The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis and today represents a worldwide health crisis with prevalence estimates of 2.2%. HCV-specific therapeutics have never been more urgently needed. One of the validated drug targets is the non-structural (NS) protein 3 (NS3) membrane-bound protease. The major aim of this thesis was characterization of NS3 allosteric activation by its viral cofactor, NS4A. We hypothesized that there would be specific residues that dominate the interaction between NS3 and NS4A, and further hypothesized that binding and activation may be separate events mediated by different residues. This thesis details the development of novel cell-based assays for detection of NS3-4A protease activity and heterocomplex formation. The protease assay substrate was a membrane-targeted intracellular protein, which upon proteolysis released a red fluorescent protein (FP) reporter, DsRed-Express, into the cytoplasm; this change was detected by microscopy or quantified by Western blotting. The complex formation assay detected fluorescence resonance energy transfer (FRET) between yellow and cyan FP-tagged NS3 and NS4A, respectively. Our data shows binding can be functionally separated from activation. We identified two NS4A residues (I25 and I29) important for NS3 binding and two NS4A residues (V23 and I25) important for NS3 activation. Therefore the binding-pockets of these residues are prime targets for small-molecule therapeutic development. In addition, I have compared the NS3-4A substrate sequence cleavage efficiencies in vivo. I have been able to show that the activation-dependent NS4B/NS5A junction is processed efficiently and the NS4A/NS4B junction is not. I have also shown NS3-4A substrate specificity is not modulated by replicase components; however the specific activity of this enzyme is increased. The strength of this thesis work stems from the novel and creative development of cell-based assays that can easily be modified to study other membrane-associated proteases. In vitro assays fall short in that they do not take into account the unique micro-environment in which these proteases are found.

The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis and today represents a worldwide health crisis with prevalence estimates of 2.2%. HCV-specific therapeutics have never been more urgently needed. One of the validated drug targets is the non-structural (NS) protein 3 (NS3) membrane-bound protease. The major aim of this thesis was characterization of NS3 allosteric activation by its viral cofactor, NS4A. We hypothesized that there would be specific residues that dominate the interaction between NS3 and NS4A, and further hypothesized that binding and activation may be separate events mediated by different residues. This thesis details the development of novel cell-based assays for detection of NS3-4A protease activity and heterocomplex formation. The protease assay substrate was a membrane-targeted intracellular protein, which upon proteolysis released a red fluorescent protein (FP) reporter, DsRed-Express, into the cytoplasm; this change was detected by microscopy or quantified by Western blotting. The complex formation assay detected fluorescence resonance energy transfer (FRET) between yellow and cyan FP-tagged NS3 and NS4A, respectively. Our data shows binding can be functionally separated from activation. We identified two NS4A residues (I25 and I29) important for NS3 binding and two NS4A residues (V23 and I25) important for NS3 activation. Therefore the binding-pockets of these residues are prime targets for small-molecule therapeutic development. In addition, I have compared the NS3-4A substrate sequence cleavage efficiencies in vivo. I have been able to show that the activation-dependent NS4B/NS5A junction is processed efficiently and the NS4A/NS4B junction is not. I have also shown NS3-4A substrate specificity is not modulated by replicase components; however the specific activity of this enzyme is increased. The strength of this thesis work stems from the novel and creative development of cell-based assays that can easily be modified to study other membrane-associated proteases. In vitro assays fall short in that they do not take into account the unique micro-environment in which these proteases are found.

