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Tese Jucimar Zacaria.Pdf (4.725Mb) UNIVERSIDADE DE CAXIAS DO SUL CENTRO DE CIÊNCIAS BIOLÓGICAS E DA SAÚDE INSTITUTO DE BIOTECNOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA DIVERSIDADE, CLONAGEM E CARACTERIZAÇÃO DE NUCLEASES EXTRACELULARES DE Aeromonas spp. JUCIMAR ZACARIA CAXIAS DO SUL 2016 JUCIMAR ZACARIA DIVERSIDADE, CLONAGEM E CARACTERIZAÇÃO DE NUCLEASES EXTRACELULARES DE Aeromonas spp. Tese apresentada ao programa de Pós- graduação em Biotecnologia da Universidade de Caxias do Sul, visando à obtenção de grau de Doutor em Biotecnologia. Orientador: Dr. Sergio Echeverrigaray Co-orientador: Dra. Ana Paula Longaray Delamare Caxias do Sul 2016 ii Z13d Zacaria, Jucimar Diversidade, clonagem e caracterização de nucleases extracelulares de Aeromonas spp. / Jucimar Zacaria. – 2016. 258 f.: il. Tese (Doutorado) - Universidade de Caxias do Sul, Programa de Pós- Graduação em Biotecnologia, 2016. Orientação: Sergio Echeverrigaray. Coorientação: Ana Paula Longaray Delamare. 1. Aeromas. 2. DNases extracelulares. 3. Termoestabilidade. 4. Dns. 5. Aha3441. I. Echeverrigaray, Sergio, orient. II. Delamare, Ana Paula Longaray, coorient. III. Título. Elaborado pelo Sistema de Geração Automática da UCS com os dados fornecidos pelo(a) autor(a). JUCIMAR ZACARIA DIVERSIDADE, CLONAGEM E CARACTERIZAÇÃO DE NUCLEASES EXTRACELULARES DE Aeromonas spp. Tese apresentada ao Programa de Pós-graduação em Biotecnologia da Universidade de Caxias do Sul, visando à obtenção do título de Doutor em Biotecnologia. Orientador: Prof. Dr. Sergio Echeverrigaray Laguna Co-orientadora: Profa. Dra. Ana Paula Longaray Delamare TESE APROVADA EM 11 DE ABRIL DE 2016 ____________________________________________ Orientador: Prof. Dr. Sergio Echeverrigaray Laguna ____________________________________________ Co-orientadora: Profa. Dra. Ana Paula Longaray Delamare ____________________________________________ Profa. Dra. Andrea Balan ____________________________________________ Prof. Dr. Henrique Bunselmeyer Ferreira ____________________________________________ Prof. Dr. Jomar Pereira Laurino iv “Duvide sempre de si mesmo, até que os dados não deixem lugar a dúvidas.” Louis Pasteur v Dedico este trabalho a todos aqueles que me incentivaram a sempre buscar algo mais e a nunca desistir dos sonhos, por mais distantes que pareçam, em especial ao meu pai, a minha mãe, a minha irmã, a minha avó Gema e a minha namorada Carol. vi AGRADECIMENTOS Agradeço imensamente ao meu orientador professor Dr. Sergio Echeverrigaray, pelo incentivo e pelo exemplo de competência e perseverança. Por acreditar na minha capacidade de pesquisador, por me ensinar a ver aquilo que ninguém viu, e, sobretudo pensar o que ninguém pensou sobre aquilo que todos veem. A professora Dra. Ana Paula Longaray Delamare pelo seu apoio, incentivo, sugestões e amizade durante todo o tempo de convívio no laboratório. A professora Dra. Mirian Salvador pela amizade, exemplo de pesquisadora e pelo acompanhamento crítico ao longo do desenvolvimento deste trabalho. Ao professor Dr. Jomar Laurino Pereira pela análise minuciosa deste trabalho, por sua ética, amizade e solidariedade, que sempre lhe foi peculiar. A todos os colegas do Laboratório de Microbiologia Aplicada do Instituto de Biotecnologia, pela colaboração, incentivo, convivência e amizade em especial ao meu amigo Maurício Schiavo. A todos os colegas do programa de Pós-graduação em Biotecnologia, em especial ao meu amigo Edegar. A todos os meus colegas e acima de tudo amigos do Laboratório Analitus, pela compreensão, apoio e incentivo. Aos professores e funcionários do INBI pelos conhecimentos transmitidos e pelos exemplos de ética e caráter, que carregarei para a minha vida. A CAPES e CNPq pelo auxilio financeiro que foi primordial na realização deste trabalho. Por fim, agradeço ao Programa de Pós-graduação em Biotecnologia do qual me orgulho enormemente de fazer parte! Muito Obrigado! vii SUMÁRIO LISTA DE TABELAS .................................................................................................... xi LISTA DE FIGURAS ................................................................................................... xiii ANEXOS ........................................................................................................................ xx RESUMO ...................................................................................................................... xxi ABSTRACT ................................................................................................................. xxii 1. INTRODUÇÃO E JUSTIFICATIVA .......................................................................... 1 2. OBJETIVO GERAL ..................................................................................................... 