Orientador: Hélia Harumi Sato / Texto em português e inglês / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-20T21:23:26Z (GMT). No. of bitstreams: 1 Castro_RuannJanserSoaresde_M.pdf: 2807315 bytes, checksum: e6965e5648d4556cb18db9b7869a58c4 (MD5) Previous issue date: 2012 / Resumo: As proteases constituem um dos mais importantes grupos de enzimas produzidos comercialmente, apresentando diversas aplicações nas indústrias de alimentos e farmacêutica. A utilização de proteases na hidrólise enzimática de proteínas para obtenção de peptídeos com propriedades antioxidantes tem recebido grande notoriedade nas pesquisas científicas. Nesse contexto, o presente trabalho visou estudar a produção e caracterização bioquímica de protease de Aspergillus oryzae LBA 01 obtida por processo fermentativo em estado sólido e avaliar a aplicação desta protease e de preparações comerciais na hidrólise de proteínas para obtenção de hidrolisados com atividade antioxidante. A maior produção de protease por A. oryzae LBA 01 foi observada em meio de cultivo composto de farelo de trigo, peptona (2,0% p/p) e extrato de levedura (2,0% p/p) sob as seguintes condições: 50,0% de umidade inicial, inóculo de 107 esporos.g-1 e incubação a 23°C por 72h. A caracterização bioquímica, realizada por planejamento experimental, mostrou que a protease apresentou maior atividade na faixa de pH 5,0-5,5 e 55-60°C, e estabilidade no intervalo de pH 4,5-6,0 após 1h de tratamento na faixa de temperatura de 35-45°C. Proteína isolada de soja, soro de leite e clara de ovo apresentaram aumento expressivo nas suas propriedades antioxidantes quando hidrolisadas com diferentes proteases microbianas. A aplicação de protease comercial Flavourzyme® 500L, obtida de A. oryzae, para a hidrólise de proteína isolada de soja, resultou na obtenção de hidrolisados com maior atividade antioxidante quando comparados aos hidrolisados preparados com as proteases de A. oryzae LBA 01 e a protease comercial Alcalase® 2.4L de Bacillus licheniformis. As condições de hidrólise, definidas a partir de delineamento composto central rotacional (DCCR), foram: concentração de substrato de 90,0 mg.mL-1 e adição de 70,0 U de protease por mL de mistura reacional (U.mL-1), resultando em 775,17 e 11,83 Trolox EQ µmol.g-1, para os ensaios de ORAC e DPPH, respectivamente. Os hidrolisados de soro de leite com maior capacidade antioxidante foram obtidos com a protease de A. oryzae LBA 01. A adição de 70,0 U.mL-1 de protease a solução de soro de leite 80,0 mg.mL-1, resultou em 424,32 e 16,39 Trolox EQ µmol.g-1, para os ensaios de ORAC e DPPH, respectivamente. Na preparação de hidrolisados de proteínas de clara de ovo, a utilização de 30,0 mg.mL-1 de substrato e 20,0 U.mL-1 da protease comercial Flavourzyme® 500L de A. oryzae, resultou em 1.193,12 e 19,05 Trolox EQ µmol.g-1 para os ensaios de ORAC e DPPH, respectivamente. Os maiores valores de atividade antioxidante, para os três substratos, foram detectados entre 30 e 180 minutos de incubação, onde o grau de hidrólise variou de 40,0 a 66,0%. Os resultados obtidos mostraram que a preparação de protease de A. oryzae LBA 01 obtida por fermentação em estado sólido e posterior concentração por precipitação com sulfato de amônio, diálise e liofilização, apresentou atividade enzimática semelhante às preparações comerciais avaliadas, tendo, portanto, potencial para aplicação na hidrólise proteica. A hidrólise enzimática, nas condições de estudo avaliadas, aumentou de 2 a 23 vezes a capacidade antioxidante de proteína isolada de soja, soro de leite e clara de ovo, mostrando-se um processo eficaz para obtenção de peptídeos com atividade antioxidante / Abstract: Proteases are one of the most important groups of enzymes produced commercially, with several applications in the food and pharmaceutical industries. The use of proteases in the enzymatic hydrolysis of proteins to obtain peptides with antioxidant properties has gained great notoriety in scientific research. In this context, the main objectives of the present study were to optimize the production of the protease from Aspergillus oryzae LBA 01 by solid state fermentation, and to determine its biochemical characteristics. The application of this protease and of commercial preparations to protein hydrolysis, and the study of the antioxidant properties of the hydrolysates obtained, was evaluated. The optimum fermentation medium was composed of wheat bran, 2.0% (w/w) peptone and 2.0% (w/w) yeast extract, and the conditions