The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis and today represents a worldwide health crisis with prevalence estimates of 2.2%. HCV-specific therapeutics have never been more urgently needed. One of the validated drug targets is the non-structural (NS) protein 3 (NS3) membrane-bound protease. The major aim of this thesis was characterization of NS3 allosteric activation by its viral cofactor, NS4A. We hypothesized that there would be specific residues that dominate the interaction between NS3 and NS4A, and further hypothesized that binding and activation may be separate events mediated by different residues. This thesis details the development of novel cell-based assays for detection of NS3-4A protease activity and heterocomplex formation. The protease assay substrate was a membrane-targeted intracellular protein, which upon proteolysis released a red fluorescent protein (FP) reporter, DsRed-Express, into the cytoplasm; this change was detected by microscopy or quantified by Western blotting. The complex formation assay detected fluorescence resonance energy transfer (FRET) between yellow and cyan FP-tagged NS3 and NS4A, respectively. Our data shows binding can be functionally separated from activation. We identified two NS4A residues (I25 and I29) important for NS3 binding and two NS4A residues (V23 and I25) important for NS3 activation. Therefore the binding-pockets of these residues are prime targets for small-molecule therapeutic development. In addition, I have compared the NS3-4A substrate sequence cleavage efficiencies in vivo. I have been able to show that the activation-dependent NS4B/NS5A junction is processed efficiently and the NS4A/NS4B junction is not. I have also shown NS3-4A substrate specificity is not modulated by replicase components; however the specific activity of this enzyme is increased. The strength of this thesis work stems from the novel and creative development of cell-based assays that can easily be modified to study other membrane-associated proteases. In vitro assays fall short in that they do not take into account the unique micro-environment in which these proteases are found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A Protease do Vírus da Imunodeficiência Humana Tipo 1 (HIV-1 PR, EC 3.4.23.16) é um alvo macromolecular de grande importância no desenvolvimento de fármacos na terapia da Síndrome da Imunodeficiência Adquirida (AIDS). As maiores indústrias farmacêuticas do mundo concentram inúmeros esforços em estudos acerca desta proteína, que desde a introdução do saquinavir (Invirase&#174) na terapêutica em 1995, tem se mantido como um alvo fundamental para a descoberta de novos fármacos anti-HIV. A protease do vírus HIV-1 possui uma história rica de enorme sucesso no processo de descoberta e desenvolvimento de fármacos. A Química Medicinal moderna, de forte caráter multidisciplinar, fornece um arsenal de alternativas e estratégias racionais úteis no processo de planejamento de novos fármacos. Uma das tecnologias muito empregadas com sucesso é o estudo das relações quantitativas entre a estrutura e atividade (QSAR) para conjuntos de dados padrões. Os estudos de QSAR visam identificar e quantificar no complexo campo de modelagem as relações entre a estrutura e atividade de uma série de moléculas. Na presente dissertação de mestrado, foram realizados estudos de QSAR bi- (2D) e tridimensionais (3D) empregando, respectivamente, as técnicas holograma QSAR (HQSAR) e a análise comparativa dos campos moleculares (CoMFA), visando à geração de modelos preditivos para um conjunto de inibidores da protease do HIV-1. Os modelos gerados, associados às informações obtidas pelos mapas de contribuição 2D e de contorno 3D, são guias químico-medicinais úteis no planejamento de novos inibidores mais potentes e seletivos da protease do HIV-1. / The Human Immunodeficiency Virus Type 1 Protease (HIV-1 PR, EC 3.4.23.16) is a macromolecular target of great importance for the therapy of the Acquired Immunodeficiency Syndrome (AIDS). Major pharmaceutical companies around the world concentrate several efforts on studies concerning this enzyme, which since saquinavir (Invirase&#174) reached the market in 1995, has maintained its status as a fundamental target for anti-HIV drug discovery. HIV-1 protease has a rich history of enormous success in the drug discovery and development process. The strong multidisciplinary character of modern Medicinal Chemistry supplies an arsenal of useful rational strategies for the design of new drugs. One such technology is quantitative structure-activity relationships (QSAR), which has been successfully applied in a number of settings. QSAR studies aim to identify and quantify the relations between structure and activity of series of bioactive molecules organized within standard data sets. In the present master\'s dissertation, 2D and 3D QSAR studies were performed employing the hologram QSAR (HQSAR) and comparative molecular field analysis (CoMFA) techniques, respectively, in order to generate predictive models for a large set of HIV-1 PR inhibitors. The final models along with the information obtained from the 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selective within the chemical diversity of the data set.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 5 uppsatser.

