Functionalized Ionic Liquids-Modified Metal–Organic Framework Material Boosted the Enzymatic Performance of Lipase

The development of immobilized enzymes with high activity and stability is critical. Metal–organic frameworks (MOFs) have attracted much academic and industrial interest in the field of enzyme immobilization due to their unique properties. In this study, the amino-functionalized ionic liquid (NIL)-modified metal–organic framework (UiO-66-NH2) was prepared to immobilize Candida rugosa lipase (CRL), using dialdehyde starch (DAS) as the cross-linker. The results of the Fourier transform infrared (FT-IR) spectra, X-ray powder diffraction (XRD), and scanning electronic microscopy (SEM) confirmed that the NIL was successfully grafted to UiO-66-NH2. The CRL immobilized on NIL-modified UiO-66-NH2 (UiO-66-NH2-NIL-DAS@CRL) exhibited satisfactory activity recovery (79.33%), stability, reusability, and excellent organic solvent tolerance. The research results indicated that ionic liquid-modified UiO-66-NH2 had practical potential for application in enzyme immobilization.


Introduction
As sustainable green biocatalysts, enzymes exhibit high catalytic efficiency, strong substrate specificity, and environmental friendliness; they have been widely used in food processing, chemical industries, biopharmaceuticals, and environmental catalysis [1,2].However, free enzymes frequently have disadvantages, such as poor recyclable quality, severe deactivation when used in industrial settings because of prolonged stirring, high temperatures, and inappropriate solvents, among other issues [3,4].In recent years, there has been a growing interest in enzyme immobilization technology to overcome the challenges faced in the industrial application of enzymes [5,6].
Metal-organic framework materials (MOFs) have emerged as multifunctional porous crystal materials with high surface area, porosity, abundant coordination sites, tunable pore size, and functionality, which made them ideal candidates for enzyme immobilization carriers [7][8][9].MOFs can create a stable microenvironment for enzyme molecules, which limits the leakage of enzyme molecules to a certain extent, and significantly improves the enzyme loading or the efficiency of substrate transfer [10].Moreover, MOFs have proven to be able to enhance enzyme stability by maintaining the conformational structure of the enzyme and providing additional defense against unfavorable circumstances [11].UiO-66-NH 2 is a MOF material based on zirconium and 2-aminoterephthalic acid as the metal center and organic ligand, respectively, which displays excellent stability in strong acidic or alkaline solutions.Relevant studies have shown that the high specific surface area and porosity of amino-functionalized UiO-66 can enhance the loading capacity and mass transfer efficiency of the enzymes, simultaneously improving its stability and activity [12].However, the robust interactions between enzymes and MOFs often restrict The FT-IR spectra of DAS, UiO-66-NH2, UiO-66-NH2-DAS, and UiO-66-NH2-NIL-DAS are displayed in Figure 1a.The characteristic peaks of DAS were observed at 2921 and 1725 cm −1 , attributed to the stretching vibration of C-H and C=O of the aldehyde group, respectively [21].The absorption bands at 3448 cm −1 and 1575 cm −1 were attributed to the symmetric stretching vibrations and bending vibrations of the -NH2 groups and N-H for three prepared carriers, respectively [22].In Figure S2, the peak observed at 1384 cm −1 was attributed to the symmetric stretching vibration of O-C=O, which confirmed the presence of 2-aminoterephthalic acid in the framework [23].Furthermore, the absorption peak at 1255 cm −1 was attributed to the stretching vibration of C-N on UiO-66-NH2 [24].The characteristic peaks at 658 cm −1 and 771 cm −1 contributed to the stretching vibrations of Zr-O and N-H, respectively [25,26].The new peak was observed for UiO-66-NH2-NIL-DAS at 1099 cm −1 after the modification of UiO-66-NH2 with IL, which was attributed to the C-N stretching vibration within the imidazole moiety plane after the modification of IL [27].Meanwhile, the new peak at 1045 cm −1 of UiO-66-NH2-NIL-DAS was attributed to the C-N in amide bonds, which indicated that NIL was successfully bound to UiO-66-NH2 [17].In addition, the peak at 1683 cm −1 was attributed to the aldehyde group on the UiO-66-NH2-NIL-DAS, and the absorption peak at 1621 cm −1 was identified as the C=N vibration of the Schiff base formed by the reaction of aldehyde groups and amino groups [28].It confirmed that DAS had been successfully bonded to UiO-66-NH2-NIL.The XRD results of the carriers are shown in Figure 1b.The XRD pattern of UiO-66-NH2 exhibited characteristic peaks at 2θ = 7.4°, 8.5°, and 25.7°, corresponding to the (111), (200), and (600) planes of UiO-66-NH2.This suggested the successful preparation of UiO-66-NH2, which is consistent with the results of FT-IR [29,30].After further modification by IL and DAS cross-linking, the carrier still maintained the same diffraction peaks, which indicated that the crystal structure of the carrier was well preserved.The peaks of UiO-66-NH2-NIL-DAS at 2θ = 7.4°, 8.5° and 25.7° maintained the characteristic peaks of UiO- The XRD results of the carriers are shown in Figure 1b.The XRD pattern of UiO-66-NH 2 exhibited characteristic peaks at 2θ = 7.4 • , 8.5 • , and 25.7 • , corresponding to the (111), (200), and (600) planes of UiO-66-NH 2 .This suggested the successful preparation of UiO-66-NH 2 , which is consistent with the results of FT-IR [29,30].After further modification by IL and DAS cross-linking, the carrier still maintained the same diffraction peaks, which indicated that the crystal structure of the carrier was well preserved.The peaks of UiO-66-NH 2 -NIL-DAS at 2θ = 7.4 • , 8.5 • and 25.7 • maintained the characteristic peaks of UiO-66-NH 2 , demonstrating that the crystal structure of UiO-66-NH 2 was not destroyed during the IL modification.