for maximum protease production were an initial moisture content of 50.0%, an inoculum level of 107 spores.g-1 and incubation at 23°C for 72h. The biochemical characterization, evaluated using an experimental design, showed that the enzyme was most active in the pH range 5.0-5.5 and 55-60°C. The enzyme was stable from pH 4.5 to 6.0 after 1h incubation at 35-45°C. Soy protein isolate, bovine whey protein and egg white protein exhibited increases in antioxidant activity when hydrolyzed with the different microbial proteases. For the hydrolysis of soy protein isolate, application of the commercial protease Flavourzyme® 500L from A. oryzae resulted in hydrolysates with greater antioxidant activity as compared to hydrolysates prepared with the protease from A. oryzae LBA 01 and the commercial protease Alcalase® 2.4L from Bacillus licheniformis. The hydrolysis conditions, as defined by a central composite rotational design (CCRD), were: 90.0 mg.mL-1 substrate concentration plus 70.0 U of protease per mL of reaction mixture (U.mL-1), which resulted in 775.17 and 11.83 Trolox EQ µmol.g-1 as determined by the ORAC and DPPH assays, respectively. For the whey protein hydrolysates, the greatest antioxidant activity was obtained with the protease from A. oryzae LBA 01. According to the CCRD, the use of 80.0 mg.mL-1 of bovine whey protein and 70.0 U.mL-1 of protease resulted in 424.32 and 16.39 Trolox EQ µmol.g-1, respectively, as determined by the ORAC and DPPH assays. For the egg white protein, hydrolysis with 20.0 U.mL-1 of Flavourzyme® 500L from A. oryzae with 30.0 mg.mL-1 substrate concentration, resulted in 19.05 and 1,193.12 Trolox EQ µmol.g-1, respectively, as determined by the ORAC and DPPH assays. The maximum antioxidant activities were obtained in the range from 30 to 180 min of hydrolysis, with a degree of hydrolysis of about 40.0-66.0%. The results showed that the protease preparation from A. oryzae LBA 01 obtained by solid state fermentation produced enzymatic activity similar to that of the commercial preparations, and was an attractive enzyme to apply in protein hydrolysis. Under the conditions evaluated in this study, enzymatic hydrolysis resulted in a 2.0- to 23.0-fold increases in antioxidant activity for the soy protein isolate, bovine whey protein and egg white protein, being an effective process to obtain peptides with antioxidant activity / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos

La Flavescence Dorée (FD) est une maladie épidémique infectant les vignes européennes causée par un phytoplasme (pFD) qui est transmis de vigne à vigne par l’insecte vecteur Scaphoideus titanus. Aucun cépage actuellement cultivé n’est totalement résistant, mais des différences de sensibilité existent. En France, les méthodes de lutte réglementaires se concentrent sur la plantation de pieds certifiés sains, l’arrachage des plants contaminés et des traitements insecticides contre le vecteur dans les zones touchées par la FD. En plus du coût économique de ces moyens de lutte, l’usage des produits phytosanitaires impacte l’environnement et la santé humaine. Une meilleure compréhension des relations entre les différents acteurs du pathosystème vigne-pFD-S. titanus est donc essentielle pour pouvoir appliquer d’autres stratégies de lutte. Dans ce contexte, les principaux objectifs de ma thèse étaient (1) d’identifier et de caractériser au niveau fonctionnel des gènes codant des protéases de surface, les FtsH, potentiellement impliquées dans la virulence du pFD, (2) de caractériser la multiplication et la diffusion du pFD dans la vigne en s’affranchissant des interactions vigne-S. titanus, (3) d’initier la caractérisation du déterminisme génétique de la résistance par analyse d’un pool de descendants issus du croisement entre un cépage sensible (CF) et un cépage peu sensible (Mag) et (4) d’identifier des différences de dérégulations géniques entre le cépage très sensible CS et le cépage peu sensible M par comparaison des transcriptomes. Les résultats indiquent que (1) huit gènes FtsH sont codés par le pFD et s’expriment de manière différentielle selon l’hôte, (2) les différences de sensibilité à la FD chez la vigne sont dues au moins en partie aux interactions vigne-pFD, (3) une ségrégation des caractères « infection des plantes » et « multiplication du pFD » dans cette descendance est obtenue et (4) le M active des voies métaboliques différentes par rapport au CS lors de l’infection. A terme, la compréhension puis l’exploitation des