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[Background] Floudas et al. (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information., [Results] With this purpose, we analyzed the evolutionary pathway leading to the most efficient lignin-degrading peroxidases characterizing Polyporales species. After sequence reconstruction from 113 genes of ten sequenced genomes, the main enzyme intermediates were resurrected and characterized. Biochemical changes were analyzed together with predicted sequences and structures, to understand how these enzymes acquired the ability to degrade lignin and how this ability changed with time. The most probable first peroxidase in Polyporales would be a manganese peroxidase (Mn3+ oxidizing phenolic lignin) that did not change substantially until the appearance of an exposed tryptophan (oxidizing nonphenolic lignin) originating an ancestral versatile peroxidase. Later, a quick evolution, with loss of the Mn2+-binding site, generated the first lignin peroxidase that evolved to the extant form by improving the catalytic efficiency. Increased stability at acidic pH, which strongly increases the oxidizing power of these enzymes, was observed paralleling the appearance of the exposed catalytic tryptophan., [Conclusions] We show how the change in peroxidase catalytic activities meant an evolutionary exploration for more efficient ways of lignin degradation by fungi, a key step for carbon recycling in land ecosystems. The study provides ancestral enzymes with a potential biotechnological interest for the sustainable production of fuels and chemicals in a biomass-based economy., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI), and the EC OpenAIRE FP7 post-grant Open Access Pilot., This work was supported by the INDOX (KBBE-2013-613549) and EnzOx2 (H2020-BBI-PPP-2015-2-720297) EU projects and the NOESIS (BIO2014-56388-R) project of the Spanish Ministry of Economy and Competitiveness (MINECO). The work conducted by JGI was supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231., EUR 1,620 APC fee funded by the EC FP7 Post-Grant Open Access Pilot

Combining a computational analysis with site-directed mutagenesis, we have studied the long-range electron transfer pathway in versatile and lignin peroxidases, two enzymes of biotechnological interest that play a key role for fungal degradation of the bulky lignin molecule in plant biomass. The in silico study established two possible electron transfer routes starting at the surface tryptophan residue previously identified as responsible for oxidation of the bulky lignin polymer. Moreover, in both enzymes, a second buried tryptophan residue appears as a top electron transfer carrier, indicating the prevalence of one pathway. Site-directed mutagenesis of versatile peroxidase (from Pleurotus eryngii) allowed us to corroborate the computational analysis and the role played by the buried tryptophan (Trp244) and a neighbor phenylalanine residue (Phe198), together with the surface tryptophan, in the electron transfer. These three aromatic residues are highly conserved in all the sequences analyzed (up to a total of 169). The importance of the surface (Trp171) and buried (Trp251) tryptophan residues in lignin peroxidase has been also confirmed by directed mutagenesis of the Phanerochaete chrysosporium enzyme. Overall, the combined procedure identifies analogous electron transfer pathways in the long-range oxidation mechanism for both ligninolytic peroxidases, constituting a good example of how computational analysis avoids making extensive trial-error mutagenic experiments. This work was supported by the INDOX (KBBE-2013-7-613549) EU project, and by the projects EnzOx2 (H2020-BBI-PPP-2015-2-720297) of the Joint Undertaking of European BioBased Industries (www.bbi-europe.eu), and BIO2014-56388-R (NOESIS) and CTQ2016-79138-R of the SpanishMinistry of Economy and Competitiveness (MINECO). F.J.R.-D. acknowledges a MINECO Ramón & Cajal contract.

An efficient heterologous expression system for Auricularia auricula-judae dye-decolorizing peroxidase (DyP) has been constructed. DNA coding for the mature protein sequence was cloned into the pET23a vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant DyP was obtained in high yield as inclusion bodies, and different parameters for its in vitro activation were optimized with a refolding yield of ∼8.5% of the E. coli-expressed DyP. Then, a single chromatographic step allowed the recovery of 17% of the refolded DyP as pure enzyme (1.5 mg per liter of culture). The thermal stabilities of wild DyP from A. auricula-judae and recombinant DyP from E. coli expression were similar up to 60 °C, but the former was more stable in the 62–70 °C range. Stabilities against pH and H2O2 were also measured, and a remarkably high stability at extreme pH values (from pH 2 to 12) was observed. The kinetic constants of recombinant DyP for the oxidation of different substrates were determined and, when compared with those of wild DyP, no important differences were ascertained. Both enzymes showed high affinity for Reactive Blue 19 (anthraquinone dye), Reactive Black 5 (azo dye), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,6-dimethoxyphenol, with similar acidic pH optima and oxidative stabilities. Oxidation of veratryl alcohol and a nonphenolic lignin model dimer were confirmed, although as minor enzymatic activities. Interestingly, two sets of kinetic constants could be obtained for the oxidation of Reactive Blue 19 and other substrates, suggesting the existence of more than one oxidation site in this new peroxidase family.