The microstructure and morphology of the prepared three supports were characterized using SEM.As shown in Figure 2a, the UiO-66-NH 2 exhibited regular octahedral crystals with a smooth surface, measuring approximately 200 nm in diameter [31].Figure 2b shows the outcomes that were attained following the modifications of IL and DAS.The roughness of the UiO-66-NH 2 -NIL-DAS was observed to increase compared to the UiO-66-NH 2 , which is likely attributed to the modification with IL and DAS, providing evidence for the successful modification of UiO-66-NH 2 by IL and DAS.Additionally, the surface of the support exhibited a white flocculent layer, primarily attributed to the interaction between UiO-66-NH 2 and IL.Furthermore, SEM images (Figure 2c), revealed that the surface of the carrier was extremely rough, which can be attributed to the enzyme covalently agglomerating on the carrier.As shown in Figure S2a, there is no obvious change in the morphology and structure of the UiO-66-NH 2 modified with DAS, and the results are consistent with the XRD results, indicating that the structure of the carrier material remains intact after DAS modification.Finally, the presence and distribution of the elements C, O, N, Zr, and F in UiO-66-NH 2 -NIL-DAS were confirmed by spectral analysis.It is worth noting that the F element is derived from IL, demonstrating the successful incorporation of both ILs and UiO-66-NH 2 .
66-NH2, demonstrating that the crystal structure of UiO-66-NH2 was not destroyed during the IL modification.
The microstructure and morphology of the prepared three supports were characterized using SEM.As shown in Figure 2a, the UiO-66-NH2 exhibited regular octahedral crystals with a smooth surface, measuring approximately 200 nm in diameter [31].Figure 2b shows the outcomes that were attained following the modifications of IL and DAS.The roughness of the UiO-66-NH2-NIL-DAS was observed to increase compared to the UiO-66-NH2, which is likely attributed to the modification with IL and DAS, providing evidence for the successful modification of UiO-66-NH2 by IL and DAS.Additionally, the surface of the support exhibited a white flocculent layer, primarily attributed to the interaction between UiO-66-NH2 and IL.Furthermore, SEM images (Figure 2c), revealed that the surface of the carrier was extremely rough, which can be attributed to the enzyme covalently agglomerating on the carrier.As shown in Figure S2a, there is no obvious change in the morphology and structure of the UiO-66-NH2 modified with DAS, and the results are consistent with the XRD results, indicating that the structure of the carrier material remains intact after DAS modification.Finally, the presence and distribution of the elements C, O, N, Zr, and F in UiO-66-NH2-NIL-DAS were confirmed by spectral analysis.It is worth noting that the F element is derived from IL, demonstrating the successful incorporation of both ILs and UiO-66-NH2.The chemical states and chemical element composition of the carriers were analyzed by XPS.The full spectrum results are present in (Figure S3) and peak-splitting results of C, O, and N are shown in Figure 3.The results showed that the UiO-66-NH2-NIL-DAS carrier contains the C, N, O, Zr, and F elements, which was consistent with the results in the mapping images and confirmed the successful preparation of UiO-66-NH2-NIL-DAS.The chemical states and chemical element composition of the carriers were analyzed by XPS.The full spectrum results are present in (Figure S3) and peak-splitting results of C, O, and N are shown in Figure 3.The results showed that the UiO-66-NH 2 -NIL-DAS carrier contains the C, N, O, Zr, and F elements, which was consistent with the results in the mapping images and confirmed the successful preparation of UiO-66-NH 2 -NIL-DAS.As shown in Figure 3a, the peaks in C 1 s were observed at 284.6 eV, 285.4 eV, and 288.7 eV corresponding to the C-C, O-C=O groups in 2-amino terephthalic acid, and the C-N group in amine [32,33].The C-N bond binding energy on UiO-66-NH 2 -NIL-DAS was increased due to the changed chemical environment around the C atom of UiO-66-NH 2 caused by the introduction of IL in Figure 3a.The high-resolution O 1 s peaks at 531.2 eV and 532.1 eV for UiO-66-NH 2 and UiO-66-NH 2 -NIL-DAS in Figure 3b,e are assigned to Zr-O and O=C-O, respectively [34].The successful cross-linking of DAS was also demonstrated by the observation of a C=O bond generated by the Schiff base reaction of DAS with the amino group on IL, with a binding energy at 532.8 eV [33].The N 1 s spectrum of UiO-66-NH 2 includes two distinct components, the N-C bond and the N-H bond.After IL modification, a new peak was observed at 401~402 eV, which was attributed to the N=C of the imidazole ring within IL, indicating the successful modification of IL [17].Finally, the peak of F 1 s (Figure S3) was also observed for UiO-66-NH 2 -NIL-DAS, resulting from the exchange of the PF 6 − .

