Dissertations / Theses on the topic 'Hyperthermophilic enzymes'

Dissertation presented to obtain the Ph.D degree in Biochemistry
While Aristotle cautioned “everything in moderation”, the Romans, known for their eccentricities, coined the word “extremus”, the superlative of exter, “being on the outside”. By the fifteenth century “extreme” had arrived to English, via Middle French. At the beginning of the 21st century, we know that Earth contains environmental extremes unimaginable to our ancestors of the 19th century. Even more unimaginable to them would be the fact that there are organisms that live, and grow, in these environmental extremes. R. D. MacElroy named these organisms lovers (from the Greek “philos”), “extremophiles” as in “lovers of extreme environments”. The discovery of extremophiles has put vitality in the biotechnology industry as this discipline has exploded in the past 20 years. Several reviews have been published on extremophiles and an increasing number of meetings and conferences are organised around the theme. Genomes of extremophiles have been sequenced, patents have been filed and several funding programmes have been launched namely the US National Science Foundation and NASA’s programmes in “Life in Extreme Environments, Exobiology and Astrobiology”, and the European Union’s “Biotechnology of Extremophiles” and “Extremophiles as Cell Factories”(...)
Fundação para a Ciência e a Tecnologia (FCT), e Fundo Social Europeu (FSE), o apoio financeiro no âmbito do Quadro Comunitário de apoio (Bolsa de Doutoramento SFRH/BD/30512/2006)

Orientadores: Glaucia Maria Pastore, Fábio Márcio Squina
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Atualmente há uma crescente demanda para o desenvolvimento de combustíveis não-fósseis alternativos. Assim, como a biomassa lignocelulósica é uma das fontes de energia mais abundantes na natureza, pode ser estabelecida uma economia verde e sustentável, com o objetivo de processar a grande quantidade de energia estocada nessas matérias-primas. O etanol de cana-de-açúcar é uma das melhores opções em biocombustíveis e sua produção pode mais que dobrar, se os açúcares constituintes da parede celular vegetal forem utililizados. No entanto, o alto custo de produção das enzimas para hidrolisar e processar os materiais lignocelulósicos é um fator altamente limitante para o uso de tecnologias verdes. Este trabalho se propôs a avaliar novos biocatalisadores e construir uma enzima quimérica na tentativa de obter glicosidases com melhor desempenho que as já relatadas. Enzimas despolimerizadoras de ß-1,3-glucanos têm consideráveis aplicações biotecnológicas, incluindo produção de biocombustíveis, insumos químicos e farmacêuticos. No segundo capítulo, mostramos a caracterização funcional e a estrutura de baixa resolução da laminarase hipertermofílica de Thermotoga petrophila (TpLam), além de seu modo de operação por eletroforese capilar de zona, mostrando que ela cliva especificamente ligações ß-1,3-glucosídicas internas. O dicroismo circular (CD) UV-distante demonstrou que TpLam é formada principalmente por elementos estruturais do tipo beta, e a estrutura secundária é preservada após incubação por 16 horas a 90 º C. A forma determinada pelo pequeno espalhamento de raios-X a baixo ângulo revelou uma arquitetura de multi-domínio da enzima, com um arranjo de envelope em forma de V, no qual os dois módulos de ligação de carboidrato estão ligados ao domínio catalítico. A engenharia de enzimas multifuncionais pode melhorar coquetéis enzimáticos para tecnologias emergentes de biocombustíveis. Dinâmica molecular através de modelos baseados em estrutura (SB) é uma ferramenta eficaz para avaliar a disposição tridimensional das enzimas quiméricas, bem como para inferir a viabilidade funcional antes da validação experimental. No terceiro capítulo, descrevemos a montagem computacional de uma quimera bifuncional xilanase-liquenase (XylLich), usando os genes xynA e bglS de Bacillus subtilis. As análises in silico da área de superfície acessível ao solvente (SAS) e da raiz quadrada média das flutuações (RMSF) previram uma quimera completamente funcional, ou seja, uma enzima cujo substrato tem acesso ao seu sítio catalítico com pequenas flutuações e variações ao longo das cadeias polipeptídicas. A quimera preservou as características bioquímicas das