This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....

La fibrose kystique (FK) est caractérisée par une inflammation pulmonaire chronique. Cette dernière est définie entre autres par une augmentation importante de la production de mucus. Celui-ci s'accumule dans les bronches empêchant ainsi l'éventuel passage de l'air. De plus, le mucus forme une barrière protectrice pour les bactéries qui va causer l'inflammation des poumons. La protéase human airway trypsin-like (HAT) a été retrouvée dans les expectorations de patients atteints d'asthme et bronchite chronique, pathologies similaires à la FK. Elle semble jouer un rôle dans l'inflammation car elle augmente entre autres la production de MUC5AC, protéine composant majoritairement le mucus. Le but de cette étude était de synthétiser la HAT dans une conformation active dans un système eucaryote pour étudier ses propriétés biochimiques, enzymatiques ainsi que son potentiel pro-inflammatoire dans la lignée cellulaire pulmonaire Calu-3. L'ADNc codant pour la partie soluble de la HAT a été inséré dans le vecteur pMT-BiP V5-His. Cet ADN recombinant fut ensuite transfecté dans les cellules de Drosophile Schneider 2 (S2). La HAT recombinante a par la suite été purifiée et son activité protéolytique testée avec un substrat fluorogénique. Ses propriétés biochimiques et enzymatiques ont été étudiées ainsi que son effet sur la production d'IL-8 et sa détection dans les expectorations de patients. La HAT purifiée était «enzymatiquement» pure, donc l'effet protéolytique observé avec le substrat fluorogénique est dû à la protéase. Les analyses de ses propriétés biochimiques montrent que l'activité protéolytique de la HAT est inhibée par un pH acide (<6.5), certains inhibiteurs synthétiques (l'aprotinine, la leupeptine et le PEFABLOC) et endogène (i.e: alpha-2 anti-plasmine (a2-AP)) de protéases à sérine. D'un autre côté, les essais enzymatiques révèlent que la HAT a une préférence pour les substrats ayant une arginine en P4, P3 et Pl plutôt que ceux ayant un glutamate en P4, P2 et P1'. Finalement, l'étude sur l'implication physiologique de l'enzyme démontre que la protéase ne semble pas avoir d'effet ou au plus peu d'effet sur la production d' IL-8. [Symboles non conformes]

The main purpose of the master thesis project is to investigate the influence of selected serine protease inhibitors (SPI) on the catalytic action of the serine proteases chymotrypsin and trypsin, in a conjugated and non-conjugated state. The inhibitors included for this study were extracted from Solanum tuberosum, i.e.common potato. The purification method included in this study consist of crude extraction by mixer, followed by a salt-out procedure with ammonium sulphate. Further purification steps were cation exchange chromatography and, finally, gel filtration to obtain SPI of high purity. The purified sample was then characterized by SDS-page and kinetic activity measurement of trypsin and chymotrypsin action on synthetic substrate derivate, N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPA) and N-Succinyl-L-phenylalanine-p-nitroaniline (SFpNA) respectively. The characterization showed inhibitory inactivation of both pancreatic proteases. This would indicate successful extraction of SPI. To investigate inhibitory action in a conjugated state, either enzyme or inhibitor was immobilized onto aluminium oxide membranes. Then two different experimental setups were tested, called experiment 1 and 2. In experiment 1, the inhibitor was immobilized and the interaction was monitored from a retention shift of enzyme flow-through compared to a blank column, using detection at 280 nm of the enzyme. In experiment 2 the enzyme was instead immobilized and a mixture of inhibitor and substrate was circulated with monitoring of the catalytic activity. The main goal was thus to measure the effects on the kinetics in the conjugated state compared to enzyme and inhibitor in the free state. The result from both experiment 1 and 2 did not yield consistent and reliable result so the discussed method should be regarded as preliminary results. The study also includes investigation of inhibitor-enzyme interaction as revealed by molecular mass data to determine complex formation. This part was conducted with static light scattering analysis to determine the stoichiometry for the interaction between pancreas proteases and the inhibitor. Results from light scattering showed promising indication of many-to-one interaction between enzyme and inhibitor, which have been seen by previous studies. It should be considered a preliminary result as complex formation does not exclude aggregation of enzymes or inhibitor in the solution.

Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues. / Dissertation

A pancreatite crônica é uma desordem complexa, na qual a interação entre fatores ambientais e genéticos resulta na enfermidade. O presente estudo incluiu 148 pacientes com diagnóstico de pancreatite crônica, 110 etilistas crônicos e 297 controles sadios com o objetivo de investigar a frequência de tabagismo e das mutações N34S e P55S do gene SPINK1 e R254W do gene CTRC nesta população. Foi aplicado questionário presencial e realizada reação de sequenciamento para a pesquisa das mutações genéticas, após assinatura do Termo de Consentimento Livre e Esclarecido. Os portadores de pancreatite crônica possuíam etiologia alcoólica em 74% das vezes e idiopática em 26%. A pancreatite alcoólica apresentou-se de maneira distinta da pancreatite crônica idiopática, sendo que o primeiro grupo é composto por maior prevalência do gênero masculino (88,18% versus 34,21%), por maior média de idade (55,64 anos versus 45,20 anos), menor frequência de caucasianos (63,89% versus 84,21%), menor escolaridade (23,30% concluíram ensino médio ou superior versus 57,89%) e maior frequência de repercussões da doença, como diarréia (54,21% versus 24,24%), emagrecimento (56,07% versus 24,24%), diabete melito (57,94% versus 36,36%) e ocorrência de pseudocistos pancreáticos (31,78% versus 12,12%), repercussões estas que não foram acompanhadas de maior frequência de alterações morfológicas, como calcificações pancreáticas ou dilatação do ducto pancreático principal. A frequência de tabagismo foi significativamente maior em pacientes com pancreatite crônica alcoólica do que em etilistas sem pancreatite crônica, podendo ser considerado cofator de risco para o desenvolvimento da pancreatite crônica entre alcoolistas (p = 0,002); a frequência da mutação N34S do gene SPINK1 em pacientes com pancreatite crônica foi de 3,38%, maior do que a frequência de 0,49% encontrada nos grupos controle (p = 0,016); a frequência de 2,03% da mutação P55S do gene SPINK1 e a frequência de 0,67% da mutação R254W do gene CTRC, encontradas nos pacientes com pancreatite crônica, não diferiram estatisticamente quando comparadas às frequências, de 0,49% de ambas mutações, encontradas nos grupos controle. (p = 0,120 e 0,751). Pela investigação da associação de tabagismo e da mutação N34S do gene SPINK1 com as características clínicas e morfológicas da pancreatite crônica, verificou-se que a mutação N34S não se associou a maior gravidade da apresentação clínica ou morfológica da pancreatite crônica; no entanto o tabagismo associou-se a maior frequência de diabete melito entre os portadores de pancreatite crônica. Concluiuse que o tabagismo e a mutação N34S do gene SPINK1 podem ser considerados cofatores de risco para o desenvolvimento da pancreatite crônica / Chronic pancreatitis is a complex disorder in which the interaction between environmental and genetic factors results in the disease. This study included 148 patients with chronic pancreatitis, 110 chronic alcoholics and 297 healthy controls in order to investigate the frequency of smoking and N34S and P55S mutation of SPINK1 gene and R254W of CTRC gene in this population. A questionnaire was applied and gene sequencing was done, after having the Informed Consent Statement. Those with chronic pancreatitis had alcoholic etiology in 74% of cases and idiopathic in 26%. Alcoholic pancreatitis presented in a distinct way of idiopathic chronic pancreatitis. The first group is composed of a higher prevalence of males (88.18% versus 34.21%), by higher mean age (55.64 years versus 45.20 years), lower frequency of Caucasians (63.89% versus 84.21%), lower education (23.30% completed secondary or higher education versus 57.89%) and worst impact from the disease such as diarrhea (54.21% versus 24.24%), weight loss (56.07% versus 24.24%), diabetes mellitus (57.94% versus 36.36%) and occurrence of pancreatic pseudocysts (31.78% versus 12 , 12%). These effects were not accompanied by increased frequency of morphological changes, such as pancreatic calcifications or dilation of the main pancreatic duct. The frequency of smoking was significantly higher in patients with alcoholic pancreatitis than in alcoholics without chronic pancreatitis, therefore tabagism may be considered as a cofactor for the development of chronic pancreatitis among alcoholics (p = 0.002); the frequency of N34S mutation of SPINK1 gene in patients with chronic pancreatitis was 