Biological systems carry inherent complexity, which pose difficulties observing behavioural properties, such as diffusion coefficients, kinetic constants and state switching occurrences. With constantly improving computing power and microscopy technologies, single molecule methods have become a viable alternative when probing the behaviour of proteins, enzymes, lipids and other molecules. Processed microscopy images and videos provide information such as particle intensities and trajectories, avoiding ensemble averaging and therefore allowing for a detailed breakdown of particle mobility and interactions. A single particle tracking (SPT) algorithm was developed which implements detection, localization and position linking on image stacks. Sub-pixel precise detection is done via either centroid determination, Gaussian fit, or radial symmetry centres, while tracking makes use of distance based global cost optimization. The detection algorithm is also used for single particle spectroscopy, where intensity information is used to determine the size of oligomers, as well as their interaction with other molecules through channel intensity cross-correlation. The algorithm underwent benchmarking with simulated videos and was applied to three different biological systems with comparison to other established methods of analysis. The first system studied was the diffusion of the fluorescent lipophilic dye DiD in a five-component mitochondria-like solid-supported lipid bilayer. Comparing line-scanning fluorescence correlation spectroscopy (FCS) and single particle tracking, the measured diffusion coefficients were found to be statistically different, with DFCS = 3 μm2s-1 and DSPT = 2 μm2s-1, indicating different operational ranges for the two methods. FCS outperforms SPT when the diffusion coefficient exceeds 1 μm2s-1, making it ideal for lipid diffusion in fluid membranes and proteins in solution with weak membrane interaction. SPT is best suited for mobile and immobile membrane inserted proteins, as well as lipid diffusion in viscous membranes. The second system studied was the interaction between the two proteins Bax and Bid when inserted in a membrane. Bax and Bid are both members of the Bcl-2 family of proteins, which plays a vital role in the apoptosis mechanism, by inducing mitochondrial outer membrane permeabilization. To study this system with single particle spectroscopy, fluorescently labelled Bax and truncated Bid (tBid) were imaged when interacting with a mitochondria-like supported lipid bilayer with confocal microscopy. Immobile and mobile particles were detected and distinguished based on the eccentricity of the observed fluorescence spot. The intensity of the particle signal was used to determine oligomer type (homo-oligomerization) while the interaction with the particles' counterpart (hetero-oligomerization) was determined by channel cross-correlation. This allowed the measurement of the 2D-KD values for mobile (0.6 μm-2) and immobile (0.08 μm-2) Bax/tBid complexes, showing that the degree of insertion of the proteins in the membrane greatly affect their affinity for each other. The third and final system studied was the motion of cellulases on cellulose fibers. Enzymatic hydrolysis of crystalline cellulose is a costly step in the generation of fermentable sugars for biofuel production. Due to the complex structure and many possible interaction states of the enzymes with cellulose, single particle tracking is a well-adapted technique to the gathering of information on the enzyme dynamics, which is essential for process optimization. The movement of cellulases on cellulose substrate was observed via labelled Thermobifidia fusca Cel5A, Cel6B and Cel9A on bacterial micro-crystalline cellulose substrate. The detected trajectories were analyzed using multiple diffusion models. A simple one-state diffusion model was insufficient to describe the observed radial displacement distributions and so a two-state model was introduced and confronted with the data using conventional least-squares fits , as well as a hidden Markov approach. The diffusion coefficients of the two states are found to be on the order of Dfast = 10-3 μm2s-1 and Dslow = 10-4 μm2s-1, with the slow state being more stable and therefore more likely to occur. Single particle tracking can give us better insight into complex