Procedimiento para la purificación de dos isoenzimas lipasa de Candida rugosa

Referencia OEPM: P9201378.-- Fecha de solicitud: 03/07/1992.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).Procedimiento para la purificación de dos isoenzimas lipasa de Candida rugosa. Procedimiento para la purificación de dos lipasas extracelulares presentes en lipasa bruta comercial de la levadura Candida rugosa. El método consta de un sólo paso de cromatografía hidrofóbica en matriz de agarosa. Condiciones de elución muy diferentes permiten la completa separación y purificación de dos enzimas con actividad lipásica (lipasas A y B) presentes en el extracto comercial de partida. Para ello se utiliza un tampón concentrado de pH 6 a 8, con el que se eluyen de la columna gran parte de los contaminantes presentes en el extracto; luego con la misma solución tampón más diluida se arrastra la lipasa B. Posteriormente, con un di o polialcohol disuelto en el tampón diluido se eluye la lipasa A, de cuya solución, mediante concentración por ultrafiltración y cromatografía de exclusión molecular con dextrano, se elimina el alcohol. Se aplica industrialmente en la purificación de enzimas.Peer reviewe

Development and sensory test of a dairy product with ACE inhibitory and antioxidant peptides produced at a pilot plant scale

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGA scale-up process was carried out to obtain potent bioactive peptides from whey protein through a simple hydrolysis process. The scale-up was satisfactory, with results similar to those obtained at lab scale: a fraction of peptides < 1 kDa with ACE inhibitory activity of 18.44 ± 2.47 μg/mL, a DPPH value of 69.40 ± 0.44%, and an ORAC value of 3.37 ± 0.03 μmol TE/mg protein. The peptide sequences responsible for the ACE inhibitory activity were also similar to those identified at lab scale: PM, LL, LF, HFKG and PT. The hydrolysate was used as a functional ingredient in a low-fat yoghurt. The consumer sensory taste panel found no significant difference (p > 0.05) between the bitterness of the control and the functional yoghurt, and about 50% of consumers would buy it. The hydrolysate maintained its bioactivities for 4 months at −20 ◦C (after thawing and pasteurisation), and for 1 week in yoghurt at 4 ◦C

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-

Purificación, caracterización molecular y propiedades catalíticas en fase acuosa y en microemulsiones W/O de la lipasa de "Candida rugosa (Candida cylindracea)"

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Agrícola. Fecha de lectura: 13-12-199

Histomorphological study of the digestive tract of the oyster Crassostrea angulata (Lamarck, 1819), and distribution of carbohydrates

8 páginas, 1 figura, 3 tablas. VIII Congreso Nacional de Acuicultura: Acuicultura y desarrollo Sostenible (2001, 22-25 mayo, Santander)[EN] We present a histomorphological description of the digestive tract of the oyster Crassostrea angulata (Lamarck, 1819), as well as a histochemical study of its carbohydrate distribution. The study shows that, in general, glycogen is found in the connective tissue, and neutral mucopolysaccharides/glycoproteins and/or acid mucosubstances in the epithelium and basal cell layers. Glycogen was not detected in the digestive gland, possibly because of its mobilization to the connective tissue for reproduction.[ES] Se realiza una descripción histomorfológica del aparato digestivo del ostión Crassostrea angulata (Lamarck, 1819) y se pone de manifiesto la distribución de carbohidratos mediante técnicas histoquímicas. La pauta general observada es la presencia de glucógeno en el tejido conjuntivo y de mucopolisacáridos/glicoproteínas neutras y/o ácidas en las membranas basales y el epitelio. En la glándula digestiva no se observa glucógeno debido, posiblemente, a su movilización hacia el tejido conjuntivo para su posterior uso con fines reproductivos.Peer reviewe

Reactivity of a recombinant esterase from Thermus thermophilus HB27 in aqueous and organic media