3 3. REVISÃO BIBLIOGRÁFICA ..................................................................................... 4 3.1 O gênero Aeromonas, características e classificação taxonômica ............................. 4 3.2 Distribuição de bactérias do gênero Aeromonas ........................................................ 8 3.3 Aspectos clínicos associados ao gênero Aeromonas ................................................ 12 3.4 Fatores de patogenicidade associados ao gênero Aeromonas .................................. 16 3.5 Nucleases .................................................................................................................. 22 3.5.1 Nucleases microbianas .................................................................................. 25 3.5.2 Nucleases de Aeromonas spp. ....................................................................... 29 3.6 Predição de estruturas proteicas empregando ferramentas de bioinformática ......... 33 4. MATERIAL E MÉTODOS ........................................................................................ 35 4.1 Linhagens bacterianas utilizadas .............................................................................. 35 4.2 Condições de cultivo ................................................................................................ 36 4.3 Obtenção dos extratos brutos de proteínas extracelulares ........................................ 36 4.4 Obtenção de extratos de proteínas periplasmáticas .................................................. 36 4.5 Avaliação quantitativa da atividade DNásica extracelular ....................................... 37 4.6 Avaliação quantitativa da atividade DNásica extracelular termoestável .................. 38 4.7 Avaliação semiquantitativa da atividade DNásica ................................................... 38 4.8 Avaliação dos perfis de nucleases extracelulares e cálculo da atividade DNásica com base na área de degradação ............................................................................................. 39 4.9 Avaliação da estabilidade térmica de DNases extracelulares empregando zimogramas ..................................................................................................................... 40 4.10 Curva de crescimento e determinação da atividade DNásica extracelular quantitativa de A. hydrophila IBAer119 ........................................................................ 40 4.11 Avaliação do efeito de proteases sobre a atividade DNásica extracelular de A. hydrophila IBAer119 ...................................................................................................... 41 viii 4.12 Curva de crescimento e determinação da atividade DNásica extracelular quantitativa empregando DNA como fonte de nitrogênio e fósforo em A. hydrophila IBAer119 ........................................................................................................................ 41 4.13 Amplificação e clonagem dos códons dos genes dns e aha3441 de A. hydrophila IBAer119 ........................................................................................................................ 43 4.13.1 Amplificação dos códons dos genes dns e aha3441.................................... 43 4.13.2 Separação eletroforética e visualização dos fragmentos amplificados ........ 44 4.13.3 Ligação e clonagem dos genes dns e aha3441 ............................................ 44 4.13.4 Obtenção de células competentes e transformação ..................................... 45 4.13.5 Sequenciamento dos genes dns e aha3441 de A. hydrophila IBAer119. .... 46 4.14 Avaliação do efeito de inibidores sobre a atividade das enzimas recombinantes Dns e Aha3441 de A. hydrophila IBAer119 .......................................................................... 47 4.15 Determinação da temperatura de ação das enzimas recombinantes Dns e Aha3441 de A. hydrophila IBAer119 ............................................................................................ 48 4.16 Determinação do pH de ação das enzimas recombinantes Dns e Aha3441 de A. hydrophila IBAer119 ...................................................................................................... 48 4.17 Avaliação do efeito de diferentes cofatores na atividade das enzimas recombinantes Dns e Aha3441 de A. hydrophila IBAer119................................................................... 49 4.18 Determinação da estabilidade térmica das enzimas recombinantes Dns e Aha3441 de A. hydrophila IBAer119 ............................................................................................ 49 4.19 Avaliação da atividade enzimática sobre DNA e RNA, do extrato contendo as enzimas recombinantes Dns e Aha3441 de A. hydrophila IBAer119 ............................ 50 4.20 Avaliação do efeito de DNA como
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