Purification of β‐Galactosidase by Ion Exchange Chromatography: Elution Optimization Using an Experimental Design

Abstract

Purification of b-Galactosidase by Ion Exchange Chromatography: Elution Optimization Using an Experimental Designb-Galactosidase is an enzyme industrially used to hydrolyze milk lactose, generating dairy products destined for people intolerant to this sugar. Its importance is due to its galactosiltransferase activity. The effects of elution pH and salt gradient volume were evaluated for purification of b-galactosidase by ion exchange chromatography using an experimental design and response surface techniques. The best conditions for purification of b-galactosidase were pH 5.5 with an elution volume of 62.8 mL, obtaining a yield of 85.5 % and a 12-fold increase in the purification factor in a one-step chromatography process. Purification of b-galactosidase by application of a single stage of ion exchange and evaluation of the important process parameters using an experimental design provided good results in the recovery and purification factor that could subsequently be scaled up. Ion exchange chromatography 911where A is the activity [U mL -1 ], P is the total protein concentration (mg mL -1 ), and V is the volume (mL).

Design Strategies for Integrated β‐Galactosidase Purification Processes

“…The enzyme was extracted by adding 1.1 g glass beads with 0.6-0.8 mm diameter to each mL of cell suspension and stirred in a vortex mixer for 40 min. The cell extract was clarified by centrifugation at 4700 g and 4°C for 10 min [18] and suspended in a 50 mmol L -1 potassium phosphate buffer pH 6.6 [3].…”

“…Thus, it is necessary to investigate purification strategies which use low-cost technologies. Traditional methods for isolation and purification of proteins generally involve several steps: ammonium sulfate precipitation, ion and affinity chromatography, dialysis, and the final concentration of the product [3]. Therefore, there is a need to study the application and optimization of alternative techniques that may increase the effectiveness of the purification process and, consequently, develop a low-cost separation process [4].…”