<p>Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet.</p><p>The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. </p><p>The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. </p><p>In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet. </p>

All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the Drosophila hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as Galleria mellonella. The Drosophila clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in Drosophila coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, in vivo when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as Galleria mellonella and Ephestia kuehniella, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.

Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet. The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet.

Cross-linking in soy proteins is hypothesised to have an impact on the texture of tofu. In vitro incubation showed soy proteins and its two fractions, glycinin and β-conglycinin, were cross-linked using glutaraldehyde, formaldehyde, glyceraldehyde and transglutaminase (TGA). Increasing concentration of these carbonyl compounds and TGA, and temperature of the carbonyl compounds treatment, increased the reactivity of cross-linking. Glutaraldehyde was the most reactive in forming aggregated proteins, followed by formaldehyde and glyceraldehyde. Both carbonyl moieties of glutaraldehyde are believed to be essential for the rapid cross-linking reaction. In the unfractionated soy proteins, β-conglycinin had a higher reactivity than glycinin. In in vitro incubation using TGA, soy proteins served as good substrates for TGA, in which β-conglycinin was more susceptible to TGA than glycinin in the unfractionated soy proteins. The addition of TGA, and 1 and 2 mM glutaraldehyde prior to soymilk boiling in situ resulted in a small number of cross-linked proteins, which correspond to an increase in fracture force. The addition of glutaraldehyde after soymilk boiling resulted in a slight decrease in fracture force compared to the control. At higher concentrations of glutaraldehyde (15 and 30 mM), soy proteins were mostly cross-linked, regardless of addition before or after soymilk boiling. Highly cross-linked proteins resulted in a significant decrease in the fracture force. For TGA treatment, the fracture force was increased with increasing TGA concentration from 1000 to 5000 ppm, added either before or after soymilk boiling. However, the TGA treatment showed only a small quantity of cross-linking. It is hypothesised that TGA hydrolysed glutamine of proteins to glutamate and changed the functional properties of proteins. Upon examination of the microstructure, it was found that the TGA treatment resulted in a fine-stranded network, compact structure and less porosity. These characteristics resulted in a higher fracture force. In contrast, in the glutaraldehyde treatment, the network consisted of a higher porosity, loose network and diffuse structure, which gave lower fracture force. Thus, it appears that substrate modification to the structure of the soy proteins may have a greater impact than the number of cross-links. These findings are likely to have implications for production of soy products with a wide range of textures by manipulating the soy protein properties.

Orientador: Hélia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T16:00:04Z (GMT). No. of bitstreams: 1 Carvalho_PriscilaHoffmann_D.pdf: 4102242 bytes, checksum: 1496382da70a395d87d5c8536317d42d (MD5) Previous issue date: 2013 / Resumo: A isomaltulose e a trealulose são dissacarídeos isômeros estruturais, que podem ser obtidos a partir da sacarose utilizando-se glicosiltransferase bacteriana. Esses dissacarídeos são considerados açúcares alternativos de grande potencial para uso nas indústrias de alimentos e farmacêutica porque são hidrolisados e absorvidos mais lentamente e apresentam baixo potencial cariogênico comparado com a sacarose. Foi estudada a imobilização de células de Serratia plymuthica ATCC 15928, produtora de glicosiltransferase por gelificação iônica em gel alginato contendo transglutaminase (TG) e também a utilização de células livres para a conversão de sacarose em isomaltulose e trealulose. Utilizando-se células livres de Serratia plymuthica ATCC 15928 foi obtido 70% de conversão em isomaltulose e 8% de trealulose a 25°C por 10 bateladas de 15 minutos, a partir de solução de sacarose 30%. Entre as cinco amostras de alginato de sódio testadas, para a imobilização das células de S. plymuthica ATCC 15928 com e sem