Characterization and biotechnological application of α-galactosidase from Saccharomyces cerevisiae

Traballo fin de mestrado (UDC.CIE). Bioloxía molecular, celular e xenética. Curso 2013/201

Régimen jurídico-administrativo de las entidades gestoras en la organización de eventos de las universidades españolas. Especial referencia al programa actual de la Universidad de Almeria.

El presente Trabajo Fin de Grado tiene por objeto el diseño e implantación de un nuevo servicio en una Universidad Pública. Podemos definir Servicio desde un punto de vista económico, según la Real Academia Española como “la prestación humana que satisface alguna necesidad social y que no consiste en la producción de bienes materiales”. Deducimos que todo servicio, por lo tanto, responde a una necesidad, a una demanda

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

[Abstract] Background: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.Xunta de Galicia; ED431C 2016–01

Temporal Bacteriostatic Effect and Growth Factor Loss in Equine Platelet Components and Plasma Cultured with Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus

The aims were (1) to evaluate the bacteriostatic effect of platelet-rich plasma (PRP), platelet-rich gel (PRG), leukocyte-poor plasma (LPP), leukocyte-poor gel (LPG), plasma, and heat-inactivated plasma (IP) on both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) over a period of 24 h; (2) to determine and to compare the concentrations and degradation over time of platelet factor 4 (PF-4), transforming growth factor beta 1 (TGF-β1), and platelet-derived growth factor isoform BB (PDGF-BB); and (3) to identify any correlations between MSSA and MRSA growth and either the cellular, PF-4, TGF-β1, or PDGF-BB concentrations in the blood components. PRP and its byproducts from 18 horses were obtained by the tube method. All blood components were cultured with either MSSA or MRSA. Bacterial growth, PF-4, TGF-β1, and PDGF-BB were determined at 6 h and 24 h. At six hours, bacterial growth was significantly inhibited by all blood components, with the exception of IP. MSSA was more sensitive to the treatments than MRSA. At 24 hours, bacterial growth was significantly higher in IP. MRSA bacterial growth was significantly higher in PRP, LPP, and plasma when compared to MSSA. Growth factor concentrations were not significantly affected by bacteria

Using a regulator for a servomotor model designed for a prosthetic joint

Through the modern control theory it is possible to face any problem situation using state equations without considering mathematical rules used from recursive algorithms. The simplification of the various developments will be reflected with new findings, giving values to new concepts such as controllability and stability. This paper seeks to find new work items of the servomotor system by using a regulator, allowing a more controllable and stable performance. The servomotor presented here is designed to generate joint movements of a robotic arm designed for movement rehabilitation of a patient.V Workshop Procesamiento de Señales y Sistemas de Tiempo RealRed de Universidades con Carreras de Informática (RedUNCI

Using a regulator for a servomotor model designed for a prosthetic joint

Through the modern control theory it is possible to face any problem situation using state equations without considering mathematical rules used from recursive algorithms. The simplification of the various developments will be reflected with new findings, giving values to new concepts such as controllability and stability. This paper seeks to find new work items of the servomotor system by using a regulator, allowing a more controllable and stable performance. The servomotor presented here is designed to generate joint movements of a robotic arm designed for movement rehabilitation of a patient.V Workshop Procesamiento de Señales y Sistemas de Tiempo RealRed de Universidades con Carreras de Informática (RedUNCI

Relación entre geomorfología e hidrodinámica: un caso de estudio de la Península Valdés, Patagonia, Argentina

