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

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

Zinc: What Is Its Role in Lung Cancer?

Recently, zinc emerged as an important signaling molecule, activating intracellular pathways and regulating cell fate, although our knowledge remains incomplete. Zinc is required in many enzymatic and metabolic pathways, playing roles as enzyme cofactors. In normal cell physiology, optimal zinc availability is essential for regular growth and proliferation. Zinc accumulation has varied effects: from stimulation to inhibition of cell growth, depending on type. There is evidence that zinc is capable of inducing apoptosis in some cancers, while others proved that zinc may act as apoptosis activator depending on the dose and cell type. Upregulation of telomerase in most cancer tissues is considered to be responsible for unlimited proliferation of cancer cells, and in some cell lines, it was induced by Zn. These suggest that Zn is highly involved in cell cycle and metabolism; whether it goes to the survival or the cancer pathway depends on the concentration and the cell type involved. Nevertheless, the conclusion is that Zn is not just another trace element; but a vital one and further studies are needed to elucidate the mechanisms involved in cancer and metastatic spread in order to identify potential therapies