Active site titration of immobilized beta-galactosidase for the determination of active enzymes

Abstract In the present study, an active site titration method is demonstrated, to determine the amount of active enzyme (β-galactosidase), immobilized on a support. Two types of supports were investigated, viz. amino acrylic resin and a mixed matrix membrane. Furthermore, 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β- d -galactopyranoside was used as an inhibitor for the active site titration of immobilized β-galactosidase obtained from Kluyveromyces lactis . Using the active site titration, approximately 8.3 mg of active enzyme was found on 1 g of dried commercially available SPRIN imibond, which is an amino acrylic resin with covalently bound β-galactosidase obtained from K. lactis . However, this method, in its present form, was not effective on the mixed matrix membranes due to the irreversible partial adsorption of the leaving group (2′,4′-dinitrophenolate) by the membrane. This observation implied that it is important to investigate interactions between the support and the used inhibitor and leaving group.