enzimas parentais, com exceção de uma pequena variação na temperatura de operação e na eficiência catalítica (kcat / Km). Também foi verificado ausência de mudanças significativas no modo de operação catalítico. Além disso, a produção de enzimas quiméricas pode ser mais rentável do que a produção de uma única enzima separadamente, comparando-se o rendimento da produção de proteína recombinante e a atividade hidrolítica da enzima quimérica com as enzimas parentais. ß-Glicosidases (BGLs) são enzimas muito úteis e com grande potencial para serem empregadas em diversos processos industriais. Entretanto, algumas características são essenciais para tornar viáveis as aplicações, como por exemplo estabilidade à temperatura e ao pH, bem como baixa inibição por íons e outros compostos químicos. Assim, no quarto capítulo buscamos estudar três BGLs dos organismos extremófilos Pyrococcus furiosus e Thermotoga petrophila. Os genes PfBgl1, TpBgl1 and TpBgl3 foram clonados no vetor pET28a e as proteínas expressadas em Escherichia coli e posteriormente purificadas em duas etapas cromatográficas. As enzimas purificadas foram avaliadas quanto ao pH e temperatura de atividade, sendo que as BGLs da família GH1 (PfBgl1 e TpBgl1) apresentaram faixas mais largas de pH e temperatura de operação do que a família GH3 (TpBgl3). As BGLs mostraram grande estabilidade ao pH e o maior tempo de meia-vida (a 99 ° C) foi verificado no pH 6, e além disso, não foram significativamente afetadas pela presença de EDTA ou de íons, exceto a TpBgl1 que foi inibida por Hg2+ e Fe2+. As atividades específicas para um conjunto de diferentes substratos sugeriram que TpBgl3 é mais específica que as BGLs GH1. O kcat e kcat / Km em 4-nitrofenol-ß-D-glicopiranosídeo (pNPG) indicam que TpBgl3 é a mais eficiente para hidrólise do substrato, embora seja a enzima que foi inibida com a menor concentração de glicose (30.1 mM). Além disso, as BGLs foram analisadas quanto à influência de seis monossacarídeos na catálise, e demonstraram serem fracamente inibidas pela maioria dos açúcares testados. Os ensaios de CD UV-distante revelaram que a estrutura secundária das BGLs não é afetada pelas variações de pH, e os estudos de desnaturação térmica evidenciaram que as BGLs são proteínas hipertermofílicas
Abstract: There is an increasing demand for the development of alternative non-fossil fuels. Thus, since the lignocellulosic biomass is the most abundant source in nature, it may be settled a green and sustainable economy, aiming to process the great amount of energy stocked in these raw materials. The ethanol from sugarcane is one of the best options concerning biofuels and its productivity could be raised more than double if the use of sugars constituents of plant cell wall is considered. However the high production cost of the enzymes to hydrolyze and process lignocellulose is a great limiting factor for green technologies. In this way, this work proposed to evaluate new enzymes and engineer a chimeric enzyme in the attempt to prospect glycosyl hydrolases with better performance than those reported up to date. 1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including the production of biofuels, feedstock-chemicals and pharmaceuticals. In the first chapter we showed the comprehensive functional characterization and low-resolution structure of hyperthermophilic laminarase from Thermotoga petrophila (TpLam), besides its mode of operation through capillary zone electrophoresis, which specifically cleaves internal ß-1,3-glucosidic bonds. Far-UV circular dichroism demonstrated that LamA is formed mainly by beta structural elements, and the secondary structure is maintained after incubation up to 16 hours at 90ºC. The structure determined by small angle X-ray scattering revealed a multi-domain structural architecture of the enzyme with a V-shape envelope arrangement of the two carbohydrate binding modules in relation to the catalytic domain. Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional disposal of chimeric enzymes as well as for inferring the functional practicability before experimental validation. In the second chapter we describe the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average surface accessible area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, i.e. the substrate has access to the catalytic pocket with minor fluctuations and variations along the polypeptide chains. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes. ß-Glucosidases (BGLs) are very useful enzymes with a great potential to be employed in several industrial processes. However, some features are required to become viable the enzyme applications, such as temperature and pH stability as well, low ions and chemicals inhibition. Thus this work aimed to study three BGLs from the extremophiles organisms Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1, TpBgl1 and TpBgl3 were cloned into pET28a vector and the proteins were expressed in Escherichia coli and further purified in two chromatographic steps. The purified enzymes were evaluated for pH and temperature of activity, which showed that BGLs from the glycosyl hydrolases family 1 (PfBgl1, TpBgl1) presented a wider range of pH and temperature operation than BGL from family 3 (TpBgl3). The BGLs showed great stability to a range of pH (4-10) and the highest time of half-life (at 99 °C) was at pH 6, besides they were not significantly affected by the presence of EDTA or ions, except TpBgl1 that was inhibited by Hg2+ and Fe2+. The specific activities in a set of different substrates suggested that TpBgl3 is more specific than GH1 BGLs. The kcat and kcat/Km in pNPG indicate that TpBgl3 is the most efficient among BGLs characterized herein, although this enzyme is inhibited with the lowest glucose concentration (30.1 mM). Furthermore, the BGLs were assayed for influence of six monosaccharides in catalysis, which the results suggested a weak inhibition by the most of those carbohydrates tested. The CD experiments revealed that the secondary structure of BGLs is not affected by the pH variations and the denaturation studies evidenced that the BGLs are indeed hyperthermophilic
Doutorado
Ciência de Alimentos
Doutor em Ciência de Alimentos

Dissertation presented to obtain the Ph.D degree in Engineering Sciences and Technology.
In response to osmotic or heat stress, marine hyperthermophiles (thriving optimally at or above 80ºC) accumulate ionic compatible solutes such as α-Dmannosyl- D-glycerate (MG). It has been proposed that these hypersolutes stabilise intracellular components, such as proteins and enzymes, allowing them to withstand high growth temperatures.(...)

Le soufre est utilisé à des fins bioénergétiques par des micro-organismes tels que la bactérie hyperthermophile Aquifex aeolicus qui nécessite pour sa croissance de l'oxygène, de l'hydrogène et un composé soufré indispensable. Une soufre réductase réduisant des chaînes de soufre, une Sulfure Quinone Oxydoréductase (SQR) oxydant l'H2S et une Soufre Oxygénase Réductase (SOR) oxydant et réduisant simultanément des chaînes de soufre ont été caractérisées chez cette bactérie. L'organisation de certaines de ces enzymes dans des supercomplexes membranaires a également été démontrée.Nous avons montré qu'Aq_477, précédemment caractérisée comme une soufre transférase de la famille des rhodanèses, est capable (i) de « charger » des chaînes de soufre ; (ii) d'interagir avec deux partenaires (la soufre réductase et la SOR) ; (iii) de leur présenter ce substrat. Ceci conduit à une optimisation du métabolisme. Nous avons ainsi démontré l'implication directe d'Aq_477, rebaptisée SbdP pour Sulfur -binding -donating Protein, dans le métabolisme énergétique du soufre de la bactérie. Une analyse poussée du génome nous a permis de construire un nouveau modèle suggérant notamment un recyclage des composés soufrés entre différents systèmes enzymatiques. La recherche de l'existence d'un niveau d'organisation des complexes respiratoires supérieur aux supercomplexes chez Aquifex aeolicus nous a conduits à développer de nouvelles méthodes d'étude permettant de proposer plusieurs pistes de recherche. Enfin, nous avons montré l'existence d'un nanocompartiment protéique constitué de l'encapsuline Aq_1760, dans lequel vient s'ancrer la ferritine atypique à domaines en tandem Aq_331
