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Orientador: Rubens Monti / Banca: Rubens Monti / Banca: Luis Henrique Souza Guimarães / Banca: Olga Luisa Tavano / Banca: Maristela de Freitas Sanches Peres / Banca: Luis Vitor Silva do Sacramento / Resumo: A descoberta de resíduos agrícolas, como fonte de energia renovável tem colaborado para o desenvolvimento industrial e preservação do meio ambiente. Desta maneira, a degradação da parede celular destes resíduos tem grande importância na fabricação de pães, alimentos, bebidas, têxtil, papel e celulose entre outros produtos. A degradação enzimática destes polímeros esta se tornando uma alternativa mais atraente do que a utilização de substâncias químicas e processos mecânicos. Alguns fungos termófilos participam desta degradação enzimática, como Humicola grisea var. thermoidea, isolado do solo, é conhecido como bom produtor do complexo xilanolítico. Além deste, um fungo termófilo isolado da polpa do fruto cupuaçuzeiro em decomposição foi estudado e produziu xilanase. Esses fungos foram mantidos em meios sólidos contendo ágar e farinha de aveia. O fungo isolado da polpa do fruto cresceu em meio liquido empregando diferentes resíduos agrícolas como fonte de carbono. Dentre os vários resíduos empregados para se otimizar a produção de xilanase, o melhor foi o sabugo de milho. Esse foi utilizado nos experimentos na proporção de 1,0% (m/v), em meio líquido contendo 0,1% CaCO3, 0,5% NaCl, 0,1% NH4Cl; 0,5% água de maceração de milho. O pH ótimo foi 5,0 e a temperatura ótima de 60ºC. A xilanase presente no extrato clarificado em caulim apresentou cinética Michaeliana com Km de 10,41 ± 0,282 mgmL-1 e Vmax 3,32 ± 0,053 U (mg protein)-1. Para acompanhar o processo de clarificação da enzima solúvel realizou-se SDS-PAGE verificando a presença de uma banda protéica com atividade xilanolítica de massa molecular de aproximadamente 30kDa e a Cromatografia Sílica Gel P60 para verificar os produtos da hidrólise da xilana em função do tempo e da associação de xilosidase do fungo termófilo Humicola com a xilanase do fungo isolado do fruto... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The researches of agricultural residues as renewable energy sources have been contributing for the industrial development and the environment conservation. Thus, the degradation of these residues' cell walls has great importance in fabrication of bread, food, drinks, textile, paper, cellulose, and other products. The enzymatic degradation of such polymers is becoming an attractive option when compared to the use of chemical substances and mechanical processes. Some thermophilic fungi take role in this enzymatic degradation, such as Humicola grisea var. thermoidea, isolated from soil, and is known to be a good producer of the xylanolytic complex. Besides it, another thermophilic fungus, isolated from the Theobroma grandiflorum decomposing fruit pulp, was studied and also produced xylanases. These fungi were kept in solid media containing agar and oat bran. The fungus isolated from the fruit pulp grew in liquid media in wich different kinds of agricultural residues were employed as carbon sources. Among the various types of residues that were used to optimize the xylanase production, corn cobs proved to be the best one. It was used in assays in a proportion of 1,0% (m/v), in liquid media containing 0.1% of CaCO3, 0.5% of NaCl, 0.1% of NH4Cl, and 0.5% of corn steep liquor. The pH optima was 5.0 and the temperature optima was 60ºC. The xylanase present in the kaolin clarified extract showed a Km of 10.41 ± 0.282 mg.mL-1, and Vmax 3.32 ± 0.053 U (mg.protein)-1. In order to follow the soluble enzyme clarification process, SDS-PAGE was run, verifying the presence of a proteic band with xylanolytic activity of, approximately, 30kDa of molecular mass. Furthermore, Silica Gel 60 Thin Layer Chromatography (TLC) was used to verify the xylan hydrolysis products in function of time and the association of Humicola sp. thermophylic fungus xylosidase with the xylanase produced by the fruit pulp... (Complete abstract click electronic access below) / Doutor