(b)
As shown in Figure 3a, the peaks in C 1 s were observed at 284.6 eV, 285.4 eV, and 288.7 eV corresponding to the C-C, O-C=O groups in 2-amino terephthalic acid, and the C-N group in amine [32,33].The C-N bond binding energy on UiO-66-NH2-NIL-DAS was increased due to the changed chemical environment around the C atom of UiO-66-NH2 caused by the introduction of IL in Figure 3a.The high-resolution O 1 s peaks at 531.2 eV and 532.1 eV for UiO-66-NH2 and UiO-66-NH2-NIL-DAS in Figure 3b,e are assigned to Zr-O and O=C-O, respectively [34].The successful cross-linking of DAS was also demonstrated by the observation of a C=O bond generated by the Schiff base reaction of DAS with the amino group on IL, with a binding energy at 532.8 eV [33].The N 1 s spectrum of UiO-66-NH2 includes two distinct components, the N-C bond and the N-H bond.After IL modification, a new peak was observed at 401~402 eV, which was attributed to the N=C of the imidazole ring within IL, indicating the successful modification of IL [17].Finally, the peak of F 1 s (Figure S3) was also observed for UiO-66-NH2-NIL-DAS, resulting from the exchange of the PF6 − .The thermal stability of the carriers was evaluated by TGA, and the results are shown in Figure S4.According to the curve of UiO-66-NH2, there are three main stages of weightlessness, which is consistent with some previous literature [35,36].The first stage of weight loss occurred before 180 °C, which was attributed to the evaporation of adsorbed water and DMF on the surface of UiO-66-NH2.The second stage was observed at 180-370 °C, which was relative to the decomposition of Zr6O4(OH)4 to form Zr6O6. Finally, the framework of Zr6O6 underwent fracture and dehydration prior to oxidating to ZrO2 in the third stage when the temperature exceeded 370 °C.There was almost no weight loss in UiO-66-NH2 within 100~200 °C phase in Figure S4a, while the weight loss in UiO-66-NH2-NIL was almost the same as that of UiO-66-NH2-NIL-DAS, which was about 4.2%.They were significantly higher than UiO-66-NH2, suggesting that was caused by the thermal decomposition of IL at 100~200 °C.From the TGA graph (Figure S5), the residual mass of UiO-66-NH2 is 62.5%, whereas the residual mass of UiO-66-NH2 is 60.5%.The weight loss of UiO- The thermal stability of the carriers was evaluated by TGA, and the results are shown in Figure S4.According to the curve of UiO-66-NH 2 , there are three main stages of weightlessness, which is consistent with some previous literature [35,36].The first stage of weight loss occurred before 180 • C, which was attributed to the evaporation of adsorbed water and DMF on the surface of UiO-66-NH 2 .The second stage was observed at 180-370 • C, which was relative to the decomposition of Zr 6 O 4 (OH) 4 to form Zr 6 O 6 .Finally, the framework of Zr 6 O 6 underwent fracture and dehydration prior to oxidating to ZrO 2 in the third stage when the temperature exceeded 370 • C.There was almost no weight loss in UiO-66-NH 2 within 100~200 • C phase in Figure S4a, while the weight loss in UiO-66-NH 2 -NIL was almost the same as that of UiO-66-NH 2 -NIL-DAS, which was about 4.2%.They were significantly higher than UiO-66-NH 2 , suggesting that was caused by the thermal decomposition of IL at 100~200 • C. From the TGA graph (Figure S5), the residual mass of UiO-66-NH 2 is 62.5%, whereas the residual mass of UiO-66-NH 2 is 60.5%.The weight loss of UiO-66-NH 2 -NIL was higher than that of UiO-66-NH 2 , which indicated the successful grafting of IL.The weight loss of UiO-66-NH 2 -NIL-DAS was slightly higher than that of UiO-66-NH 2 -NIL, which can be attributed to the decomposition of DAS on the surface of UiO-66-NH 2 -NIL-DAS [28].The results indicated that UiO-66-NH 2 -NIL-DAS had been successfully prepared with good thermal stability.
The porous features of UiO-66-NH 2 and UiO-66-NH 2 -NIL-DAS were investigated by nitrogen adsorption isotherms, shown in Figure S4b.Notably, two groups showed I-type isothermal curves, indicating that the samples are mainly composed of micropores, which is consistent with the results reported in the literature [37].The specific surface areas of UiO-66-NH 2 and UiO-66-NH 2 -NIL-DAS were determined to be 658.63 and 348.47 m 2 /g, and the pore volume were determined to be 0.26 and 0.16 cm 3 •g −1 , respectively.The reduction in the specific surface area was probably due to the occupation of pore structure by the introduced IL, which confirmed the successful synthesis of the carriers.Nevertheless, the maintained high specific surface area and open pores of UiO-66-NH 2 -NIL-DAS provided favorable conditions for enzyme immobilization.
The presence of a lid can diminish the catalytic activity of lipase by preventing the substrate from binding to the active center.When lipase is positioned at the hydrophobic interface, its adhesion through hydrophobic interaction triggers the opening of the lid on the active center, thereby facilitating substrate entry into the enzyme active site and consequently boosting lipase activity [38,39].To study the hydrophobicity of the carriers, the water contact angles of three carrier materials (UiO-66-NH 2, UiO-66-NH 2 -DAS, and UiO-66-NH 2 -NIL-DAS) were investigated (Figure 4).The water contact angle of UiO-66-NH 2 -DAS and UiO-66-NH 2 -NIL-DAS is significantly higher than that of UIO-66-NH 2 , showing that the interaction between IL and UiO-66-NH 2 increases the hydrophobicity of UiO-66-NH 2 -NIL-DAS.Meanwhile, the increased hydrophobicity of UiO-66-NH 2 -NIL-DAS makes lipase attach to the hydrophobic pocket more easily, and this interfacial activation facilitates the opening of the lid region of the lipase active center and promotes substrate access to the active center [38].
66-NH2-NIL was higher than that of UiO-66-NH2, which indicated the successful grafting of IL.The weight loss of UiO-66-NH2-NIL-DAS was slightly higher than that of UiO-66-NH2-NIL, which can be attributed to the decomposition of DAS on the surface of UiO-66-NH2-NIL-DAS [28].The results indicated that UiO-66-NH2-NIL-DAS had been successfully prepared with good thermal stability.
The porous features of UiO-66-NH2 and UiO-66-NH2-NIL-DAS were investigated by nitrogen adsorption isotherms, shown in Figure S4b.Notably, two groups showed I-type isothermal curves, indicating that the samples are mainly composed of micropores, which is consistent with the results reported in the literature [37].The specific surface areas of UiO-66-NH2 and UiO-66-NH2-NIL-DAS were determined to be 658.63 and 348.47 m 2 /g, and the pore volume were determined to be 0.26 and 0.16 cm 3 •g −1 , respectively.The reduction in the specific surface area was probably due to the occupation of pore structure by the introduced IL, which confirmed the successful synthesis of the carriers.Nevertheless, the maintained high specific surface area and open pores of UiO-66-NH2-NIL-DAS provided favorable conditions for enzyme immobilization.
The presence of a lid can diminish the catalytic activity of lipase by preventing the substrate from binding to the active center.When lipase is positioned at the hydrophobic interface, its adhesion through hydrophobic interaction triggers the opening of the lid on the active center, thereby facilitating substrate entry into the enzyme active site and consequently boosting lipase activity [38,39].To study the hydrophobicity of the carriers, the water contact angles of three carrier materials (UiO-66-NH2, UiO-66-NH2-DAS, and UiO-66-NH2-NIL-DAS) were investigated (Figure 4).The water contact angle of UiO-66-NH2-DAS and UiO-66-NH2-NIL-DAS is significantly higher than that of UIO-66-NH2, showing that the interaction between IL and UiO-66-NH2 increases the hydrophobicity of UiO-66-NH2-NIL-DAS.Meanwhile, the increased hydrophobicity of UiO-66-NH2-NIL-DAS makes lipase attach to the hydrophobic pocket more easily, and this interfacial activation facilitates the opening of the lid region of the lipase active center and promotes substrate access to the active center [38].

