Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...

The non-ribosomal peptide synthase (NRPS) blue pigment synthase (BpsA) has been shown in several heterologous hosts to mediate the production of the blue pigment indigoidine from two molecules of L-glutamine. Activation of BpsA is mediated by transfer of a coenzyme A (CoA) by a 4′-phosphopantetheinyl transferase (4′-PPTase). In this thesis, I explored heterologous co-expression of BpsA and the Pseudomonas aeruginosa 4′-PPTase (PaPcpS) and their co- localization to either cytoplasm or chloroplast stroma of the green model microalga Chlamydomonas reinhardtii. The alga represents a potentially sustainable production host for indigoidine, as it is able to grow using CO2 as a sole carbon source and (sun)light for its energy. Both heterologous proteins (BpsA and PaPcpS) could be expressed as full-length fusion proteins with either the mVenus yellow fluorescent reporter or spectinomycin resistance (aadA) selection marker in both subcellular localisations. Dual transformants were identified and subjected to multiple growth conditions to determine whether indigoidine was produced. Under no condition tested was indigoidine detected, indicating that either activation of BpsA or the catalysis of L-glutamine to indigoidine was not occurring in alga. Future work will be required to determine whether it is possible to activate the BpsA in C. reinhardtii. However, this represents the first documented example of expression of a heterologous NRPS in a eukaryotic alga and may serve as foundational work for other target NRPS expression projects.

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

<p> Tissue plasminogen activator (tPA), is commonly used as thrombolytic agent for the treatment of various cardiovascular diseases. This thesis constitutes the first report on cloning and expression of tPA in the methylotrphic yeast Pichia pastoris. </p> <p> The tPA gene was first cloned into an E. coli/Pichia shuttle plasmid and then integrated into the Pichia genome. The recombinant Pichia was able to express tPA intracellularly, under methanol induction. The tPA produced by the Pichia had a similar molecular weight as native tPA and it had serine protease activity. At the shake flask scale, the recombinant Pichia strain was able to produce twice as much tPA as reported for E. coli. </p> <p> Elastin-like polypeptides (ELP) are proteins that have a peculiar characteristic: they are able to undergo a reversible inverse phase transition temperature within a very narrow temperature range. On a second aspect of heterologous protein, a construct composed of thioredoxin-intein-ELP was used to provide direct evidence, for the first time, that protein folding and activity, in this case the intein, was maintained when the tripartite fusion was present in the aggregated state. These results are important, since they provide the necessary degree of confidence to stimulate future work directed towards expression and maintenance of proper folding of aggregation-prone proteins when expressed in-vivo E. Coli as ELP directed inclusion bodies. It is also shown that the intein-ELP system may be a very interesting system to be used as a drug delivery vehicle. </p> / Thesis / Master of Applied Science (MASc)

