Glicose e oxigênio são moléculas essenciais para a maioria dos organismos vivos. Além de sua importância nos processos de produção de energia - glicose como fonte de carbono e energia e oxigênio como aceptor dos elétrons doados por NADH e FADH2 - estes dois compostos funcionam como efetuadores, modulando vários processos metabólicos e fisiológicos nas células. Visto que a mitocôndria é um dos alvos afetados pelas disponibilidades destas duas moléculas, nós isolamos e seqüenciamos o genoma mitocondrial de Trichoderma reesei, um fungo multicelular empregado neste trabalho como sistema modelo. Foi estudado o efeito da variação de concentração de glicose e oxigênio sobre a expressão de transcritos do genoma mitocondrial, bem como sua implicação no metabolismo de glicose. São apresentadas análises da expressão gênica de aproximadamente 2000 transcritos de T. reesei submetido a concentrações limitantes de oxigênio dissolvido, realizadas com o emprego da técnica de microarrays de cDNA. Pelo menos 330 transcritos foram diferencialmente expressos em função da disponibilidade de oxigênio. Aqueles envolvidos nos processos de síntese protéica e divisão celular foram regulados negativamente, enquanto transcritos relacionados com funções de defesa celular e síntese de RNA foram positivamente regulados. Uma fração substancial de outros genes afetados pela baixa disponibilidade de oxigênio não possui, atualmente, funções celulares conhecidas. Esta observação deve contribuir para a posterior anotação funcional do genoma de T. reesei. Também foram identificados reguladores transcricionais diferencialmente expressos em baixas tensões de oxigênio. O perfil de expressão destes reguladores aponta-os como potenciais candidatos ao envolvimento com a expressão de genes afetados pela disponibilidade de oxigênio. / Glucose and oxygen are essential molecules in most of living organisms. In addition to their importance in production of energy - glucose as a carbon and energy source and oxygen as an acceptor of electrons donated by NADH and FADH2 - both molecules function as effectors modulating various metabolic and physiological processes in the cell. Because one of the targets affected by both molecules is the mitochondrion, we isolated and sequenced the mitochondrial genome of Trichoderma reesei, a multicellular fungus that is used in this study as a model system. The effect of varying the concentration of glucose and oxygen on the expression of the transcripts of the mitochondrial genome, and its implication on the metabolism of glucose, was studied. Gene-wide expression analyses of nearly 2000 transcripts of T. reesei under limited concentration of dissolved oxygen, using cDNA microarry technique, are presented. At least 330 transcripts were differentially expressed with respect to oxygen availability. Those involved in protein synthesis and cell division processes were downregulated, while transcripts involved in cell defense and RNA synthesis were upregulated. A substantive fraction of other anaerobically affected genes have currently unknown cellular roles, and these results should therefore contribute to further functional annotation of the genome. ln addition, we have identified transcriptional regulators that are differentially expressed at a low oxygen tensions. The expression profile of these regulators points them out as potential candidates involved in the expression of genes affected by oxygen availability.

Não consta resumo na publicação. / The gene encoding actin in the cellulolytic filamentus fungus Trichoderma reesei has been isolated and sequenced. The nucleotide sequence reveals that the gene is composed of 6 exons separated by 5 introns within the coding region. The positions of the introns were predicted by comparison of sequence homology to the genes coding for actin with known amino acid sequence and by identification of splice-site signal sequences. The actin protein of Trichoderma reesei shows extensive homology to the actins of other fungi E. nidulans, 95% , T. lanuginosus, 92% and S. pombae. The T. reesei actin promoter has a CT-rich region, CAAT and GC. There is no obvious TATA sequence in the T. reesei actin promoter. The absence of TATA-like sequence were also observed in anothers genes of T. reesei. An important aspect in molecular biology of filamentous fungi is the analysis, under a specific metabolic events, of the mechanism(s) regulating the expression of constitutive and induced genes. The filamentous fungus Trichoderma reesei is considered to be one of the most efficient producer of cellulase, and it serves as a model system for enzymatic cellulose hydrolysis. Expression of the cellulase genes are stringently regulated by the carbon source. Growth on cellulose results in induction of the cellulase transcripts, whereas glucose strongly represses their expression. The availability of a constitutive expressed genes of T. reesei provides not only important information regarding the molecular biology of the fungi, but also is essential for a better understanding of the mechanism(s) controlling the expression of the cellulase transcripts. Under inductive process of the of the major cellulase transcript (cbh1) and its repression by glucose, actin mRNA is constitutively expressed. The present results should be useful for further structural and functional analysis of the elements involved in inductive and constitutive expression of cellulase and actin transcripts.