adição de TG, foram obtidos melhores resultados (médio de três bateladas) de conversão de sacarose (37,4% de isomaltulose) utilizando o alginato de sódio B, de alta viscosidade (14.000cP Sigma ¿ A 7128) em presença de TG. Nas condições estudadas (1,7% de alginato de sódio, 30% de massa celular úmida, solução de cloreto de sódio 0,2Mol/L, 2% de TG e 35% de sacarose) também houve maior facilidade de formação de grânulos uniformes. A presença de TG como agente de reticulação na matriz de imobilização melhorou a estabilidade de conversão por três bateladas onde observou-se resultado médio 27% maior com relação a matriz com o mesmo tipo de alginato (B) em ausência de TG. A composição da matriz de imobilização com adição de TG foi otimizada por metodologia de planejamento experimental, assim como a adição de gelatina como fonte de proteína adicional para promoção de ligações cruzadas catalisadas pela TG. Os melhores resultados de conversão de sacarose (solução 35%) em isomaltulose (72,66% de isomaltulose e 8% de trealulose em 4 bateladas de 24horas) foram obtidos utilizando-se matriz de polissacarídeo-proteína composto de 1,7% de alginato de sódio 14.000cP (Sigma®-A7128), 0,25mol/L de CaCl2, 0,5% de gelatina, 3,5% de TG e concentração de massa celular úmida superior a 35% (m:v). Verificou-se que a adição de ALMP na matriz de alginato de cálcio-gelatina-TG para imobilização de S. plymuthica, testada por planejamentos experimentais seqüenciais, não aumentou a estabilidade da taxa de conversão de sacarose em isomaltulose quando comparada com as células imobilizadas em matriz de alginato de cálcio-gelatina-TG. Em processo contínuo utilizando-se coluna empacotada com células de S. plymuthica imobilizadas em matriz otimizada e descrita acima, foi obtida taxa de conversão média de 64% de sacarose em isomaltulose durante 200 horas de processo, equivalente a 0,27g de isomaltulose/g de células imobilizadas/hora em coluna a 25°C e fluxo de substrato (35% de sacarose) 0,2mL/min / Abstract: The isomaltulose and trehalulose are disaccharides and structural isomers, which can be obtained from sucrose using bacterial glycosyltransferase. These disaccharide are considered alternative sugars with great potential for use in the food and pharmaceutical industries because they are hydrolyzed and absorbed more slowly and have a low cariogenic potential compared with sucrose. The conversion of sucrose to isomaltulose and trehalulose was estudied using immobilized and free cells of Serratia plymuthica ATCC 15928. The cells were immobilized by ionic gelation in alginate gel containing transglutaminase. Using free cells of Serratia plymuthica ATCC 15928 was obtained 70% isomaltulose conversion and 8% trehalulose conversion at 25° C in 10 batches of 15 minutes from a 30% sucrose solution. Among the five samples of sodium alginate tested for S. plymuthica ATCC 15928 cells immobilization, with or without the addition of TG, the best results (average of three batches) were obtained using sodium alginate B, high viscosity (14.000cP Sigma - A 7128) in the presence of TG, leading to 37.4% isomaltulose conversion from sucrose. In the studied conditions (1.7% sodium alginate, 30% wet cell mass solution of sodium chloride 0.2 Mol/L, 2% TG, 35% sucrose) was also easier to form uniform granules. The presence of TG as a crosslinking agent in the immobilization matrix improved the stability during three batches, resulting in an 27% higher average conversion with respect to a same type of alginate (B) matrix in absence of TG. Immobilization matrix compositions with addition of TG was optimized by experimental design methodology, as well as the addition of gelatin as a protein source for promoting additional crosslinking catalyzed by TG. The best results conversion of sucrose (35% solution) into isomaltulose (72.66% of isomaltulose and 8% of trehalulose in 4 batches of 24 hours) were obtained using proteinpolysaccharide matrix composed of 1.7% alginate 14.000cP sodium (Sigma® A7128), 0.25 Mol/L CaCl2, 0.5% gelatin, 3.5% TG, and wet cell mass concentration of 35% (w:v). It has been found that the addition of ALMP (amidated low methoxyl pectin) into the calcium alginate-gelatin-TG matrix for immobilization of S. plymuthica, tested by sequential experimental design, do not increase the stability of sucrose to isomaltulose conversions rate when compared with cells immobilized in calcium alginate -gelatin-TG matrix. In continuous process using a packed column with S. plymuthica cell's immobilized in the optimized matrix described above, it was obtained an average