Preparation and characterization of Fe3O4@COF-OMe nanoparticles and immobilized RML.
The facile synthesis of magnetic core-shell COFs is based on the room-temperature synthesis of COF-OMe (Fig S1-S4). The detailed preparation and immobilization process is illustrated in Fig.1, which involved two main steps: (1) coprecipitation synthesis of magnetic Fe3O4 nanoparticles and rapid room-temperature synthesis of the core-shell structured magnetic Fe3O4@COF-OMe composites in a one-pot process by mixing Fe3O4 nanoparticles (30 mg, 0.13 mmol) as the magnetic core and 2,5-dimethoxyterephthalaldehyde (DMTP, 0.24 mmol) and 1,3,5-tris(4-aminophenyl)-benzene (TPB, 0.16 mmol) as building units of COF-OMe in the acetonitrile according to the result of morphology (Fig. S5). (2) immobilization process of RML by physical absorption in PBS buffer. The as-prepared biocomposites could be applied in the production of biodiesel.
The morphologies of COF-OMe, Fe3O4@COF-OMe, and [email protected]3O4@COF-OMe are verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), shown in Figure 2. It can be seen that COF-OMe has good dispersity and exhibits a uniform nanosphere structure (Fig. 2(A)) with a size of 500-600 nm. The core-shell structure of Fe3O4@COF-OMe with the thickness of the COF shell about 70 nm is confirmed by the TEM image (Fig. 2(B)). The Fe3O4@COF-OMe particles display similar spherical morphology as COF-OMe (Fig. 2(C)). And after enzyme immobilization, the composite morphology remains unchanged, only the surface becomes rough (Fig. 2(D)).
Fourier transforms infrared (FT-IR) spectroscopy is carried out to prove that the successful synthesis of Fe3O4@COF-OMe. As shown in Figure S6, The FTIR spectrum of Fe3O4 contains a band at 579 cm-1, which is assigned to characteristic Fe-O-Fe stretch. The characteristic absorption bands at 1610cm-1 assigned to the C=N stretch mode observed in the curve of Fe3O4@COF-OMe means the successful synthesis of COF-OMe by condensation of aldehydes and amines. Along with the characteristic Fe-O-Fe stretch found in the curve of biocomposite, the combination of COF-shell and magnetic Fe3O4 was proved, demonstrating the successful preparation of Fe3O4@COF-OMe.41
The crystalline structure of Fe3O4@COF-OMe is examined by PXRD patterns (Fig. 3(A)). XRD image exhibits 6 peaks with 2θ at 30.12°, 35.42°, 43.18°, 53.64o, 56.96°, and 62.60°, corresponding to (220), (311), (400), (422), (511) and (440),41 which matches well with magnetite, indicating that the Fe3O4@COF-OMe are well crystallized after coating COFs. As to COFs, the characteristic peak appears about 2.0° of 2θ (Cu Kα1), attributed to the (100) facet of a primitive hexagonal lattice.29 This is found in the PXRD image of COF-OMe pattern, shown inset. The peak at 2.75° is attributed to the plane (100) of COF-OMe. The other planes, like (110), (200), (210), (220) corresponds to peaks at 4.8°, 5.5°, 7.3° and 9.2°.45 This pattern confirms the formation of the crystalline form of COF-OMe. These successful and facile preparation represents it an alternative way of traditional synthesis of them, which provides guidance for the exploration of other COFs.
Thermogravimetry analysis (TGA) expounds on the thermal stability and different components of biocomposites, shown in Fig. 3(B) and Fig. S7 (DTG). For COF-OMe, there is a distinct decrease in weight that occurs at 300-400 ℃, which means its structure begins to disintegrate. In other words, a long plateau under 419 oC demonstrates the high thermal stability of COF-OMe. As for [email protected]3O4@COF-OMe, the weight loss at about 280 oC can be attributed to the removal of lipase. The two parts of mass losses occurred at 48 oC and 410 oC is consistent with it of bare Fe3O4 (4 % at 48 oC) and COF-OMe (12 % at 410 oC) respectively. The sharp weight-loss at over 700 oC may due to the reaction between melt COF-OMe and Fe3O4 of core-shell structure. In a word, the support Fe3O4@COF-OMe displays such satisfactory thermal stability as COF-OMe, where the TG curve runs smoothly under 400 oC. At the same time, the core-shell structure doesn’t react mutually under 700 oC, which means it is qualified to be a good carrier of an enzyme.
The magnetic property of these nanospheres is characterized by a vibrating sample magnetometer (VSM). The magnetic hysteresis curve (Fig. 3(C)) of nanomagnetic Fe3O4 has an excellent magnetic property, with a saturated magnetization value of 46.07 emu g-1. There is a drop observed in Fe3O4@COF-OMe (~20 emu g-1) and [email protected]3O4@COF-OMe (~6 emu g-1), which are attributed to the loading of COF shell and enzyme. Despite this, rapid aggregation of biocomposites from the suspension is obtained with the help of an external magnet, which could reduce the desorption of the enzyme by centrifugation in this way.