Superóxido dismutases (SODs) são metaloenzimas que convertem o ânion superóxido em oxigênio molecular (O2) e peróxido de hidrogênio (H2O2). A presença de metal nessas enzimas está diretamente relacionada com seus mecanismos de catálise e com suas estruturas tridimensionais. Evolucionariamente, FeSOD e MnSOD podem ter evoluído de um gene ancestral comum, porque possuem sequências homólogas e estruturas cristalográficas sobreponíveis. Entretanto, a nível catalítico, ambas as proteínas divergiram o suficiente para que seus metais não possam ser intercambiáveis, produzindo uma enzima funcional, indicando que essas proteínas possuem alta especificidade por metal. O objetivo deste projeto de pesquisa é Identificar os determinantes estruturais do ajuste fino da especificidade por metal de MnSOD e FeSOD. Inicialmente, pretendese selecionar resíduos para mutagênese sítio-dirigida em TrMnSOD e TbFeSODB2, a partir de análise de acoplamento estatístico (SCA). Em seguida, mutantes serão construídos, expressos, purificados e cristalizados. A estrutura tridimensional dos mutantes será resolvida por cristalografia e sua atividade enzimática determinada, bem como a acomodação estrutural dos metais por Resonância Paramagnética Eletrônica. Nossa hipótese de trabalho é que através de SCA é possível elencar resíduos de aminoácidos candidatos para mutagênese sítio-dirigida para desenhar novas SODs, com características intermediárias de ligação por Fe/Mn, como possibilidade de interconversão de especificidade, caminhando na história evolutiva dessas moléculas. / Superoxide dismutases (SODs) are metalloenzymes that convert the superoxide anion in molecular oxygen (O2) and hydrogen peroxide (H2O2). The metal in the catalytic center of such enzymes is directly related to their catalysis mechanisms and tridimensional structures. Evolutionarily, FeSOD and MnSOD may have evolved from a common ancestor, because both proteins have homologous primary sequences and superposable crystallographic structures. However, at the catalytic level, both proteins diverged sufficiently to prevent interchange of their metallic centers, which would generate non-functional enzymes, indicating that these proteins have high metal specificity. The objective of this research project is to identify structural determinants of Fe/MnSODs necessary to metal specificity. We intend to use statistical coupling analysis (SCA) to select amino acid residues for site-directed mutagenesis in TrMnSOD e TbFeSODB2. Mutant genes will be constructed and their proteins expressed, purified and crystallized. The tridimensional structure of such mutants will be solved by X-ray crystallography and their enzymatic activities determined, as well as their electron paramagnetic resonance spectra. We hypothesize that SCA is useful to identify amino acid candidates for site-directed mutagenesis to design new SODs with intermediated Fe/Mn specificity, and even metal specificity interconversion, by studying the evolutionary history of these proteins.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Complete degradation of cellulose produces mainly glucose, which can be fermented to ethanol. Therefore cellulose presents an abundant renewable energy resource for the production of an alternative, environmentally friendly, transportation fuel. Enzymatic degradation of cellulose is achieved by the synergistic action of three cellulase enzyme groups: endoglucanases, exoglucanases and -glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol. Therefore, a need has arisen to develop a recombinant microorganism with the ability to produce cellulolytic enzymes, hydrolyze cellulose and ferment the resulting sugars to ethanol in a single process step, referred to as “Consolidated Bioprocessing” (CBP). This would provide a cost-effective, economically feasible strategy for the production of bioethanol. The naturally fermentative yeast, Saccharomyces cerevisiae, is often