3.1. Synthesis of GO-Asp-Fe3O4 nanoparticles
The synthesis of GO-Asp-Fe3O4 was investigated using FT-IR, XRD, and VSM. The FT-IR spectrum of GO (Fig. 2) revealed characteristic peaks at 3427 cm− 1, 1724 cm− 1, 1617 cm− 1, and 1050 cm− 1 that could be assigned to the O–H, the C = O carboxylic group, the C = C and C–O stretching vibrations. After treatment with L-Asp, the intensity of these peaks decreased. The FT-IR spectrum of GO-Asp also displays two peaks at 1622 cm− 1 and 1400 cm− 1, which correspond to the amide carbonyl group and C-O stretching of the carboxylic acid group, respectively. Moreover, the peak at 2998 cm− 1 corresponds to the alkane group of aspartic acid, which confirms the functionalization of the GO nanosheets. Similar bands are also observed in the case of GO-Asp- Fe3O4. In addition, the appearance of two peaks at 559 cm− 1 and 630 cm− 1 suggests the presence of the Fe-O bond, which corresponds to the existence of Fe3O4 nanoparticles on the graphene oxide surface.
Figure 3 illustrates the XRD pattern of GO, GO-Asp, and GO-Asp-Fe3O4. GO shows a sharp diffraction peak at 2θ value of 11.06, attributing to the (002) reflection. Following the chemical functionalization with L-Asp, the peak shifted to 2θ values of 13.04. Moreover, a reduction in the intensity of the (002) reflection peak was observed, suggesting the introduction of amino acid molecules between the layers (23). The XRD pattern of the GO-Asp-Fe3O4 nanocomposite exhibits six characteristic peaks of Fe3O4 at around 2θ = 30.42, 35.72, 43.54, 53.92, 57.36, and 62.96, corresponding to the crystallographic indices (220), (311), (400), (422), (511), and (440), respectively. These peaks closely match the positions and intensities of pure magnetite as per the JCPDS card no.75-1610. Moreover, the disappearance of the (002) reflection peak of GO is attributed to the inability of the GO sheets to form crystalline structures once they are coated with the magnetic nanoparticles.
The magnetization curve of GO-Asp-Fe3O4 (as shown in Fig. 4) demonstrates a specific saturation magnetization (Ms) of 70 emu. This characteristic high magnetic property of GO-Asp- Fe3O4 is desirable for various applications, including industrial enzyme immobilization, magnetic resonance imaging, and targeted delivery. Additionally, the absence of coercivity indicates the superparamagnetic nature of the immobilized Fe3O4 nanoparticles.
3.1.2 Morphology and zeta potential measurement
To investigate the surface morphological changes of GO upon functionalization and magnetization, electron microscopy was applied. According to the FE-SEM micrograph (Fig. 5), the GO surface sheets exhibit a smooth texture characterized by a typical wrinkled structure. Following L-Asp immobilization, the graphene nanosheets display a rougher and more wrinkled surface compared to unmodified GO. Furthermore, the transparency of the graphene layers notably reduced, suggesting the successful GO modification procedure. A notable difference in morphology was observed between GO-Asp and GO-Asp-Fe3O4, with the presence of multiple tiny spots on the nanosheets, indicating the uniform coating of Fe3O4 nanoparticles throughout the GO sheets. Consistent with these findings, TEM images of GO-Asp-Fe3O4 depicted the presence of Fe3O4 nanoparticles on the graphene layers in the form of spherical particles (Fig. 6). These particles exhibited a uniform distribution and an average size of 24.4 nm.