Nitrogen sorption isotherms measured at 77 K indicates the BET surface of Fe3O4@COF-OMe decreases from 232 cm2 g-1 to 28 cm2 g-1 after immobilization of RML (Fig. 3(D)). The pore-size distribution analyses of Fe3O4@COF-OMe and [email protected]3O4@COF-OMe calculated by the density functional theory have shown that both of the samples have a pore size centered at about 3.1 nm, whereas the pore volume drops from 0.223 cc g-1 to 0.036 cc g-1 after RML absorption (Figure S8 & Table S1). The result indicates the successfully loading of lipase, and it suggests that the magnetic COFs may serve as a promising carrier for lipase immobilization.
To further verify the distribution of RML on the support, the Fluorescein-labelled enzyme is an optical way to prove its existence and determine its distribution. Fluorescent probe fluorescein isothiocyanate (FITC) is used to label the enzyme molecules (green) generally.44, 46 However, it is not available to use in [email protected] in this work. This is because the support, COF-OMe itself, is fluorescent. Under an excitation λ=488 nm (the parameter of FITC-labelled protein), the long emission at λ=490-690 nm is got by the COF-OMe itself, which interferes with the detection of the FITC-labelled enzyme, so it is incapable to prove the existence of enzyme on the surface of the carrier and determine its distribution (Figure S9). In this case, Rhodamine B isothiocyanate (RBITC)-labeled RML was prepared. The RBITC-labelled RML (red) is present throughout Fe3O4@COF-OMe (green), which is observed by CLSM analysis at excitation wavelengths of 488 nm for Fe3O4@COF-OMe and 543 nm for RBITC-RML, demonstrating that the enzyme accommodated in this composite (Figure S10).
The optimization of the immobilization process
RML is immobilized on the carrier by physical absorption and different conditions of immobilization will affect the loading of enzymes. Then, the effect of time, concentration of lipase, and temperature were studied during the immobilization process. The enzyme loading of Fe3O4@COF-OMe increased with the time at the beginning (Figure 4(A)), and decreased after 8h. It can be explained that long-term shaking caused leakage of lipase after absorption saturation. The temperature made an influence on both immobilization efficiency and activity of RML. In the immobilization process, we mixed the support and lipase in 4oC and 25oC respectively. We found there was an improvement in RML attaching with stirring at room temperature (Figure 4(B)). The lipase solution with the initial concentration of 10, 20, 40, 80 mg/L was prepared. Though the relative immobilization efficiency decreased as the ratio of the enzyme increased, the total amount of immobilized RML further increased with a higher concentration of RML (Figure 4(C)). According to the results, the immobilization process was undergoing at 25oC, mixing 80mg/L of lipase with 10mg support for 8 hours.
After the immobilization, the hydrolysis of p-NPA (see details in Support information) was adapted to examine whether the immobilized RML is active and its stability. In this work, we employed the core-shell magnetic COFs (Fe3O4@COF-OMe) to enhance the recovery efficiency. Here, we also compared this strategy with the common mixing method. This tactic is to make COFs magnetic by mixing COFs and magnetic Fe3O4 nanoparticles in solutions (Fe3O4-COF-OMe), where the Fe3O4 nanoparticles are attached on the surface of COFs, shown in Scheme 2. The SEM & EDS mapping images display these two magnetic strategies in detail (Fig. S11&S12).
As shown in Fig. 5. After immobilization, there was a shuttle decrease in activity in hydrolysis of p-NPA of both immobilized RML (Fig. 5(A)). The best outcome could recover to 60% of the free enzyme (Fe3O4@COF-OMe) as the time prolonged. The Fe3O4@COF-OMe also showed good thermal and pH stability. The stability of the activity for both free RML and immobilized enzyme in different pH ranging from 5.0 to 10.0 was studied and plotted in Fig. 5(B). The result showed that the optimal pH altered slightly, from about 7.0 to 8.0. Thermal stability was investigated, which the biocomposites were stored at 60 ℃ for 12h ahead of tests. It was observed that there is a decrease in activity for all of them, but the range of decrease was not significant for Fe-COFs immobilized RML (Fig. 5(C)). We found that although the Fe3O4-COF-OMe has a higher RML uptake, the enzyme activity of it did not perform well. It is due to the non-uniform and solid two-phase framework prepared by physical mixing and adhesion strategy, which was not enough to maintain the RML activity.
Based on the outcomes, Fe3O4@COF-OMe with better thermal stability and activity can indeed be optimal support for the subsequent study of transesterification reaction.
Activity Assay of transesterification reaction
The activities of both free lipase and immobilized RML were determined by a transesterification reaction between 2-phenyl ethanol and vinyl acetate (Figure S13). First of all, both Fe3O4@COF-OMe and Fe3O4-COF-OMe were adapt to catalyze under the same conditions. We found that this result was consistent with it of hydrolysis of p-NPA (Fig. 5) where it was Fe3O4@COF-OMe that performed better than Fe3O4-COF-OMe (Fig. 6(A)). At the same time, in the transesterification reaction, the yields of both immobilized enzymes were higher than that of free enzymes, which proved the excellent protective effect of carriers on the enzyme.
The solvent effect of the reaction in which n-Hexane functioned as a solvent was shown in Fig. 6(B), with the highest yield up to 80%. Interestingly, according to the results, we found that the yields altered by the trends of the polarity of different solvents. In detail, the more hydrophobic the solvent was, the higher yields we got. As for the optimized solvent, whose log P value was largest, the yield was much higher than the others at the same time. Carbon tetrachloride, trichloroethylene, and toluene, whose polarity was similar, had almost the same yields of 20%. However, if the solvent was hydrophile, such as THF, acetone, the transesterification didn’t happen in it. To furtherly verify the hydrophobic solvents were conducive to this reaction, several homologous liquids of n-Hexane were adapted (Fig. S14). n-Hexane, c-Hexane, and n-Heptane had similar yields, which indicated the hydrophobic solvents were beneficial in this work.
Then we investigated the influence of temperature on the reaction (Fig. 6(C)). Immobilized RML did better in the transesterification than the free enzyme. With the rise of temperature, the yield of the immobilized enzyme increased gradually and reached a peak at about 50 ℃, while of liquid enzyme decreased continuously. The loss of activity may due to the conformation change of RML caused by high temperature, which affected the binding of the active center and substrate. It demonstrated that the carrier could effectively protect the enzyme from heat and kept its catalytic activity.
The dosage of RML has also played an important role in a transesterification reaction, where excessive enzyme not only causes waste but also reduces the rate due to the aggregation. So here, we studied the yields of the reaction with different amounts of RML. As we can see, in Fig. 6(D), the yield of free RML still went up along with the increase of amount. For Immobilized RML, the yield didn’t show a significant rise when the RML on carrier changed to 0.8 mg. Considering the efficiency and economy of this reaction, 0.5 mg RML was used in every single sample assay. At the optimal conditions, n-Hexane as a solvent, the transesterification yield can reach about 80% with 0.5 mg of immobilized RML at 50 ℃.
The preservation of activity by the protection of support in organic solvents and high temperature were shown above, where the immobilized RML always did better in different organic solvents and at over 30 ℃ than free lipase. To furtherly assess the function of COFs in protecting the catalytic ability of RML, the tolerance of immobilized RML against ultrasonic operation was investigated. As shown in Figure S15, the yields of immobilized RML did not change significantly after ultrasonic treatment, and always higher than that of free RML at the same time. Here we also studied the leakage ratio of RML by washing the immobilized lipase (Fig. 7). As we can see, the amount of loss of RML for every single wash was about 2% and the total leakage ratio was less than one-fifth after the 8 wash cycle, which indicated a good ability to preserve the lipase from washing operation.
Production of biodiesel
Having established the efficiency of [email protected]3O4@COF-OMe in the transesterification reaction of 2-phenol ethanol and vinyl acetate, then we studied its catalytic ability in the production of biodiesel from inedible Jatropha curcas Oil (Table 1). The outcomesa catalyzed by immobilized RML are much better than those by free RML, with a yield of 67.8% and 5.1% respectively. It is noticed that there is an obvious loss in enzymatic activity when the amount of methanol exceeds the stoichiometric ratio (3:1). This is due to the inhibitory effect of methanol, and the activity is irreversibly inactivated.36 Compared to free RML, magnetic Fe3O4 nanoparticles could protect RML from the methanol, as the product could be detected with a satisfactory yield. It is noticed that the protective effect is not permanent although the activity could be maintained if the concentration of methanol is doubled (Entry 4, Table 1). But if the amount of methanol is excessive too much (15:1 and 30:1), there is a huge loss in yield. In a word, the nanoparticle efficiently improved the stability and maintained the activity of the enzyme in practical application.
Table. 1 [email protected]3O4@COF-OMe-catalyzed production of Biodiesel by Jatropha curcas Oila.
aReaction conditions: Jatropha curcas Oil (0.15 mmol), methanol (0.45 mmol, 10 μL), n-Hexane (3 mL), [email protected]3O4@COF-OMe/RML (5mg of RML), and 50 °C at 100 rpm for 48 h. The yields were determined by GC.