Energising the E-factor: The E+-factor

[EN] The E-factor has become an important measure for the environmental impact of (bio)chemical reactions. However, summing up the obvious wastes generated in the laboratory neglects energy-related wastes (mostly greenhouse gases) which are generated elsewhere. To estimate these wastes, we propose to extend the E-factor by an energy-term (E-factor). At the example of a lab-scale enzyme fermentation, we demonstrate that the E-factor can constitute a multiple of the classical E-factor and therefore must not be neglected striving for a holistic estimation of the environmental impact.This workwas supported by the European Union Project H2020-BBI-PPP-2015-2-720297-ENZOX2 and F.H. gratefully acknowledges funding by European Research Council (ERC Consolidator Grant No. 648026) and the for financial support through a Netherlands Organisation for Scientific Research VICI grant (no. 724.014.003). J.M.R, B.R and A.S.B. gratefully acknowledge support from the United States National Science Foundation grant IIP-1540017

Rational Enzyme Engineering Through Biophysical and Biochemical Modeling

Due to its importance in the pharmaceutical industry, ligand dynamic simulations have experienced a great expansion. Using all-atom models and cutting edge hardware, one can perform non-biased ligand migration, active site search and binding studies. In this letter we demonstrate (and validate by PCR mutagenesis) how these techniques, when combined with quantum mechanics, open new possibilities in enzyme engineering. We provide a complete analysis where: 1) biophysical simulations produce ligand diffusion and, 2) biochemical modeling samples the chemical event. Using such broad analysis we engineer a highly stable peroxidase activating the enzyme for new substrate oxidation after rational mutation of two non-conserved surface residues. In particular, we create a new surface-binding site, quantitatively predicting the in vitro change in oxidation rate obtained by mutagenic PCR and achieving a comparable specificity constant to active peroxidases.This work was supported by the INDOX (KBBE-2013-7-613549 to ATM) European project, and the CTQ2013-48287 (to VG) and BIO2014-56388-R (to FJR-D) projects of the Spanish Ministry of Economy and Competitiveness (MINECO). FJR-D acknowledges a MINECO Ramón&Cajal contract.Peer ReviewedPostprint (author's final draft

Аналіз стійкості вибійних компоновок на проектній траєкторії

Рассмотрены основные причины, оказывающие дестабилизирующее влияние на роботу компоновок низа бурильной колонны. Проведён анализ процесса износа опорноцентрирующих элементов забойной компоновки и его влияния на изменение её конструктивных параметров. Получены графические зависимости, позволяющие оценить степень стойкости различных типов забойных компоновок на проектной траектории. Сделаны основные выводы, касающиеся поведения различных типов компоновок при воздействии на них дестабилизирующих факторовThe basic reasons, causing destabilizing influence on the work of drilling string assembly are reviewed. The analysis of wear out process of strong centralizing elements bottom drilling string assembly and its influence to change of its constructive properties is done. The graphic dependences, giving an opportunity to value the stage of firmness of different types of bottom drilling string assemblies on projected trajectory are given. The conclusion about conduct of different types of drilling string assemblies during influence on it destabilizing factors is draw

Escuela secundaria posible

REPENSAR LA EDUCACIÓN SECUNDARIA Colección de infografías II Parte Seguimiento de medios de comunicación 2017-2019Fil: Ferreyra, Horacio Ademar. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Di Francesco, Adriana Carlota. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Equipo de investigación en educación de adolescentes y jóvenes. Unidad Asociada CONICET. Universidad Católica de Córdoba. Facultad de Educación; Argentina

Identificación, expresión y caracterización de peroxidasas ligninolíticas de interés en genomas de basidiomicetos

