Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2006. / Submitted by Diogo Trindade Fóis (diogo_fois@hotmail.com) on 2009-11-06T19:28:31Z No. of bitstreams: 1 dissertação total01.pdf: 714792 bytes, checksum: fdfd3d22be39f9a1cbc64666ef7e68ae (MD5) / Approved for entry into archive by Gomes Neide(nagomes2005@gmail.com) on 2010-01-15T18:14:51Z (GMT) No. of bitstreams: 1 dissertação total01.pdf: 714792 bytes, checksum: fdfd3d22be39f9a1cbc64666ef7e68ae (MD5) / Made available in DSpace on 2010-01-15T18:14:51Z (GMT). No. of bitstreams: 1 dissertação total01.pdf: 714792 bytes, checksum: fdfd3d22be39f9a1cbc64666ef7e68ae (MD5) Previous issue date: 2006 / O diclofenaco sódico apresenta efeitos deletérios sobre a cicatrização e em vários estudos foi demonstrada a eficácia do Fator XIII da coagulação sanguínea sobre a cicatrização nas mais variadas situações no pós-operatório. Este estudo foi idealizado no intuito de avaliar se o Fator XIII da coagulação sanguínea poderia bloquear estes efeitos deletérios provocados pelo diclofenaco sódico. Este ensaio experimental randomizado duplo cego foi realizado com a utilização de 96 ratos da linhagem Winstar. Os animais foram distribuídos em quatro grupos de 24 animais de acordo com a utilização da drogas em estudo: DS (diclofenaco sódico), DSF (diclofenaco e Fator XIII da coagulação), SF (solução de NaCl a 0,9%) e F (Fator XIII da coagulação). Estes grupos foram realocados em três novos grupos cada de acordo com o dia de eutanásia (terceiro, sétimo e décimo - quarto) ficando cada grupo então com oito animais. Foi realizada incisão de 04 cm no dorso à esquerda com posterior sutura em pontos separados. A cicatrização foi avaliada quanto aos aspectos microscópicos do processo inflamatório cicatricial assim como a variação da resistência tênsil da ferida operatória no terceiro, sétimo e décimo - quarto dia de pós-operatório. Os resultados foram submetidos à análise estatística, tendo sido utilizado os testes Mann-Whitney Rank Sum Test e o t-test. Foi considerado estatisticamente significante p < 0,05. O Fator XIII da coagulação sanguínea diminuiu o edema no pós-operatório no 3º. dpo em relação ao grupo controle (p=0,015). O grupo do Fator XIII da coagulação sanguínea apresentou no 3º. dpo aumento da resistência tênsil em relação ao grupo do diclofenaco sódico (p=0,024). O grupo do diclofenaco sódico apresentou diferença estatisticamente significante com maior presença de leucócitos polimorfonucleares no sétimo dia de pós-operatório (p=0,04; p=0,01; p=0,04), maior presença de células mononucleares (p=0,021; p=0,021; p=0,021), maior neoformação vascular (p=0,038; p=<0,01; p=0,021) e maior quantidade de tecido de granulação (p=0,03; p=0,001; p=0,03) no décimo quarto dia de pós-operatório em relação aos demais grupos avaliados. O Fator XIII da coagulação sanguínea foi eficaz em bloquear parte dos efeitos deletérios na cicatrização provocados pelo diclofenaco sódico. ____________________________________________________________________________________ ABSTRACT / Diclofenac presents deleterious effects on the healing process and many studies showed effectiveness of Factor XIII in the healing of different kinds of post-operatory situations. The aim of this study was to detect if Factor XIII could block this pernicious effects in the healing process promoted by diclofenac. 96 rats was randomized in four groups: group DS (diclofenac), group DSF (diclofenac + Factor XIII), group SF (NaCl 0,9%) and group F (Factor XIII). Each group was divided again in another three groups of eight animals. Four groups of each initial group were killed in the third, seventh and fourteenth postoperatory day. The microscopic inflammatory healing process and the breaking strength of wound was examined in the third, seventh and fourteenth postoperatory day. The Mann-Whitney Rank Sum Test and the T-test was utilized in the statistical analysis. It was considered statistical significant p < 0,05. In third post-operatory day the Factor XIII decreased edema in relation to the control group (p=0,015) and increased the breaking strength in relation to the diclofenac group (p=0,024). In the seventh post-operatory day diclofenac increased the number of polymorfonuclear cells in relation to the other groups: DSF (p=0,04), SF (p=0,01) and F (p=0,04). In the fourteenth post-operatory day diclofenac increased the number of mononuclear cells in relation to the other groups: DSF (p=0,021), SF (p=0,021) and F (p=0,021). Also diclofenac increased vascular neofomation in relation to the other groups respectively (p=0,038; p=<0,01 and p<0,021) and granulation tissue (p=0,03; p=0,001 and p=0,03). The coagulation Factor XIII was efficient in prevent part of deleterious diclofenac effects in the normal healing process.

