Glucose-6-phosphate dehydrogenase purification by two-phase aqueous micellar systems with affinity ligands

Author(s)

André Moreni Lopes

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2006-03-27

Examining board members

Adalberto Pessoa Júnior; Cristina Northfleet de Albuquerque; Pedro de Oliva Neto

• Advisor: Adalberto Pessoa Júnior

Abstract

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. (AU)