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pH Dependence of Catalysis by Pseudomonas aeruginosa Isochorismate-Pyruvate Lyase: Implications for Transition State Stabilization and the Role of Lysine 42

Olucha, Jose; Ouellette, Andrew N.; Luo, Qianyi; Lamb, Audrey L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H sidechain at high pH, the enzyme retains lyase activity at approximately 100-fold lowered catalytic efficiency, but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid sidechains.

Bifunctionality of the thiamin diphosphate cofactor: assignment of tautomeric/ionization states of the 4′-aminopyrimidine ring when various intermediates occupy the active sites during the catalysis of yeast pyruvate decarboxylase

Balakrishnan, Anand; Gao, Yuhong; Moorjani, Prerna; Nemeria, Natalia S.; Tittmann, Kai; Jordan, Frank
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Thiamin diphosphate (ThDP) dependent enzymes perform crucial C-C bond forming and breaking reactions in sugar and amino acid metabolism and in biosynthetic pathways via a sequence of ThDP-bound covalent intermediates. A member of this superfamily, yeast pyruvate decarboxylase (YPDC) carries out the non-oxidative decarboxylation of pyruvate and is mechanistically a simpler ThDP enzyme. YPDC variants created by substitution at the active center (D28A, E51X, E477Q) and on the substrate activation pathway (E91D and C221E) display varying activity, suggesting that they stabilize different covalent intermediates. To test the role of both rings of ThDP in YPDC catalysis (the 4′-aminopyrimidine as acid-base, and thiazolium as electrophilic covalent catalyst), we applied a combination of steady state and time-resolved circular dichroism experiments (assessing the state of ionization and tautomerization of enzyme-bound ThDP-related intermediates), and chemical quench of enzymatic reaction mixtures followed by NMR characterization of the ThDP-bound intermediates released from YPDC (assessing occupancy of active centers by these intermediates and rate-limiting steps). Results suggest that: (1) Pyruvate and analogs induce active site asymmetry in YPDC and variants. (2) The rare 1′...

Catalytic Asymmetric α-Acylation of Tertiary Amines Mediated by a Dual Catalysis Mode: N-Heterocyclic Carbene and Photoredox Catalysis

DiRocco, Daniel A.; Rovis, Tomislav
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Cross-coupling reactions are some of the most widely utilized methods for C-C bond formation; however, the requirement for pre-activated starting materials still presents a major limitation. Methods that take direct advantage of the inherent reactivity of the C-H bond offer an efficient alternative to these methods, negating the requirement for substrate pre-activation. In this process two chemically distinct activation events culminate in the formation of the desired C-C bond with loss of H2 as the only by-product. Herein we report the catalytic asymmetric α-acylation of tertiary amines with aldehydes facilitated by the combination of chiral N-heterocyclic carbene catalysis and photoredox catalysis.

Role of Trp19 and Tyr200 in Catalysis by the γ-Class Carbonic Anhydrase from Methanosarcina thermophila

Zimmerman, Sabrina; Domsic, John F.; Tu, Chingkuang; Robbins, Arthur H.; McKenna, Robert; Silverman, David N.; Ferry, James G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Although widely distributed in Nature, only two γ class carbonic anhydrases are reported besides the founding member (Cam). Although roles for active-site residues important for catalysis have been identified in Cam, second shell residues have not been investigated. Two residues (Trp19 and Tyr200), positioned distant from the catalytic metal, were investigated by structural and kinetic analyses of replacement variants. Steady-state kcat/Km and kcat values decreased 3- to 10-fold for the Trp19 variants whereas the Y200 variants showed up to a 5-fold increase in kcat. Rate constants for proton transfer decreased up to 10-fold for the Trp19 variants, and an increase of ~2-fold for Y200F. The pKa values for the proton donor decreased 1–2 pH units for Trp19 and Y200 variants. The variant structures revealed a loop composed of residues 62–64 that occupies a different conformation than previously reported. The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis. Trp19 is important for both CO2/bicarbonate interconversion, and the proton transfer step of catalysis.

