3.1 Characterization of ZIFs and ZIF-immobilized cellulases
As shown in Fig. 1, the XRD image reflects changes in the position and intensity of diffraction peaks. ZIF-8-NH2 typically exhibits characteristic peaks at specific 2θ values, such as 7.3°, 10.3°, 12.7°, 16.4°, and 18.0°, corresponding to the (011), (002), (112), (022), and (013) planes, respectively. After modification with magnetic nanoparticles, the ZIF-8-NH2 showed additional characteristic peaks at 30.2°and 35.5° besides the original peaks. This indicates that Fe3O4 has been successfully modified onto the carrier. The ZIF-immobilized cellulases showed minimal differences compared to the original ZIF carrier. This is because the cellulase does not possess a crystalline structure, and hence no additional characteristic peaks exist.
Figure 2 shows the FTIR spectra of the ZIF carrier and cellulase enzyme immobilized on ZIF. Typically, ZIF-8 materials exhibit a strong peak around 420 cm− 1, attributed to the stretching vibration of Zn-N bonds. Fe3O4@ZIF-8-NH2 composite materials simultaneously exhibit characteristic peaks of both ZIF-8 and Fe3O4. There is a peak at 580 cm− 1corresponding to the Fe-O bond of Fe3O4. Another peak at 3300 cm− 1 corresponds to the stretching vibration of the N-H bond[14]. Cellulase@ZIF-8 and cellulase@ZIF-8-NH2 composite materials should exhibit both the characteristic peak of cellulase and the characteristic peak of the ZIF carrier after cellulase immobilization. For physical adsorption on ZIF-8-NH2 and Fe3O4@ZIF-8-NH2, the peaks characteristic of cellulase, such as amide I (C-O stretching) at around 1660 cm− 1 and amide II (N-H bending and C-N stretching) vibrations at approximately 1540 cm− 1, are observed. The presence of these additional peaks confirms the successful loading of cellulase onto the ZIF carriers[9, 15].
Figure 3 displays SEM images of the ZIF carriers and the ZIF carrier with immobilized cellulase. In Fig. 3a and b, it can be observed that ZIF-8 typically exhibits a rhombic dodecahedron shape, and ZIF-8-NH2 exhibits an overall roughened rhombic dodecahedron shape after amino functionalization. Compared to ZIF-8-NH2, Fe3O4@ZIF-8-NH2 features a core-shell structure with Fe3O4 nanoparticles as the core and ZIF-8 as the shell. Due to the presence of -NH2 groups, Fe3O4@ZIF-8-NH2 also exhibits a similar rough surface. From Fig. 3c and d, it can be observed that enzyme molecules create small protrusions on the surface of ZIF materials after immobilization. Cellulase@ZIF-8-NH2, due to the presence of -NH2 groups and cellulase molecules, displays an even rougher surface. Cellulase@Fe3O4@ZIF-8-NH2, also due to the presence of amino groups and cellulase molecules, exhibits an even rougher surface.
As shown in Fig. 4, the adsorption isotherm of ZIF-8-NH2 exhibits a type I curve, indicating its microporous structure. However, the adsorption isotherm of Fe3O4@ZIF-8-NH2 displays a type IV curve, suggesting that the magnetic MOF material possesses both microporous and mesoporous structures[16]. As seen in Table 1, the specific surface area of ZIF-8-NH2 is 434.75 m²/g, while that of Fe3O4@ZIF-8-NH2 is 1166.86 m²/g. Thus, Fe3O4@ZIF-8-NH2 has a much larger surface area than ZIF-8-NH2, indicating a stronger loading capacity.
Table.1. Specifific Surface Area and pore characteristics of ZIF carriers and ZIF-immobilized cellulases
Carriers
|
BET Surface area(m2/g)
|
average pore size(nm)
|
ZIF-8-NH2
|
434.75
|
2.04
|
Fe3O4@ZIF-8-NH2
|
1166.86
|
1.62
|
3.2 Optimization of immobilization process parameters
Figures 5a and b demonstrate that under a 2 h immobilization time, different ratios of enzyme to carrier significantly affect the loading capacity and immobilized enzyme activity Generally, the loading capacity and activity of the enzyme first increase with the enzyme to carrier ratio, and then decrease. This may be due to the limited binding sites and loading capacity of the ZIF carrier. When reaching a maximum loaded protein, the tight and compact loading causes protein interactions and conformational changes[17]. Excessive enzyme can lead to aggregation, hindering the contact between the enzyme and the substrate, ultimately reducing loading capacity and relative enzyme activity. For ZIF-8-NH2, the highest loading capacity and enzyme activity were observed at a ratio of 1.25, being 263.3 mg/g and 66.3% respectively, while for Fe3O4@ZIF-8-NH2, the maximum loading capacity and activity were achieved at a mass ratio of 1.5, amounting to 339.8 mg/g and 71.39%.
