Membrane Technology as a Strategy for Improving β-Galactosidase Concentration Processes: The Influence of the pH, Membrane Molecular Weight, Pressure, and Ionic Strength in the Process

The enzyme beta-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, although for its effective application it is necessary to establish techniques for purification, concentration, or polishing, such as membrane separation processes, in particular ultrafiltration. The present study aimed to investigate ultrafiltration and diafiltration applied as initial steps for concentration and salt removal, respectively, in the beta-galactosidase purification processes. Additionally, the influence levels of the pH (6.5, 7.7, or 7.5), membrane molecular weight cut-off (30, 50, 60, or 100 kDa), operating pressure (1.5, 2.0, or 2.5 kgf/cm(2)), and ionic strength of the ultrafiltration using NaCL or KCl (0.01-0.1 M) were evaluated considering the enzyme recovery, purification, retention, and concentration factors in relation to the proteins, volume, activity, and protein flux and yield of the processes. The ultrafiltration of the crude enzyme extract at pH 7.5 and 1.5 kgf/cm(2) with a 50 kDa polyethersulfone membrane resulted in a volume concentration of the beta-galactosidase extract up to 7.1-fold greater, a purification factor 1.2-fold greater, and an enzyme recovery rate of 108.9% by eliminating metabolites during the purification process. In addition, the lowest flux variation range (16.0 to 13.1 L/m(2)center dot h) was observed under these same conditions, thereby representing a decrease of 18.0%. An increase in the operating pressure and the addition of salts results in reduced enzyme recovery (up to 38% of the process yield (734.1 to 453.7 U/h) and up to 40% of the enzyme recovery rate (108.9 to 60.6%) during the ultrafiltration using NaCl, respectively). The operation in the diafiltration mode allowed salt removal after the purification of beta-galactosidase (enzymatic recovery rates above 93.4%) via precipitation and ion-exchange chromatography elution and as part of an aqueous two-phase system using 6 diafiltration cycles, thereby revealing its application potential.

Automated summary

The study investigated ultrafiltration and diafiltration as initial steps for concentration and salt removal in beta-galactosidase purification processes. Results showed that ultrafiltration of the crude enzyme extract at pH 7.5 and 1.5 kgf/cm(2) with a 50 kDa polyethersulfone membrane achieved a 7.1-fold volume concentration, 1.2-fold purification factor, and 108.9% enzyme recovery rate. Increased operating pressure and salt addition resulted in reduced enzyme recovery. Diafiltration allowed for salt removal after beta-galactosidase purification, demonstrating potential application.