A case study is presented to assess the relevance of geomorphology in hydrogeological phenomena in an arid coastal area in the Argentinean extra-Andean Patagonia (Peninsula Valdes) with an average rainfall of 232 mm/year and a soil moisture deficit of about 472 mm/year. Various geomorphic units were identified by interpreting Landsat 7 satellite images processed with ER Mapper software and then surveyed in the field, as well as by geological characterization. The hydrodynamic analysis was based on a survey of 89 wells, the construction of equipotential maps, and the interpretation of pumping-test results by a non-equilibrium method. The hydrochemical characterization was based on chemical tests analyzed with the Easy_Quim 6.0 application. The combination of geomorphological, geological, hydrodynamic and hydrochemical elements allowed the definition of hydromorphological units that are typical of recharge, circulation and discharge areas, the latter both for coastal and inland areas in wetlands (salt pans) with elevations to −40 m relative to sea level. These units and the criteria used for their definition allow immediate recognition of hydrogeological phenomena in arid regions such as the extra-Andean Patagonia, with low information density but with near-optimal satellite imaging of landforms due to the lack of vegetation cover.Se presenta un caso de estudio en el cual se evalúa la importancia de la geomorfología en los fenómenos geohidrológicos, en una región costera árida de la Patagonia Extrandina de Argentina (Península Valdés), con lluvias medias de 232 mm/año y un déficit hídrico del orden de 472 mm/año. Se identificaron distintas unidades geomórficas en base a la interpretación de imágenes satelitales Landsat 7 procesadas con el software ERMapper y luego chequeadas en el campo, junto con la caracterización geológica. El análisis hidrodinámico se basó en el relevamiento de 89 pozos, el trazado de mapas equipotenciales y la interpretación de ensayos de bombeo por métodos de no-equilibrio. La caracterización hidroquímica se basó en análisis químicos, procesados utilizando el código Easy_Quim 6.0. La conjunción de elementos geomorfológicos, geológicos, hidrodinámicos e hidroquímicos permitió definir unidades hidromorfológicas, que son típicas de las áreas de recarga, circulación y descarga, esta última tanto en el litoral como en áreas interiores en humedales (salinas) con cota de hasta −40 m en relación al nivel del mar. Estas unidades y los criterios utilizados para su definición permiten un reconocimiento inmediato del fenómeno geohidrológico en regiones áridas, como la Patagonia Extrandina, con baja densidad de información pero con buena percepción satelital de las geoformas debido a la falta de cobertura vegetal.Facultad de Ciencias Naturales y Muse

Optimización de la producción heteróloga de la enzima α-galactosidasa de "Saccharomyces cerevisiae" a partir de residuos agroindustriales