Sulfur can be used in energy metabolism by microorganisms as electron donor and acceptor. The hyperthermophilic bacterium Aquifex aeolicus, which need oxygen, hydrogen and an essential sulfur compound for its growth presents sulfur reduction and oxidation pathways linked to the energy synthesis. A sulfur reductase (reduction of sulfur chains), a Sulfide Quinone Oxidoreductase (SQR, oxidation of H2S) and a Sulfur Oxygenase Reductase (SOR, simultaneous oxidation and reduction of sulfur chains) have been characterized in this bacterium. It has also been shown that some of these enzymes are organized in membrane-bound supercomplexes.We have demonstrated that Aq_477, previously characterized as a sulfurtransferase belonging to the rhodanese superfamily, can load long sulfur chains and acts as a sulfur donor for its partners (sulfur reductase and SOR) which use these sulfur chains as substrate, thus optimizing the metabolism. These results show that Aq_477, renamed SbdP for Sulfur -binding -donating Protein, is involved in the sulfur energy metabolism of Aquifex aeolicus. The identification in the genome of some new proteins potentially involved in this metabolism permitted us to propose a new model which suggests a recycling of sulfur compounds between different enzymatic systems. We also looked for an organization level of respiratory complexes higher than supercomplexes, which led us to develop new study methods and propose several research trails. Finally, we have shown the existence of protein nanocompartment constituted by the encapsulin Aq_1760, in which the atypical tandem-domain ferritin Aq_331 is anchored

Cette these comporte trois aspects concernant les enzymes impliques dans la biosynthese et l'utilisation du carbamylphosphate chez l'archaebacterie hyperthermophile et barophile pyrococcus abyssi. Dans un premier temps nous avons purifie et caracterise la carbamylphosphate synthetase (cpsase) de ce microorganisme. Cet enzyme est unique pour les voies de biosynthese de l'arginine et des nucleotides pyrimidiques. Son poids moleculaire est particulierement reduit par rapport aux enzymes homologues (environ 47 000 au lieu de 120 000). Il est actif sous forme de monomere et utilise uniquement l'ammonium comme donneur de groupement amine. L'activite de cette cpsase est sensible a des effecteurs allosteriques, presentant uniquement une inhibition par les derives nucleotidiques triphosphates a l'exception du gtp et pas de stimulation par les activateurs des cpsases. L'inhibition semble competitive par rapport a l'atp, ce qui n'exclut pas l'existence de site(s) regulateur(s). L'enzyme presente une thermostabilite remarquable ; toutefois, les hautes pressions ont un leger effet negatif sur sa stabilite et sur son activite. Une serie de proprietes cinetiques de cet enzyme le distingue des carbamates kinases, suggerant qu'il represente une forme ancestrale des carbamylphosphate synthetases. La deuxieme partie du travail concerne l'etude de l'aspartate transcarbamylase (atcase) de p. Abyssi. L'enzyme presente des phenomenes de cooperativite entre les sites catalytiques pour ses deux substrats. Contrairement aux enzymes homologues, cette atcase n'est pas inhibee par les analogues du carbamylphosphate, ce qui suggere une configuration ou une accessibilite particuliere du site de fixation de ce substrat. L'atcase de p. Abyssi est soumise a la regulation allosterique: elle est activee par l'atp et retroinhibee par le ctp et l'utp. A l'exception du ctp, cette sensibilite est dependante de la temperature. La thermostabilite ainsi que la stabilite sous haute pression de l'atcase de p. Abyssi sont tres elevees. La purification partielle de cet enzyme entraine la perte de certaines de ses proprietes de regulation homotrope et heterotrope. Dans la troisieme partie de cette these nous avons mis en evidence l'existence d'une canalisation du carbamylphosphate entre les sites catalytiques de la cpsase et ceux de l'atcase et de l'ornithine transcarbamylase (otcase) chez p. Abyssi. Le phenomene a ete mis en evidence a l'aide des experiences de dilution isotopique et de cinetique de reactions couplees et verifie par les criteres cinetiques proposes par ovadi. Son role n'est pas de favoriser une des voies metaboliques de l'arginine ou des nucleotides pyrimidiques, mais probablement de proteger le carbamylphosphate, intermediaire tres instable. C'est, de plus, la premiere fois que la canalisation du carbamylphosphate vers la voie de biosynthese de l'arginine a ete mise en evidence. Cette canalisation est imparfaite et semble avoir lieu lors d'une association transitoire de ces enzymes lors de la catalyse. Aucune des conditions experimentales de temperature ou de presence de substrats n'ont permis de mettre en evidence cette association transitoire entre les enzymes de couplage