A Transglutaminase (TGase; proteína-glutamina γ-glutamil-transferase; EC 2.3.2.13) é uma enzima que catalisa reações de acil-transferência introduzindo ligações cruzadas entre cadeias protéicas. Em função de suas características, as TGases microbianas têm ampla e crescente aplicação na indústria alimentícia e em outras áreas. Portanto, o objetivo desse trabalho foi aumentar o conhecimento existente sobre a produção e as aplicações dessas enzimas. A composição do meio de cultura para a produção da enzima em cultivo submerso (CSm) pelo Bacillus circulans BL32, um isolado Amazônico, foi otimizada através de uma estratégia em três etapas. A otimização do meio resultou numa atividade de TGase que é 60 % maior que a máxima obtida utilizando um meio de cultura previamente citado na literatura para a produção dessa enzima, além da redução dos custos dos constituintes do mesmo. Metodologias de planejamento experimental foram utilizadas para otimizar a temperatura de incubação e o pH do meio de cultura. As melhores condições de cultivo para a produção da TGase pelo B. circulans BL32 para a produção da enzima em CSm foram 30 °C e pH 8.5, sendo que a máxima produção foi obtida no final da fase estacionária de multiplicação. Os efeitos da agitação e aeração sobre a produção de TGase e esporulação do B. circulans BL32 em sistema de CSm também foram estudados. Os resultados demonstraram que as condições ótimas de processo para a formação de biomassa e esporulação são diferentes. Portanto, foi adotada uma estratégia de controle da taxa de aeração em dois estágios, com formação de biomassa, no primeiro estágio, nas condições ótimas de crescimento seguido por um segundo estágio sob as condições de esporulação. Também estudou-se a produção de TGase pelo B. circulans BL32 em cultivo no estado sólido (CES). Vários resíduos agroindustriais foram usados como substrato para crescimento do microrganismo e produção da enzima. As melhores condições de cultivo foram 0,6 L/min de ar, 33 °C e 10 log 10 UFC/g de substrato para a concentração celular do inóculo, em resíduo fibroso de soja como substrato. A determinação das condições de extração para a efetiva recuperação da TGase produzida em CES foi realizada através do emprego de ferramentas de planejamento experimental. A melhor condição de extração da enzima foi quando utilizou-se água a 7 ºC como solvente, por 5 minutos, 250 rpm e uma relação de sólidos/líquidos de 1:6. Caseína, proteína isolada de soja e proteína hidrolisada de carne foram tratadas com essa TGase microbiana. A redução do número de grupos aminos livres após o tratamento com a enzima, principalmente, na caseína, demonstrou a formação de ligações cruzadas catalisadas por essa TGase. As propriedades emulsificantes dessas proteínas foram melhoradas após o tratamento com a TGase do B. circulans BL32. Além disso, a fim de investigar o mecanismo de inativação térmica, incubou-se a enzima por diferentes períodos de tempo em temperaturas entre 30 e 70 °C. As cinéticas de termoinativação desta TGase seguiram o modelo de Lumry-Eyring e a enzima mostrou-se estável até 50 °C, sendo que após 12 h nessa temperatura a mesma ainda mantém 50 % da sua atividade enzimática. Os resultados sugerem que esta TGase microbiana apresenta um grande potencial de uso em aplicações alimentícias e não alimentícias. / Transglutaminase (TGase; protein-glutamine γ-glutamyltransferase; EC 2.3.2.13) is an enzyme capable of catalyzing acyl transfer reactions by introducing covalent cross-links between proteins. Microbial TGases have found widespread and growing applications in the food and non-food industry. In this work, an effort has been made in order to increase available knowledge about microbial TGases. Medium composition for TGase production on submerged cultivations by Bacillus circulans BL32, a recently isolated strain from the Amazon basin, was optimized using a stepwise strategy. The optimization of the medium resulted not only in a 60 % higher TGase activity than the obtained in a media previously cited in the literature but also in a reduction of constituents