3.38%, higher than the rate of 0.49% found in the control groups (p = 0.016); the frequency of 2.03% of the P55S mutation of SPINK1 gene and the frequency of 0.67% of the CTRC gene R254W mutation found in patients with chronic pancreatitis were not statistically different when compared to the frequencies of 0.49% of both mutations, found in the control groups. (p = 0.120 and 0.751) For the investigation of the association of smoking and N34S mutation of SPINK1 gene with the clinical and morphological features of chronic pancreatitis, it was noticed that the N34S mutation did not determine a greatest severity in the presentation of chronic pancreatitis, however smoking was associated with a higher frequency of diabetes mellitus in patients with chronic pancreatitis. It was concluded that smoking and the N34S mutation of SPINK1 gene are positively correlated with chronic pancreatitis

A soja vem ganhando destaque, pois alguns estudos apontam uma série de potenciais benefícios para a saúde. Estão presentes na soja dois grupos de inibidores de proteases. Alguns estudos demonstram que o tratamento térmico pode não ser suficiente para a inativação completa desses inibidores. Esses inibidores podem afetar a digestibilidade da proteína e a absorção e biodisponibilidade de alguns minerais, como o ferro. Realizaram-se cinco tratamentos: T1 e T2 - com secagem prévia + 10 e 15 minutos de forno, respectivamente; T3, T4 e T5 - sem secagem prévia + 45, 50 e 60 minutos de forno. O controle foi seco em estufa (55ºC) durante 31 horas. A quantidade de ferro nas amostras analisadas variou de 83,67 a 58,8 mg/kg e a porcentagem de ferro dialisado foi de 0,95% a 1,38%, estando de acordo com o encontrado na literatura. Os valores da digestibilidade da proteína in vitro variaram de 91,21% a 93,14%, sem que houvesse correlação com os inibidores de tripsina, sendo a melhora da digestibilidade devida ao tratamento térmico. A atividade dos inibidores de tripsina para os tratamentos com secagem prévia variou de 25,24 a 23,94 UTI/mg de proteína e para os sem secagem prévia de 46,39 a 38,33 UTI/mg de proteína. A atividade dos inibidores para o controle foi de 55,81 UTI/mg de proteína. Os valores estão de acordo com a literatura, sendo, portanto, considerados como adequados, mostrando que a tostagem em forno convencional é bastante eficaz quanto à inativação do inibidor de tripsina. Após a proteólise in vitro, verificou-se reativação dos inibidores de tripsina em todos os tratamentos analisados. Os valores variaram de 5,48 UTI/mg de proteína (controle) a 7,65 UTI/mg de proteína (T1). As maiores porcentagens foram para os tratamentos com secagem prévia, 30,31% e 28,45%, enquanto que para os sem secagem prévia foi de 14,68%, 14,12% e 19,38%. O controle apresentou 9,82% de reativação. Concluiu-se que a tostagem em forno convencional inativou os inibidores de tripsina, sendo o tratamento sem secagem prévia o mais eficaz, pois, com esse tratamento, houve menor reativação dos inibidores após a proteólise in vitro. A tostagem foi responsável pela melhora da digestibilidade da proteína e da diálise de ferro in vitro. O tratamento que promoveu a maior aceitabilidade dos grãos de soja tostados foi o que recebeu o tempo intermediário de 50 minutos, sem secagem prévia. / The soy comes gaining prominence, therefore some studies point a series of potential benefits with respect to the health. Two groups of inhibitors of proteases are present in the soy. Some studies demonstrate that the thermal treatment can not be enough for the complete inactivation of these inhibitors. These inhibitors can affect the digestibility of the protein and the absorption and availability of some minerals, as the iron. Five treatments had been do: T1 and T2 - with previous drying + 10 and 15 minutes of oven, respectively; T3, T4 and T5 - without previous drying + 45, 50 and 60 minutes of oven. The control was dry in oven (55ºC) during 31 hours. The amount of iron in the analyzed samples varied of 58,8 the 83,67 mg/kg and percentage of dialysed iron was of 0,95% the 1.38%, being in accordance with the found one in literature. The values of the digestibility of protein in vitro had varied of 91,21% the 93.14%, without that it had correlation with trypsin inhibitors, being the improvement of the digestibility due to the thermal treatment. The trypsin inhibitors activity for the treatments with previous drying varied of 25,24 the 23,94 UTI/mg of protein and for the ones