Dans la nature, la réduction de l'oxygène et l'oxydation de l'hydrogène sont catalysées par des enzymes oxydoréductases. Ces catalyseurs spécifiques, efficaces, renouvelables et biodégradables constituent une alternative séduisante au platine dans les piles à combustible. L'immobilisation à des interfaces nanostructurées de l'hydrogénase membranaire tolérante à l'oxygène de la bactérie hyperthermophile Aquifex aeolicus, et de la bilirubine oxydase thermostable de la bactérie Bacillus pumilus, a été étudiée dans ce sens.L'électrochimie et la dynamique moléculaire ont permis d'affiner le modèle d'orientation de l'hydrogénase sur les surfaces planes. L'efficacité de l'immobilisation de l'hydrogénase sur différents nanomatériaux carbonés (nano-particules, tubes et fibres de carbone) structurant la surface de l'électrode a été évaluée. Les nanofibres de carbone (CNFs) ont permis de former une bioanode efficace pour l'oxydation de l'H2 en l'absence de médiateurs redox. L'étude a souligné l'importance d'un transport efficace du substrat dans le film carboné mésoporeux. Les CNFs ont également été utilisées comme matériau d'électrode pour réaliser la 1ère connexion directe de la bilirubine oxydase. L'existence d'une forme resting alternative de l'enzyme, influencée par les ions chlorures, le pH et la température, a été mise en évidence. Une biocathode efficace pour la réduction de l'oxygène a été développée.Les deux électrodes thermostables ont permis le développement de la 1ère biopile H2/O2 qui délivre des densités de puissance supérieures au mW.cm-2 sur une large gamme de température. Ce résultat ouvre la voie à l'alimentation électrique de dispositifs de faibles puissances
The oxygen reduction and the hydrogen oxidation reactions are realized in nature by oxidoreductase enzymes. These highly efficient, specific, renewable and biodegradable catalysts appear as a seducing alternative to platinum in fuel cell devices. The immobilization at nanostructured interfaces of the membrane-bound oxygen-tolerant hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus, and of the thermostable bilirubin oxidase from Bacillus pumilus, has been studied within this objective.Electrochemistry and molecular dynamics have been used to validate the orientation model of the hydrogenase at planar electrodes. Hydrogenase immobilisation in 3D-networks based on various carbon materials (nanoparticles, nanotubes and nanofibers) has been especially studied. Fishbone carbon nanofibers were demonstrated to provide an efficient platform for mediatorless H2 oxidation. Mass transport inside the carbon mesoporous film has been especially studied and demonstrated to be one of the limitations of the catalytic efficiency. Direct electrical connection of bilirubin oxidase has also been realized for the first time thanks to its immobilization on carbon nanofiber films. An alternative resting form of the enzyme, influenced by chlorides, pH and temperature, has been evidenced. An efficient biocathode for the oxygen reduction reaction has been developed. Thanks to the two thermostable electrodes, the first H2-O2 bio fuel cell able to deliver power densities over 1 mW.cm-2 over a large temperature range has been developed. This result paves the way for the electrical alimentation of low-power devices

Les ADN topoisomérases jouent un rôle primordial dans le maintien de la fonctionnalité des molécules d'ADN. Ce rôle est encore plus essentiel chez les organismes vivant à haute température. Le travail décrit dans cette thèse a donc été entrepris afin d'appréhender certaines bases de l'adaptation des systèmes de contrôle du surenroulement de l'ADN aux hautes températures. De même, cette étude a tenté de contribuer à une meilleure connaissance de la réaction de topoisomérisation catalysée par les topoisomérases de type IA. Dans ce but, nous avons entrepris l'analyse enzymatique de la topoisomérase I de la bactérie hyperthermophile Thermotoga maritima. Cette analyse a permis d'identifier la tyrosine catalytique de l'enzyme ainsi que la présence d'un motif doigt de zinc. Ces expériences ont également mis en évidence une impressionnante activité de relaxation indépendante de la fixation de zinc par