Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin

Background: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility.Results: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k 3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20–30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used.Conclusions: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer

Cyber-Physical Vulnerability Assessment in Smart Grids Based on Multilayer Complex Networks

This article belongs to the Special Issue Cybersecurity and Privacy-Preserving in Modern Smart GridIn the last decade, the main attacks against smart grids have occurred in communication networks (ITs) causing the disconnection of physical equipment from power networks (OTs) and leading to electricity supply interruptions. To deal with the deficiencies presented in past studies, this paper addresses smart grids vulnerability assessment considering the smart grid as a cyber-physical heterogeneous interconnected system. The model of the cyber-physical system is composed of a physical power network model and the information and communication technology network model (ICT) both are interconnected and are interrelated by means of the communication and control equipment installed in the smart grid. This model highlights the hidden interdependencies between power and ICT networks and contains the interaction between both systems. To mimic the real nature of smart grids, the interconnected heterogeneous model is based on multilayer complex network theory and scale-free graph, where there is a one-to-many relationship between cyber and physical assets. Multilayer complex network theory centrality indexes are used to determine the interconnected heterogeneous system set of nodes criticality. The proposed methodology, which includes measurement, communication, and control equipment, has been tested on a standardized power network that is interconnected to the ICT network. Results demonstrate the model’s effectiveness in detecting vulnerabilities in the interdependent cyber-physical system compared to traditional vulnerability assessments applied to power networks (OT).This research was funded by Fundación Iberdrola España, within the 2020 research support scholarship program

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [a-2H2]-p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s-1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H+ transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H+ from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes

Switching the substrate preference of fungal aryl-alcohol oxidase: towards stereoselective oxidation of secondary benzyl alcohols

Oxidation of primary alcohols by aryl-alcohol oxidase (AAO), a flavoenzyme that provides H2O2 to fungal peroxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.This work was supported by the INDOX (KBBE-2013-7-613549) EU project and by the BIO2017-86559-R (GenoBioref), CTQ2016-79138-R and BIO2016-79106-R projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds. Pedro Merino (University of Zaragoza, Spain) is acknowledged for his suggestions on chiral HPLC analyses.Peer ReviewedAward-winningPostprint (published version

Deciphering the unique structure and acylation pattern of Posidonia oceanica lignin

Copyright © 2020 American Chemical Society. Lignins from different parts of the seagrass Posidonia oceanica-namely sheaths, rhizome, and roots-as well as from fibrous balls from P. oceanica detritus were isolated and thoroughly characterized by pyrolysis coupled with gas chromatography/mass spectrometry, derivatization followed by reductive cleavage, two-dimensional nuclear magnetic resonance spectroscopy, and gel permeation chromatography. The lignins of P. oceanica were enriched in guaiacyl (G) over syringyl (S) units, with S/G ratios ranging from 0.1 (fibrous balls) to 0.5 (rhizome). β-O-4′ ethers and phenylcoumarans were the most abundant lignin substructures, followed by resinols, and minor amounts of dibenzodioxocins and spirodienones. Moreover, all lignins were found to be highly I-Acylated (up to 44% of total units), mainly with p-hydroxybenzoates but also, to a lesser extent, with acetates. The data indicated that this acylation extensively occurred in both G-and S-lignin units, contrary to what happens in palms, poplar, and willow, where p-hydroxybenzoates overwhelmingly appear at the I-position of S-units

Theoretical-Experimental Study of the Effect of the External Plates on the Thermal Performance of a Plate Heat Exchanger

Abstract In this work we present a theoretical-experimental research on the thermal phenomena that the endplates present on the performance of the plate heat exchangers in a single step and with a U type configuration. The endplate effect is reflected in a reduction of the temperature difference for heat transfer. The model discusses a theoretical focus that includes the elongation factor, which is a parameter that relates the actual area to the projected area of a plate. This model was performed to 2, 3, 4, 5, and 6 channel flow; therefore the experimentation was carried out to validate the theoretical model, it was carried out for 4, 5, 6, 7, 8 and 9 flow channels. Such results show that the theoretical model deviates in a 5% of the experimental values. The results obtained for theorical temperature correction factor presents a maximum deviation of ± 0.0245 with respect to the experimental values

Efecto del contenido del agente de refuerzo en materiales cerámicos compuestos de hidroxiapatitacircona

En este trabajo se estudia el efecto del contenido partículas de circona parcialmente estabilizadas con magnesia (Mg-PSZ) en el comportamiento mecánico de cerámicas de hidroxiapatita (HA) reforzadas con circona. Se emplearon cantidades de 10, 20 y 30% (en peso) de circona. Los materiales fueron obtenidos mediante prensado uniaxial y sinterización en oxígeno húmedo a una temperatura de 1250°C por 4 horas. La caracterización mediante difracción de rayos X y espectroscopía infrarroja reveló que la hidroxiapatita fue la única fase fosfato cálcica. Los mejores valores de densidad relativa y de resistencia mecánica (tenacidad a la fractura y resistencia a la flexión en tres puntos) se alcanzaron para un contenido de 20% en peso de Mg-PSZ. El estudio fractográfico permitió afirmar que un 10% de circona resulta insuficiente para mejorar significativamente la resistencia mecánica del material compuesto mientras que un 30% inhibe la correcta sinterización de la matriz de hidroxiapatita.Peer Reviewe