We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS-­activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized.

In the cell, as in in vitro, the final conformation of a protein is determined by it's amino acid sequence (1). Some isolated proteins can be denatured and refolded in vitro in absence of extrinsic factors. However, in order to fold in the cell, the newly synthesized polypeptide chain has to negotiate an environment far more complex than that faced by the unfolded chain in vitro. Cells have evolved proteins called “chaperones” to assist folding and assembly of polypeptides (2). Thus, the linear sequence of a protein not only contains information that specifies the final three-dimensional functional form, but also recognition motifs, which can be recognized by the cellular folding machinery. The work reported in this thesis is aimed at understanding some aspects of recognition of target substrates by the cytosolic chaperone, SecB, which forms part of the protein translocation machinery in E. coli. The sec pathway is involved in both translocation of precursor proteins across and the insertion of integral membrane proteins into the cytoplasmic membrane (3). Chapter one discusses some general aspects of protein folding and briefly describes chaperone systems, which have been extensively characterized in literature. Chapter two discusses the effect of chaperone SecB on the refolding pathway of a model substrate protein barstar, whose folding pathway has been extensively characterized (4,5). The effect of SecB on the refolding kinetics of the small protein barstar (wild type) and fluorescein labeled C82A (single Cys mutant) in 1 M guanidine hydrochloride at pH 7.0 at 25 °C has been investigated using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to late near-native intermediate (s) along the folding pathway. ESR studies and fluorescence anisotropy measurements show that SecB forms stable complexes with the near-native intermediate (s). For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. Steady state polarization measurements indicated the presence of stable complexes of barstar bound to SecB. Studies on the spin labeled C82A show an immobilization of the spin label adduct at the 40th position of barstar, suggesting that the binding of SecB to barstar occurs in that region. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models (6). Chapter three discusses the energetics of substrate:SecB interactions using the following model protein substrates: unfolded RNase A, BPTI, partially folded disulfide intermediates of alpha-lactalbumin,. The thermodynamics of binding of unfolded polypeptides to the chaperone SecB were investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29 and -0.41 kcal mol-1 K-1 respectively and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft (7). Chapter four discusses the thermodynamics of unfolding to gain insights into the mechanism of assembly and stability of the tetrameric structure. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. The value of ACP obtained was 10.7 ± 0.7 kcal mol-1 K-1, which is amongst the highest measured for a multimeric protein. At 298 K, pH 7.4. the AG°U for the SecB tetramer is 27.9 ± 2 kcal mol-1. Denaturant mediated unfolding of SecB was found to be irreversible. The reactivity of the 4 solvent exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four Cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well folded and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity (8). Chapter five discusses the bound state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI), as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58 residue protein and contains 3 disulfide groups between residues 5 and 55, 14 and 38, and 30 and 51. Single disulfide mutants of BPTI were reduced and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four, solvent accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The ESR data suggest that these cysteine residues are in close proximity when no substrate protein is bound, but move away from each other when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein. Chapter six discusses the mechanism of dissaggregation of a model peptide aggregate by chaperone SecB. The Hspl04, Hsp70 and Hsp40 chaperone system are capable of dissociating aggregated state(s) of substrate proteins, though little is known of the mechanism of the process. The interaction of the B chain of insulin with chaperone SecB was investigated using light scattering, pyrene excimer fluorescence and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B chain of insulin. We show that SecB is capable of dissociating soluble B chain aggregate as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B chain aggregate by SecB has also been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B chains, rather it binds the small population of free B chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate towards the individual B chains. Thus SecB can rescue aggregated, partially folded /misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B chain to chaperone SecB. The data suggests that the B chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate (9).