différences de sensibilités des cépages pourront contribuer à l’élaboration de pistes alternatives à la lutte actuelle contre la FD dans le cadre d’une réduction des intrants. / Flavescence Dorée (FD) is a severe epidemic disease of grapevine caused by a wall-less bacteria, a phytoplasma (pFD), transmitted by the insect vector Scaphoideus titanus. There is no resistant variety, but differences in susceptibility between Vitis exist. In France, the actual control consists in insecticide treatments against the vector, up-rooting of diseased plants and sanitary control of plants. It has high economic and environmental impacts. A better understanding of the pathosystem FDp-grapevine-S. titanus is essential to develop alternative measures. In this context, the main objectives of my thesis were (1) to identify and characterize the expression of genes encoding surface proteases, FtsH, potentially involved in the virulence of the pFD, (2) to characterize the FDp multiplication and diffusion in Vitis without taking into account grapevine-S. titanus interactions, (3) to initiate the characterization of genetic determinism by phenotyping a pool of progeny from the cross between the susceptible variety CF and the poorly susceptible variety Mag and, (4) to identify differences in gene expression between the very susceptible variety CS and the poorly susceptible variety M by transcriptomes comparison. Results indicate that (1) eight FtsH genes are encoded by the FDp and are differentially expressed between plant and insect hosts, (2) differences in FD susceptibility in Vitis are caused, at least in part, by Vitis-FDp interactions, (3) a segregation of the characters "plant infection" and "pFD multiplication" is obtained in the progeny and, (4) M activates different metabolic pathways than CS to respond to FDp infection. Such knowledge can contribute to the development of alternative methods for limiting phytosanitary inputs.

Doctor of Philosophy / Department of Entomology / C. M. Smith / Brenda Oppert / Coleopteran insects compensate for dietary protease inhibitors by a number of mechanisms. To study this compensation response at the molecular level, the digestive proteases of Tenebrio molitor were studied. Biochemical studies of the pH optima and inhibitor sensitivity of proteases indicated the cysteine proteases were mostly in the anterior and serine proteases were in the posterior midgut of T. molitor larvae. Expressed Sequence Tags (ESTs) from T. molitor larval midgut cDNA libraries contained sequences encoding putative digestive proteases. Of a total of 1,528 cDNA sequences, 92 cDNAs encoded proteases, and 50 full-length cDNAs were grouped into serine, cysteine and metallo protease classes. Sequences tmt1a, tmt1b and tmt1c were identified as genes encoding isoforms of T. molitor trypsin, and tmc1a encoded T. molitor chymotrypsin. The general distribution cysteine protease transcripts in the anterior and serine protease transcripts in the posterior midgut, of T. molitor larvae, was in agreement with the biochemically-characterized compartmentalization of proteases. Expression analyses of selected transcripts demonstrated varied expression patterns across five developmental stages of T. molitor, with maximal expression of most protease transcripts in first instar larvae. Dietary serine and cysteine protease inhibitors fed in combination to early-instar T. molitor larvae caused a significant delay in larval growth in 21-day-old larvae. Real-time quantitative PCR analysis of RNA isolated from larvae fed different protease inhibitor treatments indicated that dietary inhibitors affected the expression of serine and cysteine proteases. Larvae fed soybean trypsin inhibitor, a serine protease inhibitor, compensated by the hyperproduction of proteases from the same class, as well as the upregulation of cysteine proteases. A cysteine protease inhibitor, E-64, caused a reduction in the hyperproduction of all proteases, and, in combination with the soybean trypsin inhibitor, lowered the compensation response of T. molitor larvae to negligible levels. These data suggest that T. molitor larvae are more sensitive to the effects of cysteine protease inhibitors, perhaps because these proteases are the first line of defense for larvae against plant protease inhibitor. The bioassay and molecular studies suggested that combinations of inhibitors that target both serine and cysteine proteases are needed to effectively control larval infestations of T. molitor.