l'enzyme, contrairement à ce qui avait été décrit pour la topoisomérase I d'E. Coli. Des expériences menées sur des molécules uniques d'ADN indiquent que malgré leurs différences d'efficacité, ces deux enzymes suivent le même modèle catalytique. L'analyse biochimique d'enzymes chimères indique que la partie C-terminale de l'enzyme de T. Maritima n'est pas impliquée dans l'étape de passage de brin. Nous pensons que ces résultats peuvent être extrapolés à toutes les topoisomérases de type IA. Des essais de cristallisation ont permis l'obtention d'une structure préliminaire du domaine "core" de la topoisomérase I de T. Maritima à une résolution de 3,5 Angströms. De plus, l'analyse des produits de protéolyse ménagée de la topoisomérase I de T. Maritima nous a permis de définir la zone charnière impliquée sans doute dans le changement conformationnel de l'enzyme. La contribution de ce travail à la connaissance du cycle de la réaction de topoisomérisation et à la compréhension de l'adaptation aux hautes températures du contrôle du surenroulement de l'ADN est discutée dans cette thèse
DNA topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperature in thermophilic organisms. The work described in this thesis, try to better understand the adaptation to the high temperatures of the DNA supercoiling control systems. Moreover, it try also to better understand the type IA DNA topoisomerase-mediated catalysis. In this goal, we start the enzymatic study of the topoisomerase I from the hyperthermophilic Thermotoga maritima. We identified the catalytic tyrosine and a zinc finger motif. We also showed that T. Maritima topoisomerase I exhibits a very high DNA relaxing activity. Zinc binding is not required for DNA relaxation activity, contrary to the E. Coli enzyme. Experiments with a unique single-stranded DNA showed that both enzymes act in the same way though there is a important difference between their activities. Biochemical study of chimerical proteins showed that the C-terminal domain does not seem to be involved in strand passage mechanism. We think that it is also true for all type IA topoisomerases. Crystallization assays allowed us to get a preliminary structure (at 3. 5 Angstrom resolution) of the T. Maritima topoisomerase I core domain. Limited proteolysis of T. Maritima topoisomerase exhibits a unique hot spot susceptible to proteolytic attack which could correspond to the hinge involved in conformational movements during type IA DNA topoisomerase-mediated catalysis. The contribution of this work to a better understanding of the adaptation to the high temperatures of the DNA supercoiling control systems and to a better comprehension of the type IA DNA topoisomerase-mediated catalysis is discussed in this thesis

Chemistry
Ph.D.
The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates.
Temple University--Theses

Les besoins de l'industrie en nouveaux outils biotechnologiques, notamment des enzymes thermostables, ont conduit précédemment à l'isolement chez une " archaea " hyperthermophile " Thermococcus hydrothermalis " de plusieurs glycosyl-hydrolases, dont une pullulanase type II. L'identification et le séquençage du gène codant pour cette dernière a abouti au clonage et à l'expression du domaine catalytique de l'enzyme chez " E. Coli ". Ce domaine, désigné Th-ApuD2, correspond à une protéine de 88469 Da, composée de 770 acides aminés. Comme toute pullulanase type II, Th-ApuD2 est capable d'hydrolyser aussi bien les liaisons a-(1,6) du pullulane que les liaisons a-(1,4) de l'amylose. Par ailleurs, elle appartient à la famille 57 des glycosyl-hydrolases, une famille pour laquelle il existe très peu d'informations et aucune description de structure tridimentionnelle. Dans la présente étude, de manière à élucider les bases de la bi-fonctionnalité de Th-ApuD2 et d'enrichir les connaissances des enzymes de la famille GH57, une caractérisation biochimique approfondie a été conduite. L'étude de la spécificité de substrat et du mode ont permis de décrire Th-ApuD2 comme une exo-enzyme qui dégrade le pullulane ou l'amylose par l'extrémité réductrice. De plus, la caractérisation des modes d'inhibition de différentes molécules ainsi que la détermination des paramètres cinétiques d'hydrolyse d'une gamme variée de substrats ont conduit non seulement à prouver l'existence d'un site actif unique, responsable des deux activités hydrolytiques, mais aussi à proposer un modèle d'organisation de ce dernier. En ce qui concerne les deux activités hydroliques de Th-ApuD2, des expériences de mutagène basées sur la comparaison de la séquence de Th-ApuD2 avec d'autres protéines notamment des enzymes de la famille GH57, ont mis en évidence l'importance de trois résidus acides (E291, D313 et D394). L'absence d'activité mesurable des mutants Th-ApuD2-E291A et Th-ApuD2-D394A sur pullulane et amylose indique que ces deux résidus jouent des rôles essentiels dans le mécanisme catalytique