227 p.-43 fig.-13 tab.-1 anexo con material suplementario[EN] The sequenced genomes of P. ostreatus and C. subvermispora allowed us to investigatethe biochemical, structural and operational properties of the different ligninolytic hemeperoxidases present in two model fungi of biotechnological interest, due to theirability to degrade lignin preferentially when growing on lignocellulosic substrates. The kinetic constants of the different class-II peroxidases (of the superfamily of plantfungal-prokaryotic peroxidases) from the P. ostreatus genome, after their heterologous expression in Escherichia coli, revealed a repertoire formed by three VPs and six MnPs and confirmed the absence of LiP, which until now has been associated with the ability of white-rot fungi to degrade lignin. The study also showed for the first time the presence in a MnP of the conserved catalytic tryptophan that oxidizes the bulky lignin polymer at the surface of VPs and LiPs. This anomaly was explained by a natural mutation in peroxidase evolution that interrupts the electron transfer pathway from the surface to the activated heme cofactor, as shown by reversed mutagenesis. The structural-functional study of the six MnPs revealed that they correspond to a novel subfamily characterized by their Mn-independent activity on some substrates and the presence of a C-terminal tail shorter than in typical (long) MnPs from Phanerochaete chrysosporium. One member of this new subfamily has been crystallized, being the first short MnP crystallized to date. Moreover, studies using 14C-labeled lignin and model dimers showed for the first time the lignin degrading ability of VPs. The above results confirm that VP plays in P. ostreatus and other species of Agaricales the role of LiP in P. chrysosporium and other species of the order Polyporales. In general the different MnP and VP isoenzymes from P. ostreatus genome present slightly differences in their catalytic constants but, surprisingly, they show significant differences in their stability against temperature (with T50 values of 43-63 ºC, after 10 min at pH 5) and pH (with residual activities of 0-96% at pH 3 and 0-57 % at pH 9, after 4 h at 4 ºC). To take advantage from all the above findings, the two isoenzymes from the P. ostreatus genome with the highest thermal and pH stabilities were crystallized, with the aim of identifying the structural bases of the above properties. The high abundance of basic residues and salt-bridge/H-bond interactions at the surface of the protein was related to the high stability of one of these peroxidases, as confirmed by directed mutagenesis. To investigate the biological meaning of these differences, specific primers were designed for each isoenzyme and their differential transcription was analyzed by quantitative PCR, by varying the temperature and pH conditions of cultures grown in a lignocellulose medium, combined with secretomic and activity studies. Although the genes of some of the most stable isoenzymes showed higher relative transcription levels at the most extreme temperature and pH conditions assayed, no correlation between the transcriptomic and secretomic results was observed for the most expressed MnP gene. This was due to impaired secretion as shown by the abundance of this isoenzyme in the intracellular proteome.These results showed the environmental regulation of isoenzyme gene expression, but also evidenced the need for careful studies to establish correlations between transcriptomic data and production of extracellular enzymes by these fungi. Although P. chrysosporium has been the model ligninolytic fungus for years, the simultaneous degradation of wood lignin and polysaccharides prevents its use in biotechnological applications, where the use of cellulose is intended. In contrast, C. subvermispora is a selective lignin degrader of interest in wood delignification (similar to Pleurotus species in delignification of agricultural wastes) but its production of lignin-degrading peroxidases (LiPs and VPs) remained unknown. The sequenced C. subvermipora genome revealed an unusually high number of MnPs, together with genes implicated in the synthesis of unsaturated fatty acids, suggesting their contribution in lignin degradation via free radicals derived from lipid peroxidation. However, in the C. subvermispora genome two genes of one putative VP and one putative LiP were also found. These genes were heterologously expressed, the obtained proteins were biochemically characterized and, as in the case of P. ostreatus peroxidases, their ability to degrade lignin was demonstrated using a radiolabeled model dimer and synthetic lignin. These studies confirm that these two new identified genes in C. subvermispora genome correspond to functionally competent LiPs, which probably represent VP-to- LiP transitional forms. This conclusion was based on the kinetics constant on aromatic substrates and the inability to oxidize Mn2+ of the enzyme initially identified as a VP. This enzyme presents an hypothetical Mn2+-oxidizing site (formed by three acidic residues close to the internal heme propionate) which was non-functional due to the presence of a neighbor forth acidic residue (absent in VPs and MnPs) as demonstrated by site directed mutagenesis.In addition to the above mentioned short and long MnP subfamilies, a new subfamily of the so-called extralong MnPs has been distinguished in recent surveys of basidiomicete genomes. The three different