General acid-base catalysis mediated by nucleobases in the hairpin ribozyme

Kath-Schorr, Stephanie; Wilson, Timothy J.; Li, Nan-Sheng; Lu, Jun; Piccirilli, Joseph A.; Lilley, David M. J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.

Biphasic catalysis using amphiphilic polyphenols-chelated noble metals as highly active and selective catalysts

Mao, Hui; Yu, Hong; Chen, Jing; Liao, Xuepin
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 18/07/2013 Português
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In the field of catalysis, it is highly desired to develop novel catalysts that combine the advantages of both homogeneous and heterogeneous catalysts. Here we disclose that the use of plant pholyphenol as amphiphilic large molecule ligand/stabilizer allows for the preparation of noble metal complex and noble metal nanoparticle catalysts. These catalysts are found to be highly selective and active in aqueous-organic biphasic catalysis of cinnamaldehyde and quinoline, and can be reused at least 3 times without significant loss of activity. Moreover, the catalytic activity and reusability of the catalysts can be rationally controlled by simply adjusting the content of polyphenols in the catalysts. Our strategy may be extended to design a wide range of aqueous-organic biphasic catalysis system.

Splicing of mRNA precursors: The role of RNAs and proteins in catalysis

Wachtel, Chaim; Manley, James L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Splicing of mRNA precursors was discovered over 30 years ago. It is one of the most complex steps in gene expression and therefore must be tightly controlled to ensure that splicing occurs efficiently and accurately. Splicing takes place in a large complex, the spliceosome, which contains approximately 200 proteins and five small RNAs (U snRNAs). Since its discovery, much work has been done to elucidate the pathway of the chemical reaction as well as the proteins and RNAs involved in catalysis. A variety of studies have established the potential for U2 and U6 snRNAs to play a role in splicing catalysis, raising the possibility that the spliceosome is a ribozyme. If correct, this would point to the spliceosomal proteins playing a supporting role during splicing. On the other hand, it may be that proteins contribute more directly to the spliceosomal active site, with the highly evolutionarily conserved Prp8 protein being an excellent candidate. This review will concentrate on recent work on splicing catalysis, and elucidating possible roles proteins play in this process.

Entropic Origin of Cobalt-Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Wang, Miao; Warncke, Kurt
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >1011-fold. The cleavage-generated 5′-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen atom transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex, and 2H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the 2H- and 1H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ±1 kcal/mol...

Advances in Nucleophilic Phosphine Catalysis of Alkenes, Allenes, Alkynes, and MBHADs

Fan, Yi Chiao; Kwon, Ohyun
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/12/2013 Português
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In nucleophilic phosphine catalysis, tertiary phosphines undergo conjugate additions to activated carbon–carbon multiple bonds to form β-phosphonium enolates, β-phosphonium dienolates, β-phosphonium enoates, and vinyl phosphonium ylides as intermediates. When these reactive zwitterionic species react with nucleophiles and electrophiles, they may generate carbo- and heterocycles with multifarious molecular architectures. This Article describes the reactivities of these phosphonium zwitterions, the applications of phosphine catalysis in the syntheses of biologically active compounds and natural products, and recent developments in the enantioselective phosphine catalysis.

Probing the chemistry of thioredoxin catalysis with force

Wiita, Arun P.; Perez-Jimenez, Raul; Walther, Kirstin A.; Gräter, Frauke; Berne, B. J.; Holmgren, Arne; Sanchez-Ruiz, Jose M.; Fernandez, Julio M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/11/2007 Português
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Thioredoxins are enzymes that catalyse disulphide bond reduction in all living organisms1. Although catalysis is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction1,2, the role of the enzyme in modulating this chemical reaction is unknown. Here, using single-molecule force-clamp spectroscopy3,4, we investigate the catalytic mechanism of Escherichia coli thioredoxin (Trx). We applied mechanical force in the range of 25–600 pN to a disulphide bond substrate and monitored the reduction of these bonds by individual enzymes. We detected two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulphide bond, causing a shortening of the substrate polypeptide by 0.79 ± 0.09 Å (± s.e.m.), and the second elongating the substrate disulphide bond by 0.17 ± 0.02 Å (±s.e.m.). These results support the view that the Trx active site regulates the geometry of the participating sulphur atoms with sub-ångström precision to achieve efficient catalysis. Our results indicate that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease5...