The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized., The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+ oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+ binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+ and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

Ministerio de Economia y Competitividad (Espana) FPA2010-22131- C02-01 (FINURA) FPA2013-47327-C2-1-R

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine–VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine–VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.

We analyze radiative processes of a quantum system composed by two identical two-level atoms interacting with a massless scalar field prepared in the vacuum state in the presence of perfect reflecting flat mirrors. We consider that the atoms are prepared in a stationary maximally entangled state. We investigate the spontaneous transitions rates from the entangled states to the collective ground state induced by vacuum fluctuations. In the empty-space case, the spontaneous decay rates can be enhanced or inhibited depending on the specific entangled state and changes with the distance between the atoms. Next, we consider the presence of perfect mirrors and impose Dirichlet boundary conditions on such surfaces. In the presence of a single mirror the transition rate for the symmetric state undergoes a slight reduction, whereas for the antisymmetric state our results indicate a slightly enhancement. Finally, we investigate the effect of multiple reflections by two perfect mirrors on the transition rates., Comment: submitted version to the journal

18 p.-8 fig., [Background]: Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures., [Results]: Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14–16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10–14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends., [Conclusions]: Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results., This work was supported by the INDOX (KBBE-2013-613549) EU project, the BIO2014-56388-R, AGL2014-53730-R, and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the ProteoRed platform of the Spanish Institute of Health Carlos III (ISCIII). The work conducted by the US DOE JGI is supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231. FJR-D thanks a Ramón y Cajal contract of the Spanish MINECO, and JR thanks a contract of the CSIC project 201440E097., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441, 487–498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h of incubation at pH 9 compared with complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability., This work was supported by HIPOP [grant number BIO2011-26694], NOESIS [grant number BIO2014-56388-R] and DEWRY [grant number BIO2013-43407-R] projects of the Spanish Ministry of Economy and Competitiveness (MINECO), and by the PEROXICATS [grant number KBBE-2010-4-265397] and INDOX [grant number KBBE-2013-7-613549] European projects. V.S.-J. and F.J.R.-D. are grateful for the financial support of an FPI fellowship and a Ramón y Cajal contract of the Spanish MINECO, respectively.

ECOS-LINCE (Martel I et al., Proceedings IPAC’14, 2014) is a proposal for a new European First Class high intensity heavy-ions accelerator for stable ions, with energies at and above the coulomb barrier. The low energy section will be achieved using a 72.75 MHz normal conducting four vanes RFQ designed to give a 460 keV/u boost for A/Q = 7 ions in about 5 m (Orduz AK et al., IPAC’14, 2014). The geometry vanes are modeled to accommodate windows in order to obtain a clear separation of the RFQ modes (Ostroumov P et al., Rev ST Accel Beams 15:110101, 2012). This article presents the experimental results of the RF test carried out on a aluminum prototype.

In this work we present the GLObal ReactIon Array (GLORIA), a compact silicon array which has been build in order to study heavy ion reactions involving radioactive beams . It has been used for the first time at the SPIRAL-GANIL facility in Caen (France), for studying the scattering of the system \(^{8}\)He + \({^{208}}\)Pb at energies around the Coulomb barrier.

Ligninolytic peroxidases are involved in natural wood decay in strict acid environments. Despite their biotechnological interest, these high-redox potential enzymes are not functional at basic pH due to the loss of calcium ions that affects their structural integrity. In this study, we have built catalytic activity at basic pH in a versatile peroxidase (VP) previously engineered for thermostability. By using laboratory evolution and hybrid approaches, we designed an active and highly stable alkaline VP while the catalytic bases behind the alkaline activation were unveiled. A stabilizing mutational backbone allowed the pentacoordinated heme state to be maintained, and the new alkaline mutations hyperactivated the enzyme after incubation at basic pHs. The final mutant oxidises substrates at alkaline pHs both at the heme channel and at the Mn2+ site, while the catalytic tryptophan was not operational under these conditions. Mutations identified in this work could be transferred to other ligninolytic peroxidases for alkaline activation., This research was supported by the European Commission project FP7-KBBE-2013-7-613549-INDOX, the COST-Action CM1303 and by the Spanish government (grants: BIO2013-43407-R-DEWRY and CAMBIOS-RTC-2014-1777-3).

In this work the preliminary results obtained during a experimental campaign for detecting neutron radiation using a single crystal diamond detector are shown. This investigation was motivated by the need of monitoring neutron radiation in radiotherapy facilities.