Methods for integrating enzyme systems with electrochemical reactions having applications to diagnostic sensing are described. Diagnostic tests that include biological molecules can be classified as biosensors. Existing testing methods often require trained technicians to perform, and laboratory settings with complex infrastructure. The theme of this dissertation is the development of methods that are faster, easier to use, and more applicable for non-laboratory environments. These goals are accomplished in systems using enzymatic catalysis and galvanic processes. Two biosensors with specific model pathologies have been designed and demonstrated in this study. The first assay senses a DNA fragment representing the Epstein Barr virus and uses enzyme-mediated Ag deposition over a v microfabricated chip. The chip contains a specially designed pair of electrodes in an interdigitated array (IDA). Detection is signaled by a change in the resistance between the two electrodes. The second biosensor discussed in this study is targeted towards the digestive enzyme trypsin. It is selfpowered due to its construction within an open-circuit galvanic cell. In this system, a small volume of blood serum is introduced onto the device over barriers made of protein and Al that block the anode from solution. In the presence of trypsin, the protein gel is rendered more permeable to sodium hydroxide. Adding hydroxide initiates the dissolution of the Al layer, closing the cell circuit and illuminating a light-emitting diode (LED). A relationship was observed between LED illumination time and trypsin concentration. Biosensors that utilize enzymes to generate or amplify a detectable signal are widely used, and the final project of this study uses a nanoparticle based approach to protect the catalytic activity of alkaline phosphatase (AlkP) from hostile chemicals. By incubating Au colloid with AlkP overnight and adding Ag+, core@shell nanoparticles of Au@Ag2O can be isolated that show AlkP activity. The resulting enzyme-metal composite material was analytically characterized and demonstrated greater activity in the presence of organic inhibitors relative to either wild type vi or Au colloid-associated AlkP without the Ag2O shell. The stabilization procedure is complete in one day using a onepot synthesis. This method may provide opportunities to carry out biosensing chemistry in previously incompatible chemical environments. / text

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

Protease-Activated Receptors (PARs) belong to an unusual family of G Protein Coupled Receptors (GPCRs). Each of the four known members is activated by its own N-terminus exposed by proteolytic cleavage and there is no other endogenous agonist known to date. PAR2 is the second member of the family and it has been implicated in wide range of pathophysiological conditions, particularly in various inflammatory diseases and cancers. In contrast, very little is known about the PAR2 receptor itself despite having been discovered more than 10 years ago. The purpose of this project was to improve our understanding of PAR2 regulation by discovering new agonists and antagonists and using them to probe the structural and functional properties of the receptor. Chapter 1 provides a brief literature overview of the initial discovery of PAR2, what is known about the mechanism of receptor activation, information on the structures and properties of current agonists and an antagonist for PAR2, and the putative physiological roles of human PAR2. As well, it summarizes the aims of this thesis. Chapter 2 investigates the regulation of gene expression by two different agonists of PAR2, a synthetic hexapeptide, 2f-LIGRLO-NH2, and the endogenous activator, trypsin, the idea being that genes up- or down- regulated by both agonists may more accurately profile PAR2-selective events. The effects of PAR2 activation on gene transcription in the human kidney HEK293 cell line were studied using a DNA microarray consisting of 19,000 human genes in an attempt to broadly cover the human genome and associated cell pathways with PAR2 activation. About 2,500 genes were regulated similarly by both agonists and, for genes expressed more than 5-fold, the mRNA results were further analyzed by quantitative RT-PCR techniques. PAR2 activation was shown to be associated with cellular metabolism, cell cycle, mitogen-activated protein kinase pathways, histone deacetylase and sirtuin enzymes, inflammatory cytokines and anti-complement function. Chapter 3 described a