Novel biocatalysts are highly demanded in the white biotechnology. Hence, the development of highly stable and enantioselective biocatalysts with novel functionalities is an ongoing research topic. Here, an osmium ligating single-site ArM was created based on the biotinstreptavidin technology for the dihydroxylation of olefins. For the creation of the artificial catalytic metal center in the streptavidin (SAV) cavity, efficient osmium tetroxide (OsO4) chelating biotin-ligands were created. The unspecific metal binding of the host scaffold was diminished through genetical and chemical modification of the host protein. The created single-site OsO4 chelating ArM was successfully applied in the asymmetric cyclopropanation, revealing a stable and tunable catalytic hybrid system for application. The structural analysis of protein-ligand complexes is essential for the advanced rational design and engineering of artificial metalloenzymes. In previous studies, a SAV-dirhodium ArM was created and successfully applied in the asymmetric cyclopropanation reaction. To improve the selectivity of the SAV-dirhodium complex, the structural location of the organometallic complex in the SAV cavity was targeted and small-angle x-ray scattering (SAXS) was used to obtain the structural information. The SAXS analysis revealed valuable information of the molecular state of the complexes; hence, the method proved to be useful for the structural analysis of protein-ligand interactions. The discovery of novel enzymes from nature is still the major source for improved biocatalysts. One of the most important enzymes used in the molecular biology are DNA polymerases in PCR reactions. The halothermophilic brine-pool 3 polymerase (BR3 Pol) from the Atlantis II Red Sea brine pool showed optimal activities at 55 °C and salt concentrations up to 0.5 M NaCl, and was stable at temperatures above 95 °C. The comparison with the hyperthermophilic KOD polymerase revealed the haloadaptation of BR3 Pol due to an increased negative electrostatic surface charge and an overall higher structural flexibility. Engineered chimeric KOD polymerases with swapped single BR3 Pol domains revealed increased salt tolerance in the PCR, showing increased structural flexibility and a local negative surface charge. The understanding of the BR3 Pol haloadaptation might enable the development of a DNA polymerase tailored for specific PCR reactions with increased salt concentrations.

The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.

Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.

La température est un paramètre crucial dans le fonctionnement du monde vivant, notamment de la machinerie moléculaire (les protéines) dont la stabilité et l’activité en dépendent sensiblement. Celles-ci sont souvent considérées comme étant équivalentes : si une protéine fonctionne, c’est qu’elle est stable, et vice-versa. Cependant, les protéines des organismes thermophiles, qui prolifèrent dans de températures élevées, sont stables à température ambiante, mais y présentent une faible activité. Cette dernière est optimale à la température de croissance de l’organisme hôte. Lorsqu’on parle de stabilité et d’activité protéique, la rigidité mécanique est souvent utilisée comme paramètre pertinent, offrant une explication simple et attractive à la fois pour la stabilité thermodynamique à haute température et au manque d’activité à des températures plus modérés. La réalité s’avère souvent plus complexe, et les mécanismes moléculaires reliant rigidité/flexibilité avec la stabilité et l’activité sont encore mal compris. Dans ce travail, nous abordons le problème au travers de trois systèmes. Nous avons examiné l’activation thermique des modes fonctionnels du domaine G de la protéine EF ainsi que les homologues mésophiles et thermophiles de la déshydrogénase Lactate/Malate. Par ailleurs, nous avons mis en évidence l’existence d’un paramètre unique (la moyenne des fluctuations atomiques) permettant d’expliquer la dynamique de la protéine lysozyme près de son point de fusion, et ce quelle que soit la nature de l’environnement autour de la protéine (qui décale le point de fusion). Nos conclusions se basent principalement sur une approche in silico où la dynamique moléculaire et des techniques d’échantillonnage améliorées sont utilisées et sont complémentées par des expériences de diffraction de neutrons / Temperature is one of the major factors governing life as demonstrated by the fine tuning of stability and activity of the molecular machinery, proteins in particular. The structural stability and activity of proteins have been often presented as equivalent. However, the thermophilic proteins are stable at ambient condition, but lack activity, the latter recovered only when the temperature increases to match that of the optimal growth condition for the hosting organism. In discussing the protein stability and activity, mechanical rigidity is often used as a relevant parameter, offering a simple and appealing explanation of both the extreme thermodynamic stability and the lack of activity at low temperature. The reality, however, illustrates the complexity of the rigidity/flexibility trade off in ensuring stability and activity through intricate thermodynamic and molecular mechanisms. Here we investigate the problem by studying three study cases. These are used to relate the thermal effects on mechanical properties and the stability and activity of the proteins. For instance, we have probed the thermal activation of functional modes in EF G-domain and Lactate/Malate dehydrogenase mesophilic and thermophilic homologues and verified a “universal” scaling of atomistic fluctuation of the Lysozyme approaching the melting in different environmental conditions. Our conclusions largely rest on an in silico approach, where molecular dynamics and enhanced sampling techniques are utilized, and are often complemented with neutron scattering experiments