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

The immune response to an infection is determined by a number of factors, which also affect the generation of memory T cells afterwards. The immune response can also affect the stability of the pre-existing memory populations. The memory developed after an infection can influence the response to subsequent infections with unrelated pathogens. This heterologous immunity may deviate the course of disease and alter the disease outcome. The generation and stability of memory CD8 T cells and the influence of the history of infections on subsequent heterologous infections are studied in this thesis using different viral infection sequences. Previous studies using mice lacking individual immunoproteasome catalytic subunits showed only modest alterations in the CD8 T cell response to lymphocytic choriomeningitis virus (LCMV). In this study, I found that the CD8 T cell response to LCMV was severely impaired in mice lacking all three catalytic subunits of the immunoproteasome, altering the immunodominance hierarchy of the CD8 T cell response and CD8 T cell memory. Adoptive transfer experiments suggested that both inefficient antigen presentation and altered T cell repertoire contribute to the reduction of the CD8 T cell response in the immunoproteasome knockout mice. Immune responses generated during infections can reduce pre-existing memory T cell populations. Memory CD8 T cells have been shown to be reduced by subsequent heterologous infections. In this study, I re-examined the phenomenon using immune mice infected with LCMV, murine cytomegalovirus (MCMV) and vaccinia virus (VACV) in different infection sequences. I confirmed that memory CD8 T cells were reduced by heterologous infections, and showed that LCMV-specific memory CD4 T cells were also reduced by heterologous infections. Reduction of the memory CD8 T cells is thought to be the result of apoptosis of memory CD8 T cells associated with the peak of type I interferon early during infection. I showed that memory CD4 T cells were similarly driven to apoptosis early during infection; however, Foxp3+ CD4+ regulatory T cells were relatively resistant to virus infection-induced apoptosis, and were stably maintained during LCMV infection. The stability of Treg cells during viral infections may explain the relatively low incidence of autoimmunity associated with infections. The history of infections can deviate the course of disease and affect the disease outcome, but this heterologous immunity is not necessarily reciprocal. Previous studies have shown the effects of heterologous immunity during acute infections. In this thesis, I showed that the history of LCMV infection led to higher viral titers during persistent MCMV infection, caused more severe immunopathology at the beginning of infection, and reduced the number of MCMV-specific inflationary memory CD8 T cells after the period of memory inflation. In a different context of infection, the history of LCMV infection can be beneficial. LCMV-immune mice have been shown to have lower viral titers after VACV infection, but VACV-immune mice are not protected during LCMV infection. I found that memory CD8 T cells generated from LCMV and VACV infections were phenotypically different, but the differences could not explain the nonreciprocity of heterologous immunoprotection. By increasing the number of crossreactive VACV A11R198-205-specific memory CD8 T cells, however, I showed that some VACV-immune mice displayed reduced viral titers upon LCMV challenge, suggesting that the low number of potentially cross-reactive CD8 T cells in VACV-immune mice may be part of the reasons for the non-reciprocity of immunoprotection between LCMV and VACV. Further analysis deduced that both number of potentially cross-reactive memory CD8 T cells and the private specificity of memory CD8 T cell repertoire played a part in determining the outcome of heterologous infections.

Cytochromes P450 are heme enzymes with very broad substrate specificity, they can oxidize tens to hundreds of different substrates including both eobiotics and xenobiotics, but with varying efficiencies and kinetics. They are responsible for the metabolic activation of many carcinogens resulting in formation of reactive intermediates, these reactive species participate in the formation of DNA adducts and also increase of oxidative stress. Eukaryotic cytochromes P450 are membrane bound enzymes found mostly in the endoplasmic reticulum. In vertebrates, they are expressed in a variety of tissues mostly in the liver, but also in kidney, lung, skin and others. The cytochrome P450 1B1 is an inducible enzyme which occurs mainly in the lung and skin. Its expression is induced predominantly by the presence of polycyclic aromatic hydrocarbons, dioxins and heterocyclic amines. The human cytochromes P450 are typically obtained using heterologous expression and isolated as a C-terminally modified hexa-histaq constructs using immobilized metal affinity chromatography. This thesis focuses on effect of C-terminal modifications on activity of human cytochrome P450 1B1. First, the two expression vectors encoding the human form of cytochrome P450 1B1 were prepared, one contained a classical C-terminal hexa-histaq...

Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis

Esse trabalho teve como objetivo a obtenção e caracterização de uma cepa transformante de Aspergilus nidulans para produção de uma lipase de Beauveria bassiana (denominada Bbl1) por meio do vetor pExpyr, utilizando xarope de milho como fonte de carbono indutora. Para isso utilizou-se o gene sob referência XP_008602131.1 (denominado bbl1) do NCBI que foi introduzido em vetores de pGEM-T, para multiplicação, e no pExpyr, para expressão. Foi feita a modelagem molecular dessa enzima, revelando cavidades hidrofóbicas para interação com substrato, e a \"tampa\", considerada característica comum das lipases \"verdadeiras\". A produção de Bbl1 em cultivos contendo maltose foi aproximadamente o dobro em relação à cepa selvagem B. bassiana. Posteriormente, com o intuito de melhorar a produção e baratear os custos de fermentação desta cepa, a maltose e o tampão HEPES (insumos caros e utilizados em escala laboratorial, porém inviáveis em padrão industrial) foram substituídos respectivamente por xarope de milho e tampão fosfato de sódio; o resultado foi a obtenção de quase 40 U.mL-1 de atividade de lipases em Erlenmeyer de 125 mL, quadruplicando a produção em relação a cepa selvagem. A Bbl1 foi purificada em colunas de Octyl-Sepharose e DEAE-celulose. Essa enzima demonstrou-se estável em solução com 0,5% de tween-20 e tween-80, e hiper-ativada na presença destes dois detergentes e de Triton X-100. Além disso, os ácidos oleico e linoleico demonstraram-se como inibidores análogos a não-competitivos. O estudo de imobilização revelou que o suporte de Octyl-Sepharose é o mais ideal para ativação de Bbl1, sendo que a atividade relativa da mesma foi cerca de 123%, em relação a mesma enzima em solução aquosa. Quanto à estabilidade térmica e ao pH, a imobilização promoveu um ganho de estabilidade nas temperaturas de 30°C até 60°C nos derivados de Octyl, Phenyl, Butyl, DEAE-celulose, MANAE e PEI, e na faixa de pH de 3 até 9 nesses mesmos suportes estudados, em comparação à enzima em solução. Por meio de estudos cinéticos, observou-se que a velocidade máxima de Bbl1 imobilizada em Octyl foi 2,39 vezes maior e o respectivo valor de K0,5, 2,7 vezes menor em relação à Bbl1 livre. O padrão de inibição pelos ácidos oleico e linoleico sofreu alteração em Bbl1 imobilizada, sendo dependente da concentração destes inibidores. Os produtos hidrólise dos óleos de açaí e buriti foram fracionados em fases polares e apolares e aplicados em culturas de monocamada de linhagens celulares de glioblastoma LN-18. Observou-se os produtos de hidrólise do óleo de buriti provocaram redução na viabilidade respiratório das células de glioblastoma até 120 horas de exposição, enquanto que nos cultivos de células de fibroblasto observou-se um \"ganho\" de viabilidade neste mesmo período de cultivo. Por fim, esse trabalho permitiu a gratificação em obter uma cepa de A. nidulans transformante para expressão de lipase - uma enzima comercialmente onerosa e com poucos trabalhos envolvendo sua produção de forma heteróloga. Além disso, tornou-se a utilização destas enzimas na hidrólise de óleos brasileiros como um potencial agente à obtenção de insumos para o tratamento de glioblastoma. / The objective of this work was to obtain and characterize a transforming strain of Aspergilus nidulans to produce a Beauveria bassiana lipase (called Bbl1) using the pExpyr vector, having corn syrup as inducing carbon source. For this purpose, the NCBI reference gene XP_008602131.1 (designated bbl1) was used, which was introduced into pGEM-T vectors for storage, and pExpyr for expression. The molecular modeling of this enzyme was performed, revealing hydrophobic cavities for the interaction with substrate, and the \"lid\", considered a common characteristic of \"true\" lipases. Production of Bbl1 in cultures containing maltose was approximately double that of B. bassiana wild strain. Later, in order to improve the production and to reduce the costs of fermentation of this strain, maltose and HEPES buffer (expensive and laboratory-grade inputs, however, not viable in an industrial standard) were replaced by corn syrup and sodium phosphate buffer; the result was the obtaining of almost 40 U.mL-1 of lipase activity in Erlenmeyer of 125 mL, quadrupling the production in relation to the wild strain. Bbl1 was purified on Octyl-Sepharose and DEAE-cellulose columns. This enzyme showed to be stable in solution with 0.5% tween-20 and tween-80, and hyperactivated in the presence of these two detergents and Triton X-100. In addition, oleic and linoleic acids have shown to be non-competitive analogues. The immobilization study revealed that Octyl-Sepharose support is the most ideal for the activation of Bbl1, and the relative enzymatic activity was about 123% in relation to the same non-immobilized enzyme. As for thermal stability and pH, immobilization promoted a stability gain at temperatures of 30 ° C to 60 ° C in the Octyl, Phenyl, Butyl, DEAEcellulose, MANAE and PEI derivatives, and in the pH range of 3 up to 9 in these same supports, compared to the enzyme in solution. By means of kinetic studies, it was observed that the maximum rate of Bbl1 immobilized on Octyl was 2.39 higher and the respective value of K0.5, 2.7 lower than the free Bbl1. The inhibition pattern for oleic and linoleic acids was altered in immobilized Bbl1, being dependent on the concentration of these inhibitors. The hydrolysis products of açai and buriti oils were fractionated in polar and nonpolar phases and applied to monolayer cultures of LN-18 glioblastoma cell lines. It was observed that the buriti oil hydrolysis products were able to cause a reduction in the respiratory viability of glioblastoma cells up to 120 hours of exposure, whereas in the fibroblast cell cultures a viability \"gain\" was observed in the same culture period. Finally, this work allowed the gratification in obtaining a strain of transforming A. nidulans for lipase expression - a commercially expensive enzyme with few studies involving its production in a heterologous way. In addition, the use of these enzymes in the hydrolysis of Brazilian oils has become a potential agent to obtain inputs for the treatment of glioblastoma.