range of molecular events surrounding the activation of the receptor. PAR2 mRNA expression was quantitated by qRT-PCR and cross-checked with an intracellular Ca2+ assay. In this way whole cell PAR2 could be correlated with cell surface expression of PAR2. Three cell lines expressing high levels of PAR2 were chosen for subsequent experiments, these being colorectal carcinoma HT29, lung carcinoma A549 and human embryonic kidney HEK293 cells. Receptor activation, internalization, desensitization and resensitization assays were carried out on these cell lines to define some key functions relevant for investigating inhibitors in subsequent chapters. Chapter 4 reports a PAR2 mutagenesis study designed to identify the location of the binding site on PAR2 for a specific peptide agonist. A homology model of PAR2 based on bovine rhodopsin was used for docking of an agonist ligand, and the docking results were then investigated via two successive rounds of PAR2 mutagenesis in which the effect of each mutation (20 in all) was separately investigated by changes in agonist potency in the intracellular calcium release assay. Five PAR2 mutants showed more than a 5-fold reduction in agonist potency, while three others showed up to a 7-fold reduction. Mutations found to be important for agonist activity were mapped back to the model. Because there was extensive clustering of these key mutated amino acids, it is likely that this study has pinpointed the precise binding site of the agonist peptide in PAR2. Interestingly, this site is within the transmembrane region of the receptor. Chapter 5 reports the design, discovery and development of novel PAR2 agonists and antagonists and their regulatory effects in a diverse array of cell types. Structure-activity relationships were used to examine influences on the first, sixth and seventh positions of a PAR2 agonist peptide. At least five compounds were found herein to be equiopotent with the most potent PAR2 agonist reported. Knowledge obtained from this study was then used to create the first non-peptidic agonists for PAR2. The most potent nonpeptidic agonist (retaining one natural amino acid) was at least equipotent with the best peptide agonists. Conversion to nonpeptidic antagonists proved to be successful and this chapter reports the most potent known nonpeptide antagonist, which was selective for PAR2 and active at low micromolar concentrations. It inhibited intracellular Ca2+ release induced by different PAR2 agonists (trypsin, 2f-LIGRLO-NH2, nonpeptide agonists) in multiple cell lines (HT29, Panc-1, A549, MKN1, MKN45, MDA-MB-231, HUVEC) that have been physiologically associated with PAR2. It also inhibited release of inflammatory cytokines IL-8 and IL-6 and shows antiproliferative activity against primary human cells. The antagonist is competitive, reversible and surmountable (pA2 6.11). This thesis summarizes a large body of work that provides valuable molecular insights to PAR2 regulation, and lays the groundwork for rational design and development of novel nonpeptidic agonists and antagonists of PAR2 as potentially valuable pharmacological probes in vivo and as useful leads to development of therapeutics for inflammatory diseases and cancers.

This study has assessed the effects of pulse type and processing (soaking and cooking) on antinutritional factors (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins, phytic acid and oxalates) in a wide range of Canadian pulses, using soybean as a control. The contents of these antinutrients in Canadian pulses varied widely, but the levels were generally lower than those found in soybean. Analysis of variance indicated that both pulse type and processing had significant effects (P < 0.0001) on the investigated seeds. Soaking markedly decreased the contents of α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins and oxalates, but had no impact on phytic acid. Cooking of presoaked seeds appeared to be more effective; all proteinaceous antinutrients (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor and lectins) were decreased by 80 – 100%, and significant reductions in phytic acid content (11 – 39%) were observed for all pulses, except common beans and soybean. / February 2016