The Hammerhead Ribozyme: Structure, Catalysis and Gene Regulation

Scott, William G.; Horan, Lucas H.; Martick, Monika
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
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The hammerhead ribozyme has long been considered a prototype for understanding RNA catalysis, but discrepancies between the earlier crystal structures of a minimal hammerhead self-cleaving motif and various biochemical investigations frustrated attempt to understand hammerhead ribozyme catalysis in terms of structure. With the discovery that a tertiary contact distal from the ribozyme’s active site greatly enhances its catalytic prowess, and the emergence of new corresponding crystal structures of full-length hammerhead ribozymes, a unified understanding of catalysis in terms of the structure is now possible. A mechanism in which the invariant residue G12 functions as a general base, and the 2′-OH moiety of the invariant G8, itself forming a tertiary base pair with the invariant C3, is the general acid, appears consistent with both the crystal structure and biochemical experimental results. Originally discovered in the context of plant satellite RNA viruses, the hammerhead more recently has been found embedded in the 3′-untranslated region of mature mammalian mRNAs, suggesting additional biological roles in genetic regulation.

Chiral poly-rare earth metal complexes in asymmetric catalysis

Shibasaki, Masakatsu
Fonte: The Japan Academy Publicador: The Japan Academy
Tipo: Artigo de Revista Científica
Publicado em /04/2006 Português
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Asymmetric catalysis is a powerful component of modern synthetic organic chemistry. To further broaden the scope and utility of asymmetric catalysis, new basic concepts for the design of asymmetric catalysts are crucial. Because most chemical reactions involve bond-formation between two substrates or moieties, high enantioselectivity and catalyst activity should be realized if an asymmetric catalyst can activate two reacting substrates simultaneously at defined positions. Thus, we proposed the concept of bifunctional asymmetric catalysis, which led us to the design of new asymmetric catalysts containing two functionalities (e.g. a Lewis acid and a Brønsted base or a Lewis acid and a Lewis base). These catalysts demonstrated broad reaction applicability with excellent substrate generality. Using our catalytic asymmetric reactions as keys steps, efficient total syntheses of pharmaceuticals and their biologically active lead natural products were achieved.

Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis

Martick, Monika; Scott, William G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 Å resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner.

Evidence for the Dimerization-Mediated Catalysis of Methionine Sulfoxide Reductase A from Clostridium oremlandii

Lee, Eun Hye; Lee, Kitaik; Kwak, Geun-Hee; Park, Yeon Seung; Lee, Kong-Joo; Hwang, Kwang Yeon; Kim, Hwa-Young
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 24/06/2015 Português
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Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.

Understanding nano effects in catalysis

Yang, Fan; Deng, Dehui; Pan, Xiulian; Fu, Qiang; Bao, Xinhe
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica Formato: text/html
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Catalysis, as a key and enabling technology, plays an increasingly important role in fields ranging from energy, environment and agriculture to health care. Rational design and synthesis of highly efficient catalysts has become the ultimate goal of catalysis research. Thanks to the rapid development of nanoscience and nanotechnology, and in particular a theoretical understanding of the tuning of electronic structure in nanoscale systems, this element of design is becoming possible via precise control of nanoparticles’ composition, morphology, structure and electronic states. At the same time, it is important to develop tools for in situ characterization of nanocatalysts under realistic reaction conditions, and for monitoring the dynamics of catalysis with high spatial, temporal and energy resolution. In this review, we discuss confinement effects in nanocatalysis, a concept that our group has put forward and developed over several years. Taking the confined catalytic systems of carbon nanotubes, metal-confined nano-oxides and 2D layered nanocatalysts as examples, we summarize and analyze the fundamental concepts, the research methods and some of the key scientific issues involved in nanocatalysis. Moreover, we present a perspective on the challenges and opportunities in future research on nanocatalysis from the aspects of: (1) controlled synthesis of nanocatalysts and rational design of catalytically active centers; (2) in situ characterization of nanocatalysts and dynamics of catalytic processes; (3) computational chemistry with a complexity approximating that of experiments; and (4) scale-up and commercialization of nanocatalysts.