Celulases têm atraído muito interesse nos últimos anos devido a sua habilidade na bioconversão de material lignocelulolítico em glucose, a qual pode, então, ser convertida a etanol por fermentação. O complexo celulolítico capaz de degradar a celulose consiste de várias enzimas (principalmente celulases e &beta;-glucosidases) e proteínas auxiliadoras, que atuam em sinergismo para eficientemente hidrolizar a biomassa. Nesse estudo, investigou-se a hidrólise enzimática do bagaço de cana-de-açúcar pré-tratado utilizando enzimas produzidas por T. harzianum e enzima comerial. O rendimento de hidrólise foi avaliado quanto a diferentes níveis de deslignificação de biomassa, graus de cristalinidade da celulose, composição dos coquetéis enzimáticos e adição de BSA. Estudos de difração de raios-X mostraram que a cristalinidade da lignocelulose não é um fator determinante na recalcitrância ao ataque enzimático. Além disso, a adição de BSA não teve qualquer efeito no rendimento da hidrólise. O mais eficiente coquetel enzimático foi obtido misturando o preparado comercial com o produzido pelo T. harzianum (rendimento acima de 97%). Esse desempenho está, provavelmente, relacionado com níveis adequados de &beta;-glucosidases e xilanases no coquetel. Devido a essa eficiente atividade celulolítica, o fungo T. harzianum tem um grande potencial em aplicação para hidrólise de biomassa. A celobiohidrolase I, uma exoglucanase, é a principal enzima secretada por esse fungo (cerca de 60% do total) e nesse estudo ela foi expressa em bioreator, purificada por cromatografia de troca iônica seguida de gel filtração e caracterizada bioquímica, biofísica e estruturalmente. Conforme confirmado por SAXS, tanto a CBHI inteira quanto seu domínio catalítico, obtido por digestão parcial com papaína, são monoméricos em solução e apresentam distância máxima (DMax) de 110 e 60 &Aring;, e raio de giro (Rg) de 20 e 27 &Aring;, respectivamente. Os resultados indicam que o linker é flexível em solução e confirmam o formato de girino da enzima. A CBHI possui atividade máxima em pH 5.0 e temperatura de 50 &deg;C, com atividade específica contra Avicel &reg e pNPC de 0,28 and 1,53 U/mg, respectivamente. Outras celulases de interesse foram também expressas para caracterização, no entanto, para essas, foi utilizado o sistema de expressão heteróloga em Aspergillus Níger ou Pichia pastoris. O domínio catalítico da endoglucanase I de T. harzianum foi expresso em A. Níger. A proteína tem atividade específica contra CMC de 15,8 U/mg e pH e temperatura ótima de 3 e 50 &deg;C, respectivamente. A proteína é estável nessas condições em até 3 dias de incubação (dados de ensaios de atividade residual). Estudos biofísicos de deslocamento térmico e dicroísmo circular apresentaram alguns parâmetros de estabilidade de estrutura terciária e secundária, respectivamente. A proteína perde estrutura terciária regular, em pH 5, em torno de 30 &deg;C mas sua estrutura secundária é desordenada somente em pH 9 (quando a 25 &deg;C). Experimentos de dicroísmo circular também indicaram a composição de estrutura secundária do domínio catalítico da EGLI de 6% de &alpha;-hélice e 42% de folhas- &beta;. / Cellulases have attracted an outstanding interest in the recent years because of its ability