Ziel der vorliegenden Arbeit war es, den Einfluss von unterschiedlich behandelten Sojabohnen in einer Broilerration (Mais und behandeltes Soja) auf die ernährungsphysiologischen Parameter beim wachsenden Küken zu prüfen. Unter Berücksichtigung der Einflussgrößen (z. B. Zeit, mechanischer Energieeinsatz und Futterstruktur), die die Effektivität der Futterbearbeitungsverfahren beeinflussen, wurde zunächst die Behandlung einer einheitlichen Charge von Vollfettsojabohnen unter folgenden technische Bedingungen vorgenommen: A: Walzenstuhl (WS) + Konditionierung (40 min./100 °C, Standardverfahren); B: WS + Konditionierung (40 min./100 °C) + Flockierung; C: WS + Konditionierung (10 min./100 °C); D: WS + Konditionierung (10 min. /100 °C) + Expandieren (20 kWh/t); E: Hammermühle (HM) + Konditionierung (10 min./100 °C) + Extrusion (15 kWh/t); F: HM + Konditionierung (10 min./100 °C), G: HM + Konditionierung (10 min./100 °C) + Expandieren (20 kWh/t); H: HM + Expandieren (20 kWh/t); I: HM + Expandieren (20 kWh/t) mit Dampfzufuhr; K: HM + Expandieren (40 kWh/t). Mit 6 ´ 10 männlichen Broilern der Herkunft Cobb wurde im Zeitraum vom 7. - 28. Lebenstag (LT) je Behandlung ein Wachstumsversuch durchgeführt. Während des Wachstumsversuchs wurden Daten zum Futterverzehr, Lebendmassezuwachs und Futteraufwand sowie Energie- und Proteinansatz über Körperanalyse erfaßt. Die ileale Aminosäureverdaulichkeit der einzelnen Mischungen wurde in jeweils 4 gepoolten Chymusproben (9 Tiere / gepoolte Probe) mit Hilfe eines Indikators (HCl-unlösliche Rohasche, Zusatz von 1 % Celite) nach Verfütterung vom 21. - 28. LT bestimmt. Parallel zum Wachstumsversuch wurde ein Stoffwechselversuch (6 männliche Küken der Herkunft Cobb je Behandlungsstufe) im Zeitraum vom 15. - 21. LT bei Fütterung der Versuchsmischung durchgeführt. Dabei erfolgte 3-mal täglich eine quantitative Exkrementsammlung. Neben der N- Bestimmung in den Exkrementen der Basis für die N-Bilanzmessung wurde der Bruttoenergiegehalt erfaßt und auf dieser Grundlage der Gehalt an N-korrigierter umsetzbarer Energie (MEn) in den Futtermischungen bestimmt. Weiterhin wurde aus den Daten der Bilanzmessungen der physiologische Nutzwert (PNu) als Kriterium der Proteinqualitätsbeurteilung sowie die Lysinwirksamkeit abgeleitet. Am Ende des Stoffwechselversuchs wurden die Tiere geschlachtet und die Trypsinaktivität im Chymus des Jejunums bestimmt. Die Ergebnisse lassen sich folgendermaßen zusammenfassen: 1. In Abhängigkeit von den gegebenen Bedingungen der Futterbehandlungsverfahren konnten unterschiedliche Trypsininhibitoraktivitäten (TIA) in den behandelten Sojabohnen erzielt werden. Eine starke Reduzierung des TIA war insbesondere bei den behandelten Sojabohnen A bis G erkennbar. Die relative Restaktivität lag bei diesen Gruppen (im Vergleich zur rohen Sojabohne) bei 16-27 %. Im Gegensatz dazu wurde eine höhere relative Restaktivität in den behandelten Sojabohnen H, I und K in Höhe von 75 %, 32 % und 48 % festgestellt . 2. Die Futteraufnahme, Lebendmassezunahme und Futterverwertung waren bei der Gruppe H infolge der höchsten Restaktivität des Trypsininhibitors signifikant niedriger als bei der Kontrolle A und anderen Gruppen (B, C, D, E, F, G, I und K). Analog dazu führte die höchste TIA in den Sojabohnen H ebenfalls zu einem signifikant verminderten Nährstoffansatz im Ganzkörper. Der Einfluss der höchsten Restaktivität des Trypsininhibitors auf den PNu war bei Gruppe H wenig ausgeprägt. 3. Die N-korrigierte umsetzbare Energie in den Futtermischungen B (14,87 MJ /kg T) und F (14,70 MJ /kg T) war signifikant höher als bei der Kontrolle A (13,96 MJ /kg). Der Grund für die Steigerung der N-korrigierten umsetzbaren Energie bei B und F kann dabei in der möglichen Verbesserung der Energieverfügbarkeit durch die Behandlungsverfahren Flockierung und Expander liegen. 4. Aufgrund der höchsten Restaktivität des Trypsininhibitors wurde bei der Gruppe H eine verminderte ileale Aminosäure- und Stickstoffverdaulichkeit im Vergleich zu Kontrolle A und den anderen Gruppen (B, C, D un E) festgestellt. Durch zusätzliche Dampfzufuhr und Erhöhung des Energieeintrages in den Gruppen I bzw. K konnten die antinutritiven Faktoren in den Sojabohnen reduziert werden und demzufolge die ileale Aminosäure- und Stickstoffverdaulichkeit bei den Gruppen I und K gegenüber der Gruppe H signifikant verbessert werden. Obwohl bei der Gruppe H die ileale Lysinverdaulichkeit gegenüber der Kontrolle A signifikant niedriger lag, wurde in Bezug auf die Lysinwirksamkeit kein signifikanter Unterschied zwischen den Gruppen H und der Kontrolle A festgestellt. 5. Die Trypsinaktivität im Chymus wurde auch in Abhängigkeit von der Restaktivität des Trypsininhibitors in den Sojabohnen unterschiedlich beeinflusst. Eine signifikant verminderte Trypsinaktivität im Chymus war insbesondere bei den Gruppen H sowie K feststellbar.