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn2+ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s−1 mm−1, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s−1 mm−1 for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower Km values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.

16 p.-8 fig.-1 tab., The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora., Supported by a Junta para Ampliación de Estudios fellowship of the Consejo Superior de Investigaciones Científicas, co-funded by the European Social Fund. Supported by a Ministry of Economy and Competitiveness (MINECO) Ramón y Cajal contract. This work was supported by the RAPERO Grant BIO2008-01533 and HIPOP Grant BIO2011-26694 from the Spanish Ministry of Economy and Competitiveness (to A. T. M. and F. J. R.-D., respectively), by PEROXICATS European Project Grant KBBE-2010-4-265397 (to A. T. M.), and by United States Department of Energy Grant DE-AI02-07ER64480 (to K. E. H.)

16 p.-8 fig.-1 tab. Courier, Marie et al., This study was also funded by the French National Research Agency (A*MIDEX project ANR11-IDEX-0001-02;ANR FUNLOCK ANR-13-BIME-0002-01). The work by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231.

12 páginas, 5 figuras, 2 tablas -- PAGS nros. 487-498, The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability

a)Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, E-41080 Seville, Spain b)University of Aveiro, CICECO, 3810-193, Aveiro, Portugal c)Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Oxidation of the manganese-substituted polyoxometalate [SiW11MnII(H2O)O39]6- (SiW11MnII) to [SiW11MnIII(H2O)O39]5-, one of the most selective polyoxometalates for the kraft pulp delignification, by versatile peroxidase (VP) was studied. First, SiW11MnII was demonstrated to be quickly oxidized by VP at room temperature in the presence of H2O2 (Km= 6.4±0.7 mM and kcat= 47±2 s-1). Second, the filtrate from eucalypt pulp delignification containing reduced polyoxometalate was treated with VP/H2O2, and 95-100% reoxidation was attained. In this way, it was possible to reuse the liquor from a first SiW11MnIII stage for further delignification, in a sequence constituted by two polyoxometalate stages, and a short intermediate step consisting of the addition of VP/H2O2 to the filtrate for SiW11MnII reoxidation. When the first ClO2 stage of a conventional bleaching sequence was substituted by the two-stage delignification with polyoxometalate (assisted by VP) a 50% saving in ClO2 was obtained for similar mechanical strength of the final pulp., This study has been supported by the EU project BIORENEW (NMP2-CT-2006-026456) and the Spanish projects AGL2008-00709 and BIO2008-01533.

Lignin removal is a central issue in paper pulp manufacture, and production of other renewable chemicals, materials, and biofuels in future lignocellulose biorefineries. Biotechnology can contribute to more efficient and environmentally sound deconstruction of plant cell wall by providing tailor-made biocatalysts based on the oxidative enzymes responsible for lignin attack in Nature. With this purpose, the already-known ligninolytic oxidoreductases are being improved using (rational and random-based) protein engineering, and still unknown enzymes will be identified by the application of the different ‘omics’ technologies. Enzymatic delignification will be soon at the pulp mill (combined with pitch removal) and our understanding of the reactions produced will increase by using modern techniques for lignin analysis., The partners of BIORENEW EU-contract NMP2-CT-2006-026456 (see at www.biorenew.org) are acknowledged for contributions. The financial support of the Spanish Biotechnology (project BIO2008-01533), Agriculture (project AGL2008-00709), and CENIT (project I + DEA on ‘Ethanol for automotion’) programs is also acknowledged.

We analyse the possibilities to detect a new Z ′ boson in di-electron events at Tevatron and LHC in the framework of the 331 model with right-handed neutrinos. We also study other fermions and Higgs events as final state at LHC. Using $p\bar{p}$ collision data collected by the CDF II detector at Fermilab Tevatron, we find that the 331 Z ′ boson is excluded with masses below 920 GeV. For an integrated luminosity of 100 fb−1 at LHC, and considering a central value $M_{Z^{\prime}}=1500$ GeV, we obtain the invariant-mass distribution in the process pp→Z ′→e + e −, where a huge peak, corresponding to 800 signal events/(20 GeV), is found above the SM background. The number of di-electron events vary from 50000 to 2400 in the mass range of $M_{Z^{\prime}}=800$ –2000 GeV. We also obtain branching ratios and cross sections in other fermion and Higgs channels at LHC, where a heavy top quark T exhibits the biggest ratio with m T =300 GeV.

Lignin-degrading peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii versatile peroxidase (VP) revealed, for the first time in a fungal peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k2app) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k2) was more affected than the dissociation constant (KD2). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k3) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.