costs. Statistical experimental methods also were used to optimize the temperature and pH parameters. The best culture conditions for TGase production by B. circulans BL32 were 30 °C, pH 8.5 and the highest production was obtained in late-stationary culture phase. And besides, the effects of agitation and aeration on TGase production and cell sporulation on submerged cultivations were studied. The results demonstrated that the optimal process conditions are different for biomass and spore production. It was adopted a two-stage aeration rate control strategy with biomass production under the growth conditions in the first stage followed by the second stage under the conditions for sporulation. The present work also dealt with the TGase production in solid state cultivations. Several agro-industrial residues were used as substrates for microbial growth and enzyme production. The best culture conditions were determined as being 0.6 L air min-1, 33 °C and 10 log 10 CFU g-1 of dried substrate to the inoculum cell concentration, on industrial fibrous soy residue as substrate. The optimization of downstream processing parameters for the effective enzyme recovery of the cultivated solids was carried out. The optimal conditions for the extraction were: water as solvent at 7 ºC; 5 min of extraction time; agitation speed of 250 rpm; and 1:6 solid:liquid ratio. Casein, soy protein isolated, and hydrolysed animal protein were treated with this microbial TGase. The decrease in the amount of free amino groups after TGase treatment, mainly in the casein, demonstrated the cross-linking catalyzed by this enzyme. The emulsifying properties of these proteins were improved after treatment with B. circulans BL32 TGase. Furthermore, in order to investigate the mechanism of thermal inactivation, the enzyme was incubated at temperatures ranging from 30 to 70 °C. The thermoinactivation kinetics of this microbial TGase followed a Lumry-Eyring model. The enzyme presented good stability until 50 °C. About 50 % of the activity still remained after heating for 12 h in this temperature. Results presented in this work suggest that this microbial TGase exhibit some interesting properties for food and non-food industrial applications.

Transglutaminases (protein-glutamine:amine y-glutamyl- transferase, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When lysine acts as the acyl-acceptor substrate, α-glutamyl lysine isopeptide bond is formed. Isopeptide catalyzation results in protein cross-linkage which is prevalent throughout biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond. Furthermore, mTGs promiscuity in donor substrate preference highlights its biocatalytic potential. To realize the potential of the enzyme, a high-reactivity tag was necessary for protein labelling. To address this, an enzyme-coupled assay was developed to characterize peptides in the hopes of developing orthogonal substrates to facilitate mTG-mediated labelling and biocatalysis. The discovery of high-reactivity peptide tags allowed the realization of in vitro protein labelling- facilitated by mTG. The 7M48 peptide was fused to a test protein, where it was subsequently propargylated with propargyl amine to fluorescently label or immobilize a test protein. Although there are endless possibilities for in vitro bio-conjugation through mTG, proteolytic activation limits any in-cell labelling strategies with this enzyme. To circumvent this issue, development of an alternative bacterial enzyme, Bacillus subtilis transglutaminase (bTG), was chosen to replace mTG. bTG maintains the advantages associated with mTG but is expressed in its active form. Unlike mTG, there is limited preliminary research associated with the enzyme or its substrate scope. To better understanding substrate reactivity, a FRET-based assay was developed allows for the discovery of new high-reactivity peptides for bTG. These peptides were then used in labelling strategies to demonstrate the potential bTG-mediated bioconjugation. This strategy includes the added advantage of potential for in-cellulo labelling.

