Abstract
Naringinase consists of two enzymes: α-L-rhamnosidase and β-D-glucosidase. The enzyme was immobilized on a carrier prepared from carob gum activated with polyethyleneimine. Cross-linking with dextran aldehyde was used to improve the stability of the immobilization. Knowledge of the characteristics of naringinase subunits is important for developing efficient and selective enzymatic reactions involving flavonoids. This study aimed to characterize two subunits of naringinase-α-L-rhamnosidase and β-D-glucosidase-free, immobilized on a magnetic polysaccharide carrier and cross-linked with dextran aldehyde. The characterization of free, immobilized, and stabilized naringinase, as well as α-L-rhamnosidase and β-D-glucosidase, included the effect of pH and temperature on enzyme activity, as well as the determination of their stability depending on the pH and temperature of the environment, and the determination of kinetic constants. Immobilization and subsequent stabilization of naringinase did not affect the optimal pH for the activity of α-L-rhamnosidase and β-D-glucosidase. Immobilization caused a change in the optimal temperature for the activity of α-L-rhamnosidase and β-D-glucosidase from 60 to 65°. Cross-linking of immobilized naringinase with dextran aldehyde increased the temperature stability of its subunits. Cross-linking also altered the pH stability profile of β-D-glucosidase. Immobilization and stabilization of naringinase slightly reduced the maximum reaction rate for α-L-rhamnosidase and β-D-glucosidase compared to the free enzyme. As a result of immobilization, the enzymes' affinity for substrates for both subunits decreased.