CRUZ, Wallace Teixeira da. Análise proteômica, purificação e caracterização de uma peptidase cisteínica oligomérica do látex de Thevetia peruviana (Pers.) Schum. 2015. 112 f. Dissertação (Mestrado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2015. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-09-02T11:47:46Z No. of bitstreams: 1 2015_dis_wtcruz.pdf: 3443910 bytes, checksum: 9220d32e8822f3355afef0df27cdbe24 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-05T20:28:16Z (GMT) No. of bitstreams: 1 2015_dis_wtcruz.pdf: 3443910 bytes, checksum: 9220d32e8822f3355afef0df27cdbe24 (MD5) / Made available in DSpace on 2016-09-05T20:28:16Z (GMT). No. of bitstreams: 1 2015_dis_wtcruz.pdf: 3443910 bytes, checksum: 9220d32e8822f3355afef0df27cdbe24 (MD5) Previous issue date: 2015 / A great number of plant species produce latex, including Apocynacea, Sapotacea, Papaveracea and Euphorbiaceae. Thevetia peruviana (Pers.) Schum is a laticifer shrub belonging to Apocynaceae family popularly known as “chapéu-de-napoleão”. It is very limited the proteomic information about this specie. Thus, a proteomic analysis of protein fraction (TpLP) from T. peruviana latex was performed using two dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86%) were identified, including storage proteins, peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. This protein fraction showed strong proteolytic activity at pH 5.0 which was increased in the presence of low concentrations of the reducing agent DTT. The inhibition this activity in the presence of specific inhibitors E-64 and IAA and the high activity with BANA showed the predominance of cysteine peptidases in latex. A cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme was inhibited by E-64 and iodoacetamide (IAA) and follows the Michaelis-Menten kinetics, showing high affinity for azocasein, with Km value of 17.6 uM, exhibiting an optimal pH and temperature of 5.0-6.0 and 25-37 °C, respectively. Two-dimensional gel electrophoresis and mass spectrometry revealed that peruvianin-I (100 kDa) possesses a pI of 4.0 and five subunits (20 kDa). The N-terminal amino acid sequence of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36) was similar to that of germin or germin-like proteins. High-resolution images from atomic force microscopy indicated the possible hexameric structure of peruvianin-I, which is organized as a trimer of dimers that form a central channel. TpLP and peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects on the spore germination of Fusarium solani. This study showed that T. peruviana latex are a rich source of pathogenesis-related proteins, including cysteine peptidases. Interestingly, these peptidases exhibit different structural and biochemical characteristics that may be related to their specific physiological functions. / Um grande número de espécies vegetais produzem látex, incluindo Apocynacea, Sapotacea, Papaveracea e Euphorbiaceae. Thevetia peruviana (Pers.) Schum é um arbusto laticífero pertencente à família Apocynaceae, popularmente conhecido como "chapéu-de-Napoleão". São bastante limitadas as informações proteômicas sobre esta espécie. Por tanto, uma análise proteômica da fração proteica (TpLP) do látex de T. peruviana foi realizada a partir de eletroforeses bidimensionais e espectrometria de massas. Um total de 33 proteínas (86%) foi identificado, incluindo proteínas de reserva, inibidor de peptidase, peptidases cisteínicas, peroxidases e osmotinas. As proteínas desta fração apresentaram uma forte atividade proteolítica no pH 5,0, a qual foi aumentada na presença de baixas concentrações do agente redutor DTT. A inibição desta atividade na presença dos inibidores específicos E-64 e IAA e a alta atividade com o substrato BANA evidenciou a predominância de