Glioblastoma is the most common and aggressive type of adult brain tumour. It is currently incurable and requires more effective treatments. Tissue transglutaminase 2 (TGM2) has previously been suggested to have a role in glioblastoma. Previous studies focused on TGM2 expression and inhibition in glioblastoma cells. Here we were interested in TGM2 expression in glioblastoma-associated microglia/macrophages and in identifying the role it plays in the tumor microenvironment. Based on data from bioinformatics, cell culture experiments, immunohistochemistry and immunofluorescence on mouse samples and human samples, we have shown that glioblastoma-associated microglia/macrophages are the major source of TGM2 in the tumor microenvironment. We also identified a novel role for TGM2 in efferocytosis in glioblastoma; this suggests a role for TGM2 in the maintenance of an immunosuppressive environment in this cancer. With this, we hope that further studies will be designed to evaluate the use of TGM2 antagonists as therapeutic agents for glioblastoma.

In an effort to mimic the continuous protein matrix found in gluten-containing breads, the effects of the enzyme microbial transglutaminase were tested in gluten-free (GF) sourdough breads containing five different GF flours: chickpea, brown rice, white rice, oat, and quinoa. The utilization of transglutaminase in GF sourdough bread applications was shown to improve some of the final bread characteristics of GF breads. White and brown rice GF sourdough loaves at 24 hours showed a 28% and 13% decrease in crumb firmness with the addition of 2 U/g dough, respectively. Quinoa-GF sourdough breads at 24 hours showed a 6% increase in specific volume with the addition of 1 U/g dough. Oat and chickpea-GF sourdough loaves did not show improvements in loaf quality with mTG addition. SDS-PAGE analysis revealed that white rice, brown rice, quinoa, and chickpea proteins are adequate substrates for mTG activity, while oat proteins, without the addition of exogenous proteins, lack the characteristics suitable for mTG action.

In this study, a sustainable packaging system was developed to provide food safety and security. Soy protein isolate (SPI) was enzymatically modified by transglutaminase under different conditions to ensure desirable and optimized enzyme crosslinking activity before film preparation. Physicochemical properties including viscosity and molecular weight distribution of the modified proteins and films were measured. Results confirmed the enzymatic treatment is an effective way to modify the SPI based biopolymeric film. Modified films with the enzyme had significant increases in tensile strength (TS), percent elongation (%E), initial contact angle, and a reduction in swelling and protein solubility properties compared to the control films. FTIR and XRD spectra revealed that the enzyme treatment modified the structure of SPI film matrix. The optimal film preparation conditions achieved in this part were protein denaturation temperature 80 °C, and enzyme incubation time 2hr. We attempted to enhance antioxidant activity of enzymatically modified SPI film with the addition of two types of lignin, alkali lignin (AL) and lignosulphonate (LSS), at different concentrations. Results indicated that AL carried higher radical scavenging ability than LSS. Films containing AL showed high absorption in the UV region, and this UV-blocking ability increased with increasing lignin concentration. Deconvoluted FTIR spectra and XRD results suggested that the addition of lignin caused some changes in secondary structure of the protein matrix. The addition of lignin improved TS and thermal stability of films, but reduced %E as a function of lignin concentration. Radical scavenging activity and UV-blocking ability alongside improvement in physicochemical properties of enzymatic modified SPI film with lignin motivated us to apply this bioplastic in two types of oil, soy oil and fish oil. Results revealed that applying enzymatically modified SPI film with AL and LSS in the inner layer of a soy oil packaging system, decreased oxidation rate to around 75%, and pentanal production to about 40% of control. UV-blocking ability of AL caused reduction in oxidation rate for more than 75% compared with the normal packaging system. The effectiveness of this active packaging system in soy oil was greater than fish oil. Thus, the developed biopolymeric materials may have application to food packaging. / Ph. D.