MnP types share a Mn-binding site, but it has been suggested that they would differ in their catalytic and stability properties. Interestingly, the genome of C. subvermispora revealed the joint presence of short, long and extralong MnPs (in addition to the above mentioned LiPs) offering a unique opportunity to compare the three proposed subfamilies. After their heterologous expression, we performed a biochemical and structural characterization, together with directed mutagenesis studies of the C-terminal tail. This tail is located at the vicinity of the conserved Mn-oxidation site and its removal from extralong and long MnPs confers them the Mn-independent activity characteristic of short MnPs. It was concluded that the short forms represent a true MnP subfamily, whose different catalytic and stability properties are related to the presence of the tail, while the so-called long and extralong MnPs did not show enough differences to be classified as two separate subfamilies. A highly stable extralong MnP (maintaining its activity at pH 2, which inactivate other peroxidases) was crystallized and its structure was solved at 1.2 Å, being the first extralong MnP crystallized to date. After its structural-functional characterization, this MnP was used as a robust scaffold to obtain stable high redox-potential peroxidases of biotechnological interest, by introducing an exposed catalytic tryptophan. This enzyme was able to act at extremely acidic pH that increases its oxidizing power against recalcitrant aromatic compounds but inactivates wild-type LiPs and VPs. The results obtained in this thesis have allowed to establish the existence of LiP type enzymes in C. subvermispora and confirm their absence in P. ostreatus, where they are substituted by VPs. On the other hand, the first structural-functional characterization of short-MnP has been performed and the results obtained for long and extralong MnPs show that they do not present enough differences to be classified as two different peroxidase subfamilies.[ES] La secuenciación de los genomas de Pleurotus ostreatus y Ceriporiopsis subvermispora nos ha permitido investigar las propiedades bioquímicas, estructurales y operacionales de las diferentes hemoperoxidasas ligninolíticas presentes en dos hongos modelo de interés biotecnológico, por su capacidad para degradar preferentemente la lignina frente a la celulosa en diferentes sustratos lignocelulósicos. Las constantes cinéticas de las diferentes peroxidasas de clase-II (de la superfamilia de peroxidasas vegetales-fúngicas-procariotas) del genoma de P. ostreatus, tras su expresión heteróloga en Escherichia coli, revelan un repertorio formado por tres peroxidasas versátiles (VPs) y seis manganeso peroxidasas (MnPs), confirmando la ausencia de lignina peroxidasa (LiP), a la que hasta ahora se ha asociado la capacidad para degradar la lignina, en los hongos ligninolíticos. Este estudio muestra por primera vez la presencia en una MnP del triptófano catalítico superficial conservado en todas las VPs y LiPs. Esta anomalía se explica por una mutación durante la evolución de estas peroxidasas que interrumpió la vía de transporte electrónico desde la superficie hasta el grupo hemo, como se demostró por mutagénesis dirigida reversa. Los estudios estructurales-funcionales de las seis MnPs de P. ostreatus revelan que éstas constituyen una nueva subfamilia, caracterizada por su actividad independiente de Mn sobre algunos sustratos y por la presencia de una cola en el extremo C-terminal más corta que en las típicas MnPs largas de P. chrysosporium. Se ha cristalizado un miembro de esta nueva subfamilia, siendo la primera MnP corta cristalizada. Además, usando dímeros modelo y lignina sintética marcados radioactivamente se ha mostrado por primera vez la capacidad de las VPs para degradar la lignina. Estos resultados confirman que la VP desempeña en P. ostreatus y en otras especies de agaricales el papel desempeñando por la LiP en P. chrysosporium y otras especies de poliporales. En general las diferentes isoenzimas de MnP y de VP del genoma de P. ostreatus no presentan grandes diferencias en sus constantes catalíticas pero, sorprendentemente, si muestran grandes diferencias en su estabilidad a la temperatura (con valores de T50 de 43-63 ºC, tras 10 min a pH 5) y al pH (con actividades residuales de 0-96% a pH 3 y de 0-57% a pH 9, en ambos casos tras 4 h a 4 ºC). La isoenzima más estable al pH y la más estable a la temperatura de P. ostreatus se cristalizaron y su estructura fue resulta a 1.0-1.1 Å para intentar identificar las bases estructurales de estas propiedades.La mayor abundancia de residuos básicos, y de puentes de hidrogeno e interacciones salinas, en la superficie de la proteina se relaciona con la estabilidad de una de estas peroxidasas, como se ha confirmado en estudios posteriores de mutagénesis dirigida. Para investigar el significado biológico de la duplicación de genes y la existencia de múltiples MnPs y VPs, se diseñaron cebadores específicos para cada isoenzima y se analizaron por RTqPCR las diferencias de expresión cuando se modifican las condiciones de temperatura y pH en cultivos de P. ostreatus sobre material lignocelulósico, en combinación con estudios proteómicos y medidas de actividad. No se encontró una correlación entre los resultados secretómicos y de transcripción de la isoenzima más expresada, debido a una secreción deficiente puesta de manifiesto mediante la comparación del secretoma