The thermoalkalophilic membrane-associated esterase E34Tt from Thermus thermophilus HB27 was cloned and expressed in Kluyveromyces lactis (KLEST-3S esterase). The recombinant enzyme was tested as a biocatalyst in aqueous and organic media. It displayed a high thermal stability and was active in the presence of 10% (v/v) organic solvents and 1% (w/v) detergents. KLEST-3S hydrolysed triglycerides of various acyl chains, which is a rare characteristic among carboxylic ester hydrolases from extreme thermophiles, with maximum activity on tributyrin. It also displayed interfacial activation towards triacetin. KLEST-3S was also tested as a biocatalyst in organic media. The esterase provided high yields for the acetylation of alcohols. In addition, KLEST-3S catalyzed the stereoselective hydrolysis of (R,S)-ibuprofen methyl ester (87% ee). Our results indicate that KLEST-3S may be a robust and efficient biocatalyst for application in industrial bioconversions.Xunta de Galicia | Ref. 10MDS373027PRXunta de Galicia | Ref. GRC ED431C 2020/08European Commision | Ref. FP7-PEOPLE 2012-IAPP, n. 32443

Biofunctionality assessment of α-lactalbumin nanotubes

The functional properties α-LA nanotubes and the gels formed during their synthesis were studied to evaluate their potential applications. The bioactivity of α-LA was improved by the synthesis of nanotubes, with significantly (p < 0.05) higher ACE-inhibitory (IACE) and antioxidant activity (μmol TE/mg protein). The IC50 (protein concentration to inhibit 50% of ACE activity) of α-LA nanotubes was 281 ± 21 μg mL−1 and the antioxidant activity 1.28 ± 0.01 μmol TE mg−1 protein. Besides, the IACE-value was significantly higher (p < 0.05) when the α-LA fragments incorporated into the nanotube’ structure were considered, and not only the low MW peptides formed during the nanotube synthesis (not incorporated into the nanotube’ structure).This study also analysed how different ion ratios in the synthesis of α-LA nanotubes changed their functionality. Thus, gels formed by long and regular α-LA nanotubes (2 mol of Mn2+/mol of α-LA) showed fluid−gel characteristics, with some time-dependent shear-thinning flow (thixotropy). At fixed recovery time, a less thixotropic behaviour was found in these fluid gels (high percentage −71.6 ± 7.4%− of structural regeneration). Additionally, they stayed in gel form at high temperatures and even increased their stiffness up to 65 °C. The increase in the ion ratio (3 mol of Mn2+/mol of α-LA) during nanotube synthesis modified the α-LA nanotubes' microstructure nanotubes resulting in short and branched nanotubes. These gels showed a higher level of pseudo-plasticity, higher consistency and thixotropy. These are characteristics of a more complex and dense network with greater time dependence and lower structural regeneration (18.6 ± 2.0%)Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGXunta de Galicia | Ref. ED431E 2018/0

Reproductive cycle of Spisula solida (L.) (Mollusca: Bivalvia) on the southwest coast of Spain

The reproductive cycle of Spisula solida (L., 1758) was investigated for the first time in the southwest of Spain, using specimens collected from Punta Umbri{dotless acute}a (Huelva), between April 2001 and May 2002. A total of 2805 individuals of shell lengths ranging from 9.1 to 38.4 mm were analysed. The gonad development was determined by means of histological methods and variations of dry weight. Five gametogenic stages were identified (I-Immature, II-Maturing, III-Ripe, IV-Spawning, V-Post-spawning). The sex-ratio obtained was 5:4 (χ2 = 6.12, df = 1, p > 0.01), males being more abundant than females. Two hermaphrodite individuals were recorded. Development of the gonad began in November and spawning occurred between December and June, with maximum peaks in April (82% males) and May (87% females). The period of sexual rest took place from July to October. Gametogenesis in this species is a response to low seawater temperatures, and spawning occurs with low temperatures that increase progressively. Copyright © 2013 Taylor & Francis.This research was supported by the IFOP project.Peer Reviewe

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