3.3 Comparison of the enzymatic properties of free and ZIF-immobilized cellulases
From Fig. 6a, it is observed that cellulase exhibits its highest activity at 50°C[9, 18]. Once the reaction temperature exceeds the optimum value, the activity of the free enzyme sharply declines, with a loss of nearly 55% of its activity at 80°C. In contrast, the cellulase immobilized on ZIF retains a good level of activity, suggesting enhanced thermal stability. This enhancement is likely due to the adsorption of cellulase onto the MOF, which strengthens the protein and restricts conformational changes during heating. Specifically, the relative enzyme activities of ZIF-8-NH2 and Fe3O4@ZIF-8-NH2 at 80°C are 71.8% and 82.1%, respectively, with Fe3O4@ZIF-8-NH2 immobilized cellulase exhibiting even better thermal stability.
Figure 6b reveals that the pH tolerance of immobilized cellulase is higher than that of the free enzyme. For example, at pH 7, the relative residual enzyme activity of the free cellulase is only 42%, while it is 77% for ZIF-8-NH2 immobilized cellulase and 80% for Fe3O4@ZIF-8-NH2 immobilized cellulase. This improved tolerance might be due to changes in ionization behavior caused by immobilization on ZIFs, protecting the immobilized cellulase from pH-induced deactivation.[8, 9].
From Fig. 6c, it is observed that the activity of free cellulase drops to 14% after 30 days, while the activities of cellulase immobilized on ZIF-8-NH2 and Fe3O4@ZIF-8-NH2 maintain at 59% and 65%, respectively. This stability could be attributed to the stable microenvironment provided by the ZIFs material, which helps protect the enzyme from external conditions. Additionally, the porous structure of ZIFs reduces enzyme aggregation, thereby minimizing deactivation[13]. The interaction between cellulase and the ZIF carrier reduces the impact of charged residues, enhancing the affinity of cellulase for the carrier. Therefore, cellulase immobilized on ZIF carriers exhibits better storage stability[19].
Figure 6d shows the leaching rates of cellulase immobilized on different ZIF carriers. Due to the reversible nature of adsorption, the leaching rates of ZIF-8-NH2 and Fe3O4@ZIF-8-NH2 within 10 days are 16.7% and 14.1%, respectively. The magnetically modified ZIF-8-NH2 shows a lower leaching rate, likely due to its more stable core-shell structure, which reduces enzyme leakage.
3.4 Cellulose hydrolysis enhancement and enzyme recovery
Figure 7a shows the MCC hydrolysis yield by cellulase immobilized on ZIF-8-NH2 and Fe3O4@ZIF-8-NH2, compared to free cellulase, under the condition of 5 FPU/g enzyme dosage. It was observed that the hydrolysis yields of the immobilized cellulases are higher than that of the free enzyme. The yield using free cellulase was 51.99%, while yields for cellulases immobilized on ZIF-8-NH2 and Fe3O4@ZIF-8-NH2 were 68.41% and 71.44%, respectively. Additionally, Fe3O4 nanoparticles provide extra surface area and binding sites for enzyme immobilization, leading to higher enzyme loading and consequently higher hydrolysis yields.
A key objective of immobilizing cellulase on carriers is to enable the recycling of this high-cost enzyme. As shown in Fig. 7b, this study evaluated the recyclability of immobilized cellulase over 10 consecutive cycles. After 10 cycles, the cellulase immobilized on ZIF-8-NH2 retained 65.71% of its original activity, while the enzyme immobilized on Fe3O4@ZIF-8-NH2 maintained 68.42% activity. This indicates that immobilization not only enhances enzyme efficiency, but also significantly improves its reusability, making the process more cost-effective and sustainable[19].
3.5 Comparative study of immobilization efficiency and reusability by various MOF supports
Figure 8 summarizes the enzyme loading capacity and reusability achieved by immobilizing cellulase on various MOF supports. The enzyme loading capacities of the two ZIF supports investigated in this research exceeded most of the previous studies, reaching 263.38 mg/g and 339.64 mg/g, respectively. Furthermore, the residual enzyme activity after 5 cycles of reuse in this study reached 82.43% and 85.14%, also surpassing most of the prior research. It is noteworthy that, compared to our previous study[24] where cellulase was immobilized using the cross-linking method (with a loading of 359.89 mg/g), this research demonstrates a relatively lower enzyme loading. However, the activity of 71.39% surpasses the previous 69.35%. This improvement is speculated to be due to the use of glutaraldehyde as a cross-linking agent, which can effectively increase the loading of cellulase on ZIF-8. Nonetheless, glutaraldehyde may have a certain toxic effect on cellulase itself, leading to partial deactivation and thus reducing the hydrolysis yield. Moreover, the adsorption method is simpler and more cost-effective than the cross-linking method. Consequently, the immobilized cellulase supports and methodologies developed in this study significantly enhanced the enzyme loading capacity and reusability, highlighting the advantages over previous research efforts.