without previous drying varied of 38,33 the 46,39 UTI/mg of protein. The inhibitors activity for the control was of 55,81 UTI/mg of protein. The values are according with the literature, being, therefore, considered as adjusted, showing that the roasting in conventional oven is sufficiently efficient how much to the inactivation of trypsin inhibitors. After proteolysis in vitro, verified reactivation of trypsin inhibitors in all the analyzed treatments. The values had varied of 5,48 UTI/mg of protein (control) the 7,65 UTI/mg of protein (T1). The biggest percentages had been for the treatments with previous drying, 30.31% and 28.45%, while that for the ones without previous drying it was of 14,68%, 14.12% and 19,38%. The control presented 9.82% of reactivation. The treatment the most efficient without previous drying was concluded that the roasting in conventional oven inactivated trypsin inhibitors, being, therefore, with this treatment, proteolysis had minor after reactivation of inhibitors in vitro. The roasting was responsible for the improvement of the digestibility of protein and dialyse of the iron in vitro. The treatment that promoted the biggest acceptability of the roasted grains of soy was what it received the time intermediate from 50 minutes, without previous drying.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

SOUZA, Pedro Filho Noronha de.Purificação, caracterização bioquímica e atividade antimicrobiana de uma nova albumina 2s da torta da mamoneira (Ricinus communis L.), com atividade inibitória contra tripsina. 2012. 114 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-27T14:14:02Z No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:11:34Z (GMT) No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) / Made available in DSpace on 2016-08-02T20:11:34Z (GMT). No. of bitstreams: 1 2012_dis_pfnsouza.pdf: 1847174 bytes, checksum: 6675a05ab248cfb741080071e366161c (MD5) Previous issue date: 2012 / Castor bean (Ricinus communis L.) is an important crop for the Northeast of Brazil, which recently has been used to produce biodiesel. Around 90% of the castor bean production in Brazil is concentrated at Northeast region and the state of Ceará is the second largest producer. During the oil extraction process from the castor bean seeds, a resulting residue, called castor cake, is underutilized. However, this byproduct is rich in protein and micronutrients such as nitrogen, phosphorus and potassium, an attribute that qualifies it as a stuff that could be used as organic fertilizer or as animal feed, for example, bringing added value to it. Unfortunately, its use as food has not been possible because of the presence of toxic elements and allergens (ricin, ricinine, complex allergens) in its composition, unless it were previously submitted to a detoxification process. To date, the existing technologies to this end are not economically viable on an industrial scale. Therefore, studies to add value to this abundant byproduct generated in the castor bean biodiesel productive chain is of paramount importance. Thus, in this context, this study was performed to identify, isolate, purify and characterize new bioactive molecules of de-oiled castor cake with biotechnological potential. Through extraction of soluble proteins with 50 mM Tris-HCl pH 7.5, fractionation with ammonium sulfate (50-75%), following by hydrophobic interaction chromatography in Phenyl-Sepharose column and ion exchange chromatography (DEAE-Sepharose), a protein, named Rc-2S-Alb, able to inhibit trypsin was purified. Rc-2S-Alb has a molecular mass of approximately 75.8 kDa, as determined by SDS-PAGE, and under reducing conditions showed a large and a small protein band of 15.8 kDa and 10.5 kDa, respectively. Its NH2-terminal sequence showed similarity with the following proteins: putative 2S albumin precursor (89%), chain A of RicC3 (89%), and chain A of mabilin-1 (89%), all from R. communis seeds; and with the short chain of a "napin-like" protein from Brassica napus (89%). In addition, these similar proteins have two highly conserved domains: QEVQRKDLS and YLRQS. Comparison of the partial primary structure of Rc-2S-Alb generated by the ESI-Q-TOF MS/MS analysis of 17 tryptic peptides, showed 43% similarity to Mabinlin-1 (pI/Mr 6.7 and 29.3 kDa) from R. communis. There were also high similarities among the three-dimensional structures of Rc-2S-Alb, RicC3 and Mabinlin-1. Rc-2S-Alb did not inhibit the spore germination of the