Academic literature on the topic 'Hyperthermophilic enzymes'

SUMMARY Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

ABSTRACT Genes encoding 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25°C), although both enzymes showed much less 2-deoxy-d-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25°C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 Å. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.

Archaeal dehydrogenases are often found to be of a specific class of dehydrogenase which has low sequence identity to the equivalent bacterial and eukaryotic counterparts. This paper focuses on two different types of hyperthermophilic dehydrogenase enzyme that have been cloned and over-expressed in Escherichia coli. The crystallographic structures of the apo form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from Sulfolobus solfataricus and the related holo form of GAPDH from Methanothermus fervidus have been solved to high resolution. The zinc-containing structure of ADH (alcohol dehydrogenase) from Aeropyrum pernix has also been solved as a quaternary complex with the cofactor NADH and the inhibitor octanoic acid. The results show that despite the low sequence identity to the related enzymes found in other organisms the fold of the protein chain is similar. The archaeal GAPDH enzymes show a relocation of the active site which is a feature of evolutionary interest. The high thermostability of these three archaeal dehydrogenases can be attributed to a combination of factors including an increase in the number of salt bridges and hydrophobic interactions, a higher percentage of secondary structure and the presence of disulphide bonds.

Protection of thermolabile metabolites and coenzymes is a somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles. Detailed information about the mechanisms used by thermophiles to protect these thermolabile metabolites and coenzymes is still scarce. A case in point is CP (carbamoyl phosphate), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. Recently we obtained the first evidence for a physical interaction between two hyperthermophilic enzymes for which kinetic evidence had suggested that these enzymes channel a highly thermolabile and potentially toxic intermediate. By physically interacting with each other, CKase (carbamate kinase) and OTCase (ornithine carbamoyltransferase) prevent thermodenaturation of CP in the aqueous cytoplasmic environment. The CP channelling complex involving CKase and OTCase or ATCase (aspartate carbamoyltransferase), identified in hyperthermophilic archaea, provides a good model system to investigate the mechanism of metabolic channelling and the molecular basis of protein–protein interactions in the physiology of extreme thermophiles.

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