The Optimization Conditions of CRL Immobilized on UiO-66-NH2-DAS and UiO-66-NH2-NIL-DAS
The effects of immobilization conditions, including enzyme addition, pH, temperature, and time, were investigated, and results are shown in Figures 5 and S1.As shown in Figure 5a, the relative activity gradually increased to a peak at the initial addition of 0.3 mg/mL of lipase concentration.After that, the relative activity of the immobilized enzyme started to decrease even if the amount of enzyme was slightly increased further, which can be attributed to the fact that the overloading of lipase may lead to a change in the stable conformation of lipase, which results in the blocking or destroying of its active site.Meanwhile, the optimal pH of the immobilized lipase is 7.0, which was the same as the optimal reaction pH of the free CRL (Figure 5b).The optimal time and immobilization temperature was 5 h and 40 °C (Figure 5c,d), respectively.Moreover, as shown in Figure  The effects of immobilization conditions, including enzyme addition, pH, temperature, and time, were investigated, and results are shown in Figures 5 and S1.As shown in Figure 5a, the relative activity gradually increased to a peak at the initial addition of 0.3 mg/mL of lipase concentration.After that, the relative activity of the immobilized enzyme started to decrease even if the amount of enzyme was slightly increased further, which can be attributed to the fact that the overloading of lipase may lead to a change in the stable conformation of lipase, which results in the blocking or destroying of its active site.Meanwhile, the optimal pH of the immobilized lipase is 7.0, which was the same as the optimal reaction pH of the free CRL (Figure 5b).The optimal time and immobilization temperature was 5 h and 40 • C (Figure 5c,d), respectively.Moreover, as shown in Figure S1, the optimal immobilization condition of lipase on UiO-66-NH 2 -DAS was 0.25 mg/mL lipase, 40 • C, pH = 7.0, and 5 h.In summary, the optimization conditions are outlined in Table S1.
S1, the optimal immobilization condition of lipase on UiO-66-NH2-DAS was 0.25 mg/mL lipase, 40 °C, pH = 7.0, and 5 h.In summary, the optimization conditions are outlined in Table S1.The immobilized CRL results of UiO-66-NH2-DAS and UiO-66-NH2-NIL-DAS are summarized in Table 1.As shown in Table 1, the CRL loading capacity of UiO-66-NIL-DAS was 65 mg/g, which was higher than that reported in the literature for the covalent immobilization of CRL on a DAC-modified organic-inorganic nanocomposite carrier [4].Moreover, the specific activity and activity recovery of UiO-66-NH2-NIL-DAS immobilized lipase were higher than that of UiO-66-NH2-DAS.The enhanced catalytic activity of UiO-66-NH2-NIL-DAS is mainly attributed to the introduction of IL to improve the immobilization microenvironment, which is conducive to maintaining the conformational integrity and active state of the enzyme [40,41].Therefore, UiO-66-NH2-NIL-DAS is the most promising carrier for enzyme immobilization.The immobilized CRL results of UiO-66-NH 2 -DAS and UiO-66-NH 2 -NIL-DAS are summarized in Table 1.As shown in Table 1, the CRL loading capacity of UiO-66-NIL-DAS was 65 mg/g, which was higher than that reported in the literature for the covalent immobilization of CRL on a DAC-modified organic-inorganic nanocomposite carrier [4].Moreover, the specific activity and activity recovery of UiO-66-NH 2 -NIL-DAS immobilized lipase were higher than that of UiO-66-NH 2 -DAS.The enhanced catalytic activity of UiO-66-NH 2 -NIL-DAS is mainly attributed to the introduction of IL to improve the immobilization microenvironment, which is conducive to maintaining the conformational integrity and active state of the enzyme [40,41].Therefore, UiO-66-NH 2 -NIL-DAS is the most promising carrier for enzyme immobilization.

