br Conclusions The results of this work showed that glucosid

Conclusions The results of this work showed that β-glucosidase could be efficiently immobilized onto hydroxyapatite nanoparticles in a single adsorption step. The enzyme adsorption was accomplished by coordination bonds between remaining Ca2+ sites of HA and COO− of amino acids. The immobilization process resulted in high-affinity interaction between the enzyme and the support over wide ranges of pH and ionic strength, with high enzyme binding capacity (around 50 mg protein g−1 support). The optimal immobilization conditions favorably resembled the optimal enzyme activity conditions, resulting in immobilization yield and recovered activity values of 90%. Furthermore, it was possible to recycle the immobilized β-glucosidase and retain 70% of the initial activity during at least 10 hydrolysis cycles. Therefore, the β-glucosidase was successfully immobilized on HA nanoparticles using a very simple adsorption protocol, showing excellent potential for applications in various industrial sectors.
Introduction β-glucosidase is an important glycoside hydrolase, which selectively catalyzes the hydrolysis of β-glycosidic linkages in disaccharides and oligosaccharides. It is commonly found in bacteria, fungi, plants and animals. β-glucosidases are members of the cellulase system and together with cellobiohydrolase and endo-β-glucanase play important roles in lignocellulosic Florfenicol degradation to produce ethanol [[1], [2], [3]]. β-glucosidase can hydrolase soy isoflavones, producing aglycones, compounds that are bioactive [4,5]. These enzymes have been used in many biotechnological applications, including the production of aromatic compounds in wine and other beverages [6,7]. In this industry, they are capable of degrading anthocyanins, producing free sugar and aglycone anthocyanidins, which are less soluble than anthocyanins, being easily removed during filtration [8]. Due to their various important roles, they are considered to be biologically and industrially important enzymes [2]. Many studies have looked for more efficient and thermostable β-glucosidases for industrial applications [1,2,9]. Most of the industrial processes occur at high temperatures, especially due to high reaction rates, decreased viscosity in fluid processes, increased solubility of the substrate and reduced contamination risk by undesired organisms. Hence, the use of thermostable enzymes is convenient as they maintain their catalytic activity [[9], [10], [11]]. Various thermophilic fungi have been reported to produce hydrolases characterized by valuable properties such as superior thermal stability and optimum activity at elevated temperatures [12]. Thermoascus aurantiacus is a thermophilic fungus, which grows efficiently on lignocellulosic biomass from agricultural wastes [[13], [14], [15]]. These substrates are attractive since they are readily available at low cost. Purification of enzymes presents one of the major challenges in order to characterize its structure and physical-chemical properties with a view to its biotechnological application. Chromatographic processes are far and away the most common technique when products with high purity and high yield are desired. Ion exchange chromatography (IEC) is the most widely used technique in industrial bioprocessing. In IEC, the pH of the samples is one of the most important parameters in determining protein binding, as it determines the change in both the protein and the ion exchanger [16,17]. The choice of the material to be used as carrier is also an important factor in separation efficiency. Among the materials available, a supermacroporous monolithic cryogel is an interesting alternative [18,19]. Cryogels are polymeric gels produced under freezing conditions and were first documented about 50 years ago [18]. During defrosting, ice or solvent crystals leave empty spaces of pores between the polymeric chains, forming a network of interconnected macropores, which allow the unobstructed passage of solutes. Pore sizes up to 200 μm may be found [20,21]. The properties such as high porosity and low flow resistance allow direct processing of crude extracts without prior clarification, optimizing time, saving water and reducing others expenses. In addition, reducing the number of steps in the process helps maintain the integrity of some compounds. Due to these features, the development of cryogels for use in purifying biomolecules is growing. They are also used in various fields such as drug delivery systems, dentistry, pharmaceuticals, agriculture [21], chromatography [[22], [23], [24]], immobilization [25,26] and tissue engineering [19].