in the bioconversion of cellulose-containing raw materials into glucose, which can then be converted into ethanol by fermentation. The cellulase complex able to degrade cellulose consists of several enzymes (mainly cellulases and &beta;-glucosidases) and auxiliary proteins, which act in synergism to efficiently solubilize the biomass. In this study, we investigated the enzymatic hydrolysis of pretreated sugarcane bagasse using crude enzyme extracts produced by Trichoderma harzianum as well as from the extract in combination with a commercial cocktail. The influence of different levels of biomass delignification, degree of crystallinity of lignicellulose, composition of enzymatic activities and BSA on enzymatic hydrolysis yields was evaluated. Our X-ray diffraction studies showed that crystallinity of lignocellulose is not a key determinant of its recalcitrance toward enzymatic hydrolysis. In fact, under the experimental conditions, an increase in crystallinity of lignocellulosic samples resulted in increased glucose release by enzymatic hydrolysis. Furthermore, under the same conditions, the addition of BSA had no significant effect on enzymatic hydrolysis. The most efficient enzyme blends were obtained by mixing a commercial enzymatic cocktail with T. harzianum cellulase preparations (above 97%). Increased hydrolytic efficiencies appeared to correlate with having an adequate level of both &beta;-glucosidase and xylanase activities in the blends. Due to its elevated cellulolytic activity, the filamentous fungus T. harzianum has a considerable potential in biomass hydrolysis applications. The cellobiohydrolase I, an exoglucanase, is the main enzyme secreted by this fungus (about 60% of total) and in this study we have expressed, purified and performed an initial biochemical, biophysical and structural characterization. As confirmed by small angle X-ray scattering (SAXS) both full-length CBHI and its catalytic core domain (CCD), obtained by partial digestion with papain, are monomeric in solution and they have Dmax of 110 and 60 &Aring;, and Rg of 20 and 27 &Aring;, respectively. The results indicate that the linker is flexible in solution and confirmed the tadpole shape of the enzyme. CBHI displays maximum activity at pH 5.0 and temperature of 50 &deg;C, with specific activities against Avicel &reg and pNPC of 0,28 and 1.53 U/mg, respectively. Other celulases were also expressed, however, for them we have used the heterologous expression system in Aspergillus niger and Pichia pastoris. The catalytic domain of endoglucanase I from T. harzianum was expressed in A. niger and partially characterized. The protein has specific activity against CMC of 15.8 U/mg and optimum pH and temperature of 3 and 50 &deg;C, respectively. The protein is stable in these conditions until 3 days of incubation. Biophysical studies of termal shift and circular dichroism (CD) assays have showed some parameters of stability of tertiary and secondary structure of the protein. It loses regulary terciary structure in pH 5 around 30 &deg;C but the secondary structure is desordened only in pH 9 at 25 &deg;C. CD experiments also indicated the secondary structure compsition of the catalytic domain of EGLI: 6% de &alpha;-helices and 42% de &beta;-sheet.