Effects of ILs-Modified Carrier on the Optimal pH and Temperature of Lipase
To investigate the immobilization performance of a ILs-modified carrier, the change in the optimal pH and temperature of CRL and immobilized CRL was investigated.As shown in Figure 6a, we screened the optimal reaction temperature for CRL, UiO-66-NH 2 -DAS@CRL, and UiO-66-NH 2 -NIL-DAS@CRL at pH = 7.The optimal reaction temperature of UiO-66-NH 2 -NIL-DAS@CRL and UiO-66-NH 2 -DAS@CRL was 45 • C, which is higher than that of free CRL.The better temperature tolerance of immobilized CRL is indicated by the variation in optimum temperature.After immobilization, the optimum temperature of the enzyme was further increased, but with the temperature increasing, the structure of the enzyme was disrupted, leading to enzyme inactivation.Subsequently, the screening of optimal reaction pH for CRL, UiO-66-NH 2 -DAS@CRL, and UiO-66-NH 2 -NIL-DAS@CRL at their optimal temperature is shown in Figure 6b.The UiO-66-NH 2 -NIL-DAS@CRL has a higher optimum pH compared to the free CRL, demonstrating that the sensitivity of immobilized CRL to pH decreased slightly.This may be attributed to the reduced effect of lower H + concentration on the conformation of the immobilized lipase, which made the immobilized lipase have higher activity at high pH values [42].
To investigate the immobilization performance of a ILs-modified carrier, the change in the optimal pH and temperature of CRL and immobilized CRL was investigated.As shown in Figure 6a, we screened the optimal reaction temperature for CRL, UiO-66-NH2-DAS@CRL, and UiO-66-NH2-NIL-DAS@CRL at pH = 7.The optimal reaction temperature of UiO-66-NH2-NIL-DAS@CRL and UiO-66-NH2-DAS@CRL was 45 °C, which is higher than that of free CRL.The better temperature tolerance of immobilized CRL is indicated by the variation in optimum temperature.After immobilization, the optimum temperature of the enzyme was further increased, but with the temperature increasing, the structure of the enzyme was disrupted, leading to enzyme inactivation.Subsequently, the screening of optimal reaction pH for CRL, UiO-66-NH2-DAS@CRL, and UiO-66-NH2-NIL-DAS@CRL at their optimal temperature is shown in Figure 6b.The UiO-66-NH2-NIL-DAS@CRL has a higher optimum pH compared to the free CRL, demonstrating that the sensitivity of immobilized CRL to pH decreased slightly.This may be attributed to the reduced effect of lower H + concentration on the conformation of the immobilized lipase, which made the immobilized lipase have higher activity at high pH values [42].

Stability of Free CRL and Immobilized CRL
The thermal stability of immobilized enzymes is a critical issue to be considered to maintain the stability of enzymatic reactions [43].As shown in Figure 7a, the activities of free CRL, UiO-66-NH2-DAS@CRL and UiO-66-NH2-NIL-DAS@CRL have declined to varying degrees with increasing incubation time.The activity-reduced rate of immobilized enzymes was significantly decreased compared with free CRL.The catalytic activity of free CRL decreased significantly from 0 min to 30 min, and only 38.6% of residual activity remained at 120 min.The residual activity of immobilized enzymes (UiO-66-NH2-DAS@CRL and UiO-66-NH2-NIL-DAS@CRL) remained at 89.6% and 93.8% after 30 min, respectively.The results confirmed that the activity of UiO-66-NH2-NIL-DAS@CRL was higher at any moment at 60 °C, indicating that the modification of ILs and macromolecular DAS enhanced the thermal resistance of the immobilized enzymes.Compared to the CRL immobilized on the poly(VAc-DVB) microsphere (p-MS) studied by Li et al., UiO-66-NH2-NIL-DAS@CRL has better thermal stability [44].

