Immobilization of a β-glucosidase and an endoglucanase in ferromagnetic nanoparticles: A study of synergistic effects

Nanoparticles can act as support materials for enzymatic immobilization, introducing a balance of characteristics that modulate the efficiency of biocatalysts, such as specific surface area, resistance to mass transfer and effective enzymatic loading. Magnetic nanoparticles can be easily separated using an external magnetic field, and in this work two recombinant enzymes, the β-glucosidase from Humicola insolens (Bglhi) and the endoglucanase from Scytalidium thermophilum (Egst) were immobilized on synthetized Fe₃O₄ nanoparticles derivatized with chitosan/glutaraldehyde/N-(5-amino-1-carboxy-pentyl) iminodiacetic acid and functionalized with NiCl₂. The immobilization yields were about 20% for Bglhi and Egst with efficiencies of 132% and 115%, respectively. The two enzymes were also co-immobilized with yield was about 49%. The optimal temperatures of the immobilized enzymes were 70 °C and 55 °C for Egst and Bglhi, respectively. Egst hydrolyzed CMC in the presence of 4 mM MnCl₂ with V_max = 625.0 ± 6.7 U mg^-1 and K_M = 6.4 ± 0.5 mg mL^-1 resulting in a catalytic efficiency (k_cat/K_M) of 107.4 ± 5.4 mg^-1 s^-1 mL. Bglhi hydrolyzed pNP-Glc with V_max = 52.7 ± 2.7 U mg^-1 and K_M = 0.23 ± 0.01 mM resulting in a catalytic efficiency (k_cat/K_M) of 214.3 ± 10.2 s^-1 mM^-1. The individually immobilized enzymes when combined showed a synergistic effect on the substrates tested and a very similar action when compared to the co-immobilized enzymes, suggesting excellent potential for application in biotechnological processes.