Controlling the Adsorption of β-Glucosidase onto Wrinkled SiO₂ Nanoparticles To Boost the Yield of Immobilization of an Efficient Biocatalyst

Abstract

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose, a substrate for fermentation to produce the carbon-neutral fuel bioethanol. Enzyme thermal stability and reusability can be improved through immobilization onto insoluble supports. Moreover, nanoscaled matrixes allow for preserving high reaction rates. In this work, BG was physically immobilized onto wrinkled SiO 2 nanoparticles (WSNs). The adsorption procedure was tuned by varying the BG:WSNs weight ratio to achieve the maximum controllability and maximize the yield of immobilization, while different times of immobilization were monitored. Results show that a BG:WSNs ratio equal to 1:6 wt/wt provides for the highest colloidal stability, whereas an immobilization time of 24 h results in the highest enzyme loading (135 mg/g of support) corresponding to 80% yield of immobilization. An enzyme corona is formed in 2 h, which gradually disappears as the protein diffuses within the pores. The adsorption into the silica structure causes little change in the protein secondary structure. Furthermore, supported enzyme exhibits a remarkable gain in thermal stability, retaining complete folding up to 90 °C. Catalytic tests assessed that immobilized BG achieves 100% cellobiose conversion. The improved adsorption protocol provides simultaneously high glucose production, enhanced yield of immobilization, and good reusability, resulting in considerable reduction of enzyme waste in the immobilization stage.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties

“…Nevertheless, the free enzyme was completely inactivated after 4 h of incubation at 60 • C. This result clearly displays the efficiency of the immobilization method to protect enzymes against heat inactivation. Other studies [34,35] confirm the improved thermal stability of the immobilized enzyme compared to the soluble enzyme.…”

Chitosan‐Based metal-organic framework for Stabilization of β-glucosidase: Reusability and storage stability

“…In a previous study by Sanchez-Ramirez et al [ 45 ], it was shown that cellulase immobilized on chitosan-magnetic nanoparticles retained 50 % of its activity after 4 h at temperatures of 60 °C and 70 °C. Similarly, Pota et al [ 46 ] demonstrated that β-glucosidase immobilized on wrinkled SiO 2 nanoparticles exhibited slightly enhanced stability when incubated at 70 °C. Furthermore, Wan et al [ 47 ] showed that β-glucosidase immobilized on a Fe 3 O 4 @POP composite displayed improved thermal stability at 70 °C.…”

“…The immobilized enzyme exhibited a km value of 13.4 mM, which was higher than that of the free β-glucosidase (km = 6.98 mM). In general, immobilization may increase enzyme resistance to the substrate and, as a result, raise the km value because immobilization restricted access to the enzyme active site [ 46 ]. Califano et al [ 53 ] and Verma et al [ 54 ] highlighted the potential influence of diffusion limitations on the km observed for the immobilized enzyme.…”

“…This could be attributed to a rise in the substrate concentration within the bulk solution, leading to acceleration of substrate diffusion towards the enzyme, which may eventually reach a threshold level. If an immobilized enzyme attains the maximum velocity (Vmax) only after achieving complete substrate saturation, then the Vmax value will be higher than that of the enzyme in free solution [ 46 ]. Supporting materials offer solvent resistance and stability for enzymes.…”