peptidases cisteínicas no látex. Uma peptidase cisteínica denominada peruvianina-I, foi purificada a partir do látex através de um único passo cromatográfico envolvendo filtração em gel. A enzima foi inibida por E-64 e iodoacetamida (IAA) e seguiu a cinética de Michaelis-Menten, apresentando alta afinidade à azocaseína, com um valor de Km de 17,6 µM, exibindo pH e temperatura ótimos de 5,0-6,0 e 25-37 °C, respectivamente. A peruvianina-I não foi reconhecida por anticorpos anti-papaína. As Eletroforeses bidimensionais e a espectrometria de massas revelaram que a peruvianina-I (100 kDa) possui um pI de 4,0 e cinco subunidades (20 kDa). A sequência de aminoácidos N-terminal da peruvianina-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36) mostrou similaridade à germinas ou “germin-like proteins”. Imagens de alta resolução a partir da microscopia de força atômica indicaram uma possível estrutura hexamérica da peruvianina-I, que está organizada como um trímero de dímeros, formando um canal central. TpLP e Peruvianina-I não exibiram atividade de oxalato oxidase e superóxido dismutase ou efeitos antifúngicos sobre a germinação de esporos de Fusarium solani. Este estudo mostrou que o látex de T. peruviana é uma fonte rica em proteínas relacionadas à patogênese, incluindo peptidases cisteínicas. Curiosamente, estas peptidases apresentam características estruturais e bioquímicas diferentes, que podem estar relacionadas com as suas funções fisiológicas específicas.

Made available in DSpace on 2015-07-22T20:49:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cancer de prostata e o segundo tipo de cancer mais frequente. Calicreinas teciduais e integrinas estao diretamente relacionadas com essa doenca, interferindo na adesao celular, diferenciacao e proliferacao. Neste trabalho utilizamos as linhagens estabelecidas DU145 e PC3 de cancer de prostata para o estudo do efeito do inibidor de calicreina recombinante, rBbKIm, o qual contem em sua sequencia primaria o motivo RGD, proveniente do inibidor BrTI isolado de Bauhnina rufa. O meio condicionado da cultura de DU145 e PC3 tratadas com rBbKIm por 48 horas inibiu cerca de 95% a atividade hidrolitica de HD-Pro-Phe-Arg- ƒÏNa por serino proteases da familia das calicreinas. rBbKIm inibiu a viabilidade celular da DU145 (56%, 100 ƒÊM, 48 horas) e da PC3 (28%, 100 ƒÊM, 48 horas) e interferiu somente em 5% da viabilidade da linhagem de fibroblastos de liquido amniotico (100 ƒÊM, 48 horas). rBbKIm nao inibiu a adesao das linhagens DU145 e PC3 sobre a fibronectina, laminina, colageno I e IV. Embora rBbKIm nao tenha interferido na migracao de DU145 o inibidor reduziu em 38% o processo migratorio da linhagem PC3 (100 ƒÊM) apos 23 horas de tratamento. Em relacao ao efeito do inibidor sobre a morte celular DU145 e PC3 tratadas com de rBbKIm (50 e 100 ƒÊM, 24 e 48 h) induziu a morte celular por apoptose. Alteracoes no ciclo celular foram observadas na linhagem PC3 com parada do ciclo nas fases G0/G1 e G2/M. Nao houve alteracao no ciclo celular na linhagem DU145. Ambas as celulas externalizaram o citocromo c para o citoplasma quando tratadas com rBbKIm (50 e 100 ƒÊM) por 24 horas. No caso da PC3 observou-se a ativacao de caspase-9 com 50 ƒÊM de rBbKIm apos 48 horas, nao havendo ativacao de caspase-3 enquanto que com a DU145 ocorreu ativacao da caspase-3 e nao houve ativacao da caspase-9. Os peptideos contendo a sequencia RGD e RGE nao inibiram a viabilidade celular de DU145 e PC3. Analises sobre o efeito de rBbKIm na viabilidade e migracao da linhagem endotelial humana (HUVEC), mostrou que rBbKIm (50 ƒÊM) inibiu 28% da viabilidade celular e 30% da motilidade celular da HUVEC. rBbKIm tambem apresentou atividade inibitoria na formacao de estruturas angiogenicas sobre Matrigel in vitro. Em resumo, o presente trabalho