The development of immobilization techniques for

The development of immobilization techniques for β-glucosidase offers a potential way to enable its reuse several times [13]. The aim of immobilization is to trap the enzyme within or on the surface of an insoluble material with retention of its catalytic activity [14]. The immobilization strategy also enables continuous operation, improves the stability of the biocatalyst, and allows use of a smaller reactor volume [15]. The feasibility of immobilizing β-glucosidase Ponceau S Staining Solution receptor on different supports has been previously demonstrated using physical and ionic adsorption, encapsulation and covalent attachment [[16], [17], [18], [19]]. However, it remains a challenge to find a support that presents the specific characteristics required for high enzyme immobilization efficiency and the robustness to endure different process conditions, at a competitive cost. Typically, the support should have a high surface area to allow immobilization of significant amounts of enzymes, hydrophilicity to ensure good diffusivity of the substrate, and low solubility in order to avoid product contamination [20]. The mechanical resistance and thermal stability of the support are important throughout the entire process, from immobilization of the biocatalyst to enzyme recovery [21]. Another important parameter to be evaluated in an immobilization process is the diffusivity of the substrate towards the catalyst. Materials synthesized at the nanometric scale have demonstrated low resistance to mass transfer, ensuring good accessibility of the substrate to the catalyst [22]. The main advantage of using nanoparticles as supports to immobilize enzymes is their high surface area, which allows higher enzymatic loading per unit mass of particles [23]. Previous studies have reported the immobilization of β-glucosidase on nanomaterials such as chitosan-multiwalled carbon nanotubes [8], magnetic Fe3O4 nanospheres functionalized with amino-silane [24], nanoscale polymeric materials including polyurethane, latex and silicone [23], and silicon oxide nanoparticles [25]. However, in these previous studies, the immobilization of enzymes usually required chemical modifications of the support and/or the enzyme, such as activation with glyoxyl groups for covalent attachment, crosslinking with glutaraldehyde, amination of the enzyme or of the support side groups for ionic adsorption, or insertion of a spacer arm, among others. Therefore, finding a nanomaterial for enzyme immobilization that does not require any additional chemical modification procedures would contribute to process viability, from both the technical and economic standpoints. A promising inorganic solid for the immobilization of enzymes is hydroxyapatite (HA), a nontoxic material that can be synthesized as nanoparticles [26,27]. The adsorption of proteins onto HA has been demonstrated for various applications such as separation of proteins by chromatography [28,29], bone regeneration in biomedicine [30,31] and drug delivery in the pharmaceutical industry [32]. The hydroxyapatite structure includes phosphate and calcium groups that are available for ionic adsorption involving protein side groups [33]. The calcium ions present in HA can also undergo chelation reactions with carboxylic acid groups present in the amino acids of enzymes, resulting in highly stable interactions for immobilization [34]. Recent studies have reported the immobilization of lipase enzymes on HA nanoparticles [35,36]. However, to the best of our knowledge, there have been no reports of the use of HA nanoparticles for immobilization of β-glucosidase enzymes. Given the vast range of industrial applications of this biocatalyst and the interesting properties of HA, which has still been little explored as a support for immobilization of enzymes, the development of a simple protocol for the immobilization of β-glucosidase onto HA could be of potential interest to several industrial sectors.
Materials and methods
Results and discussion