Immobilization of β-glucosidase in fixed bed reactor and evaluation of the enzymatic activity

Adsorption of β-glucosidase from almonds, an enzyme with big molecular size (130 kDa, 6.7 nm molecular diameter), on mesoporous SBA-15 silica in fixed bed column was studied. Previously, zeta potential analysis confirmed that the electrostatic interactions between β-glucosidase and SBA-15 were the driving force of the immobilization process. The maximum difference in the zeta potential was 25 mV at pH 3.5. Adsorption isotherm was classified as an L3 (Langmuir type 3) curve according to the Giles classification and fitted to a double Langmuir equation. The adsorbed amount in a fixed bed column was around 3.5 times higher than the amount reached in the adsorption in batch. In addition, the β-glucosidase was strongly immobilized on SBA-15 with only 7 % of leaching in the washing step with buffer solution. Immobilized β-glucosidase was catalytically active in a continuous process, reaching 100 % substrate conversion and maintaining this activity level for more than 10 h without deactivation of the enzyme. Adsorption-desorption isotherms at 77 K before and after the adsorption were carried out, concluding that the adsorption of β-glucosidase was produced blocking the pore mouth, so that a part of the enzyme penetrates inside and another part stays outside the pore.