Immobilization of β-galactosidase from Bacillus circulans onto epoxy-activated acrylic supports

The enzyme β-galactosidase from Bacillus circulans was immobilized onto the epoxy-activated acrylic supports Sepabeads EC-EP and Sepabeads EC-HFA. The influence of immobilization conditions on the process yield was studied. Optimal conditions for Sepabeads EC-EP were 1.4 M potassium phosphate buffer pH 8.5, enzyme loads in the range 3–30 mg/g of gel, and an incubation period of 48 h, achieving protein immobilization yields of 98–100% with activity yields of 85–98%. For Sepabeads HFA, low ionic strength (0.02 M potassium phosphate buffer), enzyme loads in the range 1.5–15 mg/g of gel, and shorter incubation periods (24 h) were required; however, when the whole process was performed at pH 8.5, maximum yields achieved were below 60%. This process was improved by performing a fast adsorption at pH 7.5 and then raising the pH to 8.5 to favor covalent coupling, achieving protein immobilization yields of 100% and enzyme activity yields of 83%.The remarkable thermal stability of derivatives obtained at low enzyme loads, and the fact that the thermal stability of medium load enzyme derivatives improved when a post-immobilization alkaline treatment was performed, support the hypothesis that stabilization was achieved through multipoint covalent attachment.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► β-Galactosidase from Bacillus circulans was immobilized onto Sepabeads EP and HFA. ► Two step protocol allows maximum immobilization yield in Sepabeads HFA. ► Immobilization at different enzyme loads revealed two modes of inactivation. ► Thermostability of medium load derivatives was improved by alkaline treatment. ► Blocking with glycine was the most stabilizing treatment for all the derivatives.