Single Chromatographic Step for β-Galactosidase Purification: Influence of Salt and Elution Parameters

Abstract

β-galactosidase purification was performed by ion exchange chromatography in which the main elution parameters and different elution salts (NaCl, KCl, and (NH 4 ) 2 SO 4 ) were evaluated through distinct purification strategies. Among the salts under analysis, KCl was the best one for β-galactosidase purification, since it reached a 95.1% recovery and an 8.2-fold purification factor. NaCl, which is the most common elution salt found in the literature, obtained a 75.1% recovery and a 7.5-fold purification factor whereas ammonium sulfate, as salt elution, yielded a 72.1% recovery and a 3-fold purification factor in the same conditions.

Recovery of β-galactosidase produced by Kluyveromyces lactis by ion-exchange chromatography: Influence of pH and ionic strength parameters

“…It was possible to observe, according to Table IV, that the fraction of β-gal in the best elution range using between 0.1 and 0.4 M NaCl had a yield of 51.65 ± 0.17% and FP of 2.00 ± 0.43. These results were similar to the study performed by Braga et al (2014) that performed the purification of the β-gal enzyme and initially reached 48% of recovery compared to the initial extract. The ion-exchange chromatography is well used in protein adsorption.…”

“…The enzyme β-gal was produced through submerged cultivation using rotary incubator (Tecnal, model TE-241), with 250 mL Erlenmeyer flasks containing 50 mL of culture medium (20 g/L of cheese whey, 1.3 g/L of (NH 4 ) 2 SO 4 , 12 g/L of yeast extract, 5.0 g/L of KH 2 PO 4 and 0.4 g/L MgSO 4 .7H 2 O), in 0.2 M potassium phosphate buffer at pH 5.5 (Braga et al 2014). The fermentation was performed with 10% (v/v) of inoculum.…”

“…The enzymatic extract was obtained from the yeast cell disruption after the fermentation. The procedure was performed in centrifuge tubes of 50 mL, containing 25 mL of the cell suspension and 27.5 g of glass pearls (diameter ranging from 0.95 to 1.05 mm) under vortex agitation for 5 min, followed by 1 minute in an ice bath (Braga et al 2014). The enzymatic extract was collected and stored at -20 °C.…”

“…The ion-exchange chromatography is well used in protein adsorption. One of the advantages of this process is that it provides smooth separation conditions allowing proteins to maintain their conformation (Medeiros et al 2012, Braga et al 2014). Cienc (2022) 94(1) e20200752 7 | 12 Polyacrylamide gel electrophoresis (PAGE)…”

“…The chromatography using an ion-exchange matrix is widely used for protein adsorption, in which the concept is based on the attraction between proteins molecules and the resin that presents opposite charges. One of the advantages of this process is that it provides smooth separation conditions, allowing proteins to maintain their conformation (Medeiros et al 2012, Braga et al 2014. It is essential to highlight that the costs of this operation can reach up to 90% of the total production costs, and the reduction of purification steps implies directly on costs and improves the yields of the targeted molecule (Sousa Junior et al 2016), which is crucial for development of new alternative biotechnological strategies regarding the use of industrial wastes.…”