phytopathogenic fungi Fusarium oxysporum and Rizoctonia solani, but promoted aggregation of their respective spores. Moreover, Rc-2S-Alb did not inhibit the mycelial growth of Fusarium oxysporum, Fusarium solani, Rizoctonia solani and Collethotricum gloeosporioides. Contrary, Rc-2S-Alb was effective in inhibiting the growth of the human pathogenic bacteria Pseudomonas aeruginosae, Klebsiella pneumoniae and Bacillus subtilis, at low concentrations. In conclusion, it was established a protocol to purify a new 2S albumin from castor bean cake, which inhibits trypsin and has important antibacterial activity. Thus, the Rc-2S-Alb should be further studied in order to verify its effectiveness as new alternative therapeutic agent against resistant bacteria to commercial antibiotics, which will contribute to improve the human health. / A mamoneira (Ricinus communis L.) é uma cultura importante para a região Nordeste do Brasil, onde, recentemente, tem sido utilizada para produção de biodiesel. O Nordeste detém 90% da produção brasileira de mamona, sendo o Ceará, o segundo maior produtor. No processo de extração de óleo das sementes de mamoneira para produção de biodiesel, o resíduo resultante, denominado de torta da mamona, representa um subproduto pouco utilizado. Apesar disso, essa torta é rica em proteínas e micronutrientes, como nitrogênio, fósforo e potássio, atributo que a qualifica como um insumo que poderia ser utilizado como adubo orgânico, ou mesmo como ração animal, o que lhe agregaria valor comercial. Todavia, seu uso como alimento não tem sido possível por causa da presença de elementos tóxicos e alergênicos (Ricina, Ricinina, Complexos Alergênicos) na sua composição, a não ser que passasse por processamento para sua destoxificação. Infelizmente, tecnologia economicamente viável para esse fim, em escala industrial, ainda é inexistente. Portanto, há necessidade de pesquisas que venham a agregar valor a esse subproduto abundante da cadeia produtiva do biodiesel. Assim, nesse contexto, o presente trabalho está inserido num projeto cujo objetivo maior é identificar, isolar, purificar e caracterizar novas moléculas bioativas da torta delipidada de sementes de mamoeira com potencial biotecnológico. Através de extração de proteínas solúveis com tampão Tris-HCl, 50 mM, pH 7,5, e fracionamento do extrato obtido com sulfato de amônio (50-75%), cromatografia de interação hidrofóbica em coluna Phenyl-Sepharose e cromatografia de troca iônica (DEAE-Sepharose) foi possível purificar uma albumina 2S, denominada Rc-2S-Alb, capaz de inibir tripsina. A Rc-2S-Alb apresentou massa molecular de, aproximadamente, 75,8 kDa, determinada por SDS-PAGE, mas, em condições redutoras, apareceu como uma banda maior de 15,8 kDa e outra menor com 10,5 kDa. Sua sequência NH2-terminal revelou haver similaridade (89%) com o precursor putativo da albumina 2S de R. communis já descrita, com a cadeia A da estrutura da RicC3 (89%), com a cadeia A da mabilin-1, ambas, também, de R. communis (89%), com a cadeia pequena de uma proteina “napin-like”de Brassica napus (89%). Em todas essas proteínas similares, dois domínios, QEVQRKDLS e YLRQS, são altamente conservados. Comparação da estrutura primária gerada por ESI-Q-TOF MS/MS, a apartir de 17 peptídeos trípticos da Rc-2S-Alb, mostrou similaridade de 43% com a Mabinlin-1 (pI/Mr de 6,7 e 29.3 kDa) de R. communis. Em relação à estrutura tridimensional da Rc-2S-Alb, ela apresentou similaridade com a de Ric C3 e Mabinlin-1. A Rc-2S-Alb foi incapaz de inibir a germinação de esporos dos fungos fitopatogênicos Fusarium oxysporum e Rizoctonia solani, mas foi capaz de promover a aglomeração dos mesmos. A Rc-2S-Alb também não foi capaz de inibir o crescimento micelial dos fungos Fusarium oxysporum, Fusarium solani, Rizoctonia solani e Collethotricum gloeosporioides. Por outro lado, a Rc-2S-Alb foi eficiente em inibir o crescimento de Pseudomonas aeruginosae, Klebsiella pneumoniae e Bacillus subtilis, todas as bactérias patogênicas a seres humanos, quando em baixas concentrações. Como conclusão, foi possível a purificação de uma nova albumina 2S da torta da mamona, capaz de inibir tripsina e com atividade antibacteriana importante. Assim, a Rc-2S-Alb deve ser explorada no sentido de verificar sua eficácia como um novo agente terapêutico alternativo no combate a bactérias resistentes aos produtos disponíveis, hoje, no mercado, o que, se confirmado, poderá contribuir para a melhoria da saúde humana.