11 páginas, 7 figuras, 4 tablas -- PAGS nros. 1685-1695 Lignin degradation by fungal peroxidases is initiated by one-electron transfer to an exposed tryptophan radical, a reaction mediated by veratryl alcohol (VA) in lignin peroxidase (LiP). Versatile peroxidase (VP) differs not only in its oxidation of Mn2+ at a second catalytic site but also in its ability to directly oxidize different aromatic compounds. The catalytic tryptophan environment was compared in LiP and VP crystal structures, and six residues near VP Trp164 were modified by site-directed mutagenesis. Oxidation of Mn2+ was practically unaffected. However, several mutations modified the oxidation kinetics of the high-redox-potential substrates VA and Reactive Black 5 (RB5), demonstrating that other residues contribute to substrate oxidation by the Trp164 radical. Introducing acidic residues at the tryptophan environment did not increase the efficiency of VP oxidizing VA. On the contrary, all variants harboring the R257D mutation lost their activity on RB5. Interestingly, this activity was restored when VA was added as a mediator, revealing the LiP-type behavior of this variant. Moreover, combination of the A260F and R257A mutations strongly increased (20−50-fold) the apparent second-order rate constants for reduction of VP compounds I and II by VA to values similar to those found in LiP. Dissociation of the enzyme−product complex seemed to be the limiting step in the turnover of this improved variant. Nonexposed residues in the vicinity of Trp164 can also affect VP activity, as found with the M247F mutation. This was a direct effect since no modification of the surrounding residues was found in the crystal structure of this variant

The gene of a peroxidase described as being involved in carotenoid degradation was cloned from a strain that was conserved as Lepista irina (CBS 458.79). Gene sequencing revealed high nucleotide an...

The readout system for the new TRACE detector requires monitorization and sampling of all pulses in a large number of channels with very strict space and power consumption restrictions for the front-end electronics and cabling. A triggerless solution has been chosen involving front-end analog memories that sample a 1 μs window of the waveform of any valid pulses at 200 MHz while performing zero suppression and serialization tasks. An analog memory ASIC named PLAS (for PipeLined Asymmetric SCA) is presented that allows pulse capture with no dead time in any channel while reducing die area requirements for high input channel counts. The circuit is based on a novel architecture where the typical Switched Capacitor Array (SCA) structure is partitioned into two asymmetric stages and FIFO queue-like control circuitry is introduced for captured data. The ASIC has been designed in 0.18 μm CMOS technology with 32 independent, self-triggered input channels and a size of 3.5 × 3.9mm2. Simulations predict figures around 100MHz bandwidth, 12 ENOB and 10 mW per channel.

One of the capabilities of diamond detectors is the measurement of neutron radiation, due to their carbon composition. In these work is described a campaign of measurements related with neutron irradiation using different sources: starting with a 252 Cf standard neutron source, continuing with a well-known nuclear reaction at a low-energy tandem accelerator and finishing with neutron radiation produced in a hospital radiotherapy machine. The aim of all these measurements is to evaluate the qualities of diamond device to detect neutron irradiation, thus proving they are good candidates to be used as neutron dosimeters at hospital accelerators. Our preliminary results show that diamond detectors are a promising tool to be implemented in such a kind of facilities.

Presented at the XXXIV Mazurian Lakes Conference on Physics, Piaski, Poland, September 6–13, 2015., The structure and dynamics of 8 He have been studied through the col- lision process with a 208 Pb target at energies of 22 and 16 MeV, above and below the Coulomb barrier, respectively. The energy and angular dis- tributions of the elastically scattered 8 He and the 6 ; 4 He fragments were measured. In this paper, we discuss the method used to determine the ef- fective position of the beam spot on the reaction target and the scattering and solid angles of each pixel of the detector array., This work was supported in part by grants FPA2010-22131-C021-01 and FPA2014-59954-C3-1-P from the Spanish Ministry of Economy and Com- petitiveness, grant N202 033637 from the Ministry of Science and Higher Education of Poland and Contract EUI2009-04163 (EUROGENESIS) from the European Science Foundation., This work was supported in part by grants FPA2010-22131-C021-01 and FPA2014-59954-C3-1-P from the Spanish Ministry of Economy and Competitiveness, grant N202 033637 from the Ministry of Science and Higher Education of Poland and Contract EUI2009-04163 (EUROGENESIS) from the European Science Foundation.