BACKGROUND:Transglutaminase-4 (TGase-4), also known as the Prostate Transglutaminase, is an enzyme found to be expressed predominately in the prostate gland. The protein has been recently reported to influence the migration and invasiveness of prostate cancer cells. The present study aimed to investigate the influence of TGase-4 on cell-matrix adhesion and search for the candidate active domains] within the protein.METHODS:Human prostate cancer cell lines and prostate tissues were used. Plasmids that encoded different domains and full length of TGase-4 were constructed and used to generate sublines that expressed different domains. The impact of TGase-4 on in vitro cell-matrix adhesion, cell migration, growth and in vivo growth were investigated. Interactions between TGase-4 and focal adhesion complex proteins were investigated using immunoprecipitation, immunofluorescence and phosphospecific antibodies.RESULTS:TGase-4 markedly increased cell-matrix adhesion and cellular migration, and resulted in a rapid growth of prostate tumours in vivo. This effect resided in the Core-domain of the TGase-4 protein. TGase-4 was found to co-precipitate and co-localise with focal adhesion kinase (FAK) and paxillin, in cells, human prostate tissues and tumour xenografts. FAK small inhibitor was able to block the action mediated by TGase-4 and TGase-4 core domain.CONCLUSION:TGase-4 is an important regulator of cell-matrix adhesion of prostate cancer cells. This effect is predominately mediated by its core domain and requires the participation of focal adhesion complex proteins.

A desnutrição é um dos principais problemas de saúde pública do mundo, que contribui significativamente para o aumento da morbidade e mortalidade. Estima-se um total de 815 milhões de pessoas subnutridas no mundo, e apesar da melhoria dos recursos alimentares o número de pessoas desnutridas ainda é alarmante. Estudos de nosso laboratório tem demonstrado, em modelo murino de desnutrição proteica, hipoplasia medular com evidências histológicas de alterações na matriz extracelular (MEC) e permanência da célula-tronco hemopoética (CTH) na fase G0/G1 do ciclo celular em camundongos desnutridos. Dados deste trabalho evidenciaram alterações nas proteínas Akt /mTOR, que podem contribuir para o aumento da expressão autofágica nas CTHs e CTPHs (célula-tronco progenitora). A literatura demonstra que desequilíbrios nutricionais e metabólicos podem induzir ativação autofágica. Autofagia é um processo catabólico que participa da manutenção da homeostase celular, da MEC e na regulação das CTHs, dados deste trabalho demonstram diminuição da quantidade de CTH e CTPH em camundongos desnutridos sem a presença do gene Atg7, proteína participativa no processo autofágico. Já camundongos com deleção da transglutaminase 2 (TG2) e submetidos a privação de nutrientes por 24 horas , apresentou diminuição da quantidade de CTH e aumento da diferenciação da CTPH. A TG2 tem participação na impulsão e formação do fagóforo (processo inicial autofágico). Considerando que a desnutrição proteica leva a comprometimento da hemopoese, alterações no ciclo celular das CTHs e hipoplasia medular com pancitopenia periférica e que privação e ou jejum prolongado de nutrientes pode aumentar a atividade autofágica, concluímos nesse projeto que autofagia é importante para regulação da CTH e diferenciação da CTPH, entretanto a desnutrição proteica e privação de nutrientes estimula de maneira diversa o mecanismo de diferenciação da CTH. / Malnutrition is one of the world\'s major public health problems, which contributes significantly to increased morbidity and mortality. An estimated 815 million people are undernourished in the world, and despite the improvement in food resources the number of undernourished people is still alarming. Studies of our laboratory have demonstrated in murine model of protein malnutrition, medullary hypoplasia with histological evidence of extracellular matrix (ECM) changes and hemopoietic stem cell (HSC) stay in the G0/ G1 phase of the cell cycle in malnourished mice. Data from this work showed alterations in Akt / mTOR proteins, which may contribute to the increase of autophagic expression in HSC and HPC (progenitor stem cell). The literature demonstrates that nutritional and metabolic imbalances can induce autophagic activation. Autophagy is a catabolic process that participates in the maintenance of cellular homeostasis, ECM and in the regulation of HSC, data from this work demonstrate a decrease in the amount of HSC and HPC in malnourished mice without the presence of the Atg7 gene, a participatory protein in the autophagic process. Mice with transglutaminase 2 deletion (TG2) and submitted to nutrient deprivation for 24 hours showed a decrease in the amount of HSC and an increase in the differentiation of HPC. TG2 plays a role in the uptake and formation of phagophore (autophagic initial process). Considering that protein malnutrition leads to hemopoiesis, alterations in the cell cycle of HSC and spinal cord hypoplasia with peripheral pancytopenia, and that prolonged nutrient starvation or fasting may increase the autophagic activity, we conclude in this project that autophagy is important for regulation of HSC and differentiation of HPC, however, protein malnutrition and nutrient deprivation stimulate in a different way the mechanism of differentiation of HSC.