used as host for the expression of recombinant proteins due to several characteristics, including its robustness in industrial processes, the well developed genetic tools available for manipulation and its proven safety status. A number of cellulase genes have previously been successfully expressed by recombinant S. cerevisiae strains. In this study, all three components of the cellulase system were co-expressed in S. cerevisiae to test the ability of the yeast to effectively produce the heterologous proteins, and consequently produce enough glucose for growth on an amorphous cellulosic substrate. The Trichoderma reesei endoglucanase gene egII (Cel5A) was successfully expressed by a S. cerevisiae Y294 strain. Recombinant EGII displayed activities of 19.6 nkat.ml-1 and 22.3 nkat.ml-1 towards CMC and barley -glucan, respectively. The major endoglucanase gene, egI (Cel7B) from T. reesei was subjected to random mutagenesis by propagating the egI-containing plasmid in an E. coli mismatch repair deficient strain. Screening of S. cerevisiae transformants revealed a strain, S. cerevisiae Y294[pLEM1], with improved levels of endoglucanase activity (21.8 nkat.ml-1), compared to S. cerevisiae Y294[pAZ40], expressing the wild type gene (10.3 nkat.ml-1). Through subcloning of the mutated ENO1 promoter region and the mutated egI gene fragment, it was established that the mutations located in both the promoter- and gene sequences were responsible for the improved levels of activity displayed by S. cerevisiae Y294[pLEM1]. The egII gene and the altered egI gene were co-expressed with a codon optimised T. reesei cellobiohydrolase (sCBHI) and a -glucosidase from Saccharomycopsis fibuligera. This resulted in a reduction in endoglucanase levels, possibly due to the metabolic burden placed on the yeast by co-expressing the different cellulases. The hydrolysis products produced by cellulase co-expressing strains were cellotriose, cellobiose and glucose, although the glucose yield was insufficient to enable growth on cellulose as sole carbon source. As the major hydrolysis product was cellobiose, it is likely that a bottleneck exists at its conversion to glucose, suggesting inadequate -glucosidase activity. This study has provided insight into co-expression of cellulase enzymes by the yeast S. cerevisiae. The knowledge obtained could be applied in optimizing cellulase cocktails for efficient cellulose degradation and eventual production of ethanol by recombinant yeast. It has also demonstrated the applicability of random mutagenesis for improving the activity of cellulases.

This project aimed investigated cellulase in-situ production for large-scale on-farm production of lignocellulosic biofuel. Cellulase activity and glucose released by T. reesei with corn stover and wheat bran as co-substrates for solid state cultivation (SSC) were examined. Co-cultivation has previously increased T. reesei cellulase, but corn stover and wheat bran have not been co-cultivated (Dhillon, Oberoi et al. 2011). This work compared cellulase activity and glucose concentration of corn stover co-cultivated with 0-40% wheat bran in high solids. Samples with at least 20% wheat bran exhibited increased cellulase activity. However, the average glucose concentration without wheat bran was 3.29 g/L compared to 16.7 g/L with wheat bran. Glucose released by T. reesei on pretreated corn stover with 0-40% wheat bran was compared at the optimal temperatures for fungal growth and for cellulase activity after SSC. Previous research has rarely used cellulase from SSC to hydrolyze lignocellulose. Following SSC of T. reesei at 30°C for seven days, samples were warmed to 50°C for five days. Glucose concentration increased to 12.1 and 32.7 g/L for samples with and without wheat bran. This strategy could reduce lignocellulosic fuel production costs by eliminating need for commercial cellulase and is promising for efficient cellulose hydrolysis.