Peroxide-activated Auricularia auricula-judae dye-decolorizing peroxidase (DyP) forms a mixed Trp377 and Tyr337 radical, the former being responsible for oxidation of the typical DyP substrates (Linde et al. Biochem. J., 2015, 466, 253-262); however, a pure tryptophanyl radical EPR signal is detected at pH 7 (where the enzyme is inactive), in contrast with the mixed signal observed at pH for optimum activity, pH 3. On the contrary, the presence of a second tyrosine radical (at Tyr147) is deduced by a multifrequency EPR study of a variety of simple and double-directed variants (including substitution of the above and other tryptophan and tyrosine residues) at different freezing times after their activation by H2O2 (at pH 3). This points out that subsidiary long-range electron-transfer pathways enter into operation when the main pathway(s) is removed by directed mutagenesis, with catalytic efficiencies progressively decreasing. Finally, self-reduction of the Trp377 neutral radical is observed when reaction time (before freezing) is increased in the absence of reducing substrates (from 10 to 60 s). Interestingly, the tryptophanyl radical is stable in the Y147S/Y337S variant, indicating that these two tyrosine residues are involved in the self-reduction reaction.

The aim is to evaluate the outcomes obtained from the implementation of a pre-hospital thrombolysis protocol in 3 rural emergency care teams, as well as delays and strategies of reperfusion applied in the treatment of the ST-segment elevation myocardial infarction.Retrospective cohort study (n=52) with historical control (n=20) of the patients assisted for ST-segment elevation myocardial infarction. Medical emergency care teams, hospital, computerized medical history and ARIAM register reports were revised, obtaining epidemiological and clinical features, off-hospital management, reperfusion, time intervals and mortality.The baseline features in both groups were not significantly different. There was a non-significant improvement of emergency care teams-hospital diagnostic concordance (85.3 versus 76.9%). We found a similar use of nitroglycerin, morphine and aspirin; significant increase (P0.0001) of clopidogrel/prasugrel (55 versus 90.4%) and enoxaparin/fondaparinux (35 versus 76.9%), as well as pre-hospital thrombolysis (5 versus 30,8%, P0.03), that was applied within the first 2h to 71.4%, with a median door-needle of 40min, whereas in-hospital thrombolysis and primary angioplasty were performed after 3h from the symptoms onset (P0.01). Delays are associated with the patient's own lateness (P0.02). Pharmaco-invasive strategy increases (62.5 versus 84.6%) more than primary angioplasty (15 versus 17.3%), reducing in-hospital thrombolysis (35 versus 19.2%), all of them non-significant. Complications are similar and one-year mortality is reduced (P0.67).The protocol is effective, safe, and reliable. It reduces delays and improves pre-hospital attention. The pharmaco-invasive strategy is a valid option.

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant., We demonstrate that an exposed tryptophan is responsible for high-turnover oxidation by DyP, a representative of a new protein superfamily. Long-range electron transfer from surface tryptophan residues forming radicals appears as a general mechanism for peroxidase oxidation of bulky substrates.

A study of the identification properties and of the energy response of a Si-Si-CsI(Tl) $\Delta$ E-E telescope exploiting a partially depleted second Si stage has been performed. Five different bias voltages have been applied to the second stage of the telescope, one corresponding to full depletion, the others associated with a depleted layer ranging from 60% to 90% of the detector thickness. Fragment identification has been obtained using either the $\Delta$E-E technique or the Pulse Shape Analysis (PSA). Charge collection efficiency has been evaluated. The $\Delta$ E-E performance is not affected by incomplete depletion. Isotopic separation capability improves at lower bias voltages with respect to full depletion, though charge identification thresholds increase.

Versatile peroxidases are heme enzymes that combine catalytic properties of lignin peroxidases and manganese peroxidases, being able to oxidize Mn(2+) as well as phenolic and non-phenolic aromatic compounds in the absence of mediators. The catalytic process (initiated by hydrogen peroxide) is the same as in classical peroxidases, with the involvement of 2 oxidizing equivalents and the formation of the so-called Compound I. This latter state contains an oxoferryl center and an organic cation radical that can be located on either the porphyrin ring or a protein residue. In this study, a radical intermediate in the reaction of versatile peroxidase from the ligninolytic fungus Pleurotus eryngii with H(2)O(2) has been characterized by multifrequency (9.4 and 94 GHz) EPR and assigned to a tryptophan residue. Comparison of experimental data and density functional theory theoretical results strongly suggests the assignment to a tryptophan neutral radical, excluding the assignment to a tryptophan cation radical or a histidine radical. Based on the experimentally determined side chain orientation and comparison with a high resolution crystal structure, the tryptophan neutral radical can be assigned to Trp(164) as the site involved in long-range electron transfer for aromatic substrate oxidation.