interactions, such as synergistic binding of proteins existing in several different states and processive enzymatic behaviour, where ensemble averaging techniques can fall short. The uses of single molecule methods are plentiful and with the current rise of machine learning, higher levels of abstraction will provide us with more detailed insights into biological processes, driving promising developments in the medical field, as well as new technologies in many sectors of industry. / Thesis / Doctor of Science (PhD) / Proteins are the motors that drive most cellular processes, for example steering a cell’s life cycle, or decomposing sources of nutrients. Being able to observe the motion of individual proteins is key to understanding their behaviour. In this work a single particle tracking (SPT) program was developed to extract protein trajectories from fluorescence microscopy experiments. With this tool-set we investigated the following two systems. The first system of interest is the Bcl-2 protein family, which is vital during the pro- grammed cell death at the end of each cell’s life span. The failure of a controlled cell death can have dire consequences, such as necrosis and cancer. The Bcl-2 family proteins Bid and Bax are active on the outer membrane of the mitochondria, where they initiate the process of terminating the cell’s functions by forming pores. For our experiments we ar- tificially mimicked the outer membrane of the mitochondria, introduced Bid and Bax and observed their preferential groupings on the membrane surface. This provided indications of the mechanisms involved during binding and pore formation. The motivation behind the investigation of the second system is the improvement of biofuel generation from a renewable source: plant-based biomass. Cellulases are enzymes from bacteria or fungi that break down cellulose – one of the main building blocks of all plant cell walls – into fermentable sugars. In fluorescence microscopy experiments a purified cellulose substrate was used to monitor the motion of three types of cellulases. The insight which we gained into the cellulase behaviour may allow the optimization of the process of cellulose decomposition.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-03T16:30:05Z No. of bitstreams: 1 2011 - Alex da Silva Santos.pdf: 1990293 bytes, checksum: b92b4e5aec031d9fcd17900f0f1ef9c8 (MD5) / Made available in DSpace on 2016-08-03T16:30:05Z (GMT). No. of bitstreams: 1 2011 - Alex da Silva Santos.pdf: 1990293 bytes, checksum: b92b4e5aec031d9fcd17900f0f1ef9c8 (MD5) Previous issue date: 2011-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES / The production of enzymes for application at different areas of food agroindustry presents promising future perspectives, due to several intrinsic properties regarded to the performance of the enzymes as natural and biodegradable compounds, responsible for achieving specific reactions with better quality. Cellulases have been the most employed enzymes on food industry, acting sinergically on the hydrolysis of the glucosidic links ?-1,4 from the molecules of cellulose, and being used on several applications in this sector as in vegetal oils extraction, fruit maceration and juice clarification. Based on this context, the present study aimed to produce, concentrate and partially characterize an enzymatic extract by a mutant fungus strain of Aspergillus niger. Production was performed by solid-state fermentation (SSF) in aerated columns, using humidified wheat bran with (NH4)2SO4 solution on 0,1N HCl as substrate and cellobiose as inducer. Cellulolytic extract was a blend of extracts from three different assays selected on previous studies as the best conditions for the enzymes caboxymethilcellulase, ?-glucosidase and filter paper cellulose (FPase). During the characterization of the enzymatic extract, besides cellulases activity, the presence of protease and other enzymes with similar action to cellulases as xylanase and poligalacturonase was evaluated. For enzymatic extract concentration, three different strategies were performed: ultrafiltration, using a stainless steel plates system through a 20 kDa molecular weight cut-off polyethersulphone membrane and 0,014 m2 area; precipitation with ammonium sulphate under 20%, 40 %, 60% and 80% saturation level and lyophiilization. The best results were achieved by the ultrafiltration process, partially purified sample and providing enzymatic activities recovery between 75% and 99%, except for FPase. SDS-PAGE analysis presented 15 visible protein bands on cellulolytic extract with molecular weights ranging from 13.3 to 104.6 kDa. Zymography test was applied for cellulases and correlate enzymes as well as to protease, however, just for the last one the conditions were considered appropriate, identifying bands on 88, 103 and 145 kDa. The effective performance of ?