Extensive research into enzyme-induced bio-conversion of lignocellulose to soluble sugars has been conducted and research continues in this area. Several approaches have been taken to attempt to alleviate the economic problems associated with utilisation of lignocellulose in fuel ethanol production. By expressing cellulase genes in planta, it is hoped that the cost of enzyme-mediated hydrolysis of cellulose to its soluble sugar monomers, will be reduced. Some accomplishments have been made in this area using nuclear genetic transformation (Abdeev et al., 2003; Abdeev et al., 2004; Austin-Phillips et al., 1999; Biswas et al., 2006; Dai et al., 2000a,b; Dai et al., 2005; Jin et al., 2003; Kawazu et al., 1999; Sakka et al., 2000; Ziegelhoffer et al., 1999; Ziegelhoffer et al., 2001; Ziegler et al., 2000), but more research is required to bring the levels of cellulase enzyme expression in plants to levels that will make the process economically competitive. Chloroplasts of N. tabacum were selected as a target for transformation for high level expression due to their extremely high rates of transcription and translation. These were transformed with two genes, the e1 gene from A. cellulolyticus, and the cbh1 gene from T. reesei. Further aims included the investigation of the effects of using different promoters, and the novel use of both nuclear and chloroplast-based expression in a single plant, on the level of protein production in the heterologous host. Heterologous expression of the cbh1 gene was not successful. This is thought to be due to toxicity of the protein in a prokaryotic environment. Future studies should focus on trying to avoid this toxicity by targeting of the chloroplast-expressed enzyme to specific tissues, such as the thylakoid membrane, for containment, creating a codon-optimised synthetic gene that better mimics the codon usage of the plant to be used for expression, or placing the expression under a reactive cascade that is only activated upon exposure to an external trigger. Heterologous expression of the full length gene for E1 from A. cellulolyticus was successful. Chloroplast homology vectors under the constitutive promoter Prrn, and the inducible promoter T7, were constructed and these were used to successfully transform N. tabacum cv. Petit Havana chloroplasts. Stable transgenic plants were produced and evaluated by a variety of means, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 3122 ± 466 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 0.35% ± 0.06 of the total soluble protein. Lastly, chloroplast transformation was combined with nuclear transformation to create novel dual-transgenic plants simultaneously expressing E1 from both the nuclear and chloroplast genomes. The combination of these technologies was very successful, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 35706 ± 955 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 4.78% ± 0.13 of the total soluble protein, and provides a new approach for increasing the accumulation levels of plant-produced cellulase enzymes.

In recent years, fungal and bacterial cellulases have gained popularity for the conversion of lignocellulosic material to biofuels and biochemicals. This study investigated properties of fungal (Trichoderma. reesei) and bacterial (Clostridium thermocellum) cellulases. Enzymatic hydrolysis was carried out with T. reesei using nine enzyme concentration and substrate combinations. Initial rates and extents of hydrolysis were determined from the progress curve of each combination. Inhibition occurred at the higher enzyme concentrations and higher solids concentrations. Mechanisms to explain the observed inhibition are discussed. Samples of C. thermocellum purified free cellulase after 98% hydrolysis were assayed to determine the total protein content (0.15 ± 0.08 mg/mL), the enzymatic activity (0.306 ± 0.173 IU/mL) and the cellulosome mass using the Peterson method for protein determination, the cellulase activity assay with phenol-sulfuric acid assay, and the indirect ELISA adapted for C. thermocellum cellulosomes, respectively. Issues regarding reproducibility and validity of these assays are discussed.

Made available in DSpace on 2014-06-11T19:35:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:07Z : No. of bitstreams: 1 adalberto_pr_dr_araiq.pdf: 786247 bytes, checksum: cf55691544529062c4d2288dd8709c7a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA. / The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA.

