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Master's Dissertation
DOI
10.11606/D.9.2006.tde-17042006-125406
Document
Author
Full name
André Moreni Lopes
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2006
Supervisor
Committee
Pessoa Júnior, Adalberto (President)
Albuquerque, Cristina Northfleet de
Oliva Neto, Pedro de
Title in Portuguese
Purificação da enzima glicose-6-fosfato desidrogenase por sistemas micelares de duas fases aquosas contendo ligantes de afinidade
Keywords in Portuguese
Enzima
Proteína
Sistemas micelares
Tensoativos
Abstract in Portuguese
O presente trabalho teve como objetivo a purificação da enzima glicose-6-fosfato desidrogenase pela tecnologia de extração líquido-líquido em Sistemas Micelares de Duas Fases Aquosas (SMDFA). Estes sistemas são constituídos por soluções de tensoativos contendo micelas e oferecem ambientes hidrofóbico e hidrofílico, o que possibilita seletividade na partição da enzima de acordo com sua hidrofobicidade e proporciona um ambiente ameno às biomoléculas. Foram estudados alguns dos fatores que influenciam a partição da G6PD, como: tipo e concentração de diferentes agentes tensoativos não-iônicos (C10E4 e Triton X-114), temperatura e adição de ligantes de afinidade (cibacron blue e procion red) e o efeito da adição dos sais sulfato de amônio ((NH4)2SO4) e sulfato de sódio (Na2SO4). Estudou-se ainda a síntese do tensoativo de afinidade TX-114-Blue. Em todos os ensaios a enzima foi recuperada preferencialmente na fase diluída, pobre em micelas, tanto em sistema Triton X-114/tampão como para C10E4/tampão, no qual existe maior volume disponível, resultando em valores de KG6PD inferiores a 1. A utilização dos ligantes de afinidade na partição da G6PD nos sistemas descritos proporcionou um pequeno aumento nos valores de KG6PD da enzima, porém com valores inferiores a 1. Os sistemas Triton X-114/Sal não influenciaram a partição da enzima para a fase micelar, apesar da existência da diferença de potencial eletrostático entre as fases destes sistemas. O efeito desempenhado pelo volume de exclusão foi dominante em todos os sistemas estudados e, portanto, a enzima foi predominantemente excluída para a fase aquosa, pobre em micelas. A tecnologia por SMDFA para a purificação do homogeneizado celular de Saccharomyces cerevisiae demonstrou ser eficiente em recuperar a biomolécula alvo na fase aquosa, pobre em micelas, permitindo separar da presença de biomoléculas ou mesmo de contaminantes com caráter hidrofóbico. Dessa forma, o SMDFA pode ser empregado como uma possível etapa de purificação num processo biotecnológico.
Title in English
Glucose-6-phosphate dehydrogenase purification by two-phase aqueous micellar systems with affinity ligands
Keywords in English
Enzyme
Micellar systems
Protein
Abstract in English
In this work, the use of two-phase micellar system was studied aiming at the purification of the enzyme glucose-6-phosphate dehydrogenase. Usually, these systems are constituted of micellar surfactants solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to the enzyme partitioning according to its hydrophobicity. Some of the factors influencing the G6PD partition in micellar systems were studied, such as: type and concentration of nonionic surfactant agents (C10E4 and Triton X-114), temperature, addition of affinity ligands (Cibacron Blue and Procion Red) and the addition of the salts ammonium sulfate ((NH4)2SO4) and sodium sulfate (Na2SO4). The synthesis of the affinity surfactant TX-114-Blue was also studied. In all the assays the experiments, G6PD partitioned preferentialy to dilute, micelle-poor phase, in which there is a higher volume available for the enzyme to be, resulting in KG6PD values lower than 1. The use of affinity ligands in G6PD partitioning in both C10E4 and Triton X-114 systems provided some increase in the KG6PD, however with values still lower than 1. Employing a methodology previously described in the literature with some alterations, it was not possible to obtain the affinity surfactant TX-114-Blue. The systems Triton X-114/salt have not shown a significant influence on the enzyme partition to the micelle-rich phase, in spite of the existence of an electrostatic potential difference between the phases of the systems. The excluded-volume effect was dominant in all the systems studied and, therefore, the enzyme predominantly excluded to the dilute, micelle-poor phase. The use of Triton X-114 two-phase aqueous micellar systems to the purification of the Saccharomyces cerevisiae cell homogenate was found to be efficient in the recovery of G6PD in the dilute, micelle-poor phase, partially separating this target molecule from other proteins and contaminants of hydrophobic character. Therefore, aqueous two-phase micellar systems can be considered useful as a possible purification stage in a biotechnology process.
 
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Capa-LOPES_A_M.pdf (72.97 Kbytes)
Publishing Date
2007-02-07
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • LOPES, André M., et al. Aqueous two-phase micellar systems in an oscillatory flow micro-reactor : study of perspectives and experimental performance [doi:10.1002/jctb.2642]. Journal of Chemical Technology & Biotechnology [online], 2011, vol. 86, n. 9, p. 1159-1165.
  • LOPES, André M., PESSOA-JR., Adalberto, and RANGEL-YAGUI, Carlota de O.. Can affinity interactions influence the partitioning of glucose-6-phosphate dehydrogenase in two-phase aqueous micellar systems? [doi:10.1590/S0100-40422008000500010]. Química Nova [online], 2008, vol. 31, n. 5, p. 998-1003.
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