β-glucosidase from Aspergillus niger was kindly provided by Novozyme (Bagsvaerd, Denmark). Exo-cellulase and Endo-cellulase from Trichoderma longibrachiatum were acquired from Megazyme (Wicklow, Ireland). 3-(aminopropyl) triethoxysilane (APTES) (99%), iron (II) chloride, glutaraldehyde (8%, v/v, aqueous), cellobiose, carboxymethyl cellulose (CMC), chitosan (medium molecular weight), sodium tripolyphosphate (TPP), 3,5-dinitrosalicylic acid, sodium hydroxide, phenol, Rochelle salts (Na-K tartrate), sodium metabisulfite, and citric acid were obtained from Sigma-Aldrich (St. Louis, MO). Hydrogen peroxide (30%, v/v) was sourced from Fisher Scientific (Pittsburgh, PA). Anhydrous methanol (ACS grade) and sodium citrate dihydrate were purchased from EMD Chemicals (Gibbstown, NJ). The protein assay kit (BCS assay) was obtained from Thermo Scientific (Rockford, IL). Neodymium magnets (14,800 G) were procured from K and J Magnetics, Inc (Jamison, PA). Deionized (DI) water (18.2 Ω) was used to prepare aqueous solutions.

MNPs were prepared by oxidative alkaline hydrolysis of ferrous ions, which was described in Figure 1 (Park et al., 2011; Park et al., 2018). Briefly, ferrous chloride (FeCl₂) was dissolved in 50 mL of deionized water to create a 0.05M FeCl₂ solution. To this solution, 1 M potassium hydroxide (KOH) was added to form an aqueous suspension of ferrous hydroxide (Fe(OH)₂). The pH of the mixture was carefully adjusted to a range of 7.9–8.0 while continuously stirring to ensure uniform mixing. The precipitation reaction was allowed to proceed for 2 h at room temperature, resulting in the formation of black precipitates. The separated MNPs were then subjected to a series of washes: three times with 50 mL of deionized water to remove any residual reactants, followed by two washes with 50 mL of ethanol to further purify the nanoparticles. After washing, the MNPs were dried at room temperature to obtain the final product.

Particle sedimentation was assessed over time. A 2 mL solution of MNP, APTES-MNP, and CS-MNP (each at 0.025 mg/mL) was placed in a PMMA cuvette, and optical density at 405 nm was measured over a 2-h period with data collected at 10-min intervals.

10 mg of surface-modified MNPs and CS-MNP were redispersed in three different enzyme solutions: exo-cellulase (0.72 mg/mL in citrate buffer at pH 5), endo-cellulase (0.87 mg/mL in citrate buffer at pH 5), and β-glucosidase (0.64 mg/mL in citrate buffer at pH 5). The mixtures were gently shaken overnight to ensure proper interaction between the enzymes and the nanoparticles. After the incubation period, the resulting enzyme-MNP bioconjugates were separated from the solution using magnetic decantation. These bioconjugates were then washed three times with 10 mL of citrate buffer to remove any unbound enzymes. After washing, the bioconjugates were redispersed in 10 mL of citrate buffer and stored at 4°C to preserve their stability. The amount of protein (enzyme) that was loaded onto the nanoparticles’ surface was determined by analyzing the supernatant from each bioconjugate preparation.

The activities of native exo-cellulase, endo-cellulase, β-glucosidase (βG), and their bioconjugates were measured using carboxymethyl cellulose (CMC) and cellobiose (CB) as substrates. The rate of reducing sugar production was determined by the 3,5-dinitrosalicylic acid (DNS) reducing sugar assay. CMC served as the substrate for exo-cellulase, endo-cellulase, and their bioconjugates, while CB was used for βG and its bioconjugate. The DNS reagent was prepared according to Ghose (1987)(Ghose, 1987). 1 mL of CMC or CB was placed in a heating block at 37°C. Native enzymes and bioconjugates were then added to the substrates and allowed to react for 30 min. Following this, 1 mL of the reaction mixture was added to 1 mL of DNS reagent and heated in boiling water for 5 min. The optical density was measured at 540 nm using a spectrophotometer.

The optimum ratio of bioconjugates to produce glucose was observed. Three different levels (100 μg, 200 μg, and 300 μg) of each bioconjugate were mixed and added to the CMC (2% (w/v)) and paper powder (2% (w/v)). Glucose production with seven different ratios of bioconjugate mixtures (exo-, endo-, βG: 111,112,113,121,131,211,311, the number represents mixing ratio, for instant, 111 represents 0.1 mg of each bioconjugate) of bioconjugates was monitored for 24 h using YSI 2007 biochemical analyzer.