L'exposition à la chaleur, notamment lors d'incendies est particulièrement dévastatrice et dans le cas d'objets du patrimoine elle entraine la destruction de tout ou partie de ces témoins du passé. Notre étude porte sur les effets de la chaleur sur le cuir, matériau largement présent dans les collections patrimoniales.A ce jour, aucune méthode de restauration permettant d'inverser les effets de la chaleur n'a été développée. Le premier objectif de notre étude est d'évaluer les effets d'une exposition à une chaleur sèche par une caractérisation systématique d'échantillons avant et après exposition à la chaleur. Des échantillons modèles, issus d'une même peau de veau de tannage végétal connu, ont été utilisés et caractérisés à différentes échelles structurales par un large ensemble de techniques physico-chimiques et biochimiques avant et après chauffage.Au-delà du brunissement et de la rétraction visible du cuir, la chaleur induit de nombreuses altérations au niveau de la structure du matériau, notamment, une perte de masse, une fonte des structures cristallines, une augmentation de l'hydrophobie ainsi qu'une rigidification. Une partie de ces changements sont attribués à l'agrégation protéique mise en évidence par cette recherche.Le second objectif était de développer une méthode de restauration innovante basée sur l'utilisation de molécules biologiques afin de respecter la nature de l'objet. Des enzymes de type protéase, capables de rompre les agrégats protéiques ont été utilisées. Un des défis est d'apporter suffisamment d'eau, nécessaire pour l'activité de l'enzyme, sans mouiller le cuir pour éviter tout dommage supplémentaire. Plusieurs supports d'application de la protéase ont été testés. Avec une émulsion enzymatique les résultats obtenus ne mettent en évidence ni coloration, ni rétraction et dans certains cas un gain de souplesse est observé. Des résultats encourageants ont également été obtenus dans le cas d'un cuir de veau historique (XIXe siècle). Des mesures complémentaires ont fait attribuer ces propriétés principalement à l'émulsion elle-même, cependant des mesures à plus long terme semblent mettre en évidence un effet positif de l'enzyme sur le gain de souplesse. Sous réserve de nouvelles caractérisations à des temps plus longs, le traitement élaboré pourrait constituer un nouveau support de restauration par voie biologique.Mots clefs : cuir - dénaturation thermique – agrégation protéique – bio-restauration – protéases / Heat, induced by fire, is particularly devastating for cultural heritage objects as it causes the destruction of all or part of these witnesses of the past. In this study, we focused on leather, a material largely present in heritage collections. Until now, no restoration method has been developed to treat the damaging effects of heat.The first aim of our study was to evaluate the effects of dry heat on leather samples through a systematic characterization. Model samples from a calf skin vegetable tanned in known conditions were used and the consequences of heat exposure was characterized at different structural scales using a range of physical, chemical and biochemical methods.Besides the visible browning and shrinkage of leather, heat induces many changes including a loss of mass, the melt of the crystalline regions, an increase in both hydrophobicity and rigidity. Some of these changes result from the protein aggregation induced by exposure to heat and evidenced by our research.Our second goal was to develop an innovative restoration method based on the use of biological molecules in order to respect the nature of the object. Enzymes