conversion rate of 64% sucrose to isomaltulose during a 200 hours process, equivalent to 0.27g isomaltulose per gram of immobilized cell per hour, in a column at 25° C and using flow substrate (35% sucrose) of 0.2 mL / min / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that natively catalyses the formation of isopeptidic bonds between protein- or peptide-bound glutamine and lysine residues. Physiologically, it is ubiquitously expressed in tissues, with roles in cellular differentiation, extracellular matrix stabilisation, and apoptosis, among others. However, its unregulated activity has been associated with various pathologies including fibrosis, cancer and celiac disease. Since most pathologies are associated with an increased transamidation activity, efforts have been directed towards the development of TG2 inhibitors. In this context, the work described in this thesis is centred on reversible inhibitors, building on recent work done within the Keillor group in two directions, namely localisation and potency. In a localisation-driven approach, we developed a photolabile derivative of a known reversible inhibitor, in order to form a covalent bond with the enzyme and determine the inhibitor’s binding site. In tandem, we optimised a protocol for the expression of TG2 incorporating ArgΔ10 and LysΔ8, amino acids that are 13C- and 15N-labelled to provide a mass shift of 10 and 8 Da, respectively, compared to the corresponding unlabelled amino acids. This “heavy” TG2 was developed as a tool for reference in the analysis of the tryptic digest of labelled protein. In a potency-driven approach, based on the observation that previous trans cinnamoyl inhibitor scaffolds were susceptible to nucleophilic attack by glutathione, we developed a bis(triazole) scaffold with reduced electrophilicity. The preparation of a small library of compounds showed that this scaffold demonstrates a preference for electron-withdrawing substituents, such as nitro groups. Continuing in a potency-driven approach, and inspired by work done in the identification of glutathione-resistant scaffolds, we studied a new alkynyl scaffold. While still susceptible to glutathione addition, these compounds showed a marked improvement in potency, with the lead compound having an IC50 of 930 nM and being established as a competitive inhibitor with a Ki of 420 nM, our most potent reversible inhibitor to date. Furthermore, this scaffold also produced an inhibitor lacking nitro groups (to limit eventual cellular toxicity), but maintaining good potency, with an IC50 value of 3.03 μM.

Orientador: Hélia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T06:55:18Z (GMT). No. of bitstreams: 1 Bagagli_MarcelaPavan_D.pdf: 5889679 bytes, checksum: 9bd456830fa35028a89bffb19885f07d (MD5) Previous issue date: 2014 / Resumo: A transglutaminase catalisa a formação de ligações cruzadas entre grupos ?-amino de resíduos de lisina e o grupo ?-carboxiamida de resíduos de glutamina de proteínas. Esta enzima pode ser usada para unir diferentes proteínas e melhorar suas propriedades funcionais. A transglutaminase obtida de micro-organismos por processos fermentativos apresenta vantagens em relação à enzima obtida de plantas e tecidos animais para as aplicações industriais. Neste trabalho, foram estudados os efeitos de diferentes subprodutos ou resíduos agroindustriais no meio de cultivo para a fermentação submersa de Streptomyces sp. CBMAI-837, visando o aumento da atividade e da produtividade da enzima. Entre os substratos proteicos avaliados, o uso de 2,5% (m:v) de farelo de algodão ou de 2,5% (m:v) de farelo de soja no meio de cultivo resultou no aumento da atividade enzimática de 1,2 U.mL-1 (214%) e 1,0 U.mL-1 (182%), respectivamente, em relação ao valor obtido pela fermentação do micro-organismo em meio de cultivo contendo 2,5% de farinha de soja (0,57 U.mL-1). Em relação às fontes de carbono principais avaliadas, a adição de 2,0% de glicerol ou de 12% de melaço de cana de açúcar permitiu o aumento da atividade de transglutaminase em 0,94 U.mL-1 (167%) e 0,88 U.mL-1 (157%), respectivamente. Foi verificado que a adição de 1% de quitina nativa no meio de cultivo favoreceu a produção da enzima elevando a atividade de transglutaminase em 181% em relação ao meio de cultivo sem quitina. Os efeitos da aplicação da TGase de Streptomyces sp. CBMAI-837 em massa de farinha de trigo mole e na fabricação de pães foram avaliados e os resultados foram comparados com a atuação de uma TGase comercial (BioBond) formulada para aplicação em cereais. De forma geral, as duas enzimas