Physical Chemistry Chemical Physics / Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity

Abstract in deutscher Sprache nicht verf\ufcgbarThis study presents the first electrochemical characterization of the pyranose oxidase (POx) variant N593C (herein called POx-C), which is considered a promising candidate for future glucose-sensing applications. The resulting cyclic voltammograms obtained in the presence of various concentrations of glucose and mediator (1,4-benzoquinone, BQ), as well as the control experiments by addition of catalase, support the conclusion of a complete suppression of the oxidase function and oxygen reactivity at POx-C. Additionally, these electrochemical experiments demonstrate, contrary to previous biochemical studies, that POx-C has a fully retained enzymatic activity towards glucose. POx-C was immobilized on a special screen-printed electrode (SPE) based on carbon ink and grafted with gold-nanoparticles (GNP). Suppression of the oxygen reactivity at N593C-POx variant is a prerequisite for utilizing POx in electrochemical applications for glucose sensing. To our knowledge, this is the first report presented in the literature showing an absolute conversion of an oxidase into a fully active equivalent dehydrogenase via a single residue exchange

Policies for an Ageing Workforce Work-life balance, working conditions and equal opportunities 2019

At a time of rapid population ageing, a key means of sustaining current welfare states is to extend the length of working lives. In 2050, the share of people over the age of 75 years will be the same as the share over 65 years today. And just as not all are able to work to the age of 65 now, not everyone will be able to work to the age of 75 in 2050; even if future older workers will in all likelihood be healthier and have better working aids at their disposal. Extending average working lives by 10 years, and at the same time ensuring an adequate social safety net for those unable to work into their late 60s and 70s, is a major social policy challenge for the coming decades. And because people are much more likely to work late in life if they had stable careers before reaching 60, tackling this policy challenge means pulling on many more social policy levers than just pension policy. While being keenly aware of these issues and how they relate to the overall agenda of active ageing, Commissioner Thyssen also reminds us in her Foreword that marked increases in life expectancy – both past and in the future – represent enormous social progress. The Commissioner makes the point that older people too contribute to society. And more so with lifelong learning and investment in skills

Semi-rational engineering of cellobiose dehydrogenase for improved hydrogen peroxide production

Abstract Background The ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH’s ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH’s low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover. Results A uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction. Conclusions Site-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase’s oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.The authors thank the European Commission (FP7 243529-2-COTTONBLEACH) for financial support. CKP thanks the Austrian Science Fund (FWF) for financial support (grant P22094). IK is a member of the doctoral program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF; W1224). MA thanks the Spanish Government for financial support (BIO2010-19697).Peer Reviewe

Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris)

Nicht verfügbarMultigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucosemethanolcholine (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.(VLID)192910

Heterologous expression of a recombinant lactobacillal -galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system

Background: Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric -galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri -galactosidase. Results: Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum -galactosidase levels obtained were 130 U/mg protein and 3540 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), -galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant -galactosidase. Conclusions: The over-expression of recombinant L. reuteri -galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.(VLID)90704

Mercury pollution for marine environment at Farwa Island, Libya

Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os­(4,4′-dimethoxy-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ or [Os­(4,4′-dimethyl-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidation. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidation using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer solution, pH 7.4, with 150 mM NaCl), higher glucose oxidation current densities, 0.41 mA cm^–2, are obtained from enzyme electrodes containing the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepared using deglycosylated enzyme coimmobilized with [Os­(4,4′-dimethyl-2,2′-bipyridine)₂(poly­(vinylimidazole))₁₀Cl]⁺ and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymatic fuel cell. A maximum power density of 275 μW cm^–2 is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva maximum power densities of 73 and 6 μW cm^–2 are obtained for the same fuel cell configuration, respectively

Revisiting the (Proto-)Feminist Traits of Leonora Christina’s Life and Work through Nina Karin Monsen’s Jammersminne

This paper argues that the novel Jammersminne by the Norwegian writer Nina Karin Monsen exhibits striking intertextual references, which are central to a proper understanding of this evidently feminist text, and that the life and work of the Danish noblewoman Leonora Christina forms a central part of Monsen’s intertextual strategy. Beyond the obvious title analogy between Monsen’s epistolary novel Jammersminne and Leonora Christina’s prison recollections Jammers Minde, Monsen’s novel refers to Leonora Christina through a number of subtle hints. A comparative look at Monsen’s Jammersminne and Leonora Christina’s writings will furthermore shed light on the latter’s (alleged) feminist viewpoints, as exhibited in the French autobiography, in Jammers Minde, and in Hæltinners Pryd

Hartmanns "Iwein" und die altnordische "Ivens saga" als produktive Rezeption

Anhand ausgewählter Themen (Alliteration, kelt. Mythos in höf. Roman, Genderthematik, König Artus und Protagonist, Löwe) werden die Schwerpunkte, Übersetzungsmethoden und Interpretationen der beiden Adaptionen Iwein und Ívens saga miteinander verglichen. Dabei werden für die jeweilige Thematik repräsentative Textstellen einander gegenüber gestellt und sowohl individuell, als auch vergleichend analysiert

Semi-rational engineering of Adh2 for improved methanol utilization in Komagataella phaffii

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