20 p.-3 tab.-9 fig., Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea’s extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes., The major portions of this work were performed under US Department of Agriculture Cooperative State, Research, Education, and Extension Service Grant 2007-35504-18257 (to DC and RAB). The US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231. This work was also supported by the HIPOP (BIO2011-26694) project of the Spanish Ministry of Economy and Competitiveness (MINECO) (to FJRD), the PEROXICATS (KBBE-2010-4-265397) and INDOX (KBBE-2013-.3.3-04-613549) European projects (to ATM), and the Chilean National Fund for Scientific and Technological Development Grant 1131030 (to LFL).

Electron transfer (ET) of versatile peroxidase (VP) was studied in the bioelectrocatalytic reduction reaction of H2O2 at peroxidase-modified graphite electrodes to obtain additional information on the kinetic characteristics of this novel ligninolytic peroxidase. Rotating disk electrodes (RDE) experiments were performed at 0 V (vs. SCE) in two different buffers (tartrate buffer, pH 5.0; and citrate buffer, pH 3.0). From measurements of the mediated and mediatorless currents of H2O2 reduction at the RDE, the percentage of VP molecules involved in direct ET (� 55%) was calculated. The peroxidase-modified electrodes were used for determination of the donor substrates in RDE mode, and the results were interpreted in terms of catalytic efficiencies. 2005 Elsevier B.V. All rights reserved.

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca(2+) and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.

Heterologous expression of Pleurotus eryngii versatile peroxidase (VP) in Escherichia coli was investigated. The cDNA encoding the mature sequence of the allelic variant VPL2 was cloned into the expression vector pFLAG1 and expressed in E. coli W3110. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant polypeptide (VPL2∗) was found to be the major protein located in inclusion bodies. In vitro folding of VPL2∗ was initially performed under conditions previously determined for folding of recombinant lignin peroxidase from Phanerochaete chrysosporium (LiPH8∗) but a very low yield of active enzyme was obtained (

12 p.-2 tab.-5 fig., [Background] Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities., [Results] In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus VP and Phanerochaete chrysosporium LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn2+-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer., [Conclusion]: A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes., This work was supported by the HIPOP (BIO2011-26694) Spanish project, and the European projects INDOX (KBBE-2013-7-613549) and PEROXICATS (KBBE-2010-4-265397). EF-F acknowledges a Junta de Ampliación de Estudios fellowship of CSIC, co-funded by the European Social Fund, and FJR-D acknowledges a Ramón y Cajal contract of the Spanish Ministry of Economy and Competitiveness

19 pag.-5 fig.-1 tab., During several forays for ligninolytic fungi in different Spanish native forests, 35 white-rot basidiomycetes growing on dead wood (16 species from 12 genera) and leaf litter (19 species from 10 genera) were selected for their ability to decolorize two recalcitrant aromatic dyes (Reactive Blue 38 and Reactive Black 5) added to malt extract agar medium. In this study, two dye decolorization patterns were observed and correlated with two ecophysiological groups (wood and humus white-rot basidiomycetes) and three taxonomical groups (orders Polyporales, Hymenochaetales and Agaricales). Depending on the above groups, different decolorization zones were observed on the dye-containing plates, being restricted to the colony area or extending to the surrounding medium, which suggested two different decay strategies. These two strategies were related to the ability to secrete peroxidases and laccases inside (white-rot wood Polyporales, Hymenochaetales and Agaricales) and outside (white-rot humus Agaricales) of the fungal colony, as revealed by enzymatic tests performed directly on the agar plates. Similar oxidoreductases production patterns were observed when fungi were grown in the absence of dyes, although the set of enzyme released was different. All these results suggest that the decolorization patterns observed could be related with the existence of two decay strategies developed by white-rot basidiomycetes adapted to wood and leaf litter decay in the field., This work was supported by the CGL2009-07316 (to JMB) and BIO2011-26694 (to FJR-D) projects of the Spanish Ministry of Economy and Competitiveness (MINECO), and by the PEROXICATS (KBBE-2010-4-265397) and INDOX (KBBE-2013.3.3-04-613549 of the European Union (to ATM). FJR-D thanks a “Ramón y Cajal” contract of the Spanish MINECO.

The partition function of a bosonic Riemann gas is given by the Riemann zeta function. We assume that the hamiltonian of this gas at a given temperature $\beta^{-1}$ has a random variable $\omega$ with a given probability distribution over an ensemble of hamiltonians. We study the average free energy density and average mean energy density of this arithmetic gas in the complex $\beta$-plane. Assuming that the ensemble is made by an enumerable infinite set of copies, there is a critical temperature where the average free energy density diverges due to the pole of the Riemann zeta function. Considering an ensemble of non-enumerable set of copies, the average free energy density is non-singular for all temperatures, but acquires complex values in the critical region. Next, we study the mean energy density of the system which depends strongly on the distribution of the non-trivial zeros of the Riemann zeta function. Using a regularization procedure we prove that the this quantity is continuous and bounded for finite temperatures., Comment: 17 pages, title changed. Typos corrected, rewritten to clarify some points, added references

We present a remarkable connection between the asymptotic behavior of the Riemann zeros and one-loop effective action in Euclidean scalar field theory. We show that in a two-dimensional space, the asymptotic behavior of the Fourier transform of two-point correlation functions fits the asymptotic distribution of the nontrivial zeros of the Riemann zeta function. We work out an explicit example, namely the nonlinear sigma model in the leading order in 1/N expansion.