avaliadas apresentaram o mesmo efeito sobre a massa quanto ao aumento da resistência à extensão, a redução da máxima extensão da massa e a redução da pegajosidade da massa. Efeito antagônico foi observado na hidrofobicidade de superfície das proteínas da massa sendo que este parâmetro foi reduzido pela adição da TGase de Streptomyces sp. CBMAI-837 e foi elevado pela enzima comercial. A adição de TGase na preparação da massa de farinha de trigo mole resultou em aumento da massa molecular das proteínas, indicantivo da formação de ligações cruzadas entre proteínas. A aplicação da transglutaminase de Streptomyces sp. CBMAI-837 em massa de pão de farinha de trigo mole promoveu a redução do volume do pão em 9% e o aumento da firmeza em 32%. O aumento da quantidade de solvente adicionado na massa de 53% para 56% permitiu o aumento do volume dos pães, no entanto, com pouca diferença dos parâmetros de textura em relação ao controle para a TGase comercial BioBond e para a TGase de Streptomyces sp. CBMAI-837 / Abstract: Transglutaminase catalyzes the cross-linking reaction between a ?-carboxyamide of a glutamine residue from a peptide bond and the ?-amino group of a lysine. TGase can bind different proteins and improve their functional properties. The microbial transglutaminase shows advantages over the enzyme extracted from plants and mammals. In the present study, the effect of different industrial wastes and byproducts in the culture medium during the submerged fermentation of Streptomyces sp. CBMAI-837 was studied aiming to increase the enzyme activity and yield. Amongst the substrates with high protein content, the use of 2,5% of cottonseed meal or 2,5% of soybean meal in the culture medium increased the transglutaminase activity to 1.2 U.mL-1 (214%) and 1.0 U.mL-1 (182%), respectively, as compared to the results obtained using 2,5% of soybean flour. With regard to the main carbon sources, both 2% glycerol and 12% sugar cane molasses increased the transglutaminase activity to 0.94 U.mL-1 (167%) and 0.88 U.mL-1 (157%), respectively. It was observed that the addition of 1% of chitin on culture medium increased the transglutaminase activity by 181% as compared to the results obtained without the addition of chitin. The effects of the TGase from Streptomyces sp. CBMAI - 837 on soft wheat flour dough and on the manufacture of bread were evaluated, and the results were compared with the performance of a commercial TGase (BioBond) formulated for specific applications in cereal products. In general, both enzymes had the same effect on the rheological properties of the doughs, increasing the resistance to extension, reducing the maximum extension and reducing stickiness of the dough. The surface hydrophobicity of the protein dough was reduced by the addition of TGase from Streptomyces sp. CBMAI 837 but increased by the addition of the commercial enzyme. In general the analysis of the protein structure indicated an agglomeration of the proteins causing an increase in molecular weight. The application of transglutaminase from Streptomyces sp. CBMAI-837 in the formulation of bread loaves decreased the bread volume by 9% and increased firmness by 32%. Increasing the amount of solvent added to the dough from 53% to 56% increased the volume of the loaves, but resulted in little difference in the texture profiles of the loaves made with the addition of the commercial BioBond and Streptomyces sp. CBMAI ¿ 837 TGases, in relation to the control / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

No description available.

Es ADCs (Antibody-Drug Conjugates) correspondent à une nouvelle stratégie thérapeutique anti-tumorale particulièrement prometteuse. Néanmoins, les ADCs actuellement utilisés en clinique sont obtenus par conjugaisons chimiques, resultant en des mixtures hétérogènes impactant négativement leurs pharmacocinétiques et leurs performances in vivo.Récemment, différentes strategies de couplage site-spécifique ont été développées afin de réduire cette hétérogénéité. Dans cette thèse, nous rapportons le développement d’une nouvelle technologie CovIsoLink™ (Covalently Isopeptide Crosslinking) permettant la génération d’ADCs par utilisation de nouveaux peptides glutamine Q-Tag présentant des affinités optimisées par rapport à des peptides disponibles (ZQG, LLQG) pour une enzyme bactérienne la transglutaminase (mTG).La preuve de concept de cette technologie a été réalisée par insertion de ces peptides Q-Tag en C-ter de la région codant pour la chaine lourde des anticorps anti-HER2 (Trastuzumab). Nous avons ainsi pu