-glucosidase and xylanase over xylan and cellobiose hydrolysis was confirmed by thin layer chromatography. A central rotational statistical design 22 with 4 central points was used for evaluating optimal temperature and pH for carboxymethylcellulase and ?-glucosidase. The analysis of the results obtained for both enzymes demonstrated that all variables were significative at a 95% confidence level. Based on the conditions studied it can be concluded that optimal pH and temperature ranges for efficient and combined action of carboxymethylcellulase and ?-glucosidase are 3.7 to 5.5 and 60 to 65?C, respectively. / A produ??o de enzimas para uso em diferentes ?reas da agroind?stria de alimentos mostra perspectivas futuras promissoras, devido ?s v?rias caracter?sticas inerentes ? a??o das enzimas que s?o compostos naturais, biodegrad?veis e capazes de desempenhar rea??es espec?ficas com melhor qualidade. Entre as enzimas mais utilizadas pelo setor de alimentos est?o as celulases, um complexo de enzimas que atuam de forma sin?rgica sobre a hidr?lise das liga??es glicos?dicas ?-1,4 das mol?culas de celulose, e possuem v?rias aplica??es industriais neste setor, como na extra??o de ?leos vegetais, na macera??o de frutas e na clarifica??o de sucos. Dentro deste contexto, este trabalho teve como objetivo produzir, concentrar e caracterizar parcialmente um extrato celulol?tico obtido por linhagem f?ngica mutante de Aspergillus niger. A produ??o foi realizada por fermenta??o no estado s?lido (FES) em colunas aeradas, utilizando como substrato farelo de trigo triturado umidificado com solu??o de (NH4)2SO4 em HCl 0,1N e celobiose, como indutor. O extrato celulol?tico consistiu de uma mistura de extratos obtidos em 3 ensaios fermentativos diferentes, selecionados em trabalhos anteriores como as melhores condi??es para produ??o de cada uma das enzimascarboximetilcelulase (CMCase), ?-glicosidase e celulase em papel de filtro (FPase). Durante a caracteriza??o do extrato enzim?tico, al?m da atividade das celulases, tamb?m era avaliado o teor de prote?na, a presen?a de protease e de enzimas correlatas ? a??o de celulases como xilanase e poligalacturonase. Para concentra??o do extrato enzim?tico foram realizadas tr?s diferentes estrat?gias: ultrafiltra??o em um sistema de quadro e placas em a?o inox, utilizando uma membrana de polietersulfona com massa molar de corte de 20 kDa e ?rea de 0,014m2; precipita??o com sulfato de am?nio utilizando satura??es de 20%, 40%, 50%, 60%, 80% e liofiliza??o. O processo de ultrafiltra??o foi o que obteve o melhor resultado, purificando parcialmente a amostra e proporcionando uma recupera??o das atividades enzim?ticas entre 75% e 99% para todas as atividades avaliadas, exceto FPase. A an?lise eletrofor?tica em SDS-PAGE demonstrou a presen?a de 15 bandas vis?veis de prote?nas no extrato celulol?tico com pesos moleculares que compreendem uma faixa entre 13,3 e 104,6 kD. O teste de zimografia foi realizado para as celulases e enzimas correlatas, bem como para protease, no entanto somente para esta ?ltima, as condi??es testadas foram adequadas tornando-se poss?vel identificar bandas em 88, 103 e 145 kDa. A efetiva a??o das enzimas ?-glicosidase e xilanase na hidr?lise de celobiose e xilana, respectivamente,foi comprovada em cromatografia de camada fina. Al?m disso, a temperatura e pH ?timos de atua??o de carboximetilcelulase e ?-glicosidase foram determinados utilizando o delineamento composto central rotacional 22, com 4 pontos centrais. A an?lise dos resultados de ambas as enzimas demonstrou que as vari?veis eram significativas, a um n?vel de confian?a de 95%. Com base nas condi??es estudadas, concluiu-se que as faixas de pH e temperatura ?timos para a atua??o eficiente e conjunta de CMCase e ?-glicosidase est?o entre 3,7 a 5,5 e 60 a 65 ?C, respectivamente.