O pão é um dos alimentos mais consumidos na dieta humana, estando presente na mesa de diferentes povos e classes sociais. Além do seu aspecto apetitoso, o pão apresenta importante valor nutricional, uma vez que é fonte de carboidratos, proteínas, vitaminas e sais minerais. Na medida em que a panificação se estendeu do processo artesanal para a escala industrial, a utilização de agentes melhoradores de farinha vem se ampliando em função da necessidade de melhorar as características de processo e a vida útil dos produtos obtidos. Durante décadas, enzimas foram adicionadas à farinha na produção de pães com a finalidade de melhorar seu volume, sabor, aroma, estrutura da casca e do miolo, maciez e vida-deprateleira. O presente trabalho teve como objetivo avaliar a influência da adição de enzimas na qualidade de pães brancos e pães ricos em fibras através do uso de associações enzimáticas de transglutaminase, xilanase e amilase. Foram preparadas 17 formulações para cada tipo de pão, com diferentes concentrações das enzimas, de acordo com o planejamento experimental 23 e para análise foi utilizada a metodologia de superfície de resposta. As etapas básicas da produção dos pães foram: pesagem e amassamento; divisão, boleamento e descanso; modelagem; fermentação; forneamento e resfriamento. As farinhas com a adição da associação enzimática e padrão foram submetidas às análises de umidade, cinzas, teor de glúten, cor, absorção de água, estabilidade, elasticidade e extensibilidade. Todas as 17 formulações e a formulação padrão para pão branco e pão rico em fibra foram analisadas sensorialmente, através da Análise Descritiva Quantitativa (ADQ), e físico-quimicamente, através das análises de umidade, cinzas, textura, cor, altura das fatias e volume específico. Os resultados obtidos no presente trabalho mostraram que a adição destas enzimas não é necessária para se ter um pão com boa qualidade e com características exigidas pelos consumidores. Observou-se que o efeito da associação das três enzimas testadas não foi significativo, pois na maioria das características avaliadas o melhor resultado foi o apresentado na amostra padrão, sem adição de enzimas. / Bread is one of the most consumed foods in the human diet. It is found on the table of people from different cultures and social classes. Besides its appetizing aspect, bread also presents important nutritional value, since it is a source of carbohydrates, proteins, vitamins and mineral salts. As bread-making has gone from the handmade process to the industrial scale, the usage of bread enhancements has increased in order to attend the necessity of improving process’s characteristics and lifespan of the obtained product. Throughout many decades, enzymes were added to the flour during bread’s production with the objective of increasing its volume, taste, aroma, crust’s and crumb’s structure, softness and lifespan. The present work is proposed to evaluate how the addition of enzymes can influence on the quality of white and wholemeal bread through the use of enzymatic associations of transglutaminase, xylanase and amylase. 17 formulations have been prepared for each type of bread, each one with different enzyme concentrations, according to the experimental planning 2³. The methodology used for the analysis was the Response Surface Methodology – RSM. The basic steps of production were: weighing and kneading; dividing, ball making and resting; molding; fermenting; baking and cooling. Both the standard flours and the ones with the addition of enzymatic associations were submitted to humidity, ashes, gluten level, color, water absorption, stability, elasticity, and extensibility analysis. All 17 formulations and the standard formulation for white bread and wholemeal bread have been submitted to sensorial evaluation using Quantitative Descriptive Analysis. They have also been physically and chemically tested through the analysis of humidity, ashes, texture, color, height of the slices and specific volume. The results obtained from this research proved that the addition of those enzymes is not necessary in order to make good quality bread with characteristics demanded by its consumers. It has been observed that the effect of the association of the 3 tested enzymes was not significant. The standard sample - free of enzyme addition - presented the best results for most of the evaluated characteristics.