such as proteases, able to hydrolyze protein aggregates, were used. One of the challenges was to provide the water necessary for the enzyme activity without wetting the leather surface in order to avoid further damage of the leather. Several enzyme supports were tested. The use of an enzymatic emulsion reveals neither darkening nor retraction and in some cases a flexibility gain is observed. Encouraging results were also obtained in the case of an ancient book cover made from calfskin and dated from the nineteenth century. Additional measurements lead to attribute its effect mainly to the emulsion itself, however longer-term measurements appear to show a positive effect of the enzyme on the flexibility gain. Although further characterizations on the long term are required, the treatment may constitute a new support for leather bio-restoration.Keywords: leather – thermal denaturation – protein aggregation – bio-restoration - proteases

Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE). Anticoagulants are very important for the treatment of thrombotic diseases. The adverse effects associated with current clinically used anticoagulants warrant the continuous search for new agents. Thrombin, being the central player in the coagulation cascade, remains a very important target for anticoagulant therapy, however drugs inhibiting its activity carry the risk of prolonged bleeding. Based on a previously identified sulfated benzofuran thrombin inhibitor, we have developed analogs with submaximal inhibition of the protease. These agents inhibit thrombin with efficacies approaching 50%, for both chromogenic and macromolecular substrates, ensuring a basal level of thrombin activity even at saturating inhibitor concentrations. The most potent of these compounds had a potency of 1.8 µM, 2-3 fold better than the lead. Additionally, these compounds utilize an allosteric mechanism for thrombin inhibition. Further, studies have revealed structural features responsible for submaximal thrombin inhibition. Fibrinolysis is an important part of hemostasis and plasmin is the most important fibrinolytic enzyme. Anti-plasmin agents are thus important for conditions such as hemophilia; however, there are no clinically used direct plasmin inhibitors. By structural modifications of a previously identified sulfated diflavonoid plasmin inhibitor, we have achieved a compound with 12-fold better potency (IC50 = 6.3 ± 0.4 µM), and a selectivity index of at least 22 over closely related serine proteases. We have shown that this compound inhibits plasmin mediated clot lysis, and further demonstrated that its activity is reversible using protamine sulfate, indicating its potential as a lead for the development of clinical anti-plasmin agents. HNE, a serine protease associated with inflammatory diseases is known to be inhibited by GAGs. However, the interactions at the molecular level have remained elusive. Using biochemical methods, and by studying the inhibitory potency of different GAGs and GAG oligosaccharides, we have shown that an octasaccharide may be the ideal GAG length for the achievement of potent HNE inhibition. Under our assay conditions, the inhibition of HNE by an octasaccharide species was only 5-fold less than that of unfractionated heparin, whereas the hexasaccharide species was 30-fold less active. The data also suggests that the inhibition of HNE by GAGs is via an allosteric mechanism and using molecular modeling, we have identified putative GAG binding sites on HNE and further identified GAG species with potential selectivity for anti-HNE activity

No description available.

