Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

Abstract

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation.

Liquid–liquid extraction of biomolecules: an overview and update of the main techniques

“…63,65,101 The triazine dyes Cibacron Blue 3GA and Procion Red HE-3B have been studied as affinity ligands for G6PD purification using chromatography. 102,103 Lopes et al 65 present a discussion, based on experimental results, about the role of affinity interactions on the partitioning of the hydrophilic enzyme G6PD in ATPMS composed of the surfactants Triton X-114 and C 10 E 4 . According to the results, in spite of the clear presence of affinity interactions, these were not strong enough to significantly overcome the excluded-volume effect and drive the enzyme preferentially to the micelle-rich phase containing the affinity ligands.…”

“…Reversed micelles, known as waterin-oil microemulsions, are aggregates of surfactant molecules formed in organic solvents, consisting of a polar inner core and an inner layer made of the surfactant hydrophilic head (nano-sized droplet of a polar liquid phase). 103 A microemulsion is a thermodynamically stable isotropic dispersion of two immiscible liquids consisting of microdomains of one or both liquids stabilized by an interfacial film of surfactant molecules. An important property of a microemulsion is its water or oil solubilization as microdroplets dispersed in the continuous phase.…”

Can affinity interactions influence the partitioning of glucose-6-phosphate dehydrogenase in two-phase aqueous micellar systems?

“…19,20 The triazine dyes Cibacron Blue 3GA and Procion Red HE-3B have been studied as affinity ligands for the enzyme glucose-6-phosphate dehydrogenase (G6PD) purification using chromatography. 21,22 In this work, we present a discussion, based on experimental results, about the role of affinity interactions on the partitioning of the hydrophilic enzyme G6PD in two-phase aqueous micellar systems. The enzyme G6PD, presents great interest as analytical reagent, being used in various quantitative assays, including the measurement of creatin-kinase activity, hexoses concentrations, and as a marker for enzyme immunoassays.…”

Utilização de sistemas poliméricos de duas fases aquosas (SPDFA) compostos por polietileno glicol/ácido poliacrílico (PEG/APA) para extração de ácido clavulânico

“…Xylose reductase was obtained from Cryptococcus flavus with 29% yield and purification of 19-fold by salt precipitation, concentrations, buffer exchange and chromatographics step (Mayr et al, 2003). G6PD industrial purification has been carried out through multiple-step processes based on chromatography technology (Chang et al, 1995). These techniques provided recovery 98% yields and a purification factor of 12 in expanded bed ion exchange chromatography (Chang et al, 1995).…”

“…G6PD industrial purification has been carried out through multiple-step processes based on chromatography technology (Chang et al, 1995). These techniques provided recovery 98% yields and a purification factor of 12 in expanded bed ion exchange chromatography (Chang et al, 1995).…”