P-Phenylene Diisothiocyanate-Based Covalent Immobilization of β-d-Galactosidase and Determination of Enzyme Activity by Cleavage of X-Gal and ONPG on Solid Support.

Abstract

Herein, we present the immobilization of a technical grade β-d-galactosidase on amino-functionalized microtiter plates. Afterward, we transferred the results to a resin-based approach. For the covalent binding of the enzyme, an amino-functionalized microtiter plate was prefunctionalized with 1,4-phenylendiisothiocyanate. The cleavage of the substrate 5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside (X-Gal) produces a deep blue dye, which was quantified in a microtiter plate reader at 595 nm. The maximum reaction rates and the Michaelis-Menten constant were calculated. In addition, the unwanted blue precipitate formed during the experiments could be minimized by optimizing the experiments. When transferring the immobilization method to Rink amide resin, o-nitrophenyl-β-d-galactopyranoside was used as the substrate and the measurement was carried out in a photometer at 420 nm.