The DLS measurement was utilized to examine the average hydrodynamic particle size distribution of the prepared samples. The average hydrodynamic diameter of GO-Asp was 132 nm, which grows to 299 nm after Fe3O4 immobilization, indicating the formation of the Fe3O4 nanoparticles on the graphene nanosheets. Moreover, the surface zeta potential changed with the alterations in the GO surface. The naïve GO sheets, due to the presence of the negatively charged oxygen-containing groups exhibited a zeta potential of -48.7 mV. The GO-Asp surface showed a higher negative charge (-61.4 mV), potentially due to the incorporation of two carboxylate groups from each L-Asp molecule onto the GO surface. However, after magnetization, the presence of Fe3O4 reduced the overall zeta potential of GO nanosheets to -54.8 mV. With a zeta potential exceeding − 30 mV, the dispersion of GO-Asp-Fe3O4 could be considered stable, attributed to the electrostatic repulsion among colloidal particles.
3.2. Optimization of ASNase immobilization on GO-Asp-Fe3O4 nanocomposite
The GO-Asp-Fe3O4 composite was investigated as a nano-support for the covalent or non-covalent immobilization of ASNase. For covalent attachment of the enzyme, the carboxylic groups of magnetic GO-Asp were first functionalized with EDC/NHS and then reacted with ASNase lysine amine groups via the nucleophilic substitution. The covalent linkage between the amine and carboxylic acid groups prevents rapid leaching of the enzyme from the support (24). The efficacy of the protein immobilization was then assayed by the Bradford method. It was expected that the carrier-to-enzyme weight ratio and GO functional group could affect the immobilization efficacy. According to Fig. 7, for the EDC/NHS activated GO-Asp-Fe3O4 at a fixed ASNase concentration, with an increase in the carrier-to-enzyme weight ratio from 1 to 20, the conjugation degree increased from 44.7–99.2%. The same trend was also observed in the case of the physical adsorption method. However, for the non-activated GO-Asp, the complete immobilization of ASNase was achieved at a carrier-to-enzyme weight ratio of 60, and at a weight ratio of 20 only about 34% of the input ASNase was attached. Proteins could be absorbed on GO non-covalently through hydrophobic, electrostatic, and π-π stacking, and Van der Waals forces interactions. Herein, the L-Asp functionalization and magnetization of GO reduced the hydrophobicity and the possibility of physical interaction of ASNase with graphene sheets which could be a contributing factor to the reduced ASNase loading on GO-Asp. Alam et al. described the maximum efficiency of ASNase immobilization on APTES-modified magnetic nanoparticles was 62% (25). In comparison, for Fe3O4-chitosan and magnetic Fe3O4@MCM-41, the immobilization efficacy was 73.2% and 63%, respectively. These findings suggest that activated GO-Asp-Fe3O4 nanocomposite have a high immobilization yield making them a promising support for immobilizing L-ASNase. Examination of enzyme activity for EDC/NHS-activated GO-Asp-Fe3O4 revealed that as the carrier-to-enzyme weight ratio rose from 1 to 20, enzyme activity increased from 5.6–38.9%, demonstrating successful enzyme immobilization. Research showed that the enzyme's catalytic activity decreased slightly after modification (26). In the current research, the recovered enzyme activity was 38.9% when the initial enzyme was fully immobilized likely due to steric hindrance of the matrix leading to decreased substrate accessibility (27). Based on these results, GO-Asp- Fe3O4/ASNase chemically prepared at the carrier-to-enzyme weight ratio of 20 was selected for the rest of the experiments.
3.3. Determination of kinetic parameters
The Km and Vmax values, kinetic parameters, of the free and immobilized enzyme, were evaluated using substrate L-asparagine (Fig. 8) and summarized in Table 1. The Vmax parameter represents the maximum rate of reaction when substrate molecules fill every active site of the enzyme. GO-Asp-Fe3O4/Enz exhibited a significant decrease in Vmax value (100.2 U/mg) compared to the free enzyme (336.7 U/mg). This reduction in activity could be attributed to limitations in substrate diffusion, hindrance caused by steric factors of the support material, or a decrease in the flexibility of the enzyme which is essential for the enzyme-substrate complex formation (28). On the other hand, the Km value of the enzyme decreased approximately 4.4-fold after immobilization, indicating the higher affinity of the ASNase towards the substrate. Similar findings were documented for immobilized ASNase over carbon xerogels and magnetic chitosan.