Le recrutement des neutrophiles qui caractérise l’inflammation observée lors de différentes pathologies pulmonaires conduit à la libération dans le milieu extracellulaire de protéases à sérine qui sont en partie responsables de la dégradation du tissu pulmonaire et/ou de la chronicité de l’inflammation. L’objectif initial de cette thèse était de développer une méthode de quantification de ces protéases, à partir des condensats d’air exhalé. En dépit de la sensibilité de la technique nous n’avons pas été en mesure de détecter des quantités significatives de protéases actives dans ces condensats. Ces résultats négatifs ont néanmoins permis de confirmer des hypothèses sur la distribution des protéases dans le milieu extracellulaire. La deuxième partie des travaux a été consacrée à la validation du modèle souris exposée à la fumée de cigarette comme modèle animal de bronchopneumopathie chronique obstructive. Nous avons purifié les trois protéases à sérine du neutrophile murin et avons construit des nouveaux substrats sensibles et spécifiques à partir des informations fournies par des études de modélisation moléculaire. Ces nouveaux outils permettent de valider l’utilisation du modèle souris pour comprendre le rôle des protéases à sérine dans la génération de peptides chimiotactiques au cours de la BPCO. / Neutrophils recruitment is a hallmark of the inflammation associated with different lung diseases. This recruitment leads to the release in the extracellular matrix of serine proteases that are responsible at least in part, of the degradation of the pulmonary tissue and/or of the chronicity of inflammation. The initial objective of this thesis was to develop a method of quantification of these proteases, based on the analysis of exhaled air condensate. But in spite of the sensitivity of the methods, we have not been able to detect any significant activity of neutrophil serine proteases in these condensates. This negative result however gave support a hypothesis we formulated on the extracellular biodistribution of proteases in lung secretions. The second part of this thesis was devoted to the validation of an animal model of chronic obstructive pulmonary disease, i.e. the mouse exposed to cigarette smoke. We have purified three murine serine neutrophil proteases and developed new sensitive and specific FRET substrates which were designed starting from molecular modeling studies. These new tools that validate the use of the mouse model of human COPD, will be of great help to understand the role of serine proteases for generating chemotactic peptides during this chronic disease.

En réponse aux attentes actuelles des consommateurs pour des produits de haute qualité nutritionnelle et environnementale, et face aux impératifs industriels conduisant à minimiser les risques et l’impact environnemental lors de l’extraction à l'hexane de l’huile de la graine de colza, l’étude de l’extraction aqueuse avec assistance enzymatique de cette huile a été reprise avec 2 objectifs principaux : déterminer les enzymes et mélanges d'enzymes adaptés à la meilleure déstructuration du tissu adipeux végétal, et d'autre part étudier les propriétés et la déstabilisation de l'émulsion formée. De l'optimisation de ces 2 séquences du process dépendent les rendements finaux en huile de l'extraction et les propriétés du tourteau, qui constituent les clés économiques de l'émergence de cette nouvelle technologie. Pour cela, après caractérisation physicochimique des constituants de la graine, protéases et polysaccharides hydrolases ont été testées seules ou en combinaison afin d’optimiser le rendement en huile libre et en huile contenue dans l’émulsion engendrée lors de l’extraction et obtenue par centrifugation. Après caractérisation de l’émulsion (rhéologie, diffusion statique de la lumière, pH, conductivité), des tests de déstabilisation physicochimiques ou thermo-mécaniques ont été mis en œuvre afin de séparer les constituants de l’émulsion formée, et obtenir ainsi la libération de l'huile. Les tests réalisés ont permis de retenir trois procédés de déstabilisation: l’addition de talcs, l’inversion de phase par addition d’huile exogène en présence de NaCl dans la phase aqueuse, et les cycles de congélation/décongélation. Afin d’apporter les premiers éléments de l’optimisation de ce dernier procédé, une étude par planification expérimentale a été mise en œuvre / Consumer's concerns about the quality and environmental impact of the products as well as the industrial requirements regarding the risk assessment and the environmental and health repercussions of the solvent extraction of rapeseed oil using hexane led us to work on the optimisation of the aqueous enzymatic extraction of this oil. The study has been carried out to determine the best combination of enzymes able to achieve the disruption of the vegetal adipose tissue, and to characterize the emulsion obtained after centrifugation. The final objective was to maximize the yields of the oil extraction and to