Comparative study on different immobilization sites of immobilized β-agarase based on the biotin/streptavidin system

Abstract

β-Agarase was biotinylated and immobilized onto streptavidin-conjugated magnetic nanoparticles to provide insights into the effect of immobilization sites on β-agarase immobilization. Results showed that, compared with free enzyme, the stability of prepared immobilized β-agarases through amino or carboxyl activation were both significantly improved. However, the amino-activated immobilized β-agarase showed higher thermostability and catalytic efficiency than the carboxyl-activated immobilized β-agarase. The relative activity of the former was 65.00 % after incubation at 50 °C for 1 h, which was 1.77-fold higher than that of the latter. Additionally, amino-activated immobilization increased the affinity of the enzyme to the substrate, and its maximum reaction rate (0.68 μmol/min) was superior to that of carboxyl-activated immobilization (0.53 μmol/min). The visualization results showed that the catalytic site of β-agarase after carboxyl-activated immobilization was more susceptible to the immobilization process, and the orientation of the enzyme may also hinder substrate binding and product release. These results suggest that by pre-selecting appropriate activation sites and enzyme orientation, immobilized enzymes with higher catalytic activity and stability can be obtained, making them more suitable for the application of continuous production.