demonstrou a ação diferencial do inibidor de protease rBbKIm em linhagens de câncer de próstata independentes de andrógenos, PC3 e DU145, e em células normais de fibroblastos de líquido amniótico e em células endoteliais humanas, HUVEC. / Prostate cancer is the second more frequent type of cancer. In this work, using DU145 and PC3 prostate cancer cellular lines, we studied the effect of recombinant kallikrein inhibitor modified form (rBbKm), in which the sequence containing the signal motif sequence RGD of the inhibitor BrTI from Bauhnina rufa was inserted. When DU145 and PC3 were incubated with rBbKIm for 48h, and the conditioned medium the culture was incubated with the HD-Pro-Phe-Arg-ñNa, an kallikrein substrate, inhibited 95% of the degradation of this in both cells. rBbKIm inhibited cell viabillity of DU145 (56%, 100 ìM, 48 h) and PC3 (28%, 100 ìM, 48 h), but affect only 5% of the viabillity of fibroblast normal cell line (100 ìM, 48h). In cell adhesion, rBbKIm did not inhibit DU145 and PC3 cell line adhesion on fibronectin, laminin, collagen I and IV coat. In cell migration, rBbKIm did not inhibit DU145, but inhibited 38% of the PC3 (100 ìM) after 23 h. To evaluate the cytotoxicity, DU145 and PC3 were incubated with rBbKIm (50 and 100 ìM, 24 and 48h) inducing cell death by apoptosis. The presence of rBbKIm arrest PC3 cell cycle at the G0/G1 and G2/M phase, but did not affect DU145. Both cells released cytocrome c to the cytosol when treated with rBbKIm (50 and 100 ìM) for 24h. Moreover, DU145 when was treated with rBbKIm for 48h activated the caspase-3. However PC3 did not activated caspase-3, but activated caspase-9. The peptides containing the RGD and RGE sequence did not inhbit the cell viabillity of DU145 and PC3. Analyzing the effect of rBbKIm on HUVEC viabillity and migration, rBbKIm (50ìM) inhibited 28% and 30%, respectively. This protein inhibited 49% of the HUVEC angiogenesis in vitro on Matrigel. In summary, we have demonstrated the differential action of the protease inhibitor rBbKIm on androgen-independent prostate cancer PC3 and DU145, normal fibroblast and endothelial HUVEC cells. / TEDE / BV UNIFESP: Teses e dissertações

A Protease do Vírus da Imunodeficiência Humana Tipo 1 (HIV-1 PR, EC 3.4.23.16) é um alvo macromolecular de grande importância no desenvolvimento de fármacos na terapia da Síndrome da Imunodeficiência Adquirida (AIDS). As maiores indústrias farmacêuticas do mundo concentram inúmeros esforços em estudos acerca desta proteína, que desde a introdução do saquinavir (Invirase&#174) na terapêutica em 1995, tem se mantido como um alvo fundamental para a descoberta de novos fármacos anti-HIV. A protease do vírus HIV-1 possui uma história rica de enorme sucesso no processo de descoberta e desenvolvimento de fármacos. A Química Medicinal moderna, de forte caráter multidisciplinar, fornece um arsenal de alternativas e estratégias racionais úteis no processo de planejamento de novos fármacos. Uma das tecnologias muito empregadas com sucesso é o estudo das relações quantitativas entre a estrutura e atividade (QSAR) para conjuntos de dados padrões. Os estudos de QSAR visam identificar e quantificar no complexo campo de modelagem as relações entre a estrutura e atividade de uma série de moléculas. Na presente dissertação de mestrado, foram realizados estudos de QSAR bi- (2D) e tridimensionais (3D) empregando, respectivamente, as técnicas holograma QSAR (HQSAR) e a análise comparativa dos campos moleculares (CoMFA), visando à geração de modelos preditivos para um conjunto de inibidores da protease do HIV-1. Os modelos gerados, associados às informações obtidas pelos mapas de contribuição 2D e de contorno 3D, são guias químico-medicinais úteis no planejamento de novos inibidores mais potentes e seletivos da protease