Preparation and characterization of Fe 3 O 4 @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 detail 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 benzidine (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 Fig. 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 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 of monomer 2,5-dimethoxyterephthalaldehyde (DMTP) at 2868 cm− 1, 2760 cm− 1 and 1677 cm− 1 demonstrate the existence of aldehyde groups, where the 1677 cm− 1 is assigned to the C = O stretching. The bands at 1207 cm− 1, 1025 cm− 1 attributed to the aromatic = C-O-C asymmetric and symmetric vibration respectively and the peak at 1610 cm− 1 assigned to the C = N stretch mode are observed in the curve of Fe3O4@COF-OMe, which 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.
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 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 ℃ demonstrates the high thermal stability of COF-OMe. As for [email protected]3O4@COF-OMe, the weight loss at about 280 ℃ can be attributed to the removal of lipase. The two parts of mass losses occurred at 48 ℃ and 410 ℃ is consistent with it of bare Fe3O4 (4% at 48 ℃) and COF-OMe (12% at 410 ℃) respectively. The sharp weight-loss at over 700 ℃ 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 ℃. At the same time, the core-shell structure doesn’t react mutually under 700 ℃, 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 the 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 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).
Immobilized RML activity assay
To facilitate recycling in the application, we employed the core-shell magnetic COFs (Fe3O4@COF-OMe) to enhance the recovery efficiency. Here, we first 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 Fig. 4. The SEM & EDS mapping images display these two magnetic strategies in detail. In Fig. S11, the element Fe (purple pots) gather in some specific places. Compared with the morphology image left, it can be attributed to smaller particles (magnetic Fe3O4). This is the evidence that the Fe3O4 is adhering to the surface of COFs. However, element Fe (the yellow pots in Fig. S12) are averagely distributed on the image with other color dots, which represents nanoparticles Fe3O4 are wrapped.
Both immobilized enzyme uptake capacities were calculated by the method of Coomassie blue staining (Table S1). And the enzymatic activity was tested as followed.
The activity of immobilized RML was studied by hydrolysis of p-NPA (see details in Support information). The catalytic activities, stability in a different environment (such as pH, heat) of an immobilized enzyme, were shown in Fig. 5. After immobilized, 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 ℃ overnight 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 in framework prepared by physical mixing and adhesion strategy, which was not enough to maintain the RML activity.
Based on the outcomes of activity assay, Fe3O4@COF-OMe can indeed be optimal support to maintain RML activity for the subsequent transesterification reaction.
Optimization of transesterification conditions
The preparation process of biodiesel is a kind of transesterification reaction which belongs to a specific catalytic reaction of lipase. So here, we studied the catalytic performance of immobilized RML on transesterification of 2-phenol with vinyl acetate as a templet reaction and improved the yields by optimizing conditions (Figure S13). First of all, both Fe3O4@COF-OMe and Fe3O4-COF-OMe were adapt to catalyze under the same feeding ratio (20 µL of 2 phenol, 40 µL of vinyl acetate, 2 mL of solvents and 0.1 mg of RML). We found that this result was consistent with it of enzyme activity. It was [email protected]3O4@COF-OMe that performed best (Fig. 6(A)). At the same time, the yields of the immobilized RML were better than that of free RML, nevertheless, the activities were lower than it. This proved that in the application, the protection functioning was far greater than the loss of enzyme activity.
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 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)). Apparently 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 it 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, in Figure, we studied the yields of the reaction with different amount of enzyme. 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 enzyme added from 0.5 to 0.8 mg. Considering the efficiency and economy of this reaction, 0.5 mg of free and immobilized RML was used in every single sample assay. At the optimal conditions, n-Hexane as a solvent, the transesterification yield can reach to 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 loss of RML for every single wash was about 2% and the total leakage ratio was less than one-fifth after 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 templet reaction, then we studied its catalytic ability in the production of biodiesel from non-edible Jatropha curcas Oil (Table 1). The outcomesa catalyzed by immobilized RML are much better than it 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 (0.5 mg), and 50 °C at 100 rpm for 48 h. The yields were determined by GC.