SILVA, Rodolpho Glauber Guedes. Purificação, caracterização bioquímica e atividade biológica de um inibidor de tripsina da torta da mamona (Ricinus communis L.). 2012. 94 f. : Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-08T14:25:06Z No. of bitstreams: 1 2012_dis_rggsilva.pdf: 2026014 bytes, checksum: e5a8d39fc94fdd25a3238ea3dc3908a1 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:19:00Z (GMT) No. of bitstreams: 1 2012_dis_rggsilva.pdf: 2026014 bytes, checksum: e5a8d39fc94fdd25a3238ea3dc3908a1 (MD5) / Made available in DSpace on 2016-08-02T20:19:00Z (GMT). No. of bitstreams: 1 2012_dis_rggsilva.pdf: 2026014 bytes, checksum: e5a8d39fc94fdd25a3238ea3dc3908a1 (MD5) Previous issue date: 2012 / The castor bean (Ricinus communis L.) culture is of great socioeconomic importance because of its seeds, which are used mainly for production of oil that can be used for various industrial purposes. The oil extraction generates a by-product of high nutritional value known as the castor cake. However, the presence of toxic and allergenic compounds hinders the use of this residue to feeding source. Another way to add value to the castor cake would be to seek bioactive molecules that could have applications in agriculture and human health. This study aimed to purify and characterize a trypsin inhibitor of castor cake and, to test this in vitro activity against phytopathogenic fungi of agricultural importance and proteases from the midgut of Aedes aegypti. The trypsin inhibitor of castor cake, named RcTI, was purified by heat treatment, affinity column in anhydrotrypsin-Sepharose® 4B and ion-exchange chromatography ResourceTM Q. RcTI has a apparent molecular mass of 14 kDa in the absence or presence of a reducing agent, pI 5.2 and isn’t a glycoprotein. The NH2-terminal sequence of purified RcTI showed 83% similarity with sulfur-rich storage protein (2S albumin) R. communis and 48% with napin-like (2S albumin) R. communis. The RcTI was stable after heat treatment at 98 °C for two hours. The inhibition on trypsin was stable in the acid pH range. However, there was a loss of approximately 20% inhibitory effect on alkaline pH (pH 8-11). The RcTI (13 µg) was unable to inhibit the germination of spores of the fungus Fusarium oxysporum, Rhizoctonia solani and Fusarium solani. However, it was able to inhibit the germination of spores of Colletotrichum gloeosporioides. RcTI 100 µg was not effective in the inhibition of vegetative growth of the fungi mentioned above. This inhibitor was effective against proteases from the midgut of Aedes aegypti with 91% inhibition. The results of the present study indicate that RcTI has biotechnological potential, can be used as an alternative to combat the important plant pathogenic fungus C. gloeosporioides and larvae of A. aegypti. / A cultura da mamoneira (Ricinus communis L.) é de grande importância socioeconômica devido às suas sementes, que são utilizadas, principalmente, na produção do óleo que pode ser empregado para diversos fins industriais. A extração do óleo gera um subproduto de alto valor nutricional, conhecido como torta da mamona. Porém, a presença de compostos tóxicos e alergênicos dificulta utilização desse resíduo como fonte de alimentação animal. Outra forma de agregar valor à torta da mamona seria buscar moléculas bioativas que pudessem ter aplicações na agricultura e saúde humana. O presente trabalho teve como objetivos purificar e caracterizar um inibidor de tripsina da torta da mamona, bem como, testar a atividade deste in vitro contra fungos fitopatogênicos de importância agrícola e proteases do intestino médio de Aedes aegypti. O inibidor de tripsina da torta da mamona, nomeado RcTI, foi purificado através de tratamento térmico, coluna de afinidade em matriz de anidrotripsina-Sepharose® 4B e cromatografia de troca iônica em ResourceTM Q. Essa proteína apresentou massa molecular aparente de 14,0 kDa na ausência ou presença de agente redutor, pI 5,2 e não é uma glicoproteína. A sequência NH2-terminal do RcTI purificado mostrou similaridade de 83% com uma proteína de armazenamento rica em enxofre (albumina 2S) de R. communis e de 48% com uma napin-like (albumina 2S) de R. communis. O RcTI mostrou-se estável após tratamento térmico a 98 ºC durante duas horas. Da mesma forma, permaneceu com atividade inibitória estável na faixa de pHs ácidos. No entanto, houve uma perda de, aproximadamente, 20% na capacidade inibitória em pHs alcalinos (pH 8-11). O RcTI (13 µg) não foi capaz de inibir a germinação de esporos dos fungos Fusarium oxysporum, Fusarium solani e Rhizoctonia solani. No entanto, foi capaz de inibir a germinação de esporos de Colletotrichum gloeosporioides. O RcTI não foi efetivo na inibição de crescimento vegetativo dos fungos mencionados acima. Esse inibidor foi efetivo contra as proteases intestinais de Aedes aegypti com 91% de inibição. A partir dos resultados obtidos neste trabalho, conclui-se que o RcTI tem potencial biotecnológico, podendo ser utilizado como uma alternativa para o combate do importante fungo fitopatogênico C. gloeosporioides e larvas de A. aegypti.