This diploma thesis studies the utilization of whey using thermophilic bacteria of the genus Thermus and Geobacillus. The whey stripped off proteins was used as a cultivation medium during experiments. The cultivation took place in the Erlenmeyer flasks, to assess the optimal conditions for microrganism’s growth. During the cultivation in the bioreactor, growth curves were established. The amount of whey utilization was assessed by analytical methods that determine the concentration of reducing saccharides (lactose) and chemical oxygen demand (COD) while studying the optimal conditions and a method determining the concentration of reducing saccharides during growth curves analysis.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18342号 / 農博第2067号 / 新制||農||1024(附属図書館) / 学位論文||H26||N4849(農学部図書室) / 31200 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 三上 文三, 教授 平竹 潤 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Group II Introns are small segments of DNA that reside in the chromosome of bacteria or the organelles of primitive eukaryotes. These elements have some very interesting properties. First, they are retrotransposons that can move from one location to a new location in DNA via a reverse transcription mechanism. Second, they form a large ribozyme that mediates self-splicing of the intron from pre-mRNA. A Group II Intron type protein with similarity to reverse transcriptase was discovered in the thermophilic bacterium Geobacillus stearothermophilus strain 10 (Vellore et al., 2004, Appl. Environ. Microbiol. 70: 7140-7147). Numerous copies of the intron, designated Gs. Int1, are present in the chromosome of strain 10 but absent from a related strain ATCC 12980. Experiments to detect the in vivo splicing of intron Gs.Int1 from G. stearothermophilus cells did not work. Plasmids to that will over-express the Gs. Int1 intron to detest splicing in vivo in Escherichia coli have been constructed.

Syntrophic relationships between fatty acid degrading bacteria and hydrogenotrophic methanogens are important for a well-functioning anaerobic degradation process during biogas production from organic waste material. This is particularly important in biogas processes fed protein-rich material as their degradation gives rise to high levels of ammonia, which inhibits many microorganisms. A common problem arising in the high-ammonia biogas process is the accumulation of volatile fatty acids, such as acetate, propionate or butyrate, which negatively affects the methane production. The aim of this study was to isolate and identify the syntrophic butyrate-oxidizing bacteria present in the enriched syntrophic communities originating from thermophilic continuously fed laboratory-scale reactors. Butyrate was added to batch assays containing the enrichment cultures. Sequencing of a 464 bp region within the 16S rRNA gene was conducted to study the change in microbial community structure over time during the butyrate degradation. The enrichment culture was also used as an inoculum source during the isolation attempts using agar cultivation, colony transfer and 16S rRNA gene sequencing of the obtained isolates. From the Illumina sequencing data, it could be concluded that the novel species of interest belonged to the genus Syntrophothermus. Two species were isolated, however neither appeared to be the butyrate-degrading bacterium. One of the species was Defluviitoga tunisiensis and the other was a novel species related to the mesophilic bacterium Schnuerera ultunensis.