Orientador: Roberto da Silva / Banca: Hamilton Cabral / Banca: Gustavo Orlando Bonilla Rodríguez / Resumo: Peptidases são enzimas responsáveis por realizar a clivagem de ligações peptídicas de outras proteínas e peptídeos. Essas enzimas apresentam grande importância, pois são utilizadas em vários segmentos industriais, desde a indústria alimentícia até mesmo no processamento de couro e pele e formulações de medicamentos. Uma das principais aplicações das peptidases é nos laticínios, na produção de queijo, que inicialmente envolve a coagulação das caseínas do leite pela ação de enzimas proteolíticas coagulantes, sendo a renina a principal delas. Enzimas microbianas coagulantes de leite são peptidases aspárticas que catalisam a coagulação do leite, substituindo o coalho de vitelo. Em trabalhos anteriores foi isolado o fungo termofílico Thermomucor indicae-seudaticae N31 que produziu em fermentação em estado sólido (FES), uma peptidase com capacidade de hidrolisar a κ-caseína do leite e produzir coágulo de boa qualidade. Entretanto, sua caracterização funcional e especificidade de ação ainda não estão claras. Assim, neste estudo, deu-se continuidade à investigação de estrutura e ação desta enzima coagulante. Foi realizado o monitoramento da hidrólise enzimática da caseína por cromatografia líquida de alta eficiência, foi feita análise da especificidade primária da peptidase utilizando substrato de fluorescência da série Abz-LSFMAIQ-EDDnp e foi determinada a sequência primária da proteína por espectrometria de massas. O monitoramento da hidrólise enzimática da caseína por cromatografia líquida de alta eficiência mostrou que o perfil do extrato bruto de Thermomucor indicae-seudaticae N31 apresenta alta similaridade com as enzimas comerciais das marcas Bela-Vista e Chr-Hansen obtidas dos fungos Rhizomucor miehei e Aspergillus niger, respectivamente. A análise de especificidade primária da peptidase demonstrou alta especificidade e afinidade pelo substrato sintético. A determinação do ... / Abstract: Peptidase enzymes are responsible for catalyze the cleavage of peptide bonds of other proteins. These enzymes present great importance, therefore, are used in several industrial segments, since the food industry even in the processing of leather and formulations of medicinal products. One major application of peptidases is in the dairy industry for cheese production, which initially involves clotting of milk caseins by the action of coagulant proteolytic enzymes being rennin the main one. Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. In previous study it was isolated a thermophilic fungus Thermomucor indicae-seudaticae N31 that produced on Solid State Fermentation (SSF), a peptidase with capacity to hydrolyses the k-casein of milk and produce good quality curd. However its functional characterization and specificity of action is not clear yet. Thus, in this study we continued the investigation of structure and action of this enzyme coagulant. Was carried out the monitoring of enzymatic hydrolysis of casein by high performance liquid chromatography-reverse phase, was analyzed the specificity of primary peptidase using substrate of fluorescence resonance energy transfer (FRET) peptide series Abz-LSFMAIQ-EDDnp and determined the primary sequence of the protein by mass spectrometry. Monitoring the enzymatic hydrolysis of casein by high performance liquid chromatography-reverse phase showed that the crude extract Thermomucor indicae-seudaticae N31 shows high similarity with commercial enzymes brands Bela-Vista and Chr-Hansen obtained from fungi Rhizomucor miehei and Aspergillus niger, respectively. The analysis of primary peptidase specificity demonstrated high specificity and affinity for the synthetic substrate. The determination of the cleavage site showed 46% hydrolysis of the bond between the amino acids phenylalanine and methionine and 54% between alanine ... / Mestre

Orientador: Raghuvir Krishnaswamy Arni / Banca: Adélia Cristina Oliveira Cintra / Banca: Patrick Jack Spencer / Abstract: Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out. / Mestre

Made available in DSpace on 2014-06-11T19:22:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-08Bitstream added on 2014-06-13T20:10:06Z : No. of bitstreams: 1 oliveira_dgl_me_sjrp.pdf: 2024541 bytes, checksum: 9e617f882421a4dd2f2cc715da6fb79f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out.