do HIV-1. / The Human Immunodeficiency Virus Type 1 Protease (HIV-1 PR, EC 3.4.23.16) is a macromolecular target of great importance for the therapy of the Acquired Immunodeficiency Syndrome (AIDS). Major pharmaceutical companies around the world concentrate several efforts on studies concerning this enzyme, which since saquinavir (Invirase&#174) reached the market in 1995, has maintained its status as a fundamental target for anti-HIV drug discovery. HIV-1 protease has a rich history of enormous success in the drug discovery and development process. The strong multidisciplinary character of modern Medicinal Chemistry supplies an arsenal of useful rational strategies for the design of new drugs. One such technology is quantitative structure-activity relationships (QSAR), which has been successfully applied in a number of settings. QSAR studies aim to identify and quantify the relations between structure and activity of series of bioactive molecules organized within standard data sets. In the present master\'s dissertation, 2D and 3D QSAR studies were performed employing the hologram QSAR (HQSAR) and comparative molecular field analysis (CoMFA) techniques, respectively, in order to generate predictive models for a large set of HIV-1 PR inhibitors. The final models along with the information obtained from the 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selective within the chemical diversity of the data set.

Orientador: Hélia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T00:05:53Z (GMT). No. of bitstreams: 1 Contesini_FabianoJares_D.pdf: 2309946 bytes, checksum: 15f6069e97515a200deb29e0577eee62 (MD5) Previous issue date: 2014 / Resumo: Proteases bacterianas são enzimas de elevada importância comercial, amplamente aplicadas em diversas áreas como nas indústrias de detergentes, de alimentos, farmacêutica e têxtil. Este trabalho teve como principais objetivos selecionar entre 59 linhagens de Bacillus sp., da coleção de culturas do Laboratório de Bioquímica de Alimentos da FEA, aquelas que apresentam potencial de maior produção de proteases com características tais como estabilidade em diferentes condições de temperatura, pH, detergentes e solventes orgânicos, atividade em ampla faixa de pH e capacidade de lisar células de Xanthomonas campestris. Em seguida, visou-se otimizar a produção de proteases pela linhagem selecionada, determinar as características bioquímicas da protease parcialmente purificada e estudar a aplicação do extrato enzimático bruto e preparação parcialmente purificada. Entre as cinquenta e nove linhagens de Bacillus sp. testadas foram selecionadas nove linhagens que produziram maior atividade de proteases. A produção de protease pelas nove linhagens foi testada em frascos agitados contendo o meio de cultura nº 1 (10g/L de caseína, 1g/L de extrato de levedura e sais), meio nº 2 (35 g/L de melaço de cana de açúcar, 20g/L de água de maceração de milho, 3g/L de extrato de levedura Prodex-Lac SD® e 20g/L de soro de queijo), e por fermentação em meio sólido nº 3 (farelo de trigo e água, na proporção 1:1, m:m). As linhagens de Bacillus sp. LBA 07, Bacillus sp. LBA 46 e Bacillus sp. LBA 08 fermentadas nos meios de cultura nº 1, nº 2 e nº 3 produziram 222 U/mL, 548 U/mL e 13480 U/grama de substrato seco (gss) respectivamente. As proteases dos extratos enzimáticos brutos obtidos das nove linhagens fermentadas nos três meios de cultura apresentaram atividade ótima na faixa de pH 7 a 9 e 60° C, estabilidade na faixa de pH 5 a 9 por 24h a 4º C , e após 1 h de tratamento a 50° C. Entre os extratos enzimáticos brutos de proteases testados, aqueles obtidas da fermentação de Bacillus sp. LBA 46 nos três meios de cultura foram as mais estáveis em detergente Ariel®. Quando incubadas em solventes orgânicos alguns extratos enzimáticos