days. The accumulation of copper and histopathological effects on the gills, digestive gland and heart were studied. The highest copper concentrations were found in the gills, with values over 2 mg g-1 dry weight for organisms exposed to the highest concentration at the end of the exposure period (day 30). In the digestive gland, the concentration was 1 mg g-1 dry weight (highest exposure on day 30). The rate of bioconcentration (BCr, defined as the concentration in the tissue at an exposure concentration at time t minus the mean concentration of the control throughout the experiment, divided by the exposure time) decreased in both tissues. The values ranged from 392 to 57 µg g-1 day-1 for gills and from 133 to 18 µg g-1 d-1 for the digestive gland. In the gills, specimens exposed to 200 µg L Cu2+ showed disorganization and apical alterations of the cilia cells and hyperplasia, lamellar fusion and lamellar loss in organisms exposed to 600 µg L-1 Cu2+. In the digestive gland, specimens exposed to 200 µg L-1 Cu2+ showed hemocytic infiltration in the underlying connective tissue and numerous brown cells compared to the control specimens. On the other hand, thinning of the epithelium of the digestive tubules, occlusion in the lumen of some primary tubules and dilation of the digestive ducts occurred in organisms exposed to 600 µg L-1 Cu2+. The heart of oysters exposed to copper showed thinning of the epithelium of the auricles and ventricle and an increase in brown cells on the walls of the auricles, as well as connective tissue destruction in the auricles and ventricle.[ES] Ejemplares de ostión Crassostrea angulata fueron expuestos a concentraciones subletales de cobre (200 y 600 µg L-1 Cu2+) durante un periodo de 2 a 30 días. Se cuantificó la concentración de cobre, así como las alteraciones histopatológicas inducidas en branquias, glándula digestiva y corazón. Las concentraciones más elevadas de cobre correspondieron a las branquias, con valores alrededor de 2 mg g-1 peso seco en los organismos expuestos a la concentración más alta, al final del periodo de exposición (día 30). En la glándula digestiva la concentración alcanzada fue del orden de 1 mg g-1 peso seco. La tasa de bioconcentración (BCr), definida como la diferencia entre la concentración en el tejido a una concentración de exposición a tiempo t y la concentración media del control a lo largo del experimento, dividida por el tiempo de exposición, disminuyó en ambos tejidos. Los valores variaron en el intervalo entre 392 y 57 µg g-1 d-1 en las branquias y entre 133 y 18 µg g-1 día-1 en la glándula digestiva. En branquias de ejemplares tratados con una concentración de 200 µg L-1 Cu2+ se observó una desorganización del tejido conjuntivo, alteraciones en la porción apical de las células ciliadas e hiperplasia y fusión de laminillas, pudiendo llegar incluso a la pérdida de estas laminillas a 600 µg L-1 Cu2+. En la glándula digestiva (hepatopáncreas) de ejemplares sometidos a 600 µg L-1 Cu2+ se detectó un adelgazamiento del epitelio y, en algunos casos, oclusión de la luz de los túbulos digestivos y dilatación de los conductos digestivos. En el corazón de los organismos expuestos a concentraciones subletales de cobre se observó un adelgazamiento del epitelio de las aurículas y del ventrículo, un incremento de las células marrones (brown cells) en las paredes de las aurículas, así como una distensión de las fibras musculares y destrucción del tejido conectivo de soporte, tanto en las aurículas como en el ventrículo.Trabajo subvencionado por el Instituto de Ciencias Marinas de Andalucía, CSIC (Unidad Asociada de Calidad Ambiental y Patología, CSIC y UCA).Peer reviewe

Influence of pH on viscoelastic properties of heat-induced gels obtained with a β-Lactoglobulin fraction isolated from bovine milk whey hydrolysates

A β-Lactoglobulin fraction (r-βLg) was isolated from whey hydrolysates produced with cardosins from Cynara cardunculus. The impact of the hydrolysis process on the r-βLg structure and the rheological properties of heat-induced gels obtained thereafter were studied at different pH values. Differences were observed between r-βLg and commercial β-Lg used as control. Higher values for the fluorescence emission intensity and red shifts of the emission wavelength of r-βLg suggested changes in its tertiary structure and more solvent-exposed tryptophan residues. Circular dichroism spectra also supported these evidences indicating that hydrolysis yielded an intermediate (non-native) β-Lg state. The thermal history of r-βLg through the new adopted conformation improved the microstructure of the gels at acidic pH. So, a new microstructure with better rheological characteristics (higher conformational flexibility and lower rigidity) and greater water holding ability was founded for r-βLg gel. These results were reflected in the microstructural analysis by scanning electron microscopy.Fil: Estévez, Natalia. Universidad de Vigo; EspañaFil: Fuciños, Pablo. International Iberian Nanotechnology Laboratory; PortugalFil: Bargiela, Verónica. Universidad de Vigo; EspañaFil: Picó, Guillermo Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Woitovich Valetti, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; ArgentinaFil: Tovar, Clara Asunción. Universidad de Vigo; EspañaFil: Rúa, M. Luisa. Universidad de Vigo; Españ

Cloning, expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

9 páginas, 7 figuras, 3 tablasThe esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His6 was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His6 preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85°C), suggesting that E34Tt-His6 has potential for industrial applications.This work was supported by the Spanish Ministry of Science and Technology (Project CTQ2005-05609/PPQ), the Consellería de Innovación e Industria from the Xunta de Galicia, Spain (Project PGIDIT06REM38302PR), and European FEDER (Project PPQ2001-3361).Peer reviewe

Producción de cultivos potencialmente probióticos a partir de lactosuero

N