The industrial demands for new biotechnological tools expecially thermostables enzymes has previously led to the isolation of several glycosyl-hydrolases from the hyperthermophilic archaea "Thermococcus hydrothermalis", one of which was a pullulanase type II. The identification and sequencing of the gene encoding this enzyme has allowed the DNA encoding the catalytic domain to be cloned and expressed in "E. Coli". This domain designated Th-ApuD2, codes for a 88469 Da protein composed of 770 amino-acids. Like other pullulanases type II, Th-ApuD2 is able to hydrolyse both a-(1,6) likages in pullulan and a-(1,4) likages in amylose. The enzyme belongs to the glycosyl-hydrolase family 57, a poorly studied family for which no structural data is available. In this study, in order to investigate Th-ApuD2 bi-functionnality and to increase our general knowledge of family GH57 enzymes, we have performed a delaited biochemical characterization of Th-ApuD2. The study of substrat specificity and mode of action has allowed Th-ApuD2 to be classified as an exo-acting enzyme which degrades both pullulan and amylose from the reducing end. In addition, the identification of the inhibition modes of different molecules and the determination of the kinetic parameters of reactions involving a wide range of substrates have not only shown that a single active is responsible for both hydrolytic activities, but have facilitated the elaboration of a shematic model for the organisation of this site. With regard to the dual hydrolic activitiesof Th-ApuD2, mutagenesis experiments based on the sequence comparison with other proteins, including those from GH57, revealed the importance of three acidic residues (E291, D313 and D394). The absence of detectable activity in the presence of either pullulan or amylose suggest that E291 and D394 are essential for the catalytic mecanism

Thesis (Ph. D.)--University of Georgia, 2001.
Includes articles published in Methods in enzymology, and Journal of becteriology, and articles submitted to Journal of bacteriology, and Journal of biological chemistry. Directed by M.W.W. Adams. Includes bibliographical references (leaves 232-234).

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.
Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.
The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.
A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.
Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.
In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

碩士
國立臺灣海洋大學
食品科學系
92
The cloned treX gene from Sulfolobus solfataricus ATCC 35092 was transformed to Escherichia coli. The recombinant treX gene was expressed well under the T7 expression system compared to those of T5, Tac, and ara expression systems. The subunit molecular weight of GDE estimated by SDS-PAGE was about 83 kDa. Since this gene was fused in frame with the His-tag coding sequence on pET-15b-treX expression vector, we purified recombinant GDE by metal chelating chromatography after heat treatment at 80oC. In order to express wild-type GDE, we transformed pET-15b-DH-treX, which containing no His-tag coding sequence, to E. coli BL21 (DE3)-CodonPlus-RIL. The wild-type GDE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The recombinant his-tagged GDE was purified by using heat treatment, and metal-affinity chromatography. The obtained recombinant GDEs in both forms showed similar enzymatic properties. They all had an apparent optimal pH of 5 and an optimal temperature at 75oC. The enzymes were quite stable at the temperature up to 80oC for 2-h incubation. These GDEs were activated by K+、Na+、Ca2+、Mg2+、Zn2+、Cu2+、Ni2+ ions. The Cu2+ ion increased the activities of wild-type and recombinant GDEs up to 225% and 185%, respectively.