Made available in DSpace on 2016-06-02T20:20:34Z (GMT). No. of bitstreams: 1 4606.pdf: 10556895 bytes, checksum: debf5633a053f88ca7dd4618edef9175 (MD5) Previous issue date: 2012-07-06 / Universidade Federal de Minas Gerais / The plant biomass is a large-scale available resource and one of its more important applications is the second-generation ethanol production. However, the enzyme cost is one of the biggest barriers for economically viable ethanol from biomass. Therefore, it is important to identify fungal strains that can produce high concentrations of plant biomass-degrading enzymes. The aim of this work was to clone, study the gene expression and characterize the plant biomass-degrading transcript set of the filamentous fungus Trichoderma harzianum IOC-3844. A total of 1,543 highquality reads from the Trichoderma harzianum IOC-3844 cellulose induced cDNA library were organized into 1,002 transcripts representing 167 contigs and 835 singlets. Of these 1,002 transcripts 646 had unknown functions and 356 showed associated functions. Among the transcripts with associated functions, we found 20 transcripts related to plant biomass deconstruction. The real time PCR analysis of Trichoderma harzianum IOC-3844 mycelia grown for 36 and 60 hours in cellulose, revealed that the levels of the following mRNAs were induced by at least 2,000-fold when compared to uninduced mycelia: cbh1, cbh2, egl1, egl2, egl3, egl7 and swo1. In some cases, the values were higher than 100,000-fold. Among the transcripts analyzed by real time PCR, cbh1, cbh2 and egl7 exhibited the highest expression levels. The Trichoderma harzianum IOC-3844 exhibited a repertoire with high expression of plant biomassdegrading transcripts. The enzymes EGIII and Xyn2 were recombinantly expressed in Pichia pastoris, showing good quality purification and good enzymatic activity. The heterologous expression assays made possible future studies aiming at the industrial application of the enzymes. Therefore, this strain showed potencial to produce biomassdegrading enzymes for second-generation ethanol production and to be a source of enzymes for the paper industry / A biomassa vegetal é um recurso disponível em larga escala e uma das suas mais imporantes aplicações é a produção de etanol de segunda geração. No entanto, o custo das enzimas é um dos maiores entraves para a produção economicamente viável deste etanol. Neste contexto, é importante encontrar organismos produtores de grandes quantidades de enzimas que degradam a biomassa. Os objetivos deste estudo foram clonar, estudar a expressão gênica e caracterizar o conjunto de enzimas que degradam a biomassa vegetal, do fungo filamentoso Trichoderma harzianum IOC-3844. Um total de 1.543 seqüências de boa qualidade, geradas a partir de uma biblioteca de cDNA do Trichoderma harzianum IOC-3844, induzido por celulose, foi organizado em 1.002 transcritos, sendo 167 representados por mais de uma seqüência e 835 representados por apenas uma seqüência. Destes transcritos, 356 tiveram função associada e 646 não tiveram. Com isso, entre os transcritos com função associada, foram listados 20 transcritos envolvidos com degradação de biomassa vegetal. Análises de PCR em tempo real do micélio de Trichoderma harzianum IOC-3844, crescido por 36 e 60 horas em celulose, mostraram níveis de mRNA mais de 2.000 vezes mais representados para os transcritos cbh1, cbh2, egl1, egl2, egl3, egl7 e swo1, quando comparados com o micélio não induzido. Em alguns casos as maiores representatividades alcançaram valores superiores a 100.000 vezes. Entre os transcritos analisados o cbh1, o cbh2 e o egl7, mostraram os mais altos níveis de expressão. O Trichoderma harzianum IOC-3844 exibiu um repertório com alta expressão de transcritos envolvidas na degradação de biomassa vegetal. As enzimas EGIII e Xyn2 foram expressas em sistema recombinante com uso da levedura Pichia pastoris, apresentando facilidade de purificação e boa atividade enzimática. Os ensaios de expressão heteróloga viabilizaram estudos posteriores que visam a aplicação industrial das enzimas. Assim, esta cepa mostrou potencial para produzir enzimas que degradam a biomassa para a produção de etanol de segunda geração, e para ser fonte de enzimas para a indústria de papel.