Stability of Free CRL and Immobilized CRL
The thermal stability of immobilized enzymes is a critical issue to be considered to maintain the stability of enzymatic reactions [43].As shown in Figure 7a, the activities of free CRL, UiO-66-NH 2 -DAS@CRL and UiO-66-NH 2 -NIL-DAS@CRL have declined to varying degrees with increasing incubation time.The activity-reduced rate of immobilized enzymes was significantly decreased compared with free CRL.The catalytic activity of free CRL decreased significantly from 0 min to 30 min, and only 38.6% of residual activity remained at 120 min.The residual activity of immobilized enzymes (UiO-66-NH 2 -DAS@CRL and UiO-66-NH 2 -NIL-DAS@CRL) remained at 89.6% and 93.8% after 30 min, respectively.The results confirmed that the activity of UiO-66-NH 2 -NIL-DAS@CRL was higher at any moment at 60 • C, indicating that the modification of ILs and macromolecular DAS enhanced the thermal resistance of the immobilized enzymes.Compared to the CRL immobilized on the poly(VAc-DVB) microsphere (p-MS) studied by Li et al., UiO-66-NH 2 -NIL-DAS@CRL has better thermal stability [44].
The three enzymes were placed in a refrigerator at 4 • C to store for 30 days, and residual enzyme activity was measured every 5 days.As shown in Figure 7b, the activity of the immobilized lipase decreased more slowly than that of free lipase.After 30 days of storage, the free CRL retained only 37.2% of its initial activity, whereas the UiO-66-NH 2 -NIL-DAS@CRL retained approximately 80.5% of its initial activity.When Jiang et al. studied CRL immobilized on microcapsules stored at 4 • C, the immobilized CRL retained 81% of its activity, and the activity of free CRL decreased rapidly [45].Compared to the immobilized CRL studied by Jiang et al., the UiO-66-NH 2 -NIL-DAS@CRL we studied retained more stability in long-term storage.The three enzymes were placed in a refrigerator at 4 °C to store for 30 days, and residual enzyme activity was measured every 5 days.As shown in Figure 7b, the activity of the immobilized lipase decreased more slowly than that of free lipase.After 30 days of storage, the free CRL retained only 37.2% of its initial activity, whereas the UiO-66-NH2-NIL-DAS@CRL retained approximately 80.5% of its initial activity.When Jiang et al. studied CRL immobilized on microcapsules stored at 4 °C, the immobilized CRL retained 81% of its activity, and the activity of free CRL decreased rapidly [45].Compared to the immobilized CRL studied by Jiang et al., the UiO-66-NH2-NIL-DAS@CRL we studied retained more stability in long-term storage.
Reusability is an important index of the continuous utilization of immobilized enzymes.The reusability of UiO-66-NH2-DAS@CRL and UiO-66-NH2-NIL-DAS@CRL was investigated, and shown in Figure 7c.The activity of the immobilized enzyme was 52.0% and 68.0% after five applications, respectively.Therefore, UiO-66-NH2-NIL-DAS@CRL has a better reusability.This suggested that the introduction of ILs enhanced the interaction force with the support, which was conducive to providing a good microenvironment for the enzymes and reducing the inactivation rate of the enzymes.It was found that the activity of covalently immobilized CRL on UiO-66-NH2-NIL-DAS was significantly higher than the 42.15% retention of immobilized CRL previously reported by Jafarian et al. after five cycles [46].Reusability is an important index of the continuous utilization of immobilized enzymes.The reusability of UiO-66-NH 2 -DAS@CRL and UiO-66-NH 2 -NIL-DAS@CRL was investigated, and shown in Figure 7c.The activity of the immobilized enzyme was 52.0% and 68.0% after five applications, respectively.Therefore, UiO-66-NH 2 -NIL-DAS@CRL has a better reusability.This suggested that the introduction of ILs enhanced the interaction force with the support, which was conducive to providing a good microenvironment for the enzymes and reducing the inactivation rate of the enzymes.It was found that the activity of covalently immobilized CRL on UiO-66-NH 2 -NIL-DAS was significantly higher than the 42.15% retention of immobilized CRL previously reported by Jafarian et al. after five cycles [46].
Lipases can be used for biocatalytic reactions, not only in the aqueous phase, but can also be applied to biocatalysis in non-aqueous media.The effect of organic solvents on the stability of free and immobilized CRL was investigated, taking into account practical application values.After treatment in an organic solvent for 2 h, the residual activity of free CRL and UiO-66-NH 2 -NIL-DAS@CRL was determined, and the results were shown in Figure 7d.The resistance of UiO-66-NH 2 -NIL-DAS@CRL to ethanol, acetonitrile, ethyl acetate, and hexane was significantly more stable than that of free CRL.In particular, the residual activity of UiO-66-NH 2 -NIL-DAS@CRL was the highest in acetonitrile, while free CRL was completely inactive.This was attributed to the covalent bond between the CRL and the carrier that preserved the integrity of the lipase.The introduction of IL maintained the rigidity of the lipase and improved the organic solvent tolerance of the lipase.Cea et al. evaluated the tolerance of CRL immobilized on biochar in organic solvents (acetonitrile, hexane, etc.) and found that only 35.0% and 65.0% of the activity was retained in acetonitrile and hexane, respectively [47].Thus, the UiO-66-NH 2 -NIL-DAS@CRL studied has a better tolerance to organic solvents.