Table 1
The Michaelis-Menten constants for free and immobilized L-asparaginase on GO-Asp-Fe3O4
Sample | Km (mM) ± SD | Vmax (U/mg) ± SD |
---|
Enz | 5.96 ± 1.39 | 336.7 ± 35.41 |
GO-Asp-Fe3O4/Enz | 1.35 ± 0.31 | 100.2 ± 4.83 |
3.4. pH and thermal stability
pH and thermal stability are two important characteristics for industrial applications and storage stability of biomedical enzymes. Figure 9 demonstrates the effect of immobilization on the pH stability of ASNase in comparison to the free enzyme. Both the free and immobilized ASNase enzymes exhibited over 80% of their catalytic activity within the pH range of 5–9. However, the activity of the free enzyme sharply decreased in extreme pH conditions, with a complete loss of activity at pH 3. Interestingly, the immobilization of ASNase on GO-Asp-Fe3O4 promoted pH stability. The activity recovery of GO was 75% and 84% in pH values of 3 and 12, respectively. The improved stability of ASNase through covalent attachment could be attributed to a reduction in enzyme leaching and protein deformation under varying pH conditions. Furthermore, the active site of the native ASNase is susceptible to elevated concentrations of H + ions. Hence, the protective effect of GO-Asp-Fe3O4 could preserve the enzyme's structure and activity under acidic pH values.
The thermal stability of both free and immobilized ASNase was explored within a temperature range of 40–70°C. According to Fig. 10, ASNase and GO-Asp- Fe3O4/Enz remained stable for up to one hour at 50°C. Elevating the temperature to 60°C led to a significant decrease in the enzymatic activity of free ASNase, with a residual enzyme activity of 24% and 6% after 30 and 60 min of incubation, respectively. In contrast, the GO-Asp-Fe3O4/Enz exhibited higher residual enzyme activity of 47% and 21% within the same incubation times, respectively. At 70°C, the free ASNase lost almost all of its enzyme activity, while the immobilized ASNase retained 16% of its original activity. According to these findings, the recovered enzyme activity of the immobilized ASNase was two- to eight-fold higher than that of the free enzyme. Previous research has also documented the elevated thermal stability of ASNase through immobilization on calcium alginate-gelatin composites and carboxymethyl cellulose matrix (29). The enhanced thermal tolerance of the immobilized enzyme could be attributed to a reduction in enzyme chain flexibility and an elevation in the activation energy required for enzyme deformation.
3.5. Reusability
The potential applications of enzymes in large-scale or industrial settings greatly rely on their reusability. Enzyme immobilization has shown to be a promise in facilitating their recovery across multiple cycles, thereby reducing overall process costs. In the present study, we noticed a slight decrease in enzyme activity for GO-Asp-Fe3O4/Enz in the second cycle, potentially due to the leaching of physically absorbed enzymes (Fig. 11). However, in the subsequent six consecutive cycles, the recovered enzyme activity remained consistently high at 80%. This suggests that the covalent attachment of ASNase to GO nanosheets can prevent protein leaching and conformational changes during repeated use. The notable reusability aligns with the positive impact of covalent immobilization on thermal stability as reported in section 3.4. In comparison with non-magnetic GO-Asp (20), the covalent immobilization of ASNase on the novel GO-Asp-Fe3O4 led to an approximately twofold increase in the final recovered activity, underscoring the high potential applicability of magnetized GO-Asp-Fe3O4 in the food and biotechnology industries. This result is in good consistency with other enzymes immobilized on magnetic GO, for example, the reported recovered activity after 8 cycles is 68% for laccase (30), 88% for lactase (31), 79% for ficin (32), and 80% for b-xylosidase (33).