Made available in DSpace on 2016-08-17T18:39:43Z (GMT). No. of bitstreams: 1 4514.pdf: 2385637 bytes, checksum: 89b533535415a993d655f83f1387c6f0 (MD5) Previous issue date: 2012-06-27 / Financiadora de Estudos e Projetos / The selection of cellulase-producing fungi is one of the possible estrategies for obtaining necessary enzymes to hydrolyze the lignocellulosic material of plant biomass and thereby contribute to the viability of cellulosic ethanol production. The aim of this study was achive a screening of isolated fungi from the Amazon region to assess the production of enzymes related to plant biomass degradation, in order to select a line for production, purification and biochemical, kinetical and and structural biology characterizationof the ß-Galactosidase enzyme. Therefore, this work was undertaken in three stages, first of all it was performed a screening of 40 fungal strains isolated from the Amazon region through the cultivation in solid state fermentation (FES) at 35ºC for 240 hours, using as substrate wheat bran. It was evaluated the production of xylanase, endoglucanase, FPase, pectinase, ß-Glicosidase and total protein, and the fungi that stood out were: P6B2, the best producer of xylanase, P47C3 (Aspergillus niger), the best producer endoglucanase and ß-Glicosidase and P40B3, the best producer of FPase. These three fungi were selected for the second phase of this work for assessment in the production of xylanase, FPase, endoglucanase, ß-Glicosidase and total protein by submerged fermentation (FSm). The fermentation took place for 5 days at 30ºC and 200 rpm with a source of carbon: 1% of wheat bran washed and nutrient medium. The fungi P47C3, which was identified as Aspergillus niger, showed the best production of these enzymes, being selected for the third stage of this project. This last step involved the selection of an enzyme that has not been elucidated its structural biology. Given this fact, we carried out a study of selection of the medium, purification and biochemicalkinetical characterization of ß-Galactosidase. The Aspergillus niger (P47C3) was subjected to submerged for 5 days at 200 rpm at 30ºC. Purification occured in three steps using: ion exchange column SP-Sephadex C-50 and SP TSK-5PW column, and gelfiltration, with the resin Sephacryl S-200. The enzyme ß-Galactosidase showed a molecular weight of 125 kDa, being stable at pH 4,0, with anoptimum temperature of 55ºC. It was evaluated theKmap e Vmáxap of two substrates, PNPG and lactose, being: 2,204 mM-0,285 mM/min and 2,101 mM-0,75mM/min, respectively. The inhibition of hydrolasis of the substrate PNPG by ß-Galactosidase in the presence of galactose inhibitor product showed a Ki value of 5,01 mM. Finally, the ß-Galactosidase was subjected to crystallization conditions, the best conditions occurred in buffer 0,2M Tris- HCl, with the precipitation agent, 12% PEG 4000 at pH 8,6. Therefore, the unpublished protocol for purification of ß-Galactosidase was efficient and it is possible to crystallize this enzyme of isolated fungi from the Amazon region, which showed great potencial for the production of this enzyme and that the future can be used in industrial application and biotechnological innovations. / A seleção de fungos produtores de celulases é uma das possíveis estratégias para a obtenção das enzimas necessárias para hidrolisar o material lignocelulósico da biomassa vegetal e com isso contribuir para a viabilização da produção de etanol celulósico. O objetivo desse trabalho foi realizar um screening dos fungos isolados da região amazônica para a avaliação da produção de enzimas relacionadas à degradação da biomassa vegetal, a fim de selecionar uma linhagem para produção, purificação e caracterização bioquímica, cinética e biologia estrutural da enzima ß-Galactosidase. Dessa forma, esse trabalho foi realizado em três etapas, primeiramente foi realizado um screening de 40 linhagens fúngicas isoladas da região amazônica, através do cultivo em fermentação em estado sólido (FES), a 35°C, por 240 horas, utilizando como substrato o farelo de trigo. Avaliou-se a produção de xilanase, endoglucanase, FPase, pectinase, ßglicosidase e proteínas totais, sendo que os fungos que mais se destacaram foram o: P6B2, melhor produtor de xilanase, P47C3 (Aspergillus niger), melhor produtor de endoglucanase e ß-glicosidase e o P40B3, melhor produtor de FPase. Esses três fungos, foram selecionados