Genomic analysis and physiological experiments conducted on the lignocellulosic biomass degrading bacterium C. phytofermentans, indicates that it can degrade and utilize a wide-range of carbohydrates as possible growth substrates. Previous experiments characterized gene expression using custom whole genome oligonucleotide microarrays. The results indicated that C. phytofermentans utilizes ATP-binding cassette (ABC) transporters for carbohydrate uptake and does not use the sole phosphoenolpyruvate-phosphotransferase system (PTS) for any of the tested substrates.Distinct sets of Carbohydrate Active Enzymes (CAZy) genes were also up-regulated on specific substrates indicative of C. phytofermentans ability to selectively degrade lignocellulosic biomass. We also identified a highly expressed cluster of genes which includes seven extracellular glycoside hydrolases and two ABC transporters with unknown specificity on a number of substrates. These results lead to the hypothesis that when grown on plant biomass, C. phytofermentansis capable of degrading and transporting all major carbohydrate components of lignocellulose biomass. To test this, C. phytofermentans was grown on three different lignocellulosic biomass substrates (Brachypodium distachyon, cornstover, and switchgrass). Gene expression and HPLC analysis indicated that C. phytofermentans is utilizing multiple substrates with multiple sugar ABC transporter clusters, glycoside hydrolases, and sugar utilization pathways being expressed. To further test this,the sugar utilization pattern for C. phytofermentans was investigated. Growth studies were performed on individual saccaharides (glucose, cellobiose, xylose, and fucose) as well as combinations of all these sugars. From these studies we determined that C. phytofermentans does not show a characteristic diauxic shift indicative of preferential sugar utilization or carbon catabolite repression (CCR). This result was supported further by HPLC analysis indicating that co-utilization of sugars was occurring, however their were differences in the rates of consumption. Expression analysis of dual sugar combinations of glucose/cellobiose, glucose/xylose, and glucose/fucose also shows that genes involved in the transport and utilization of each sugar are expressed. We also noted glucose repression of some of the glyocside hydrolases which are normally expressed on xylose and fucose. The results from this study indicate that C. phytofermentans can utilize multiple sugars simultaneously.

As enzimas hidrolíticas são consideradas, por plena definição, aquelas com a capacidade de realizar a reação de hidrólise. Devido à sua capacidade de degradar substâncias naturais, as enzimas hidrolíticas são de grande uso industrial e podem ser aplicadas nas indústrias têxtil, alimentar, farmacêutica, biocombustíveis- agrícola, assim como na produção de derivados biotecnológicos de maior valor agregado. Conhecidas como enzimas que liberam arabinose e arabinofuranose, as arabinofuranosidases da família 62 são capazes de quebrar as conexões de arabinoxilanos. A importância biotecnológica de tais proteínas é desde o uso na agricultura à indústria. Elas podem ser utilizadas, por exemplo, em coquetéis na digestão de alimentos para animais, vinhos e para melhorar a eficiência de clarificação de sucos. Além disso, sua ação de deslignificação é promissora na degradação da biomassa para a produção de bicombustíveis. Este projeto procura realizar a clonagem, expressão heteróloga em bactérias e caracterização estrutural de uma α-L-arabinofuranosidase do fungo termofílico Thielavia terrestris. Esta enzima pertence à família 62 das hidrolases de glicosídeos, que é conhecida por melhorar a ação das celulases e, portanto, de interesse na degradação da biomassa lignocelulósica, como o bagaço de cana-de-açúcar. Estudos biofísicos, bioquímicos e estruturais apresentados neste estudo buscam promover a compreensão dos mecanismos de ação desta enzima e sua aplicação na hidrólise do bagaço de cana-de-açúcar. O trabalho destaca uma estrutura tridimensional inédita dentro da família 62 portadora de um fenômeno conhecido como domain swapping, possibilitando maiores investigações dentro do grupo das arabinofuranosidases. / Hydrolytic enzymes are considered, for the full definition, those with the ability to perform the hydrolysis reaction. Because of their ability of degrading natural substances, hydrolytic enzymes are of wide industrial use and can be applied in the textile, food, pharmaceutical, and agricultural industries, in addition to being used in the production of biofuels. Known as enzymes that release arabinose, the arabinofuranosidases from family 62 are able to break arabinoxylans connections. The biotechnological importance of these proteins are used in the agricultural and industry. These enzymes are employed to process as cocktails for animal feed digestion, wines and to improve the efficiency of juices clarification. Moreover, its delignification action is promising in the biomass degradation for biofuel production. This project seeks to carry out cloning, heterologous expression in bacteria and characterization of an α-L-arabinofuranosidase from thermophilic fungus Thielavia terrestris. This enzyme belonging to the glycoside hydrolase family 62, which is known to enhance the action of cellulases, and thus of interest in the lignocellulosic biomass degradation such as sugar cane bagasse. This study will characterize the protein structure and biochemistry in order to understand the mechanism and optimal conditions of action. The biophysical, biochemical and structural tests seek to promote an understanding of the mechanisms of action of this enzyme and its application in the hydrolysis of sugarcane bagasse. The work shows a new three- dimensional structure within the family 62 caused by a phenomenon known as domain swapping. Thus, it is possible to predict further investigations on the arabinofuranosidases group.

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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...