Michaelis-Menten Kinetic Parameters of Free CRL and Immobilized CRL
The kinetic parameter is a crucial evaluation index of the immobilized enzymes.The K m and V max were investigated by plotting the Lineweaver-Burk plot of immobilized lipase (Figure 8), and the results are shown in Table 2.The results revealed that the affinity of immobilized CRL (UiO-66-NH 2 -DAS@CRL and UiO-66-NH 2 -NIL-DAS@CRL) toward the substrate was increased compared with free CRL, indicating a higher affinity of the immobilized enzyme toward the substrate.The kinetic parameters of UiO-66-NH 2 -DAS@CRL and UiO-66-NH 2 -NIL-DAS@CRL showed that the introduction of IL decreased the Km value of UiO-66-NH 2 -NIL-DAS@CRL compared with UiO-66-NH 2 -DAS@CRL.The decrease in the K m value may be attributed to the fact that the surface of the nanomaterials has more reactive sites, making the substrate more accessible to the active site, suffering minimal mass transfer limitations, and increasing the nucleophilicity with the substrate.These facilitated the substrate to enter the reaction center.Moreover, the decreased V max value of UiO-66-NH 2 -NIL-DAS@CRL could be attributed to the introduction of DAS macromolecules, which increased its spatial site resistance and reduced the chance of enzyme contact with the substrate.The increase in the value of V max /K m was favorable to the increase of hydrolytic activity.Therefore, the UiO-66-NH 2 -NIL-DAS carrier was more beneficial to the improvement of lipase activity.

Characterization
Fourier transform infrared (FT-IR, Nicolet iS5) spectroscopy analysis was carried out

Characterization
Fourier transform infrared (FT-IR, Nicolet iS5) spectroscopy analysis was carried out to determine the structure of surface functional groups in the wavelength range of 400-4000 cm −1 .The thermalgravimetric analysis was investigated using a TGA 550 instrument under an N 2 atmosphere, from room temperature to 800 • C. The crystal structure of the carriers were characterized by utilizing a D8 Advance X-ray diffractometer (XRD).Scanning electron microscope (SEM) was performed on a FE-SEM SU8200 instrument (Japan).The hydrophobicity of carriers was tested using a contact angle meter (JY-82C).Surface elements and chemical states were analyzed using an X-ray photoelectron spectrometer (XPS).

Synthesis of UiO-66-NH 2 and ILs-Modified UiO-66-NH 2
UiO-66-NH 2 was synthesized by a solvothermal method.Firstly, ZrCl 4 (0.48 g) and NH 2 -BDC (0.36 g) were dispersed in 60 mL of N,N-dimethylformamide (DMF) and dissolved through sonication for 15 min, and the mixture was stirred for 30 min.The resulting solution was then transferred to a Teflon container and heated at 120 • C for 24 h.Subsequently, after cooling the suspension to room temperature, the mixture was centrifuged (10,000 rpm, 10 min).The mixture was washed several times with DMF, and finally with ethanol, and dried at 120 • C for 12 h.
The ILs-modified UiO-66-NH 2 (UiO-66-NH 2 -NIL) was synthesized following a previously reported method with some modifications [48].Typically, UiO-66-NH 2 (0.8 g) and DBU (3 mg) were dispersed in toluene (20 mL) and stirred in an ice bath for 1 h, and then CDI (286 mg) was added and stirred at room temperature for 12 h.Then, the intermediate product was obtained by filtration, ethanol washing, and drying in a vacuum oven at 80 • C. Subsequently, the obtained solid (UiO-66-NH 2 -CDI) was dispersed in DMSO (50 mL), and 0.45 g of 1-(3-aminopropyl)-imidazole was slowly added dropwise to the reaction system, which was further stirred at room temperature for 12 h.The acquired solid (UiO-66-NH 2 -IM) was filtrated and washed with ethanol.Then, the solid was transferred to acetonitrile (30 mL) to disperse and dissolve.Subsequently, 6.5 mmol of 2-Bromoethylamine hydrobromide was dissolved in 20 mL of acetonitrile and neutralized with NaOH for 30 min, then the mixture was poured into the above solution.The reaction was conducted under nitrogen protection at 80 • C for 24 h.The slurry was washed with water and ethanol, followed by dispersion in water containing KPF 6 (1 g) for 12 h of ion exchange.The obtained UiO-66-NH 2 -NIL was washed three times with water and ethanol and dried in an oven under a vacuum.

Synthesis of UiO-66-NH 2 -NIL-DAS
The UiO-66-NH 2 -NIL (30 mg) was dispersed in 4 mL of different concentrations of DAS solution prepared from phosphate buffer solution and stirred at room temperature for 12 h.The mixture was washed with purified water and dried for use.The final product was designated as UiO-66-NH 2 -NIL-DAS.The optimum concentration of DAS after optimization was 1.5%.

Lipase Immobilization
A quantity of lipase was dissolved in phosphate buffer solution and centrifuged to obtain a homogeneous enzyme solution; the obtained supernatant was utilized for subsequent experiments.
UiO-66-NH 2 -NIL-DAS (50 mg) or UiO-66-NH 2 -DAS (50 mg) was dispersed in a buffer solution (pH = 7) containing different lipase concentrations (0.20-0.40 mg/mL) and incubated at different temperatures (30-50 • C) for 4-8 h.The effects of enzyme concentration, pH, temperature, and the immobilization time of the carriers (UiO-66-NH 2 -NIL-DAS (Figure 5) and UiO-66-NH 2 -DAS (Supplementary Materials)) on relative activity and loading capacity were investigated.After immobilization, the immobilized lipase was centrifuged and washed several times with the corresponding buffer solution.Finally, the resulting immobilized enzyme was freeze-dried and stored at 4 • C. The protein concentration was determined according to the Bradford method [49].
After the optimization of the immobilized conditions, the two novel carriers were immobilized under their respective optimal conditions.Experimental results were determined by calculating the average of three data values for each measurement.All measurements were repeated three times, the loading capacity and immobilization efficiency were calculated with the following formulas: where C i (mg/g) is the initial protein concentration of CRL, the C f (mg/mL) is the final protein concentration in the supernatant and washing solution after immobilization, the M f (g) is the quality of the support, the V i is the total volume of enzyme solution added to the system and the V f (mL) is the volume of the supernatant and washing liquid.