para a segunda fase do trabalho para avaliação na produção de xilanase, FPase, endoglucanase, ß-glicosidase e proteínas Totais por fermentação submersa (FSm). A fermentação ocorreu por 5 dias, à 30ºC e 200 rpm tendo como fonte de carbono: 1% de farelo de trigo lavado e meio nutriente. O fungo P47C3, identificado como Aspergillus niger, apresentou melhor produção dessas enzimas, sendo selecionado para a terceira etapa desse projeto. Essa última etapa, envolveu a escolha de uma enzima que não estivesse sua biologia estrutural elucidada. Diante desse fato, realizou-se um estudo de seleção do meio de cultivo, purificação e caracterização bioquimico-cinética da ß-Galactosidase. O fungo Aspergillus niger (P47C3) foi submetido a fermentação submersa, durante 5 dias, à 200 rpm em 30ºC. A purificação ocorreu em três etapas utilizando: colunas de troca iônica SP - Sephadex C-50 e a coluna SP -TSK 5PW; e gel filtração, com a resina Sephacryl S-200. A enzima ß-Galactosidase apresentou uma massa molecular de 125 kDa, sendo estável em pH 4,0, e com temperatura ótima de 55ºC. Avaliou-se a Kmap e Vmáxap de dois substratos, o PNPG e a lactose, sendo: 2,204 mM - 0,285 mM/min e 2,101 mM 0,750 mM/min, respectivamente. A inibição da hidrólise do substrato PNPG pela ß-Galactosidase na presença do produto inibidor galactose apresentou um valor de Ki de 5,01 mM. Por fim, a ß-Galactosidase foi submetida a condições de cristalização, as melhores condições ocorreram em tampão 0,2M Tris-HCl, tendo como agente precipitante, PEG 4000 12% em pH 8,6. Portanto, o protocolo inédito de purificação da ß-Galactosidase foi eficiente, sendo possível cristalizar essa enzima do fungo isolado da região amazônica, o qual apresentou grande potencial para a produção dessa enzima e que futuramente possa ser utilizado em aplicações industriais e inovações biotecnológicas.

Made available in DSpace on 2017-07-10T18:08:19Z (GMT). No. of bitstreams: 1 Fabiano Bisinella Scheufele.pdf: 1716907 bytes, checksum: fee6288858ac3d92f0d2e7f9f8d02ba2 (MD5) Previous issue date: 2012-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lignocellulosic biomass has high yields of cellulose which can be hydrolyzed to fermentable carbohydrates. Global generation of agro-industrial wastes grows simultaneously with the sector development resulting at the accumulation of lignocellulosic residues leading environmental pollution and loss of potential materials for the bioconversion to a wide range of high added value products, such as biofuels. Recently, the search of renewable sources of energy has grown, due to the depleting of fossil fuels, increasing the possibility at the conversion of the lignocellulosic biomass via hydrolytic enzymes. The aim of this work was evaluate cellulases production by lignocellulolytic fungi from the Amazonic biome aiming at the bioconversion of the agro-industrial residues. Submerged and solid-state fermentations were performed to select the microorganism with superior cellulase productive capacity. The influence of parameters such as pH, surfactant induction (Tween 80), aeration and agitation, besides the alkaline oxidative treatment of the sugarcane bagasse. Statistical design were carried out to estimate the influence of the moisture and the initial pH at cellulases production by solid-state fermentation. Trichoderma sp. and Aspergillus niger performed the best production of enzymes, where the highest yields of total cellulase were obtained by agitated submerged fermentation with sugarcane bagasse pretreated with H2O2 (1%) reaching 0.265 U.mL-1 (12.915 U.g-1) by Trichoderma sp. at the sugarcane bagasse, and 0.155 U.mL-1 (7.549 U.g-1) by Aspergillus niger. Through solid state fermentations with the pretreated sugarcane bagasse the influence of initial pH and the moisture were evaluated by statistical design. In the case of the Trichoderma sp. both parameters were significant at the cellulase production, as well as the synergistic interaction, within the confidence interval of 95%, yielding 0.167 U.mL-1 (2.695 U.g-1), at the pH 7.0 and 1:9 solid-liquid ratio. For Aspergillus niger only pH was significant and the cellulase content obtained was 0.098 U.mL-1 (1.695 U.g-1) at pH 7.0. Finally, a cellulase produced by Trichoderma sp. at solid state fermentation and a commercial enzyme were used at enzymatic hydrolysis tests. The parameters hydrolysis time, enzyme dilution, concentration of