Reducciones asimétricas y oxidaciones selectivas catalizadas por deshidrogenasas inmovilizadas y estabilizadas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Biología Molecular. Fecha de lectura: 22-03-201

Application of Phenolic Compounds for Food Preservation: Food Additive and Active Packaging

Phenolic compounds are well known for their health benefits related to antioxidant activity. In addition, this kind of compounds can be extracted from natural sources, such as olives, grapes, fruits, vegetables, rice, spices, herbs, tea and algae, among others. In this way, these compounds have increased their popularity and, little by little, the consumers are more interested in these compounds due to the fact that they come from natural sources and because they have health biological activity. In fact, other important characteristics associated to phenolic compounds are the antimicrobial activity, because phenolics have the capacity of retarding the microbial invasion in some products and avoiding the putrefaction of others, mainly fruits and vegetables. These properties allow phenolic compounds to be suitable for numerous food preservation applications. Therefore, different kinds of products can be fortificated with phenolic compounds to extend the shelf life of some foods, to turn them in functional food or to incorporate them in food packaging. Active packing is an innovative strategy where phenolic compounds can play an important role for improving the global assessment and extend the shelf life of commercial products

Protein hydrolysis by immobilized and stabilized trypsin

El pdf del artículo es la versión pre-print.-- et al.The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pKa amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pKa amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins. © 2011 American Institute of Chemical Engineers (AIChE).Peer Reviewe

High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces

© 2020 by the authors.Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these supports present highly hydrophobic surfaces, which might promote very undesirable effects on enzyme activity and/or stability. The hydrophilization of the support surface after multipoint enzyme immobilization is proposed here as an alternative to reduce these undesirable effects. The remaining aldehyde groups on the support are modified with aminated hydrophilic small molecules (glycine, lysine or aspartic acid) in the presence of 2-picoline borane. The penicillin G acylase from Escherichia coli (PGA) and alcohol dehydrogenase from Thermus thermophilus HB27 (ADH2) were immobilized on glyoxyl-functionalized agarose, Relizyme and Relisorb. Despite the similar density of aldehyde groups displayed by functionalized supports, their stabilization effects on immobilized enzymes were quite different: up to 300-fold lower by hydrophobic supports than by highly hydrophilic glyoxyl-agarose. A dramatic increase in the protein stabilities was shown when a hydrophilization treatment of the hydrophobic support surface was done. The PGA immobilized on the glyoxyl-Relisorb hydrophilized with aspartic acid becomes 280-fold more stable than without any treatment, and it is even more stable than the PGA immobilized on the glyoxyl agarose.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012-36861 and CTQ2015-70348) and the EU FP7 project SuSy (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions, C-KBBE/3293). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19260) funded by the Spanish Ministry of Economy, Industry and Competitiveness.Peer reviewe

New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

<p>Abstract</p> <p>Background</p> <p>The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD⁺pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD⁺. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.</p> <p>Results</p> <p>The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.</p> <p>Conclusion</p> <p>We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of <it>rac</it>-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.</p

The PDZ-adaptor protein syntenin-1 regulates HIV-1 entry

Syntenin-1 is a cytosolic adaptor protein involved in several cellular processes requiring polarization. Human immunodeficiency virus type 1 (HIV-1) attachment to target CD4(+) T-cells induces polarization of the viral receptor and coreceptor, CD4/CXCR4, and cellular structures toward the virus contact area, and triggers local actin polymerization and phosphatidylinositol 4,5-bisphosphate (PIP(2)) production, which are needed for successful HIV infection. We show that syntenin-1 is recruited to the plasma membrane during HIV-1 attachment and associates with CD4, the main HIV-1 receptor. Syntenin-1 overexpression inhibits HIV-1 production and HIV-mediated cell fusion, while syntenin depletion specifically increases HIV-1 entry. Down-regulation of syntenin-1 expression reduces F-actin polymerization in response to HIV-1. Moreover, HIV-induced PIP(2) accumulation is increased in syntenin-1–depleted cells. Once the virus has entered the target cell, syntenin-1 polarization toward the viral nucleocapsid is lost, suggesting a spatiotemporal regulatory role of syntenin-1 in actin remodeling, PIP(2) production, and the dynamics of HIV-1 entry