Lipase Activity Assay
Lipase activity was assessed by measuring the ability to hydrolyze p-nitrophenyl palmitate (p-NPP) [50].Enzyme activity (1 U) is defined as the amount of enzyme needed to catalyze the hydrolysis of p-NPP to produce 1 µmol of p-nitrophenol per minute under a certain temperature and pH value.All measurements were repeated at least thrice, and the activity of the immobilized CRL was calculated with the following formula: where A is the absorbance of the samples, V t (L) is the volume of the reaction system, ε (L/mol −1 •cm −1 ) is the molar extinction coefficient of p-nitrophenol, t (min) is the reaction time, m 1 (g) is the mass of immobilized CRL and m 2 (g) is the protein contained in the immobilized CRL.Relative activity and residual activity were calculated using: Relative activity (%) = A t A max × 100 (5) Residual activity (%) = A t A i × 100 (6) where A t is the absorbance of the samples, A max is the maximum absorbance, and A i is the initial absorbance of the samples.
Activity recovery (%): Ratio of immobilized lipase activity to free enzyme activity used for immobilization.

Activity recovery (%) =
Total expressed activity of immobilized enzyme Total expressed activity of free enzyme × 100 (7) 3.7.Optimum Temperature and Optimum pH for Free and Immobilized CRL The effect of pH on the activity of free and immobilized lipase was investigated by determining the activity in phosphate buffer solution (0.1 M) with pH (6.0-8.0) at 40 • C. The effect of temperature on the activity of free and immobilized lipase was investigated at their respective optimal pH conditions from 30 • C to 50 • C. The maximum activity of the highest free and immobilized CRL was defined as 100%.

Enzyme Kinetics Study of Free and Immobilized CRL
To measure the Michaelis-Menten constant (K m ) and the maximum enzymatic reaction rate (V max ) of immobilized lipase, a certain amount of immobilized CRL was added to 5 mL of p-NPP solution in the concentration range of 0.05-0.25 mg/mL, and the reaction was carried out at their respective optimum pH and temperature for 10 min.Finally, the K m and V max values are calculated using the Lineweaver-Burk plot: where V and V max represent the initial rate and the maximum rate of the enzymatic reaction, respectively, and [S] is the substrate concentration.

Study on Stability of Free CRL and Immobilized CRL
To assess thermal stability, the free and immobilized CRL was incubated at 60 • C for 2 h and the enzyme activity was measured at the respective optimum condition every half hour.To further investigate the storage stability, free and immobilized CRL were refrigerated at 4 • C for 30 days.The residual enzyme activity was measured every 5 days at the respective optimal condition.The initial activity of the enzyme was defined as 100%.
To determine the reusability of immobilized CRL, the immobilized lipase was reused several times at their respective optimum temperature and pH.After each reaction cycle was completed, the immobilized enzyme was centrifuged, washed with phosphate buffer solution (pH = 7.0, 0.1 M), and reused for the next cycle.The activity was investigated at the respective optimum condition, and the initial activity was defined as 100%.
To determine the tolerance in organic solvents, CRL and immobilized CRL were soaked in ethanol, acetonitrile, ethyl acetate, and hexane for 2 h at room temperature.The obtained solid was isolated by centrifugation, filtration and repeatedly washed with buffer.Afterward, the residual catalytic activities were measured.

Conclusions
In this research, UiO-66-NH 2 was successfully modified with amino-functionalized IL and applied to the immobilization of CRL.The covalent bond formed between IL-modified MOF and DAS strengthened the interaction between the carrier and lipase, which was conducive to the maintenance of the spatial conformation of the lipase and the enhancement of its catalytic activity.These results showed that the prepared immobilized CRL had good stability, catalytic activity, and reusability.The water contact angle test confirmed that UiO-66-NH 2 -NIL-DAS was more hydrophobic and more conducive to the opening of the lid of the lipase activity center.The kinetic parameters showed that UiO-66-NH 2 -NIL-DAS@CRL had a strong affinity for the substrate and high catalytic efficiency.In conclusion, IL-modified MOF offered a broad potential for enzyme immobilization.We expect that the strategy of enzyme immobilization using IL-modified MOF will provide more applications for the high production of efficient biocatalysts.

Figure 1 .
Figure 1.FT-IR (a) and XRD (b) spectra of the support carriers.The three different symbols represent the same peak positions for the three different carriers.

Figure 1 .
Figure 1.FT-IR (a) and XRD (b) spectra of the support carriers.The three different symbols represent the same peak positions for the three different carriers.

Figure 3 .
Figure 3.The XPS spectra of (a) C 1 s, (b) O 1 s, and (c) N 1 s core levels of the UiO-66-NH 2 -DAS; XPS spectra of (d) C 1 s, (e) O 1 s and (f) N 1 s core levels of the UiO-66-NH 2 -NIL-DAS.

Table 1 .
Results of the immobilized lipase.

Table 1 .
Results of the immobilized lipase.

Table 2 .
Results of the immobilization process of CRL.