démontrer la conjugaison homogène et reproductible de différentes drogues sans contamination par des chaines d’anticorps non conjuguées. Nous avons pu montrer que l’immunoréactivité et la capacité d’internalisation de ces ADCs n’étaient pas altérées par la conjugaison et qu’ils présentaient in vitro et in vivo, des propriétés de lyse de cellules tumorales similaires au Trastuzumab emtansine (Kadcyla®), actuellement en clinique. Par ailleurs, afin de généraliser notre technologie à différents formats d’anticorps nous avons générés des fragments Fab et scFv et évalué leur fonctionnalité. Ainsi, nous avons pu prouver que l’utilisation de nouveaux peptides optimisés Q-Tag substrat de la transglutaminase permettait une stratégie de couplage alternative plus homogène par couplage de différentes molécules sans espèce contaminante non couplée / Antibody-drug conjugates (ADCs) are a powerful class of therapeutic agents, demonstrating success in the treatment of several malignancies. The currently approved ADCs are produced by chemical conjugations and exist as heterogeneous mixtures that negatively influence the pharmacokinetics and in vivo performance. Recently many of site-specific conjugation technologies have been developed to reduce heterogeneity and batch-to batch variability. Microbial transglutaminase (mTG) has been demonstrated as efficient tool for site-specific conjugation. In this thesis we report the development CovIsoLink™ (Covalently Isopeptide Crosslinking) technology for the generation of homogenous immunoconjugates using a novel glutamine donor peptides (Q-tag) with improved affinity compared to the known peptides (ZQG, LLQG). As a proof of concept, the peptides sequences were engineered into the heavy chain C-terminal of Trastuzumab antibody. We demonstrated the reproducible and homogeneous conjugation of Q-tagged Trastuzumab with different payloads, without any unconjugated species. The ADCs were evaluated in series of in vitro and in vivo assays. We confirmed that the immunoreactivity and internalisation are not altered by the conjugation. Furthermore similar in vitro and in vivo tumor cell killing potency was demonstrated than Trastuzumab emtansine (Kadcyla®), which is already used in the clinic. Morover we extend our site-specific conjugation technology to antibody fragments (Fab and scFv), evaluating their functionality by conjugation with AlexaFluor488-cadaverine and in antigen binding assays. Thus, using novel glutamine donor peptides, our technology provides an alternative enzymatic conjugation strategy for the engrafment of different payloads resulting in homogeneous batches, without unconjugated species

Orientadores: Helia Harumi Sato , Lara Durães Sette / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-13T13:26:40Z (GMT). No. of bitstreams: 1 Macedo_JulianaAlves_D.pdf: 1354680 bytes, checksum: f0751667b78c8b7e3db22d9ceb16adbf (MD5) Previous issue date: 2009 / Resumo: A transglutaminase (TGase) (EC 2.3.2.13; proteina-glutamina ?-glutaminiltransferase) é uma enzima capaz de catalisar reações de transferência de grupos acil utilizando resíduos de glutamina das ligações peptídicas de proteínas como doadores de grupos acil, e diversas aminas primárias como receptores. As ligações covalentes cruzadas entre inúmeras proteínas e peptídeos pela transglutaminase promovem mudanças nas propriedades de proteínas de alimentos. Por essa razão, a transglutaminase é amplamente utilizada nas indústrias de processamento de alimentos para o desenvolvimento de novos produtos e modificação de características como: viscosidade, capacidade emulsificante e valores nutricionais. Uma cepa de Actinomyceto, isolada de amostras de solo brasileiro, foi investigada taxonomicamente por uma combinação de técnicas moleculares e morfológicas, resultando na conclusão de que a cepa pertence ao gênero Streptomyces sp. A cepa, chamada de Streptomyces sp. CBMAI 837 produziu transglutaminase quando cultivada a 30°C por cinco dias, em agitador rotatório, no meio de fermentação otimizado, composto por: 0,2% KH2PO4, 0,1% MgSO4.7H2O, 2% farinha de soja, 2% amido de batata, 0,2% glicose, e 2% peptona, atingindo uma atividade enzimática de 1,37 U.mL-1. A transglutaminase foi purificada cerca de 5 vezes através de duas passagens cromatográficas sucessivas em uma coluna de filtração em gel Sephadex G-75, com 17% de recuperação. A purificação da proteína foi comprovada por homogeneidade eletroforética em SDS-PAGE. A massa molar da TGase foi estimada em cerca de 45 kDa. A transglutaminase de Streptomyces sp CBMAI 837, tanto na forma