The thermal stabilities of native enzymes and bioconjugates were assessed by subjecting 1–2 mL of each sample to varying temperatures (45°C, 65°C, and 85°C) in a heating block. At hourly intervals, samples were removed from the heating block and mixed with substrates (CMC and cellulose powder). These reaction mixtures were then incubated at 45°C for 1 hour. Glucose production from each reaction mixture was subsequently quantified using a YSI 2007 biochemical analyzer.

The recycling stability of CS-MNP bioconjugates, including Exo-CS-MNPs, Endo-CS-MNPs, and βG-CS-MNPs, was assessed by measuring glucose production from these bioconjugates over 20 consecutive cycles. To achieve this, CMC in citrate buffer served as the substrate. For each cycle, the bioconjugates were mixed with 1 mL of a 2% (w/v) CMC solution and incubated for 1 h at 45°C in a heating block to allow the reaction to proceed. After the reaction, the glucose concentration was determined using the YSI 2700 glucose analyzer. Following glucose measurement, the bioconjugates were collected by placing a magnet at the bottom of a 1.5 mL Eppendorf tube, causing the magnetic nanoparticles to aggregate at the tube’s base. The CMC solution was then carefully removed using a syringe and transferred to a separate 45 mL tube for further analysis or disposal. To prepare the bioconjugates for the next cycle, they were washed with 1 mL of citrate buffer. The magnetic decantation process was repeated to ensure that the bioconjugates were thoroughly cleaned and ready for reuse. This process of reaction, measurement, collection, and washing was repeated for a total of 20 cycles, enabling the evaluation of the bioconjugates’ stability and effectiveness in repeated uses. The consistency in glucose production across these cycles would indicate the robustness and durability of the bioconjugates in catalytic applications.

The Characterization process of MNPs and bioconjugates was similar methods used in previous research (Park et al., 2011; 2018) In brief, the size and morphology of fabricated MNPs were determined by Transmission Electron Microscopy (TEM, Hitachi H-7000) at 75 kV and BET. Samples for each step of the surface modifications on Endo-CS-MNPs were confirmed by surface analysis with X-ray Photoelectron Spectroscopy (XPS, 5,800 series, Multi-Technique ESCA systems). Survey and high resolution scans were collected and analyzed by XPSPeak 4.1 software to ensure their proper surface modifications.

A characteristic TEM image of synthesized MNPs shows a high degree of aggregation (Figure 3) which is explained by the strong magnetic dipole–dipole attractions feature between particles. This aggregation phenomenon for the MNPs is reported in other studies (Tan et al., 2018) it could also be related to the drying process of the samples prior to the morphological analyses. MNPs were spherical with an average diameter of 26 nm, as calculated from the BET analysis (Table 1). Although discrete nanoparticles of 26 nm in size were derived from BET method, the bulk of MNP preparations consisted of aggregated complexes of >140 nm in average size corroborated by dynamic light scattering and TEM results usually show higher aggregation causing solution drying effect on the specimen grids. The particle size was consistent with previously reported results (Kouassi et al., 2005; Mürbe et al., 2008; Amstad et al., 2009; Amstad et al., 2009; Sillu and Agnihotri, 2020; Dong et al., 2024). The average size of the MNPs from TEM (D_TEM) analysis showed good agreement with BET results.

The saturated magnetization (M_S) of nanoparticles is an important measure of their magnetic strength under a fully saturated magnetic field., The Ms value is reported to be 62.3 emu/g, which aligns with data from Park et al. (2018). This is lower than the Ms of bulk magnetite, a phenomenon that is consistent with other studies (Hwang et al., 1997; Tada et al., 2003). The reduction in Ms for nanoparticles compared to bulk material can be attributed to two key factors: decreased particle size and increased surface area (Tada et al., 2003). The study also examines how surface modifications impact the stability of MNPs in solution over time. Three types of nanoparticles were evaluated: unmodified MNPs, APTES-modified MNPs (APTES-MNPs), and chitosan-modified MNPs (CS-MNPs). The results (Figure 4) are summarized as follows.

• CS-MNPs retained over 95% of their original optical density after 2 hours. This indicates excellent colloidal stability, which is crucial for applications like drug delivery or medical imaging, where nanoparticles need to remain dispersed for extended periods.

The findings imply that surface modification, particularly with materials like chitosan, can dramatically improve the colloidal stability of MNPs. Surface coatings reduce the magnetic interaction between particles and provide steric or electrostatic repulsion, preventing rapid aggregation. This enhancement of stability is particularly important for biomedical applications where nanoparticles need to maintain dispersion in biological fluids for effective performance.

