Figure 1 . SEM images of OSMD (A, B, C) and CRL@OSMD (D, E, F)
Figure 2. A shows the FTIR spectra of the original diatomite, MD, and OSMD. According to the FTIR diagram of the raw diatomite, the absorption peaks of 3622.3 cm-1, 3450.0 cm-1, and 1639.7 cm-1 are formed by the stretching vibration of -OH. The absorption peaks at 1025.2 and 792.4 cm-1 correspond to the stretching vibration of Si-O-Si; the bending vibration of the Si-O bond forms the absorption peak at 451.2 cm-1. Instead, there are some new peaks in the FTIR of modified diatomite. The absorption peaks at 2925.5 cm-1, 2850.1 cm-1, and 1463.1 cm-1 in the infrared spectrum of modified diatomite correspond to the stretching vibration and bending vibration of the C-H bond in -C18, which is consistent with the study of Li et al[35]. The peak at around 1396.5 cm-1 and 1140.0 cm-1 is caused by the vibration of S=O in -SO3H. The surface modification of the diatomite with alkyl groups can be demonstrated by the Si-C associated peaks appearing at 720.3 cm-1, which is consistent with the work reported by Xu et al[36]. All the above results indicated that the diatomite was successfully modified.
The magnetization of OSMD and CRL@OSMD was measured using a Vibrating Sample Magnetometer (VSM) at room temperature (Figure 2. B ). The remanent magnetization and coercivity of OSMD and CRL@OSMD were almost zero, indicating that the sample materials are soft magnetic materials with superparamagnetic and outstanding magnetic properties[37]. The specific saturation magnetization of OSMD and CRL@OSMD were 12.2 emu·g-1and 12.1 emu·g-1, respectively. There is almost no difference between the two, which indicates that the immobilization process does not affect the magnetic properties of the support. The exceptional magnetic response helps to recover the immobilized enzyme under the application of a magnetic field, providing valuable support for the reuse of biocatalysts.
In addition, we employed the N2 adsorption-desorption isotherms to provide evidence for the successful immobilization of CRL to the OSMD (Figure 2. C ). As shown in Figure 2.D , the overall pore size of CRL@OSMD was significantly decreased compared to OSMD which may be attributed to that the CRL immobilized into the pore of OSMD and thus decreased the pore size of it. Moreover, the results showed that the Brunauer-Emmett-Teller (BET) surface area of OSMD decreased from 53.6 m2·g-1to 27.2 m2·g-1 after the immobilization process, accompanied by a corresponding reduction in pore volume from 0.227 cm3·g-1to 0.176 cm3·g-1 (Table S1 ). These findings indicate that a substantial portion of the enzyme occupies the diatomite channels, thereby achieving the effective immobilization of CRL on OSMD.
Figure 2 . FTIR spectrum of diatomite before and after modification (A), VSM plot (B), N2 adsorption and desorption isotherms of OSMD and CRL@OSMD (C), BJH-plot (D)
Figure 3 compares the XPS spectra of OSMD, MD, and raw diatomite. The peaks of 100.1 eV (Si 2p), 259.2 eV (O 1s), and 150.5 eV (Si 2s) are formed by the diatomite, and the peak intensity is significantly weakened after modification, which corroborates the change in the surface groups of the diatomite. Relatively, the peak intensity of C 1s (281.8 eV) was significantly enhanced after modification. Despite the weak intensity, the peak at around 161.0 eV (S 2p) and 225.0 eV (S 2s) is caused by -SO3H. Compared to raw diatomite, nanomagnetic Fe3O4increased the peaks located at 710.5 eV and 719.9 eV[35].Table 1 shows that the C peak intensity of modified diatomite is significantly enhanced, and the content of C is increased from 15.8% to 66.1%. However, the intensity of the O1s peak weakened, and the O content decreased from 61.8% to 22.6%. It is proved that the coating contains a large amount of -C18, and -SO3H successfully modified diatomite. In the high-resolution diagram of Si 2p, the electron absorption peaks at 100.4 eV and 99.8 eV correspond to the Si-O bond and Si-Si bond in diatomite[38]. Compared with unmodified diatomite and MD, the absorption peak at 98.9 eV corresponds to the Si-C, which demonstrates the surface modification of the diatomite with octadecyl groups[39].