y el proteoma intracelular. Sin embargo, los genes de algunas de las enzimas más estables mostraron niveles de transcripción relativa mayores en las condiciones más extremas de pH y temperatura ensayadas. Estos resultados muestran una regulación ambiental de la expresión de las isoenzimas, pero también ponen de manifiesto la necesidad de realizar estudios cuidadosos para establecer relaciones entre los datos transcriptómicos y la producción extracelular de enzimas por estos hongos.Aunque Phanerochaete chrysosporium ha sido durante años el hongo ligninolítico modelo, su patrón de degradacion simultanea de la lignocelulosa, impide su uso en aplicaciones biotecnológicas donde se busca el aprovechamiento de la celulosa. Por el contrario, C. subvermispora es un degradador selectivo de interés en la deslignificacion de la madera (como las especies de Pleurotus en la deslignificación de residuos agricolas) pero en sus cultivos nunca se habían identificado peroxidasas degradadoras de lignina, como LiP y VP. La secuenciación del genoma de C. subvermipora mostró un número inusualmente elevado de MnPs, junto con genes implicados en la síntesis de ácidos grasos insaturados, lo que sugirió su contribución en la degradación de la lignina a traves de los radicales derivados de la peroxidación de los lípidos. Sin embargo, en el genoma de C. subvermispora también se identificaron dos genes de una putativa LiP y una putativa VP. Estos genes se expresaron, las proteínas obtenidas se caracterizaron bioquímicamente y, al igual que en el caso de P. ostreatus, se determinó su capacidad de degradar directamente la lignina usando dímeros modelo y lignina sintética marcada radioactivamente. Estos estudios confirman que los dos nuevos genes identificados en el genoma de C. subvermispora corresponden a LiPs funcionales, que posiblemente representan formas de transición entre VP y LiP. Esta última conclusión se deduce de la incapacidad de oxidar Mn2+ y de las constantes cinéticas sobre sustratos aromáticos por parte de la enzima inicialmente identificada como VP. Esta enzima presenta un sitio hipotético de oxidación de Mn2+ (constituido por tres residuos ácidos junto al propionato interno del hemo) que resultó ser no-funcional por la presencia de un cuarto residuo ácido contiguo (ausente en VPs y MnPs) tal como se mostró mediante mutagénesis dirigida.En los últimos estudios de los genomas de basidiomicetos se ha diferenciado una nueva subfamilia de MnPs, además de las MnP de tipo corto y largo mencionadas anteriormente, que se han denominado MnP extralargas. Estos tres tipos de MnPs comparten el sitio de unión del Mn, y se ha sugerido que presentarían propiedades catalíticas y de estabilidad diferentes. Los tres tipos de MnP están presentes en el genoma de C. subvermispora, lo que ha supuesto una oportunidad única para comparar las tres subfamilias propuestas, sin las interferencias derivadas de diferentes historias evolutivas (en diferentes hongos). Tras su expresión heteróloga, se caracterizaron bioquímica y estructuralmente y se realizaron estudios de mutagénesis dirigida en la cola C-terminal. Esta cola se localiza próxima al sitio de oxidación del Mn y su eliminación confiere a las MnPs largas y extralargas la actividad independiente de Mn que caracteriza a las MnPs cortas. Estos estudios permitieron concluir que las MnP de tipo corto constituyen una subfamilia verdadera, con diferentes propiedades catalíticas y de estabilidad debidas a la presencia de una cola de tipo corto, mientras que las MnPs llamadas largas y extralargas no muestran suficientes diferencias para considerarse dos subfamilias diferentes. Una MnP extralarga extremadamente estable (que mantiene la actividad a pH 2, que inactiva a otras peroxidasas ligninolíticas) fue cristalizada y su estructura resuelta a 1.2 Å, siendo la primera peroxidasa de este tipo de la que se obtiene la estructura molecular. Tras su caracterización estructural-funcional, esta MnP se usó como una base "robusta" para, mediante la introducción de un triptófano catalítico, construir una peroxidasa de interés biotecnológico. Ésta es capaz de actuar a pH extremadamente ácido, lo que incrementa el poder oxidante sobre compuestos aromáticos recalcitrantes pero inactiva a las LiPs y VPs naturales. Los resultados obtenidos en la presente tesis han permitido establecer la existencia de enzimas de tipo LiP en C. subvermispora y confirmado su ausencia en P. ostreatus, donde estarían sustituidas por VPs. Por otro lado, aportan la primera caracterización estructural-funcional de las MnP cortas, y muestran que las descritas como MnPs largas y extralargas no presentan suficientes diferencias para establecer dos subfamilias.Proyecto HIPOP (BIO2011-26694, a F.J.R.-D.) del Ministerio de Economía y Competitividad (MINECO) y por el PEROXICATS (KBBE-2010-4-265397 a A.T.M.) y INDOX (KBBE-2013-3-613549, A.T.M. y V.G.) Proyectos europeos. Contratos Nº DE-AC02-05CH11231. E.F.F. y Ramón y Cajal del MINECO. Beca JAE del CSIC, y F.J.R.D.Peer reviewe

Engineering a fungal peroxidase that degrades lignin at very acidic pH

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 CompetitivenessPeer reviewe

Multienzymatic in situ hydrogen peroxide generation cascade for peroxygenase-catalysed oxyfunctionalisation reactions

[EN] There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.Financial support by the European Research Council (ERC Consolidator Grant No. 648026) is gratefully acknowledged