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Master's Dissertation
DOI
10.11606/D.87.2009.tde-17042009-174554
Document
Author
Full name
Silvia Maria Ferreira Albani
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Crespo, Joaquín Cabrera (President)
Cheng, Elisabeth
Lucarini, Adriana Celia
Title in Portuguese
Métodos alternativos de purificação do polissacarídeo capsular de Haemophilus influenzae tipo b.
Keywords in Portuguese
Haemophilus influenzae tipo b
Cromatografia
Hidrólise enzimática
Microfiltração tangencial
Purificação de polissacarídeo
Ultrafiltração tangencial
Abstract in Portuguese
Haemophilus influenzae tipo b é uma bactéria Gram-negativa, patogênica causadora de meningites em crianças. A cápsula polissacarídica (PSb) é o principal fator de virulência e é usado como antígeno vacinal. O método clássico de purificação do PSb envolve várias etapas de precipitação com etanol, fenol e detergente catiônico (inflamável, corrosivo e tóxico), e etapas de ultracentrifugação. O objetivo deste estudo foi substituir total ou parcial as precipitações e/ou uso das centrífugas por cromatografia, digestão enzimática, microfiltração e ultrafiltração tangencial. As cromatografias de troca iônica e de filtração em gel não apresentaram boas purificações, entretanto a hidrofóbica pode eliminar as proteínas contaminantes. As precipitações com etanol foram necessárias para obter a pureza requerida. O etanol de alguma forma favoreceu a ação enzimática e facilitou a posterior ultrafiltração. A separação com etanol em fibra-oca de microfiltração tangencial mostrou melhores purificações do que a centrifugação, mas com uso repetido verificou-se redução na eficiência.
Title in English
Alternative methods for purification of capsular polysaccharide produced by Haemophilus influenzae type b.
Keywords in English
Haemophilus influenzae type b
Polysaccharide purification
Chromatography
Enzymatic digestion
Tangencial microfiltration
Tangencial ultrafiltration
Abstract in English
Haemophilus influenzae type b is Gram-negative pathogenic bacterium cause meningitis in children. The capsular polysaccharide (PSb) is the main virulence factor and it is used as vaccine antigen. The classical PSb purification process includes ethanol, phenol and cationic detergent precipitations (explosion prone, corrosive, toxic) and ultracentrifugation steps. The aim of this work was to replace total or partial ethanol precipitations steps and/or elimination of centrifugation by chromatography methods, enzymatic digestion and ultrafiltration (UF) or microfiltration. The results have showed that ion exchange chromatography and gel filtration did not result in good purification, however the hydrophobic can be used for proteins elimination. The ethanol precipitation steps are necessary to achieve the required purity of PSb. In some way ethanol contributed for enzymes action and further improvements in the UF. The ethanol separation with hollow fiber microfiltration exhibited better purification than centrifugation, but after some uses the efficiency has reduced.
 
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Publishing Date
2009-04-27
 
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