The structure and properties of carrier materials have great influence on the properties of immobilized cellulase, such as internal geometry (e.g., flat surfaces or thin fibers), specific surface area, superficial activation degree, mechanical resistance, and pore diameter (Begum et al. 2019; Malar et al. 2020; Santos et al. 2015). Meanwhile, partitioning and mass transport limitations may yield spatial variations in local reaction rates in porous materials (Neira and Herr 2017). Therefore, to improve the stability and catalytic activity of immobilized cellulase, various materials, such as chitin, chitosan, nylon, and polyvinyl alcohol, have been widely used as carriers (Cherian et al. 2015; Priydarshani et al. 2018).
The physical effects of nanocarriers on immobilized cellulase are as follows: 1) The pore size and effective surface area of the nanocarriers. Not all porous carriers can be used for immobilization of cellulase due to the limitation of pore size, which should be larger than or equal to that of the cellulase to reduce steric hindrance. The effective surface area occupied by the enzyme determines the maximum load of the immobilized cellulase (Blanco et al. 2004; Brady and Jordaan 2009; Santos et al. 2015). When a stable surface area is maintained, the amount of immobilized or absorbed cellulases is related to the pore size because the pore diameter determines the size of the protein that can be immobilized on that carrier (Trevisan et al. 2000); 2) the number of carrier-bound active groups (CAGs) is another key factor controlling the enzyme-carrier multi-interaction (Cristina et al. 2011; Santos et al. 2015); 3) the size of carriers plays a very important role in the preparation of immobilized cellulase, in that a smaller carrier size with larger specific surface area will be better for the cellulase immobilization load, and the higher surface porosity of the carriers providing numerous binding sites for cellulase is one of the most important factors influencing the activity of immobilized cellulase (Chen et al. 2010; Malar et al. 2020; Santos et al. 2015); 4) the mechanical properties of the carriers need to be controlled considering the final configuration of the reactor. If the reactor is a fixed-bed reactor, it should possess very high rigidity to withstand high pressures without pressure problems, but the situation is different if a stirred-tank reactor is used (Cristina et al. 2011; Santos et al. 2015); 5) after the cellulase penetrates the carriers, the internal morphology of carriers will determine the possibility of obtaining a very intense or very limited enzyme-carrier interaction (Santos et al. 2015). When the diameter of the carriers is smaller than that of the enzyme, it is difficult to obtain an intense enzyme-carrier interaction (Cristina et al. 2011), but if the carriers have sufficiently large internal surfaces, it is possible to get an intense interaction with a similar flat surface (e.g., agarose beads, porous glass, or silicates) (Malar et al. 2018).
In particular, the special superparamagnetism of magnetic nanocarriers has attracted increasing interest as they allow easy recycling and separation of catalysts and biomolecules from high-viscosity liqueurs and high-solid-content broths. This unique characteristic has been well-applied to immobilization of cellulase, and a better hydrolysis efficiency and recycling feasibility have been observed (Alftrén et al. 2014; Cao et al. 2016; Cipolatti et al. 2014; Xing et al. 2015). During immobilization of cellulase, magnetic chitosan microspheres (C-MNPs) are used as carriers because of their significant biological (i.e., biodegradable, biocompatible, bioactive) and chemical properties (polycationic, hydrogel, contains reactive groups, such as hydroxyl [OH] and NH2). Moreover, the hydrophilic properties of the C-MNPs play an important role in the preparation of oriented-immobilized cellulase based on the SRM system. The main process of immobilizing cellulase molecules on a single magnetic nanocarrier is shown in Fig. 2. Chitosan was first coated on the magnetic nanocarriers for further combination with cellulase. Fe3O4 nanocarriers have received extensive attention in cellulase immobilization to improve enzyme activity, loading, and stability because of their low toxicity, biocompatibility, and easy synthesis (Jordan et al. 2011; Zhang et al. 2014b). Magnetite nanocarriers coated with silica and modified by organic-silanes, biocompatible, and with hydrophilic properties, are promising for cellulase immobilization.
The binding sites of enzymes on the surfaces of carriers depend on the chemical properties of the carriers. For non-covalent immobilization, the chemical structure of the skeleton and surface determines the applicability of carriers. The functional groups play a key role in the activity, stability, and selectivity of the enzyme, and the size, charge, polarity, and hydrophilicity/hydrophobicity of groups can affect their binding functions (Watanabe et al. 2010). Different properties of the ionic groups on the surfaces of carriers may result in different enzyme activities and further determine the structure of immobilized cellulase (Berlin et al. 2016; Frančič et al. 2016; Hui et al. 2016; Santos et al. 2015; Zhou et al. 2018). The conformational change of the enzyme caused by the chemical properties of carriers during the immobilization process is shown in Fig. 3. In this process, the CAGs directly participate in binding with enzyme molecules, but the carrier-bound inert groups are not directly involved. This interaction inevitably disturbs the maintenance of the natural conformation of the enzyme, leading to structural and functional changes in the enzyme molecules. No obvious stability change has been observed when the newly formed conformation is similar to that of the natural enzyme. The covalent binding between carriers and active sites of the enzyme not only causes pore plugging of the surface, but also leads to the drag increment of in-diffusion. Although an initial high dosage of cellulase is added, the inhomogeneous distribution of the carrier surface structure results in the uncontrollable immobilization site, and ineffective immobilization may lead to a significant loss of enzymatic activity and reduce the accessibility of the substrate to the functional site. Moreover, the partition and mass transport limitations of nanocarriers may cause spatial variation in local reaction rates and further affect enzymatic hydrolysis (Du et al. 2017). The chitosan molecules are mostly used because of the large number of -OH and amino groups (-NH3), which are easier to co-precipitate with cellulase (Bindhu and Abraham 2010; Mo et al. 2020; Saha et al. 2019; Urrutia et al. 2018). Moreover, surface modification is an important strategy for tuning the properties of nanocarriers. Surface modification can either alter the existing property or introduce new properties onto nanoparticles using various agents, such as organ siloxane, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and carbodiimide as well as amino silanes, such as 3-aminopropyltriethoxysilane, aminoethyl aminopropyl polydimethylsiloxane, and silica (Chang et al. 2011; Gokhale et al. 2013; Malar et al. 2018; Malar et al. 2020; Riedel et al. 2017; Zhang et al. 2014a).