A semente da Araucaria angustifolia, denominada pinhão, é consumida no Sul e Sudeste do Brasil, como farinha em pratos regionais ou cozida. Há relativamente pouca informação a respeito da composição química e do valor nutricional da semente e de sua farinha. Neste trabalho, a farinha de pinhão, obtida através de diferentes tratamentos térmicos foi avaliada como complemento protéico em um experimento biológico com ratos recém-desmamados. Os animais experimentais consumiram cinco dietas (n=6) com diferentes fontes de proteína: dieta com caseína (CAS), dieta com 80% caseína e 20% (w/w) proteína de farinha de pinhão (PF) sem tratamento térmico (NATPIN), considerando 33.1 ± 1.4 g de proteína/kg desta farinha, dieta com 80% caseína e 20% PF seca a 50°C por 16 horas (PF50), considerando 49.2 ± 0.6 g de proteína/Kg desta farinha, dieta com 80% caseína e 20% PF seca a 80°C por 16 horas (PF80), considerando 50.5 ± 0.8 g de proteína/kg desta farinha e dieta aprotéica (APROT). Os valores de ganho de peso, ingesta de alimento, Coeficiente de Eficácia Alimentar (PER) e Razão Protéica Líquida (NPR) foram similares para as dietas CAS e FP80. O escore químico de aminoácidos corrigido pela digestibilidade protéica (PDCAAS) da PF80 foi o mais alto nas farinhas de pinhão testadas. O escore químico (CS) da farinha de pinhão se mostrou semelhante ao encontrado em outros cereais, sendo a lisina o principal aminoácido limitante, seguida da histidina. Valores mais baixos em todos os parâmetros nutricionais foram encontrados nos animais alimentados com dietas onde foi usada a farinha de pinhão. A farinha de pinhão seca a 80°C por 16 horas mostrou resultados similares nos parâmetros nutricionais ao grupo CAS, e pode ser utilizada, substituindo até 20% de uma proteína de alto valor biológico (AVB) em formulações alimentares. / The seeds of Araucaria angustifolia, namely “pinhão”, are consumed in South and Southeast of Brazil, like flour in regional dishes or baked. There is relatively few information about the chemical composition and nutritional value of the seed and its flour. In this work, “pinhão” flour obtained by different heat treatments was evaluated as an additive in biological experiment for growing rats. Wistar rats were fed five experimental diets (n=6) containing different protein sources: only casein (CAS), diet with 80% casein supplemented with 20% (w/w) “pinhão” flour (PF) protein without heat treatment (NATPIN), considering 33.1 ± 1.4 g of protein/kg of this flour, diet with 80% casein and 20% PF dried at 50°C for 16 hours (PF 50), considering 49.2 ± 0.6 g of protein/kg of this flour and diet with 80% casein and diet with 20% PF dried at 80°C for 16 hours (PF80), considering 50.5 ± 0.8 g of protein/kg of this flour and one aproteic diet (APROT). Values for weight gain, feed ingest, Protein Efficiency Ratio (PER) and Net Protein Ratio (NPR) were similar for diets CAS and PF80, and acoording to this, the Protein Digestibility Corrected Amino acid Score (PDCAAS) of PF80 was the highest of all the “pinhão” flours tested. The Chemical Score (CS) of “pinhão” flour showed similarity to the results found for other cereals, being lysine the limiting amino acid, folowed by histidine. Lowest values for all nutritional parameters were observed for diets complemented with “pinhão” flour. “Pinhão” flour heated at 80°C for 16 hours and used as supplementary in diet had the most similar results in all nutritional parameters to casein-based diets, and can be used as complementary source, substituting until 20% of a high biological value protein in food formulations.

