Purification of polygalacturonases produced by Aspergillus niger using an aqueous two-phase system

Abstract

The partitioning and purification of polygalacturonases (PG) produced by Aspergillus niger URM 5162 were investigated in aqueous two-phase systems (ATPS), formed by polyethylene glycol and phosphate salts (PEG/phosphate). To evaluate the effect of the 4 independent variables-molar mass of polyethylene glycol (PEG) (400-8000 g/mol-M PEG), PEG concentration (12.5-17.5%, w/w-C PEG), phosphate concentration (15-25%, w/w, C PHOS) and pH (6.0-8.0)-on the 4 response variables: partition coefficient (K), activity yield (Y), purification factor (PF) and selectivity (S), a factorial design (2 4) was used. The endopolygalacturonases (endo-PG) and exo-polygalacturonase (exo-PG) were preferentially partitioned in the top phase. For endo-and exo-PG, the highest values for the response variables K (1.23 and 2.40), Y (74.04% and 17.97%), PF (8.18 and 1.98) and S (24.68 and 48.07), respectively, were obtained for a C PEG of 12.5% (w/w), M PEG of 8000 g/mol, and C PHOS of 25% (w/w) at pH 6.0. These conditions were considered the most suitable for the purification of PG produced by A. niger URM 5162. Furthermore, the most important independent variables for endo-and exo-PG were C PHOS and M PEG , respectively. All independent variables studied and their interactions significantly influenced the response variables. According to these results, the PEG/phosphate system is a useful cost-effective alternative for purification of PG produced by A. niger URM 5162.

Phase diagrams of aqueous two-phase systems formed by polyethylene glycol+ammonium sulfate+water: equilibrium data and thermodynamic modeling

“…Aqueous two-phase systems (ATPS) are efficient and environmentally safe approaches that have attracted considerable attention in recent years as a technique for the extraction, separation and determination of several strategic analytes, such as: metallic ions [1], phenolic compounds [2], stem cells [3], small organic molecules [4], enzyme [5], DNA [6], antibodies [7], nanoparticles [8], and membranes [9]. ATPS is traditionally formed by a mixture of aqueous solutions of two mutually incompatible polymers [10], by a polymer and a salt [11] ,or even by two salts [12].…”

Lipase purification using ionic liquids as adjuvants in aqueous two-phase systems

“…This characteristic is usually attributed to the high water content and low interfacial tension of the systems which protects the proteins 10 . Despite the well-known advantages offered by these systems, ATPS are typically composed polymers (such as polyethylene glycol, PEG, or dextrane) [11][12][13][14] , being their applicability restricted due to the limited range of polarities of the coexisting phases. Recently, to overcome this limitation and enlarge the range of polarity differences between the coexisting phases the use of ionic liquids (ILs) in ATPS was proposed [15][16][17][18][19][20][21] .…”

Aqueous two-phase systems based on cholinium salts and tetrahydrofuran and their use for lipase purification

“…Aqueous two-phase systems (ATPS) have been used for the separation and purification of a great number, of biological products as amino-acids [9,10], proteins [11] and enzymes [8,[12][13][14][15][16] as their two phases having a rich water environment are favorable to the preservation of activity of biomolecules. These systems form two aqueous phases that coexist in equilibrium due to the dissolution, at appropriate concentrations, of pairs of solutes in water [17].…”