obtain adequate nutritional properties of the cake. After the physicochemical characterization of the rapeseed raw material, several proteases and polysaccharide hydrolases have been tested individually and in combination in order to optimize the removal of free oil and the emulsion oil yield occurring during the aqueous extraction process. The physicochemical properties of the emulsion have been determined: rheological properties, pH, conductivity, spectroscopy by Short Angles Light Scattering). Thereafter some physicochemical and thermo-mechanical treatments have been carried out to destabilize the oil-in-water emulsion obtained after the centrifugation, which contained a large part of the total oil of the reaction mixture. Three destabilization processes appeared particularly interesting to increase the free oil removal from the emulsion: talc addition before centrifugation, phase inversion by addition of exogenous oil in presence of NaCl in the aqueous phase, and freezing/thawing cycles. Finally, an optimisation trial of the freezing/thawing process using a Doehlert experimental design has been done as an example

O escorpião amarelo é uma das principais espécies de interesse médico no Brasil e o tratamento recomendado em caso de acidentes é o uso do antiveneno. Pouco se sabe sobre os componentes proteolíticos de seu veneno e seus efeitos no envenenamento. O objetivo deste trabalho foi isolar peptidases do veneno de T. serrulatus e as caracterizar bioquimicamente, além de avaliar o potencial neutralizante de antivenenos. O veneno total foi caracterizado, buscando atividades proteolíticas relevantes. Após isso, foram isoladas duas metalloserrulases (ms3 e ms4) e uma ACE-like do veneno. Estudos bioquímicos como determinação de temperatura e pHs ótimos, influência de sais, determinação de constantes catalíticas e pontos de hidrólise dos substratos foram determinados para as proteases purificadas. Conclui-se que ativação/inativação de peptídeos bioativos in vitro pelas proteases são informações importantes e que deverão continuar sendo estudadas no futuro. / The yellow scorpion is one of the main species of medical interest in Brazil and the recommended treatment in case of accidents is the use of antivenom. Little is known about the proteolytic components of its venom and its effects on envenomation. The objective of this work was to isolate peptidases from T. serrulatus venom and to characterize them biochemically, besides to evaluating the neutralizing potential of antivenoms. The total venom was characterized, in searching for relevant proteolytic activities. After that, two metalloserrulases (ms3 and ms4) and one ACE-like venom were isolated. Biochemical studies such as determination of temperature and optimum pHs, influence of salts, determination of catalytic constants and hydrolysis points of the substrates were determined for the purified proteases. It was concluded that the activation / inactivation of bioactive peptides in vitro by proteases are important information and should be further studied in the future.

O escorpião amarelo é uma das principais espécies de interesse médico no Brasil e o tratamento recomendado em caso de acidentes é o uso do antiveneno. Pouco se sabe sobre os componentes proteolíticos de seu veneno e seus efeitos no envenenamento. O objetivo deste trabalho foi isolar peptidases do veneno de T. serrulatus e as caracterizar bioquimicamente, além de avaliar o potencial neutralizante de antivenenos. O veneno total foi caracterizado, buscando atividades proteolíticas relevantes. Após isso, foram isoladas duas metalloserrulases (ms3 e ms4) e uma ACE-like do veneno. Estudos bioquímicos como determinação de temperatura e pHs ótimos, influência de sais, determinação de constantes catalíticas e pontos de hidrólise dos substratos foram determinados para as proteases purificadas. Conclui-se que ativação/inativação de peptídeos bioativos in vitro pelas proteases são informações importantes e que deverão continuar sendo estudadas no futuro. / The yellow scorpion is one of the main species of medical interest in Brazil and the recommended treatment in case of accidents is the use of antivenom. Little is known about the proteolytic components of its venom and its effects on envenomation. The objective of this work was to isolate peptidases from T. serrulatus venom and to characterize them biochemically, besides to evaluating the neutralizing potential of antivenoms. The total venom was characterized, in searching for relevant proteolytic activities. After that, two metalloserrulases (ms3 and ms4) and one ACE-like venom were isolated. Biochemical studies such as determination of temperature and optimum pHs, influence of salts, determination of catalytic constants and hydrolysis points of the substrates were determined for the purified proteases. It was concluded that the activation / inactivation of bioactive peptides in vitro by proteases are important information and should be further studied in the future.