Tween 80 and solid-liquid ratio of sugarcane bagasse were evaluated. The significant variables were then optimized by a central composite rotational design. The strain of Trichoderma sp. from the Amazon biome showed potential at the cellulase production and the treated sugarcane bagasse was a fine substrate for the enzymatic production. / A biomassa lignocelulósica contêm altos teores de celulose e outros polissacarídeos em sua constituição química, podendo ser hidrolisados em açúcares fermentescíveis. A geração de resíduos agroindustriais anual tem crescido resultando no acúmulo de resíduos que contribuem para a poluição do meio ambiente e na perda de materiais que possuem potencial na bioconversão a produtos de alto valor agregado, como por exemplo, biocombustíveis. Recentemente, há a necessidade de fontes energéticas de origem renovável, devido à diminuição dos combustíveis fósseis, viabilizando a conversão das biomassas lignocelulósicas via enzimas hidrolíticas. O objetivo deste trabalho foi avaliar a produção de enzimas celulases por fungos lignocelulolíticos provenientes do bioma amazônico visando a bioconversão de resíduos agroindustriais. Diferentes fermentações foram realizadas, tanto em meio submerso quanto em estado sólido, através das quais selecionou-se os micro-organismos com melhor capacidade produtiva de celulases. Estudou-se a influência de parâmetros como pH, utilização de surfactante como indutor (Tween 80), aeração e agitação, além do tratamento alcalino oxidativo do bagaço de cana. Os micro-organismos que apresentaram melhor desempenho na produção das enzimas foram o Trichoderma sp. e o Aspergillus niger, sendo que os maiores níveis de celulase total foram obtidos por fermentação submersa nos ensaios agitados com bagaço de cana pré-tratado com H2O2 (1%), com 0,265 U.mL-1 (12,915 U.g-1) pelo Trichoderma sp., e 0,155 U.mL-1 (7,549 U.g-1) com Aspergillus niger. A partir de fermentações em estado sólido com o bagaço de cana pré-tratado avaliou-se a influência dos parâmetros pH inicial e umidade por planejamentos experimentais, verificando-se que para o Trichoderma sp. ambos os parâmetros, bem como a interação sinergética entre si, foram significativos dentro do intervalo de confiança de 95%, obtendo-se 0,167 U.mL-1 (2,695 U.g-1) no pH 7,0 e relação sólido-líquido 1:9. No caso do Aspergillus niger apenas o pH foi significativo e o teor de celulase obtido foi de 0,098 U.mL-1 (1,695 U.g-1) para um pH 7,0. Finalmente, a partir de uma celulase produzida por fermentação em estado sólido do Trichoderma sp. e uma enzima comercial foi realizada a avaliação da influência dos parâmetros tempo de hidrólise, diluição da enzima, concentração de Tween 80 e razão sólido-líquido do bagaço de cana sobre a hidrólise do mesmo. As variáveis significativas foram, posteriormente, otimizadas por um delineamento composto central rotacional. A cepa Trichoderma sp. proveniente do bioma amazônico apresentou potencial na produção de celulases e o o bagaço de cana submetido ao tratamento alcalino oxidativo apresentou-se como um bom substrato para a produção enzimática.

Microbial enzymes implicated in plant cell hydrolysis may have several potential aplications such as biomass degradation biocatalysts or with biofuel production. Bark beetles establish symbiosis with several microbial strains which play different roles benifitting the beetle, as the production of hydrolytic enzymes to degrade the ingested wood, the protection against mirobial antagonist or the detoxification of the environment. Fungal symbionts have been traditionally the best studied, but several recent research with bacterial symbionts of several bark beetle species show that bacterial also display important functions for the host. In this study, the bacterial communities of the bark beetle species Cryphalus piceae and Pithophtorus pithophtorus, collected in the Czech Republic from pine and fir trees, respectively, were isolated and 55 out of 89 samples were identified by 16S rRNA gene amplification and sequencing. Members of the genera Erwinia, Pantoea, Curtobacterium, Yersinia, Pseudomonas and Staphylococcus were detected. The isolates were object of study for their possible biotechnological potential in (ligno)cellulose materials degradation by screening several enzymes implicated in plant cell hydrolysis, as cellulases, xylanases, amylases, laccases, as well as their capability for colorant...