brutos proteases mantiveram mais de 60% de atividade residual após 24h em acetona (Bacillus sp. LBA 8 e 44), hexano (Bacillus sp. LBA 19, 29, 44, 46 e 60), clorofórmio (Bacillus sp. LBA 44 and 60) e etanol (Bacillus sp. LBA 60). Os extratos enzimáticos brutos de proteases obtidos do cultivo da linhagem de Bacillus sp. LBA 46 nos meios n° 2 e n° 3 foram as mais eficientes na lise de células de Xanthomonas campestris, aumentando cerca de 30% a transmitância a 620 nm (Trans 620nm) do meio fermentado de goma xantana. A linhagem de Bacillus sp. LBA 46 foi selecionada como melhor produtora de protease e estudos preliminares de identificação biomolecular indicam que se trata de uma linhagem de Bacillus licheniformis. Utilizando-se a linhagem de Bacillus sp LBA 46 e o meio de cultura otimizado (meio n° 4) por metodologia de superfície de resposta (MSR), composto de 40g/L de melaço de cana de açúcar, 6g/L de água de maceração de milho, 2g/L de extrato de levedura Prodex-Lac SD® e 20g/L de soro de queijo, foi obtido 3000 U/mL de protease após 96h de fermentação a 30° C e 200 rpm. No estudo da aplicação da enzima para a remoção de manchas de tecidos de algodão foram obtidos melhores resultados de remoção de manchas de sangue e molho de tomate com carne moída, utilizando-se a combinação de extrato bruto de protease (100 ou 1000U) com o detergente Omo®. O extrato enzimático bruto da linhagem de Bacillus sp. LBA 46 foi parcialmente purificado por fracionamento com sulfato de amônio (80% de saturação), diálise e cromatografia de filtração em gel (Sephadex G100), resultando em fator de purificação de 3,69. Após caracterização com MSR observou-se que a protease da preparação parcialmente purificada apresentou atividade ótima a 55° C e pH 7,5 e considerável estabilidade (95% de atividade residual) na faixa de pH 5,7 ¿ 9,3 após 1h de incubação a 30 ¿ 36° C, e acima de 78,9% quando incubadas por 1h em pH 7,5 e 50° C. A condição ótima de lise das células de X. campestris do meio fermentado de goma xantana utilizando-se o extrato enzimático bruto de protease e a preparação parcialmente purificada de proteases, foi observada utilizando 42 U de protease /mL de suspensão celular de X. campestris a 60° C, resultando em aumento de mais de 20% da Trans 620nm do meio fermentado de goma xantana. Um aumento de quase 40% de Trans 600nm foi observado após 2h de reação utilizando extrato enzimático bruto de protease (42 U de protease/mL de suspensão celular de X. campestris) a 65° C. A produção de proteases de Bacillus sp. LBA 46 por fermentação em estado sólido foi otimizada utilizando MSR, sendo obtido 5000 U/grama de substrato seco utilizando-se meio de cultura composto de farelo de trigo e água (60%:40%) após 96h de fermentação a 30° C / Abstract: Proteases are commercially relevant enzymes widely applied in several industrial areas, such as in detergent, food, pharmaceutical and textile industries. Proteases from Bacillus sp. can present advantages compared to the proteases from other sources, including better thermostability, stability in pH range from slightly acid to alkaline pH values and stability in organic solvents. The aims of this work were selecting Bacillus sp. strains with capability of producing proteases with better biochemical properties, such as stability in different conditions of temperature, pH, detergents and organic solvents, activity in a wide range of pH and capability of lysing cells of Xanthomonas campestris. Afterwards, it was aimed the optimization of the production of proteases by the selected Bacillus sp. strain and the determination of the biochemical characteristics of the partially purified protease and the application of the crude and partially purified protease. Nine Bacillus sp. strains were selected as the best protease producers