A highly segmented Double-Sided Silicon Strip Detector has been characterized using mono-energetic pulsed beams of protons (1 and 3 MeV) and carbon (13 MeV). The detector was manufactured from a Neutron Transmutation Doped (NTD) silicon wafer yielding a bulk resistivity of 2.3 kOhm cm. In this work we show the interstrip effects caused by the beam striking only one strip and the beam on both the strip and the interstrip gap. The pre-amplified output waveforms of the strips on both detector sides have been digitized at 100 MS/s. Charge sharing, induced signal and charge loss have been investigated for different incident positions between adjacent strips, for beam entering from the junction side and the ohmic side, and as a function of the depletion voltage.

28 p.-5 fig.-3 tab., Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25 °C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25 °C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3 > vp2/vp3/mnp1/mnp2/mnp6 > mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10 °C or 37 °C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10 °C treatment, while many of them were alternatively upregulated (often 6 h after the thermal shock) and downregulated (12 h) at 37 °C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion., This work was supported by the PEROXICATS (KBBE-2010-4-265397; www.peroxicats.org) and INDOX (KBBE-2013-7-613549; www.indoxproject.eu) EU projects, and by the BIO2011-26694 and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO). EF-F acknowledges a Junta de Ampliación de Estudios fellowship of the CSIC, co-funded by the European Social Fund, and FJR-D acknowledges a MINECO Ramón y Cajal contract.

AT Martínez et al... 16 páginas.-- 5 figuras.-- 62 referencias.-Artículo de acceso abierto con licencia Creative Commons Attribution-NonCommercial-NoDerivs, Most industrial enzymes are hydrolases, such as glycosidases and esterases. However, oxidoreductases have an unexploited potential for substituting harsh (and scarcely selective) chemical processes. A group of basidiomycetes are the only organisms degrading the aromatic lignin polymer, enabling the subsequent use of plant polysaccharides. Therefore, these fungi and their ligninolytic peroxidases are the biocatalysts of choice for industrial delignification and oxidative biotransformations of aromatic and other organic compounds. The latter also include oxygenation reactions, which are catalyzed with high regio/stereo selectivity by fungal peroxygenases. In search for novel and more robust peroxidases/peroxygenases, basidiomycetes from unexplored habitats were screened, and hundreds of genes identified in basidiomycete genomes (in collaboration with the DOE JGI). The most interesting genes were heterologously expressed, and the corresponding enzymes structurally-functionally characterized. The information obtained enabled us to improve the enzyme operational and catalytic properties by directed mutagenesis. However, the structural-functional relationships explaining some desirable properties are not established yet and, therefore, their introduction was addressed by ‘non-rational’ directed evolution. Then, over 100 oxidative biotransformations were analyzed. Among them, it is noteworthy to mention the regio/stereo selective hydroxylation of long/short-chain alkanes (a chemically challenging reaction), epoxidation of alkenes, and production of hydroxy-fatty acids. Concerning aromatic oxygenations, the regioselective hydroxylation of flavonoids, and stereoselective hydroxylation/epoxidation of alkyl/alkenyl-benzenes were among the most remarkable reactions, together with enzymatic hydroxylation of benzene (as an alternative for harsh chemical process). Finally, peroxidases and peroxygenases also showed a potential as delignification biocatalysts and in the decolorization of contaminant dyes from textile industries, This work was funded by the PEROXICATS European project (KBBE-2010-4-265397), and by the BIO2011-26694, AGL2011-25379 and BIO2010-19697 projects of the Spanish Ministry of Economy and Competitiveness (MINECO). Thee work conducted by the US DOE JGI is supported by the O ce of Science of the US DOE under contract number DE-AC02-05CH11231.

In view of the construction of novel and high-sensitive instrumentation for the emerging ISOL facilities new prototypes have being implemented and tested. The contri- bution focuses at the investigation of the detection efficiency of an innovative silicon-pad prototype, which is the key element for the construction of the TRACE array, pursued for the SPES facility based at the Legnaro National Laboratories (Italy). The inter-pad size has been estimated by using a commercial 100-MHz-14-bit CAEN digitizer for sampling the signals obtained by an alpha-source scan over the inter-pad region.