bruta quanto purificada, apresentou atividade enzimática ótima em pH 6,0-6,5, e em 35-40°C. Um segundo pico de atividade ótima foi observado em pH 10,0 na enzima no estado bruto. Ambas as formas da enzima foram estáveis na faixa de pH de 4,5 a 8,0 e até 45°C. A transglutaminase na forma bruta e purificada mostrou-se independente de íons cálcio, mas foi ativada na presença de K+, Ba2+, e Co2+; e inibida por Cu2+ e Hg2; o que sugere a presença de um grupo tiol no sítio ativo da enzima. A TGase purificada apresentou um Km de 6,37 mM e um Vmax de 1,70 U/mL, enquanto a enzima bruta apresentou Km de 6,52 mM e um Vmax de 1,35 U/mL para o substrato N-carbobenzoxi-L-glutaminil-glicina. A influência da transglutaminase de Streptomyces sp CBMAI 837 bruta, nas propriedades de géis ácidos de caseinato de sódio foi investigada, tendo como parâmetro géis preparados com a TGase comercial (Ajinomoto Inc.). Os géis com a enzima comercial tiveram um valor de módulo de elasticidade maior, porém, dependendo da concentração de proteína, estes foram menos deformáveis. Os géis com enzima bruta de Streptomyces sp. CBMAI 837 se mostraram muito mais rígidos e menos elásticos. Resultados da eletroforese indicaram que a enzima comercial promoveu a formação de polímeros de proteínas de massa molecular mais alta do que a enzima de Streptomyces sp. CBMAI 837. Os testes de microscopia eletrônica de varredura e da capacidade de retenção de água mostraram que características particulares de cada um dos géis poderiam estar associadas ao tipo específico de interação promovida por cada uma das amostras enzimáticas testadas / Abstract: Transglutaminase (EC 2.3.2.13; protein-glutamine ?-glutaminyltransferase) is an enzyme that catalysis an acyl transfer reaction using protein or peptide-bond glutamine residues as acyl donors and several primary amines as receptors. The covalent cross-links between a number of proteins and peptides introduced by transglutaminase promote modification of the functional properties of the food proteins. Therefore, transglutaminase are widely used by food-processing industries for the purpose of new product development, modification of the product properties such as viscosity, emulsification foaming and nutritional values. An actinomycete strain, isolated from Brazilian soil, was taxonomically investigated using a combination of molecular and morphological basedmethods, resulting on the conclusion that the strain belongs to the genus Streptomyces sp. The strain, named Streptomyces sp. CBMAI 837, produce transglutaminase when cultivated at 30°C for 5 days at 200 rpm in a rotatory shaker, on the optimized fermentation medium composed of 0.2% KH2PO4, 0.1% MgSO4.7H2O, 2% soybean flour, 2% potato starch, 0.2% glucose, and 2% peptone, with a enzymatic activity of 1.37 U.mL-1. The enzyme purification was performed by of two successive chromatographies on Sephadex G-75 columns with yields of 48% and 17%, respectively. The protein purification was successfully achieved to electrophoretical homogeneity on SDS-PAGE. The molecular mass of the MTGase was estimated to be about 45 kDa. The enzyme from Streptomyces sp., in both crude and pure forms, exhibited optimal activity in the 6.0-6.5 pH range and at 35-40°C. A second maximum of activity was observed at pH 10.0 on the crude Streptomyces sp. enzyme. Both forms of transglutaminase were stable over the pH range from 4.5 to 8.0 and up to 45°C. The activities of all the TGase samples were independent of Ca+2 concentration, but they were elevated in the presence of K+, Ba2+, and Co2+ and inhibited by Cu2+ and Hg2+, which suggests the presence of a thiol group in the TGase¿s active site. The purified enzyme presented Km of 6.37 mM and Vmax of 1.7 U/mL, while the crude enzyme demonstrated Km of 6.52 mM and Vmax of 1.35 U/mL. The influence of the transglutaminase on acid-gel properties was studied. Texture parameters showed that the commercial TGase (Ajinomoto Inc.) gels had greater values of elasticity modulus and could promote the formation of more elastic and soft food systems, while addition of the crude TGase of Streptomyces sp. CBMAI 837 to the gel led to the formation of more rigid and less elastic gels. The electrophoresis showed that the commercial TG enzyme in this system promoted higher molecular mass protein polymers than the enzyme from Streptomyces sp. CBMAI 837. Microscopy and water holding capacity (WHC) observations showed that all the gel characteristics could be associated to specific interactions promoted by each TGase tested / Doutorado / Doutor em Ciência de Alimentos