Finding immunological differences to help diagnosis and early treatment of Kawasaki Disease and MIS-C (Multisystem Inflammatory Syndrome in Children)

Resumen del trabajo presentado en el 43 Congreso De La Sociedad Española de Inmunología, celebrado en León (España) del 22 al 24 de septiembre de 2022.The recent COVID-19 pandemic was first thought to spare children from health deprivation caused by infection with SARS-CoV-2. However, soon a new syndrome resembling Kawasaki Disease (KD) was reported: Multisystem Inflammatory Syndrome in Children (MIS-C). The aim of this study is to provide new biomarkers for both diseases in order to facilitate diagnosis and reduce the time-lapse until treatment is provided – which will reduce the risk of developing severe cardiovascular complications. An extensive immune system characterization by flow cytometry and serum protein characterization by a multiplex technology (Olink) was performed from fresh blood samples of patients with acute MIS-C (n=19) and KD (n=10). For protein characterization we also analysed recovery samples for these groups (n=19 and n=8, respectively). Besides the already described lymphopenia in MIS-C, we found additional significant immune differences in both groups. Although lymphocyte numbers (cells/ml) were lower in MIS-C, percentages of activated T-CD4+ and T-CD8+ cells were higher compared to KD. Moreover, when comparing activated T cells in MIS-C and KD individually, regulatory T cells (Treg) showed the highest levels. These data suggest a stronger response of T cells in MIS-C, and higher Treg activity in both groups, which could reflect the response to the excessive inflammation. Ratios previously described in other inflammatory conditions were evaluated: MIS-C showed higher neutrophil/lymphocyte and Th17/Treg ratios than KD, suggesting higher inflammatory conditions in this group. In addition, monocyte and dendritic cells (DCs) numbers were decreased in MIS-C relative to KD. Parallel to these inflammatory cellular profiles, we identified increased levels of inflammatory cytokines in plasma of patients during the acute phase of the disease compared to recovery samples. Moreover, IL-6, which is one of the main cytokines involved in cytokine storm in adult COVID-19, was higher in MIS-C suggesting, again, stronger inflammatory conditions in this pathology compared to KD

Identifying sustainability priorities among value chain actors in artisanal common octopus fisheries

The United Nations (UN) Decade of Ocean Science highlights a need to improve the way in which scientific results effectively inform action and policies regarding the ocean. Our research contributes to achieving this goal by identifying practical actions, barriers, stakeholder contributions and resources required to increase the sustainability of activities carried out in the context of artisanal fisheries to meet UN Sustainable Development Goals (SDG) and International Year of Artisanal Fisheries and Aquaculture (IYAFA) Global Action Plan (GAP) Pillar targets. We conducted a novel ‘social value chain analysis’ via a participatory workshop to elicit perspectives of value chain actors and fisheries stakeholders associated with two Spanish artisanal common octopus (Octopus vulgaris) fisheries (western Asturias—Marine Stewardship Council [MSC] certified, and Galicia—non-MSC certified) about their priorities regarding sustainable octopus production and commercializationOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The authors acknowledge the financial support from the Cephs and Chefs Project (https://www.cephsandchefs.com/) funded by the European Regional Development Fund (https://ec.europa.eu/regional_policy/en/funding/erdf/) through the Interreg Atlantic Area Programme grant number EAPA_282/2016. CP, TF, KR and DC would also like to acknowledge financial support to CESAM by FCT/MCTES (UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020), through national funds. CP acknowledges the FCT research contract 2020.02510.CEECIND. SV and PP acknowledge the financial support from the Xunta de Galicia (https://www.xunta.gal/portada) (RECREGES II project under Grant 1400 ED481B2018/017 and Grupo de Referencia Competitiva GI-2060 AEMI, under Grant 1401 ED431C2019/11). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS

Atlas QR de procedimientos básicos en clínica hospitalaria equina

Los códigos QR se utilizan actualmente como herramienta docente de gran utilidad ya que permiten al alumno emplear las nuevas tecnologías para el aprendizaje. Con este proyecto pretendemos acercar la actividad clínica equina al alumno de veterinaria