Programa Oficial de Doutoramento en Biotecnoloxía Avanzada. 5012V01[Resumo]As α-galactosidasas son enzimas que actúan sobre os galactósidos presentes en moitas prantas que están destinadas á elaboración de alimentos e pensos. Polo tanto, o uso da actividade catalítica destas enzimas é de gran interese en moitas aplicacións biotecnolóxicas, especialmente naquelas dirixidas na industria alimentaria á degradación de oligosacáridos derivados da rafinosa (RFOs). Nesta Tese de Doutoramento propónse a caracterización enzimática e máis estudos de sistemas de produción heteróloga da enzima α-galactosidasa de Saccharomyces cerevisiae para avaliar as aplicacións de uso en procesos produtivos industriais. Confírmase que a enzima pertence ao grupo 1 de α-galactosidasas segundo a súa acción sobre galactomano-polisacáridos sintéticos e ten unha forte resistencia a proteasas acedo-neutras, calidade desexable industrialmente. A mellora xenética dunha cepa de S. cerevisiase e o uso dun sistema de expresión en Kluyveromyces lactis permitiu a produción da enzima a partir de residuos agro-industriais de difícil de reciclaxe, como as melazas de remolacha e o soro de leite. Empregouse a Metodoloxía de Superficie de Resposta (MSR) para levar a cabo a optimización estatística da produción da enzima utilizando medios de cultivo convencionais e medios alternativos máis sostibles, acadando os maiores resultados descritos ata agora expresados en termos de actividade α-galactosidasa extracelular. Finalmente, tendo en conta o interese actual pola reutilización e valorización deste tipo de subproductos da industria, realizáronse estudos de inmobilización enzimática empregando preparados enzimáticos a partir destas cepas recombinantes. O deseño de diferentes estratexias de inmobilización permitiu aumentar a estabilidade operativa de diferentes derivados da enzima que conleva a unha vantaxe adicional no seu campo de actuación segundo as necesidades do uso industrial.[Abstract] α-galactosidases are enzymes that act on galactosides present in many plants that are destined to the elaboration of food and feed. It is for this reason that the use of the catalytic activity of these enzymes is of great interest in many biotechnological applications, especially those directed in the food industry to the degradation of oligosaccharides derived from raffinose (RFOs). In this Doctoral Thesis, studies of enzymatic characterization and systems of heterologous production of the α-galactosidase of Saccharomyces cerevisiae are proposed to evaluate the possibilities of its use in industrial production processes. Enzyme belongs to Group 1 of α-galactosidases according to the action on synthetic galactomano-oligosaccharides and has a strong resistance to acid-neutral proteases, an industrial desirable quality. Genetic improvement of a strain of S. cerevisiae and the use of an expression system in Kluyveromyces lactis allowed the production of the enzyme from agroindustrial residues of difficult recycling such as beet molasses and whey. The Response Surface Methodology (RSM) was used for statistical optimization of enzyme production using conventional and sustainable alternative culture media, reaching the highest results described so far expressed in terms of extracellular α-galactosidase activity. Finally, given the current interest in the reuse and valorization of this type of agroindustrial residues, studies of enzymatic immobilization were carried out using enzymatic preparations from recombinant strains. The design of different immobilization strategies allowed to increase the operational stability of different derivatives of the enzyme, which represent an additional advantage according to the needs of the industrial use.[Resumen] Las α-galactosidasas son enzimas que actúan sobre galactósidos presentes en muchos vegetales que son destinados a la elaboración de alimentos y piensos. Es por ello, que el aprovechamiento de la actividad catalítica de estas enzimas es de gran interés en numerosas aplicaciones biotecnológicas, especialmente aquellas dirigidas, en la industria alimentaria, a la degradación de oligosacáridos derivados de la rafinosa (RFOs). En la presente Tesis Doctoral se proponen estudios de caracterización enzimática y sistemas de producción heteróloga de la α-galactosidasa de Saccharomyces cerevisiae para valorar las posibilidades de su uso en procesos productivos industriales. Se confirma que la enzima pertenece al Grupo 1 de α-galactosidasas según la acción sobre galactomano-oligosacáridos sintéticos y presenta una fuerte resistencia a proteasas ácido-neutras, cualidad deseable a nivel industrial. La mejora genética de una cepa de S. cerevisiase y el uso de un sistema de expresión en Kluyveromyces lactis permitió la producción de la enzima a partir de residuos agroindustriales de difícil reciclaje, tales como las melazas de remolacha y el lactosuero. Se utilizó la Metodología de Superficie de Respuesta (MSR) para la optimización estadística de la producción de la enzima empleando medios de cultivo convencionales y medios alternativos más sostenibles, alcanzando los resultados más altos descritos hasta el momento expresados en términos de actividad α-galactosidasa extracelular. Finalmente, dado el interés actual en la reutilización y valorización de este tipo de residuos agroindustriales, se realizaron estudios de inmovilización enzimática utilizando preparaciones enzimáticas procedentes de dichas cepas recombinantes. El diseño de diferentes estrategias de inmovilización permitió aumentar la estabilidad operacional de diferentes derivatizados de la enzima que posibilita una ventaja adicional en su campo de acción según las necesidades del uso industrial

Cycloidal Domains in the Magnetization Reversal Process of Ni80Fe20/Nd16Co84/Gd12Co88 Trilayers

The magnetization reversal of each individual layer in magnetic trilayers ( permalloy / Nd Co / Gd Co ) is investigated in detail with x-ray microscopy and micromagnetic calculations. Two sequential inversion mechanisms are identified. First, magnetic vortex-antivortex pairs move along the field direction while inverting the magnetization of magnetic stripes until they are pinned by defects. The vortex-antivortex displacements are reversible within a field interval which allows their controlled motion. Second, as the reversed magnetic field increases, cycloidal domains appear in the permalloy layer as a consequence of the dissociation of vortex-antivortex pairs due to pinning. The field range where magnetic vortices and antivortices are effectively guided by the stripe pattern is of the order of tens of mT for the Ni Fe layer, as estimated from the stability of cycloid domains in the sample