DIAS, Lucas Pinheiro. Purificação e caracterização de um inibidor de tripsina das flores de Cassia fistula Linn. com atividade antimicrobiana. 2012. 83 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-19T13:34:23Z No. of bitstreams: 1 2012_dis_lpdias.pdf: 2004862 bytes, checksum: cd42d65281498494806d7ca987015da7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:29:09Z (GMT) No. of bitstreams: 1 2012_dis_lpdias.pdf: 2004862 bytes, checksum: cd42d65281498494806d7ca987015da7 (MD5) / Made available in DSpace on 2016-08-02T20:29:09Z (GMT). No. of bitstreams: 1 2012_dis_lpdias.pdf: 2004862 bytes, checksum: cd42d65281498494806d7ca987015da7 (MD5) Previous issue date: 2012 / Plants synthesize proteins that have antimicrobial properties, which can be used to substitute chemical pesticides in agriculture and as new drugs for the control of bacterial infections in humans. Among the various plant structures, the flowers seem to be a promising source of active molecules against pathogens, particularly if considered its important physiological role, which should be preserved. Therefore, this experimental research aimed at the prospection of novel proteins with antimicrobial activity in wild flowers and to subsequent purification, biochemical characterization and evaluation of antimicrobial activity of a trypsin inhibitor present in flowers of Cassia fistula Linn (the golden shower tree). The total extract of C. fistula flowers was prepared in 50 mM sodium phosphate buffer, pH 7.5. This extract presented trypsin inhibitory activity (42.41 ± 0.35 IU/mgP) and papain (27.10 ± 0.23 IU/mgP), besides to the presence of peroxidase (20.0 ± 0.18 UAP/mgP) and chitinase (1.70 ± 0.21 ηkatal/mgP). On the other hand, the hemagglutinating, β-1,3-glucanase, protease and urease activities were not detected. The trypsin inhibitor of C. fistula, named CfTI, was purified by fractionating the crude extract with trichloroacetic acid (2.5%) followed by affinity (anidrotripsina-Sepharose-4B) and reverse phase (Vydac C-18TP 522) chromatographies. CfTI is a glycoprotein with an apparent molecular mass of 22.2 kDa, pI 5.0 and NH2-terminal sequence showing high similarity with Kunitz soybean trypsin inhibitor (SBTI). The inhibitor was not stable to heat, and loss 23.4% when incubated at 60 °C for 15 minutes. However, it proved to be stable to changes of pH. CfTI (100 µg/mL) slowed the growth of pathogenic fungi of agricultural importance, Colletotrichum lindemuthianum and Fusarium solani, and also presented antibacterial activity against the human pathogenic bacteria, Staphylococcus aureus and Enterobacter aerogenes. The results demonstrate the potential of the flowers as a source of diverse bioactive proteins, as the trypsin inhibitor present in C. fistula flowers, promoting its biotechnological potential application against fungi and bacteria of relevance to Agriculture and human health. / As plantas sintetizam proteínas que possuem propriedades antimicrobianas, podendo ser utilizadas em substituição aos defensivos químicos no campo e como fonte de novas drogas para o controle de infecções bacterianas em humanos. Dentre as diversas estruturas vegetais, órgãos reprodutivos como as flores parecem ser uma fonte promissora de tais moléculas ativas contra patógenos, particularmente se considerado o seu relevante papel fisiológico, o qual deve ser preservado. Nesse contexto, a presente pesquisa experimental teve como objetivos a prospecção de proteínas com ação antimicrobiana em flores silvestres e posterior purificação, caracterização bioquímica e avaliação da atividade antimicrobiana de um inibidor de tripsina presente em flores de Cassia fistula Linn. (Chuva-de-ouro). O extrato total das flores de C. fistula foi preparado em tampão fosfato de sódio 50 mM, pH 7,5. Esse extrato apresentou atividade inibitória de tripsina (42,41 ± 0,35 UI/mgP) e de papaína (27,10 ± 0,23 UI/mgP), além de mostrar a presença de peroxidase (20,0 ± 0,18 UAP/mgP) e quitinase (1,70 ± 0,21 ηkatal/mgP), estando ausentes as atividades hemaglutinante, β-1,3-glucanásica, ureásica e proteásica. O inibidor de tripsina de C. fistula, denominado CfTI, foi purificado do extrato total por fracionamento com ácido tricloroacético (2,5%), seguido de cromatografias de afinidade (anidrotripsina-Sepharose-4B) e fase reversa (Vydac C-18TP 522). CfTI é uma glicoproteína com massa molecular aparente de 22,2 kDa, pI 5,0 e sequência NH2-terminal exibindo alta similaridade com o inibidor de tripsina da soja do tipo Kunitz (SBTI). O inibidor mostrou-se pouco estável ao calor, reduzindo sua atividade inibitória para 23,4% quando incubado a 60 °C, durante 15 minutos. No entanto, ele se mostrou bastante estável a variações no pH. CfTI (100 µg/mL) retardou o crescimento dos fungos fitopatogênicos de importância agrícola, Colletotrichum lindemuthianum e Fusarium solani, além de apresentar atividade antibacteriana frente às bactérias patogênicas ao homem, Staphylococcus aureus e Enterobacter aerogenes. Os resultados obtidos demonstram o potencial das flores como fonte diversificada de proteínas bioativas, evidenciando o inibidor de tripsina presente em flores de C. fistula, fomentando sua aplicação biotecnológica frente a fungos e bactérias de relevância para a Agricultura e Saúde.