Resumo: A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA. / Abstract: The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA. / Orientador: Antonio Carlos Massabni. / Coorientador: Rubens Monti. / Doutor

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-09-19T13:51:14Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-09-19T13:51:26Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2018-09-19T13:51:26Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2014-05-06 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cellulolytic enzymes are highly specific biocatalysts. These are classified according to the place where they work in the cellulosic fiber: endoglucanases initiate hydrolysis exoglucanases act on reducing terminals produced by endoglucanases and β-glucosidases act on the product of catalysis of exoglucanases releasing glucose monomers . Penicillium fungi are widely reported as a great producer of cellulolytic enzymes. Aiming to contribute to the catalytic process and obtaining new sources of enzymes , this study evaluated the physicochemical properties of enzymes produced cellulolytic fungus Penicillium citrinum isolated from bagasse cane sugar mill Jayoro of S / A, located in the municipality of Presidente Figueiredo -AM - Brazil . And the enzyme production profile was repaired during submerged fermentation with the strain of Trichoderma reesei QM9414 which is widely reported as a great producer of cellulolytic enzymes. P. citrinum showed equivalent performance to T. reesei QM9414 in volumetric enzyme activity (U/mL) and superior performance in specific activities (U/mg). About properties of endoglucanases and β-glucosidase from P. citrinum, the optimum temperature for activity was 60 ° C for both enzymes. The optimum pH for endoglucanases activity was pH 4.2 and the activity of β-glucosidase pH 6.0. P. citrinum produced at least two endoglucananes with apparent molecular masses of ~ 50kDa and ~ 35kDa , visualized on SDS-PAGE gels and in gel activity ( zymogram ) . An elution fraction obtained from size exclusion chromatography, showed only β-glucosidase activity and revealed bands of approximately 100 kDa . The performance and characteristics of these endo and β- glucosidases demonstrate that P. citrinum represents a new alternative for production of cellulolytic enzymes for hydrolysis of fiber. / As enzimas celuloliticas são biocatalizadores altamente específicos. Estas, são classificadas de acordo com o local em que atuam na fibra celulósica: endoglucanases iniciam a hidrólise, exoglicanases agem nos terminais redutores produzidos pelas endoglicanases e as beta-glicosidases atuam no produto da catálise das exoglicanases liberando monômeros de glicose. Fungos do gênero Penicillium são amplamente reportados como ótimos produtores de enzimas celuloliticas. Visando contribuir para os processos biocataliticos e obtenção de novas fontes de enzimas, neste trabalho avaliou-se as propriedades físico-químicas de enzimas celuloliticas produzidas fungo Penicillium citrinum isolado do bagaço de cana-de-açucar da Usina Jayoro S/A, localizada no município de Presidente Figueiredo-AM-Brasil. E comparou-se o perfil de produção enzimática durante a fermentação submersa com o da estirpe Trichoderma reesei QM9414 que é amplamente reportado como um otimo produtor de enzimas celulolíticas. P. citrinum apresentou desempenho equivalente à T. reesei QM9414 nas atividades enzimáticas volumétricas (U/mL) e desempenho superior nas atividades especificas (U/mg). Quanto as propriedades das endoglicanases e beta-glicosidase de P. citrinum, a temperatura ótima de atividade foi 60ºC para ambas as enzimas. O pH ótimo para a atividade endoglicanásica foi pH 4,2 e para a atividade de beta-glicosidase pH 6,0. P. citrinum produziu pelo menos duas endoglicananes com massas moleculares aparente de ~ 50kDa e ~ 35kDa, visualizadas em gel de SDS-PAGE e em gel de atividade (zimograma). Uma fração obtida da eluição de cromatografia de exclusão molecular, apresentou somente atividade de beta-glicosidase e revelou bandas de aproximadamente 100kDa. O desempenho e as características dessas endo e beta-glicosidades demonstram que P. citrinum representa uma alternativa para produção de novas enzimas para hidrolise da fibra celulolitica.