Fixation of bioactive compounds to the cuticle of Artemia

Artemia is extensively used in aquaculture to feed early stages of cultured marine species. A problem associated with this practice is that Artemia fails to supply some essential nutrients. As a possible solution, we have devised a procedure to make Artemia a vehicle for exogenous nutrients and other bioactive compounds. It consists of the construction of chimeric proteins composed of a chitin-binding domain, which binds to the cuticle of Artemia, and a carrier domain that conveys a functional property. As confirmatory examples, we describe the successful fixation to Artemia's metanauplii of two hybrid proteins: a β-galactosidase from the thermophilic bacterium Thermotoga maritima and the jellyfish green fluorescent protein (GFP), both linked to the CBM2 chitin-binding domain from the hyperthermophilic archaeon Pyrococcus furiosus. Positive results of experiments carried out ex vivo and in vivo show the validity of this approach. The methodology used could become a general procedure for the attachment of different kinds of bioactive compounds, such as enzymes, hormones, antibiotics, etc., to the cuticle of Artemia as well as other arthropods. Statement of relevance: Our results overcome shortcomings of Artemia as a feedstock.En prensa2,04

Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

BACKGROUND: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. RESULTS: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g(-1) dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg(-1) dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g(-1) dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. CONCLUSIONS: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Fructo-oligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase

10 pags, 6 figs, 3 tabsEfficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (β-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of β(2,6) linkages. © 2011, American Society for Microbiology.This work was supported by Spanish Ministerio de Ciencia e Innovación grants QLK3-CT-2002-01945 and BIO2007-67708-C04-02. Julia Marín-Navarro was funded by a JAE-DOC fellowship from CSI

Preventive dietary supplements of chitin and yeast wall in gilthead seabream

To determine the immunomodulatory effect of the dietary intake of commercial chitin and yeast cell walls in gilthead seabream, Sparus auratus Linnaeus, 1758, specimens were fed diets containing different concentrations of chitin or yeast cell walls for 1, 2, 4 or 6 weeks. Subsequently, their main humoral (lysozyme and complement activities) and cellular (respiratory burst, phagocytic and citotoxic activities) immune responses were analysed. Our results show an increase in most of these immune parameters after 2 weeks of administration, suggesting that specimens fed such supplemented diets would be better prepared to confront an unfavourable situation (infection or stress).Se ha estudiado el efecto de la quitina comercial y de paredes de levadura suministradas en dieta sobre el sistema inmunitario de ejemplares de dorada, Sparus auratus Linnaeus, 1758, sometidos a engorde. Tras 1, 2, 4 y 6 semanas de suministro de dichas dietas, los ejemplares fueron sacrificados y se estudiaron las principales actividades humorales (actividad de la lisozima y del complemento) y celulares (explosión respiratoria, fagocitosis y citotoxicidad) de su sistema inmunitario inespecífico. Los resultados obtenidos indican que, tras dos semanas de suministro de dichas dietas, se observa un incremento de la mayoría de las variables del sistema inmunitario controladas. Esta situación hace prever que los ejemplares están mejor preparados para enfrentarse a una situación desfavorable (infección o estrés).Instituto Español de Oceanografí

Crystallization and preliminary X-ray diffraction analysis of the invertase from Saccharomyces cerevisiae

4 pags, 4 figsSaccharomyces cerevisiae invertase (ScInv) is an enzyme encoded by the SUC2 gene that releases β-fructose from the nonreducing termini of various β - d-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which presents a high degree of oligomerization, was crystallized by vapour-diffusion methods. The crystals belonged to space group P3121, with unit-cell parameters a = 268.6, b = 268.6, c = 224.4 A. The crystals diffracted to 3.3 A resolution and gave complete data sets using a synchrotron X-ray source. © 2012. © 2012 International Union of Crystallography All rights reserved.This work was supported by grant Nos. BIO2010-20508-C04-02 and BIO2010-20508-C04-03 from Dirección General de Investigación, MICINN. This is a product of the Project ‘Factoría Española de Cristalización’ Ingenio/Consolider 2010. MAS-P is supported by a JAE-PreDoc fellowship from CSIC. We also thank the ESRF for beam time and the ID23-1 staff for providing assistance with data collection

In vitro evolution of a fungal laccase in high concentrations of organic cosolvents

Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase-R2 variant-was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic coppers varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur

Structural insights into the specificity of Xyn10B from paenibacillus barcinonensis and its improved stability by forced protein evolution

13 pags, 6 figs, 2 tabsPaenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)8 barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His249-Glu250, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Spanish Ministry of Education and Science Grant BIO2007-67708-C04-0

Three-dimensional structure of Saccharomyces invertase: Role of a non-catalytic domain in oligomerization and substrate specificity

© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv)and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Aring; resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.This work was supported in part by Grants BIO2010-20508-C04-02 and BIO2010-20508-C04-03 from the Spanish Ministry of Education and Science.Peer Reviewe

Non-catalytic domain of invertases: the key for oligomerization and specificity

1 pag. -- XXII Congress and General Assembly of the International Union of Crystallography Madrid, Spain 22- 30 August 2011Peer reviewe