Made available in DSpace on 2014-07-29T15:16:35Z (GMT). No. of bitstreams: 1 dissertacao parte 1 lidiane biologia.pdf: 35041 bytes, checksum: c58f88ce1be4655ee71c7b5ab0bef247 (MD5) Previous issue date: 2008-04-03 / Paracoccidoides brasiliensis is a human pathogenic dimorphic fungus. The recombinant chitinase from P. brasiliensis, Pbcts1r, was overexpressed in Escherichia coli using pET-32a (+) as vector. The enzyme was produced as inclusion bodies and became soluble by Sarkosyl being purified by a single step using a Ni-NTA resin. Pbcts1r showed activity against 4-MU-(GlcNAc)3 and 4-MU-(GlcNAc)2, presenting a endochitinase activity. Immunoblot reaction with anti-Pbcts1r identified two proteins in yeast crude extract. A partial purification of P. brasiliensis yeast crude extract by cationic-exchange chromatography on HPLC revealed two different chitinases, Pbcts1 and Pbcts2, with molecular mass of 45 kDa and 34 kDa, respectively. Pbcts2 has exochitinase activity and Pbcts1 has endochitinase activities. Reactions with anti- Pbcts1r showed the presence of Pbcts1 and Pbcts2 in crude extracts of yeast and transition from mycelium to yeast. On mycelium crude extracts was found only Pbcts1 and on yeast cell wall extract only Pbcts2. Both proteins were found to be secreted by yeast parasitic phase showing their probable importance in the permanence of the fungus in the human host. Phylogenetic relationships between the orthologs Pbcts1 and the putative Pbcts2 indicated the presence of a common ancestral. During evolution, P. brasiliensis could have acquired Pbcts2 and Pbcts1 playing distinct roles in order to growth and survive in diverse environment on saprophytic and parasitic phases / Paracoccidioides brasiliensis é um fungo dimórfico patogênico humano. A quitinase recombinante de P. brasiliensis, Pbcts1r, foi superexpressa em Escherichia coli utilizando pET-32(a)+ como vetor. A enzima foi produzida em corpos de inclusão e se tornou solúvel pela adição de sarkosyl, sendo purificada em um único passo com a utilização da resina Ni-NTA. Pbcts1r mostrou atividade diante de 4-MU-(GlcNAc)3 e 4- MU-(GlcNAc)2, apresentando atividade de endoquitinase. A reação de imunoblot com anti-Pbcts1r identificou duas proteínas no extrato bruto de levedura. A purificação parcial do extrato bruto de P. brasiliensis por cromatografia de troca-catiônica em HPLC revelou duas quitinases diferentes, Pbcts1 e Pbcts2, com massas moleculares de 45 e 34 kDa, respectivamente. Pbcts2 tem atividade de exoquitinase e Pbcts1 de endoquitinase. Reações com anti-Pbcts1r mostraram a presença de Pbcts1 e Pbcts2 no extrato bruto de levedura e transição de micélio para levedura. No extrato bruto de micélio foi encontrado somente Pbcts1 e no extrato de parede celular de levedura somente Pbcts2. Ambas as proteínas foram encontradas secretadas pela fase parasitária (levedura), mostrando a provável importância dessas proteínas na permanência do fungo no hospedeiro. Relações filogenéticas entre os ortólogos Pbcts1 e a provável Pbcts2 indicam a presença de um ancestral comum. Durante a evolução, P. brasiliensis poderia ter adquirido Pbcts2 e Pbcts1 desempenhando diferentes papéis para o crescimento e sobrevivência do fungo na fase saprofítica e parasitária

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144