[EN] Versatile peroxidases (VP) are promiscuous biocatalysts with the highest fragility to hydroperoxides yet reported due to a complex molecular architecture, with three catalytic sites and several oxidation pathways. To improve the VP resistance to H2O2, an evolved version of this enzyme was subjected to a range of directed evolution and hybrid strategies in Saccharomyces cerevisiae. After five generations of random, saturation, and domain mutagenesis, together with in vivo DNA recombination, several structural determinants behind the oxidative destabilization of the enzyme were unmasked. To establish a balance between activity and stability, selected beneficial mutations were introduced into novel mutational environments by the in vivo exchange of sequence blocks, promoting epistatic interactions. The best variant of this process accumulated 8 mutations that increased the half-life of the protein from 3 (parental type) to 35 min in the presence of 3000 equiv of H2O2 and with a 6 °C upward shift in thermostability. Multiple structural alignment with other H2O2-tolerant heme peroxidases help to understand the possible roles played by the new mutations in the overall oxidative stabilization of these enzymes., European Commission Projects (Peroxicats-FP7-KBBE-2010-4-26537; Indox-FP7-KBBE-2013-7-613549; COST-Action CM1303: Systems Biocatalysis) and the National Projects (Evofacel) [BIO2010-19697], (Dewry) [BIO2013-43407-R] and Hipop (BIO2011-26694). F.J.R.-D. is grateful for the award of a “Ramón y Cajal” contract of the Spanish MINECO.

A sequence with a high homology (39% residue identity) with that of the copper-transport CopZ protein from Enterococcus hirae and with the same MXCXXC metal-binding motif has been identified in the genome of Bacillus subtilis, and the corresponding protein has been expressed. The protein, constituted by 73 amino acids, does bind copper(I) under reducing conditions and fully folded in both copper-bound and copper-free forms under the present experimental conditions. The solution structure of the copper-bound form was determined through NMR spectroscopy on an 15N-labeled sample. A total of 1508 meaningful nuclear Overhauser effects, 38 dihedral phi angles, and 48 dihedral psi angles were used in the structural calculations, which lead to a family of 30 conformers with an average rmsd to the mean structure of 0.32 +/- 0.06 A for the backbone and of 0.85 +/- 0.07 A for the heavy atoms. NMR data on the apoprotein also show that, also in this form, the protein is in a folded state and essentially maintains the complete secondary structure. Some disorder is observed in the loop devoted to copper binding. These results are compared with those reported for CopZ from E. hirae whose structure is well-defined only in the apo form. The different behaviors of copper-loaded E. hirae and B. subtilis are tentatively accounted for on the basis of the presence of dithiothreitol used in the latter case, which would stabilize the monomeric form. The comparison is extended to other similar proteins, with particular attention to the copper-binding loop. The nature and the location of conserved residues around the metal-binding site are discussed with respect to their relevance for the metal-binding process. Proposals for the role of CopZ are also presented.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) have been investigated in Phanero-chaete chrysosporium. A third ligninolytic peroxidase has been described in Pleurotus and Bjerkandera. Two of these versatile peroxidases (VPs) have been cloned, sequenced and characterized. They have high affinity for Mn2+, hydro-quinones and dyes, and also oxidize veratryl alcohol, dimethoxybenzene and lignin dimers. The deduced sequences show higher identity with Ph. chrysosporium LiP than MnP, but the molecular models obtained include a Mn2+-binding site. Concerning aromatic substrate oxidation, Pl. eryngii VP shows a putative long-range electron transfer pathway from an exposed trytophan to haem. Mutagenesis and chemical modification of this tryptophan and the acidic residues forming the Mn2+-binding site confirmed their role in catalysis. The existence of several substrate oxidation sites is supported further by biochemical evidence. Residue conservation in other fungal peroxidases is discussed.

We study the effects of light-cone fluctuations on the renormalized zero-point energy associated with a free massless scalar field in the presence of boundaries. In order to simulate light-cone fluctuations we introduce a space-time dependent random coefficient in the Klein-Gordon operator. We assume that the field is defined in a domain with one confined direction. For simplicity, we choose the symmetric case of two parallel plates separated by a distance $a$. The correction to the renormalized vacuum energy density between the plates goes as $1/a^{8}$ instead of the usual $1/a^{4}$ dependence for the free case. In turn we also show that light-cone fluctuations break down the vacuum pressure homogeneity between the plates., 29 pages, 4 figures, title changed, typos corrected, references and comments added. Accepted for publication in IJMPA