Études Structurales par Résonance Magnétique Nucléaire (RMN) du Site Actif du Ribozyme VS de Neurospora.

Desjardins-Séguin, Geneviève
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
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Nous étudions le ribozyme VS de Neurospora, en tant que système modèle, pour augmenter nos connaissances sur la relation entre la structure et la fonction chez les ARNs, ainsi que pour mieux comprendre le mécanisme de clivage de ce ribozyme. Il a été proposé précédemment que la boucle interne A730 dans la tige-boucle VI (SLVI) contient le site actif du ribozyme et lie un ou plusieurs ions métalliques qui pourraient participer au mécanisme réactionnel. Nous avons déterminé par spectroscopie RMN la structure de la tige-boucle SLVI contenant la boucle A730 afin d’éclaircir ce mécanisme. La structure obtenue est en accord avec les études biochimiques antérieures et présente un ou plusieurs sites de liaison au magnésium associé à la boucle interne. Suite à des études de cinétique et de mutagenèse, il a été proposé qu’une adénine localisée dans le site actif, A756, participe à la catalyse par acide/base générale. Des études de pH effectuées précédemment ont identifié un pKa catalytique (5.2-5.8) qui correspond probablement à l’équilibre de protonation du A756. À l’aide de méthodes utilisant le carbone-13, nous avons identifié un pKa modifié appartenant au A756, ce qui supporte le rôle de ce résidu dans la catalyse par acide/base générale. Les études structurales présentées ici aident donc à augmenter notre compréhension du mécanisme de clivage chez le ribozyme VS.; We are studying the Neurospora VS ribozyme as a model system to increase our knowledge of the structure-function relationship in RNA and to better understand the mechanism of the cleavage reaction. It has been previously postulated that the A730 internal loop of stem-loop VI (SLVI) forms the active site of the VS ribozyme and binds magnesium ion(s) that may participate in catalysis. To get insights into the catalytic mechanism...

RECENTES APLICAÇÕES EM SÍNTESE ORGÂNICA DE CATÁLISE FOTO REDOX MEDIADA POR LUZ VISÍVEL

Almeida,Angelina M. de; Almeida,Mauro V. de; Amarante,Giovanni W.
Fonte: Sociedade Brasileira de Química Publicador: Sociedade Brasileira de Química
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2015 Português
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In the past few years, photoredox catalysis has become a powerful tool in the field of organic synthesis. Using this efficient method, it is possible to excite organic compounds from visible light and attain alternative mechanistic pathways for the formation of chemical bonds, a result which is not obtainable by classical methods. The rapid growth of work in the area of photoredox catalysis is due to its low cost, broad chemical utility protocols, and, especially, its relevancy from the green and sustainable chemistry viewpoints. Thus, this study proposes a brief theoretical discussion of and highlights recent advances in visible-light-induced photoredox catalysis through the analysis of catalytic cycles and intermediates.

