Characterization and biotechnological application of α-galactosidase from Saccharomyces cerevisiae

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

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

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

Combinations of single-top-quark production cross-section measurements and vertical bar f(LV)V(tb)vertical bar determinations at root s=7 and 8 TeV with the ATLAS and CMS experiments

This paper presents the combinations of single-top-quark production cross-section measurements by the ATLAS and CMS Collaborations, using data from LHC proton-proton collisions at = 7 and 8 TeV corresponding to integrated luminosities of 1.17 to 5.1 fb(-1) at = 7 TeV and 12.2 to 20.3 fb(-1) at = 8 TeV. These combinations are performed per centre-of-mass energy and for each production mode: t-channel, tW, and s-channel. The combined t-channel cross-sections are 67.5 +/- 5.7 pb and 87.7 +/- 5.8 pb at = 7 and 8 TeV respectively. The combined tW cross-sections are 16.3 +/- 4.1 pb and 23.1 +/- 3.6 pb at = 7 and 8 TeV respectively. For the s-channel cross-section, the combination yields 4.9 +/- 1.4 pb at = 8 TeV. The square of the magnitude of the CKM matrix element V-tb multiplied by a form factor f(LV) is determined for each production mode and centre-of-mass energy, using the ratio of the measured cross-section to its theoretical prediction. It is assumed that the top-quark-related CKM matrix elements obey the relation |V-td|, |V-ts| << |V-tb|. All the |f(LV)V(tb)|(2) determinations, extracted from individual ratios at = 7 and 8 TeV, are combined, resulting in |f(LV)V(tb)| = 1.02 +/- 0.04 (meas.) +/- 0.02 (theo.). All combined measurements are consistent with their corresponding Standard Model predictions.Peer reviewe

New insights into the genetic etiology of Alzheimer's disease and related dementias

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M&gt;70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0&lt;e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Palbociclib combined with endocrine therapy in heavily pretreated HR+/HER2- advanced breast cancer patients: Results from the compassionate use program in Spain (PALBOCOMP).

This study evaluated efficacy and safety of palbociclib, a CDK4/6 inhibitor, in heavily-pretreated hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (mBC) patients during the compassionate use program in Spain from February 2015 to November 2017. Patient data were collected retrospectively from 35 hospitals in Spain. Patients with HR+/HER2- mBC who had progressed on ≥4 treatments for advanced disease were eligible. A total of 219 patients received palbociclib in combination with aromatase inhibitors (110; 50.2%), fulvestrant (87; 39.7%), tamoxifen (8; 3.6%) or as single agent (10; 4.6%). Mean age of the patients was 58 years; 31 patients (16.1%) were premenopausal and 162 (83.9%) were postmenopausal at the beginning of treatment with palbociclib. Patients had received a median of 3 previous lines of endocrine therapy (ET) for advanced disease. Real-world tumor response (rwTR) and clinical benefit rate were 5.9% (n = 13) and 46.2% (n = 101), respectively. The median real world progression-free survival (rwPFS) was 6.0 months (95% CI 5.7-7.0) and the median overall survival was 19.0 months (95% CI 16.4-21.7). Subgroup analysis revealed a significant difference in median rwPFS in patients treated with palbociclib plus fulvestrant depending on the duration of prior treatment with fulvestrant monotherapy (>6 versus ≤6 months; HR 1.93, 95% CI 1.37-2.73, p 6 versus ≤6 months; HR 1.93, 95% CI 1.37-2.73, p  Palbociclib can be an effective and safe treatment option in patients with heavily pretreated endocrine-sensitive mBC, especially in those with longer PFS to previous ET