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

• A quantitative active site titration method is described.
• This method is effective for determining the amount of immobilized β-galactosidase of K. lactis on beads.
• 8.3 mg of active enzyme was found on 1 g of dried SPRIN imibond galactosidase beads.

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.