Développement de nouveaux sels Binol-imidazoliums : de la catalyse asymétrique aux applications biologiques

Vidal, Marc
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
Português
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Le 1,1'-bi-2-naphtol ou Binol, présentant une chiralité axiale, est un ligand très utilisé en catalyse asymétrique. Au cours des vingt dernières années, le Binol a servi de synthon à l’élaboration de très nombreux ligands permettant la catalyse asymétrique de tous types de réactions, allant de l’hydrogénation, à l’alkylation, en passant par diverses réactions péricycliques. Le grand intérêt pour ce ligand vient de sa versatilité et des nombreuses possibilités de fonctionnalisation qu’il offre, permettant d’altérer ses propriétés catalytiques à volonté, aussi bien en modifiant son caractère électronique, qu’en introduisant des facteurs stériques autour du site catalytique. Parallèlement aux développements de la catalyse par des dérivés de Binol, le domaine des liquides ioniques a connu un intérêt croissant ces dernières années. Les liquides ioniques, sels dont le point de fusion est inférieur à 100°C, cumulent de nombreuses qualités convoitées : faible pression de vapeur, stabilité thermique et chimique et fort pouvoir de solvatation. Dû à ces propriétés, les liquides ioniques ont principalement été étudiés dans l’optique de développer une gamme de solvants recyclables. Alors que les propriétés des liquides ioniques sont facilement modulables en fonction de l’anion et du cation choisi...

Produção de biodiesel etílico por via enzimática a partir de óleo de milho transgénico

Sousa, Igor Rúben Brito Gomes de
Fonte: Instituto Politécnico do Porto. Instituto Superior de Engenharia do Porto Publicador: Instituto Politécnico do Porto. Instituto Superior de Engenharia do Porto
Tipo: Dissertação de Mestrado
Publicado em //2010 Português
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Mestrado em Engenharia Química; A necessidade de reduzir a dependência dos combustíveis fósseis e a emissão dos gases com efeito de estufa, implicou a aposta em diferentes tipos de matérias-primas capazes de serem usadas na produção de biodiesel, sendo este produzido industrialmente por transesterificação de óleos com um álcool de cadeia curta. Este trabalho teve por objectivo a produção de biodiesel por via enzimática, a partir de um óleo de milho transgénico e etanol, sendo usada a catálise química como procedimento de referência. Iniciou-se o trabalho com a caracterização da matéria-prima, o óleo de milho transgénico. Para o efeito, foram avaliados os seguintes parâmetros: índice de acidez (0,26 mg KOH/gamostra), o número de iodo (130 giodo/100 gamostra), a viscosidade a 40 °C (36,27 mm2/s), a massa específica (919 Kg/m3) e o teor em água (749 mg/Kg). Posteriormente, realizou-se a transesterificação do óleo por via química, usando como álcoois o metanol absoluto e o etanol absoluto. As condições usadas foram uma razão molar álcool/óleo de 6:1, 1% (m/m) catalisador/óleo, um tempo de reacção de 2 h e uma temperatura de reacção de 60 ºC e 75 ºC, usando metanol e etanol, respectivamente. Os resultados obtidos nesta fase do trabalho correspondem a um rendimento médio do processo por via metílica de 83...

Thumb-loops up for catalysis: a structure/function investigation of a functional loop movement in a GH11 xylanase

Paës, Gabriel; Cortés, Juan; Siméon, Thierry; O'Donohue, Michael J.; Tran, Vinh
Fonte: Research Network of Computational and Structural Biotechnology (RNCSB) Organization Publicador: Research Network of Computational and Structural Biotechnology (RNCSB) Organization
Tipo: Artigo de Revista Científica
Publicado em 01/07/2012 Português
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Dynamics is a key feature of enzyme catalysis. Unfortunately, current experimental and computational techniques do not yet provide a comprehensive understanding and description of functional macromolecular motions. In this work, we have extended a novel computational technique, which combines molecular modeling methods and robotics algorithms, to investigate functional motions of protein loops. This new approach has been applied to study the functional importance of the so-called thumb-loop in the glycoside hydrolase family 11 xylanase from Thermobacillus xylanilyticus (Tx-xyl). The results obtained provide new insight into the role of the loop in the glycosylation/deglycosylation catalytic cycle, and underline the key importance of the nature of the residue located at the tip of the thumb-loop. The effect of mutations predicted in silico has been validated by in vitro site-directed mutagenesis experiments. Overall, we propose a comprehensive model of Tx-xyl catalysis in terms of substrate and product dynamics by identifying the action of the thumb-loop motion during catalysis.