O pão é um dos alimentos mais consumidos na dieta humana, estando presente na mesa de diferentes povos e classes sociais. Além do seu aspecto apetitoso, o pão apresenta importante valor nutricional, uma vez que é fonte de carboidratos, proteínas, vitaminas e sais minerais. Na medida em que a panificação se estendeu do processo artesanal para a escala industrial, a utilização de agentes melhoradores de farinha vem se ampliando em função da necessidade de melhorar as características de processo e a vida útil dos produtos obtidos. Durante décadas, enzimas foram adicionadas à farinha na produção de pães com a finalidade de melhorar seu volume, sabor, aroma, estrutura da casca e do miolo, maciez e vida-deprateleira. O presente trabalho teve como objetivo avaliar a influência da adição de enzimas na qualidade de pães brancos e pães ricos em fibras através do uso de associações enzimáticas de transglutaminase, xilanase e amilase. Foram preparadas 17 formulações para cada tipo de pão, com diferentes concentrações das enzimas, de acordo com o planejamento experimental 23 e para análise foi utilizada a metodologia de superfície de resposta. As etapas básicas da produção dos pães foram: pesagem e amassamento; divisão, boleamento e descanso; modelagem; fermentação; forneamento e resfriamento. As farinhas com a adição da associação enzimática e padrão foram submetidas às análises de umidade, cinzas, teor de glúten, cor, absorção de água, estabilidade, elasticidade e extensibilidade. Todas as 17 formulações e a formulação padrão para pão branco e pão rico em fibra foram analisadas sensorialmente, através da Análise Descritiva Quantitativa (ADQ), e físico-quimicamente, através das análises de umidade, cinzas, textura, cor, altura das fatias e volume específico. Os resultados obtidos no presente trabalho mostraram que a adição destas enzimas não é necessária para se ter um pão com boa qualidade e com características exigidas pelos consumidores. Observou-se que o efeito da associação das três enzimas testadas não foi significativo, pois na maioria das características avaliadas o melhor resultado foi o apresentado na amostra padrão, sem adição de enzimas. / Bread is one of the most consumed foods in the human diet. It is found on the table of people from different cultures and social classes. Besides its appetizing aspect, bread also presents important nutritional value, since it is a source of carbohydrates, proteins, vitamins and mineral salts. As bread-making has gone from the handmade process to the industrial scale, the usage of bread enhancements has increased in order to attend the necessity of improving process’s characteristics and lifespan of the obtained product. Throughout many decades, enzymes were added to the flour during bread’s production with the objective of increasing its volume, taste, aroma, crust’s and crumb’s structure, softness and lifespan. The present work is proposed to evaluate how the addition of enzymes can influence on the quality of white and wholemeal bread through the use of enzymatic associations of transglutaminase, xylanase and amylase. 17 formulations have been prepared for each type of bread, each one with different enzyme concentrations, according to the experimental planning 2³. The methodology used for the analysis was the Response Surface Methodology – RSM. The basic steps of production were: weighing and kneading; dividing, ball making and resting; molding; fermenting; baking and cooling. Both the standard flours and the ones with the addition of enzymatic associations were submitted to humidity, ashes, gluten level, color, water absorption, stability, elasticity, and extensibility analysis. All 17 formulations and the standard formulation for white bread and wholemeal bread have been submitted to sensorial evaluation using Quantitative Descriptive Analysis. They have also been physically and chemically tested through the analysis of humidity, ashes, texture, color, height of the slices and specific volume. The results obtained from this research proved that the addition of those enzymes is not necessary in order to make good quality bread with characteristics demanded by its consumers. It has been observed that the effect of the association of the 3 tested enzymes was not significant. The standard sample - free of enzyme addition - presented the best results for most of the evaluated characteristics.