among fifty nine Bacillus sp. strains tested. The protease production by the nine strains was carried out in Erlenmeyer flasks containing medium no. 1 (10g /L of casein, 1g/L of yeast extract and salts), medium no. 2 (35 g/L of sugar cane molasses, 20g/L corn steep liquor, 3g/L of yeast extract Prodex-Lac SD® and 20g/L of dried whey), e by fermentation using solid substrate medium no. 3 (wheat bran and water, 1:1, m:m). The strains Bacillus sp. LBA 07, Bacillus sp. LBA 46 and Bacillus sp. LBA 08 when fermented in medium no. 1, no. 2 e no. 3 produced 222 U/mL, 545 U/mL and 13480 U/gram of dried substrate (gds) respectively. Proteases from the crude enzymatic extracts obtained from the fermentation of the nine Bacillus sp. strains in the three media showed optimal activity in pH range 7-9 and 60° C, stability in pH range 5-9 for 24 hours at 4° C and after 1h at 50° C. The protease preparations from the fermentation of Bacillus sp. LBA 46 in the three media were the most stable when incubated in detergent Ariel®, among the proteases tested from the Bacillus sp. strains. In addition, some proteases presented more than 60% residual activity after 24h in the organic solvents acetone (Bacillus sp. LBA 8 and 44), hexane (Bacillus sp. LBA 19, 29, 44, 46 and 60), chloroform (Bacillus sp. LBA 44 and 60) and ethanol (Bacillus sp. LBA 60). The protease preparations obtained from the cultivation of Bacillus sp. LBA 46 in medium no. 2 and no. 3 presented the best results on the lysis of Xanthomonas campestris cells, resulting in an increase of approximately 30% in transmittance at 620 nm (Trans 620nm) of the fermented broth of xanthan. Bacillus sp. LBA 46 strain was selected as the best protease producer and after preliminary biomolecular analysis of identification, the results indicate that this microorganism correspond to a Bacillus licheniformis strain. Protease preparation containing 3000 U/mL was obtained from Bacillus sp. LBA 46 cultivated in Erlenmeyer flasks containing medium no. 4 composed of 40g/L of sugar cane molasses, 6g/L of corn steep liquor, 2g/L of yeast extract Prodex-Lac SD® and 20 g/L of dried whey after 96h of fermentation at 30° C and 200 rpm, optimized with response surface methodology (RSM). In the the washing tests, the best results of the removal of blood and tomato sauce with ground beef stains from cotton fabrics were observed using the combination of crude extract of protease (100 or 1000U) with detergent Omo®. Crude protease extract of the Bacillus sp. LBA strain was partially purified by ammonium sulfate fractionation (80% saturation), dialysis and gel filtration chromatography (Sephadex G100), resulting in the purification fold of 3.69. After characterization with RSM it was observed that the crude protease extract and partially purified proteases presented optimal activity at 55° C and pH 7.5 and considerable stability (95% of residual activity) in pH range 5.7 ¿ 9.3 after 1h incubation at 30-36° C and more than 78.9% when incubated at pH 7.5 and 50 °C for 1h. The optimal conditions of the lysis of X. campestris cells contained in the fermentation broth using crude and partially purified protease preparations were observed using 42 U of protease/mL of cell suspension of X. campestris at 60° C, resulting in a increase of more than 20% in Trans 620 nm of the fermented broth of xanthan. It was observed an increase of almost 40% in Trans 620 nm after 2h reaction using crude protease (42 U de protease/mL of cell suspension of X. campestris) at 65° C. The production of proteases by Bacillus sp. LBA 46 under solid state fermentation was optimized using RSM, resulting in 5000 U/gram of dry substrate utilizing wheat bran and water (6g:4g) after 96h of fermentation at 30° C / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos