Atomic force microscopy (AFM)
Atomic force microscopy (AFM) height profile of the spin-coated sample on the silicon oxide substrate was shown in figure 2. The profile revealed a successful exfoliation of graphite layers and demonstrated the few-layer graphene oxide structure. Therefore, the results conﬁrm the synthesis of nanoscale few-layer graphene oxide. As shown in the graph, the thickness of the GO pieces is approximately 2.5 nm. Considering that the thickness of every layer in the graphite structure is 0.8 nm, the achieved GO pieces have almost three layers of carbon sheets 41.
Figure 3 shows the FT-IR spectra of a) MNP, b) GO, and c) MGO. For the bare MNP (Figure 3a), two dominant peaks were appeared at 567 cm-1 and 3400 cm-1 which are related to Fe-O and O-H bonds, respectively. In the GO spectrum (Figure 3b), the broad peak around 3400 cm-1 is attributed to O-H stretching vibration indicating the presence of O-H and COOH functional groups in the GO structure. The presence of oxygen-containing functional groups, such as C=O stretching vibration of carboxylic and carbonyl groups, C–O stretching vibration of an epoxy group and C–OH group was demonstrated with the peaks appeared at 1732, 1252 and 1040 cm-1, respectively. Furthermore, the presence of C=C bonds in aromatic rings on GO plates was demonstrated with the peak appeared at 1611 cm-1 42. The spectrum of MGO showed a peak that appeared at 574 cm-1. It is related to the Fe-O bond and can be detected in all the magnetic nanostructures spectrums (Figures 3 and 4).
Figure 4 shows the FTIR spectra of a) MGO-AP, b) MGO-AP-GA, c) ROL, and d) ROL/ MGO-AP-GA. In the first functionalization step of MGO (Figure 4a), the appearance of peaks at 1094 and 1034 cm-1 are related to Si–O–C and Si–O–Si bonds and confirmed the functionalization of MGO with amine groups. The bonds in the range of 2850–2920 cm-1 ascribed to CH2, provide another evidence of AP attachment 43. In the MGO-AP-GA spectrum (Figure 4b), enhancement of absorption peaks at 1635 cm−1 can be related to the N=C covalent bond formation between glutaraldehyde and AP. Also, the peak at 1740 cm−1 can be related to C=O aldehyde groups of glutaraldehyde. The FTIR spectrum of ROL (Figure 4d) indicated two characteristic peaks in the wavenumbers of 1610 cm-1 and 1454 cm-1. The peak at 1610 cm-1 associated with the C=O stretching vibration in amide-type I and 1454 cm-1 associated with the N-H bending and C-N stretching vibrations in amide-type II 44. The related peaks of amide-type I and amide-type II are attributed to the protein backbone of the enzyme. Broadening and increasing intensity of the characteristic absorption peaks of ROL in the FTIR spectrum of ROL/MGO-AP-GA (Figure 4d) confirmed successful immobilization of ROL on MGO-AP-GA 45.
Typical XRD patterns for synthesized supports are presented in figures 5 and 6. The existence of six characteristic peaks at 2θ of 30.0◦, 35.4◦, 43.1◦, 53.5◦, 57.0◦, and 62.6◦ in the spectrum of MNP (Figure 5a) indicated the formation of the crystalline structure of MNP. The XRD pattern of GO in figure 5b showed a strong and sharp peak at 2θ=10.74°, which is related to the exfoliated GO with a d-spacing of 0.84 nm 46. All diffraction peaks in the XRD pattern of MGO (Figure 5c) are related to the MNP crystalline phase. The characteristic peak of GO was broadened and weakened due to the exfoliation of GO during the MGO synthesis process. As shown in figure 6, the appeared peaks in XRD patterns of MGO-AP, MGO-AP-GA, and ROL/MGO-AP-GA are similar to the MGO peaks which means that functionalization and immobilization process has no effect on the crystallinity of MGO.
The FESEM was employed to characterize the morphology of the prepared MNP, GO and MGO. Also, the FESEM and EDS elemental mapping were used for evaluation of morphology and elemental distribution on ROL/MGO-AP-GA, as the nano-bio catalyst with the best performance (Figure 7). As shown in figure 7a, the spherical shape of MNP is obvious and the particle size is in range of 20-30 nm. The smooth and sheets-like structure of GO can be observed in figure 7b. Figure 7c shows the FESEM micrograph of MGO. The created wrinkles and the spherical-shape MNP nanoparticles are clearly obvious on the surface of GO. Furthermore, the size range of MNP on the GO sheets is relatively as same as the bare MNP. Figure 7d illustrates the FESEM micrograph of ROL/MGO-AP-GA. The brighter ﬁne zones that are visible on the surface of MGO in FESEM micrograph can be related to ROL immobilization on MGO-AP-GA. The EDS elemental mapping of ROL/MGO-AP-GA shows a good distribution of elements on the nano-bio catalyst (Figure 7c).
Vibrating sample magnetometer (VSM)
Vibrating sample magnetometer (VSM) analysis was used for the evaluation of magnetic properties of synthesized supports. As shown in figure 8, the magnetic hysteresis loop for all curves was S-like shape over the applied magnetic field at room temperature, indicating that the samples were superparamagnetic. The magnetic response of MNP, MGO, MGO-AP, and MGO-AP-GA were 63.67, 23.19, 17.56 and 16.06 emu.g-1, respectively. Depletion in saturation magnetization (Ms) of MGO compare with the bare MNP can be attributed to the relatively low MNP mass ratio in the MGO hybrid. The hybrid magnetization changes with the different MNP to GO mass ratios. After AP and GA grafting on MGO, the Ms value was decreased due to the introduction of non-magnetic components to the magnetic one.
Zeta potential measurement
The zeta potential measurement was carried out in phosphate buffer medium (100 mM, pH 7.5) to evaluate the surface charge of synthesized supports. As shown in Table 1, the zeta potential of MGO was -33.58 which more negative than of MNP. The more negative charge of MGO than MNP is related to hydroxyl and carboxyl groups on MGO surface. After the modiﬁcation of MGO with AP, a slight increase of zeta potential was observed. It can be related to this fact that amine groups contain less negative charge than hydroxyl and carboxyl groups 47. The zeta potential of the MGO-AP-GA decreased to -20.24 when the GA was added to the MGO-AP.
The loading capacity of immobilized ROL on the supports was evaluated using Bradford’s method and results were reported in figure 9. For this purpose, the supports were exposed to enzyme solutions with different ROL initial concentrations. As shown in figure 9a, loading capacity increased with increasing of initial ROL concentration and then reached a maximum value for each support. However, the enzyme loading showed no significant increase for the solutions with concentrations upper than 21mg.mL-1 and 14 mg.mL-1 for MGO-based supports and bare MNP, respectively. Saturation of supports because of their limited capacity is the reason of this observation 48. The optimum loading percentage of ROL on the supports was varied from 24.23±0.55 to 70.20±0.69 wt.% (Figure 9b). The MGO-AP-GA showed the highest ROL loading capacity and the lowest was attributed to MNP. It can be related to the lower surface area of MNP compared with MGO-based supports. Also, the functionalization of MGO with AP and GA increased loading capacity due to creating a wider spherical area for enzyme attachment.
Figure 10 shows the effect of the ROL initial concentration on the relative activity of nano-biocatalysts. The prepared nano-biocatalysts in the process of loading capacity tests were used for relative activity evaluation. The relative activity was defined as the ratio of each sample activity to its maximum activity 49. The increasing of initial ROL concentration caused relative activity enhancement up to the maximum value (100%). The more loaded enzyme and subsequently, more enzyme-substrate contacts in the reaction increased the relative activity. In the case of MNP, when the ROL initial concentration was 14.78 mg.mL-1, the loading capacity and relative activity were maximum. For MGO-based nano-biocatalysts, the highest relative activity was obtained for ROL initial concentration of 10.61 mg.mL-1 and was not equal to the maximum loading capacity. More increasing in ROL initial concentration causes the accumulation of enzyme molecules on supports surface. Therefore, the substrate accessibility to enzymes is limited and the relative activity decreases 48.
The Michaelis-Menten kinetic parameters for the hydrolysis of p-PNP in the presence of free and immobilized ROL were measured and presented in table 2. The Km is the substrate concentration in which the reaction rate is at half of its maximum velocity and shows an affinity of attachment between enzyme and substrate. The ROL immobilization on the supports caused an obvious reduction in Km value which indicating more enzyme-substrate affinity of attachment. The Km was decreased by more than 4-fold after attachment of AP and GA to MGO. The less negative ζ potential of MGO-AP than MGO which is due to the existence of amine groups in AP structure, as well as the increasing of surface hydrophilicity due to the GA attachment can be responsible.
The maximal velocity (Vmax) is defined as the maximum rate of reaction when the enzyme active sites are saturated with substrate and the kcat is calculated by dividing the Vmax to the ROL concentration in the reaction mixture. As shown in table 2, Vmax and kcat decreased after ROL immobilization on the supports due to the substrate diffusion limit. This behavior has been observed for enzymes which immobilized on different carriers50-52. However, because of the larger surface area of MGO, the mass transfer limit is reduced when MGO is used as support and Vmax and kcat are increased compared with MNP. According to the kcat to Km ratio which is an estimation of catalytic efficiency, a significant increase was observed in ROL immobilized on functionalized MGO supports indicating higher catalytic efficiency. The kcat/Km value for ROL/MGO-AP-GA was 25.83 which was about 4-fold more than ROL/MGO. It indicates that covalent bonding between the ROL and MGO-AP-GA can be considered as an efﬁcient method for lipase immobilization.
The time-course thermal stabilities of all nano-biocatalysts were studied by incubating them at 60 °C in different time durations (10 to 60 min) and evaluation of their residual activities (Figure 11). The residual activity was measured at the optimum conditions after cooling in ice for 30 min and mentioned as thermal stability. Nano-biocatalysts residual activity which incubated at 60 °C for 0 min considered as control (with 100% activity). As shown in figure 10, the residual activity of free ROL was decreased by increasing the incubation time. According to the figure, the thermal stability of ROL/MGO is more than ROL/MNP which is due to the more functional groups on MGO compare with MNP. After the functionalization of MGO with AP, electrostatic attractions between ROL and support increased and led to a noticeable improvement in the thermal stability of nano-bio catalyst. The residual activities of ROL/MNP, ROL/MGO, ROL/MGO-AP, and ROL/ MGO-AP-GA after 60 min incubation and at 60 °C were 13.09 %, 27.20 %, 30.59%, and 33.44% respectively. The ROL/MGO-AP-GA has the highest thermal stability and it demonstrated that enzyme more attraction with support causes more thermal stability. It can be related to the lower flexibility of the enzyme structure when it has more attractions with the support. The lower flexibility makes the enzyme more resistant to the deformation if the temperature keeps on increasing 53.
To evaluate the immobilization efﬁciency, the storage stability of the immobilized enzyme should be considered as an important requirement. The storage stabilities of the free and immobilized ROL were evaluated at two different incubation temperatures (4 ◦C and 25 ◦C). The results are presented in figure 12. As shown, for the free ROL at 4 ◦C, almost 39.13% of initial activity remained after 30 days, while the activities reached about 51.63 %, 64.27%, 70.72% and 74.49% of initial activity for ROL/MNP, ROL/MGO, ROL/MGO-AP, and ROL/MGO-AP-GA, respectively. Maximum storage stability was related to ROL/MGO-AP-GA which can be attributed to ROL covalent bonding to support. This covalent bonding prevents the conformational change of enzyme and consequently helps to preserve its catalytic activity 54. Figure 12 also shows the storage stability of nano-biocatalysts at room temperature. The behavior was similar to the examination at 4oC. However, a total decreasing of storage stability occurred at 25oC compare with 4oC.
GC-MS technique was used for products to analyze. Four FAMEs (Methyl palmitate, Methyl stearate, Methyl (7E,10E)-7,10-hexadecadienoate and Methyl (9Z,12Z,15Z)-9,12,15-octadecatrienoate) as biodiesel were detected in the product mixture via GC-MS. The results of Chlorella vulgaris oil conversion to biodiesel in the presence of immobilized ROL represents in figure 13. The reaction in the presence of ROL/MNP and ROL/MGO showed the lowest biodiesel conversion of 54.14% and 57.05%, respectively. Since ROL is attached to MNP and MGO via comparatively weak physical adsorption, it is prone to enzyme leaching. Hence it can result in low FAMEs production in comparison with two other nano-biocatalysts. However, a slightgeneral increase of ROL/MGO biodiesel conversion was occurred in order to higher catalytic efficiency of ROL/MGO compared with ROL/MNP.
Biodiesel conversion increased in the presence of ROL/MGO-AP. Amine groups of AP improve the electrostatic interactions due to the negative charge decreasing of the MGO surface as well as decreases the steric hindrance of the enzyme. Therefore, enzyme leaching is less likely to occur and subsequently higher productivity. The highest FAMEs conversion was attributed to ROL/MGO-AP-GA. The more stable and resistant biocatalyst in the harsh reaction conditions was prepared by the functionalization of MGO by AP and GA. The aldehyde groups in GA react with amine groups of ROL to create covalent double bond C=N. The bonds prevent changing ROL conformation during reaction time. In addition, the introducing of AP and GA to the supports facilitates the formation of an active complex between enzyme active sites and substrate via creating a larger polar area around the the support surface.
Nano-bio catalysts reusability
The reusability of nano-biocatalysts was evaluated and results were represented in figure 14. The best reusing performance was obtained for ROL/MGO-AP-GA among other nano-biocatalysts. The 58.77 % of FAMEs conversion was maintained after five cycles of reactions in the presence of ROL/MGO-AP-GA. The results have proved that covalent bonding between the ROL and support helps the physical strength of nano-bio catalyst to be better. So, the nano-bio catalyst stability was increased and consequently higher biodiesel conversion was obtained over multi-cycle reuse.