Colloidal stability refers to a system’s ability to keep particles dispersed without aggregating (Dobiáš, 1993). In conventional systems, stability is often explained by DLVO (Derjaguin-Landau-Verwey-Overbeek) theory, which considers the balance between attractive van der Waals forces (V_vw) and repulsive electrostatic forces (V_ES). However, for magnetic nanoparticles, this explanation is insufficient because MNPs have an additional magnetic dipole-dipole interaction (VM) due to their inherent magnetic properties. Thus, an extended or modified DLVO theory has been proposed to account for these magnetic interactions (Dobiáš, 1993; Elimelech et al., 1998; de Vicente et al., 2000). Surface modification of MNPs is a crucial strategy to improve their colloidal stability. By altering the surface chemistry, it's possible to change the balance of forces acting on the nanoparticles. In this study, two types of surface modifications, APTES and CS, were tested. Despite the fact that zeta potential values, which indicate the surface charge and are typically used as a measure of colloidal stability, were not significantly different between APTES-MNP (−20 mV ± 3.3) and CS-MNP (−19.3 mV ± 1.1), their settling behaviors showed notable differences. The DLS size measurements confirmed that the aggregation sizes for both types of modified particles were similar (140 nm), further supporting that there were no major differences in the forces described by DLVO theory (VvdW, VES, and VM). However, the fact that the two modified particles settled differently suggests that the buoyancy effect introduced by the surface modifications may be responsible for their different behaviors. This implies that surface modification alters not just electrostatic interactions but also other factors such as hydrodynamic forces or steric stabilization, which can influence colloidal stability in more subtle ways. The findings highlight the complexity of colloidal stability in magnetic nanoparticles. While traditional metrics like zeta potential and aggregation size give useful insights, they may not capture all the relevant factors, particularly for modified surfaces. The buoyancy effect, for example, might play a more significant role than previously considered, and this warrants further investigation to fully understand how surface chemistry impacts the overall stability and performance of MNPs in various applications.

Throughout the fabrication process of Endo-CS-MNPs, we conducted X-ray photoelectron spectroscopy (XPS) to analyze and verify the chemical composition at each step. Specifically, we collected both survey spectra and high-resolution spectra focusing on the Fe2p1, C1s, O1s, N1s, P1s, and Si2p orbitals. These measurements were crucial for confirming the presence and proper bonding of elements, thus ensuring the successful creation of the bioconjugates. For the fabrication of Endo-APTES-MNPs, we employed a method involving APTES and glutaraldehyde for the immobilization process. Previous studies conducted by our research group demonstrated that this method effectively modified the nanoparticles, supporting the successful attachment of the desired functional groups (Park et al., 2011; 2018). The shift in the peak position of the high-resolution O1s spectra, as depicted in Figure 5, suggests a modification in the surface properties of the material. For the bare MNPs, the most prominent peak was observed at a binding energy of 530.4 electron volts (eV). This value is characteristic of oxide crystal structures, indicating the presence of such structures on the surface of the MNPs (Kloprogge et al., 2006). Following the application of the chitosan coating, a significant peak was detected at 533.6 eV. This peak is attributed to the C-O-C bond present in chitosan. Additionally, there was a slight increase in intensity at 531.6 eV, which can be attributed to the presence of O=P and O-P bonds from sodium tripolyphosphate. When endo cellulase, an enzyme, binds to the magnetic nanoparticles, it induces notable shifts in the peaks. These shifts are caused by various functional groups within the protein structure of endo cellulase, specifically O-H, O-C, O=C, O-N, and O-C-N groups.

After immobilizing the enzymes onto MNPs, the amounts of residual protein (unbound) in the supernatant were measured to determine the levels of bound protein (binding efficiency). The specific activity of the immobilized enzymes was also measured to assess the activity loss during the immobilization process. Both the APTES + glutaraldehyde method and the CS + glutaraldehyde method showed no significant differences in binding efficiency and specific activity, indicating that both APTES and CS provided similar amounts of amine functional groups for the bi-functional glutaraldehyde to attach. The enzyme loading on MNPs of exo- and endo-cellulase was between 417 μg/mg and 417 μg/mg. This values is 6–7 times higher than the previous study (Kaur et al., 2023) However, β-glucosidase (βG) exhibited a relatively lower binding efficiency of 9%–17%, and these results align with earlier studies (Park et al., 2011; Park et al., 2018). The specific activities of bioconjugates are shown in Table 2. In particular, the activity retention of endo-cellulase in both APTES and CS coating bioconjugates was around 50% which is comparable to the previous study (Kaur et al., 2023). This diminished activity retention can be explained by the high native efficiency of endo-cellulase, which naturally produces substantial amounts of reducing sugars from carboxymethyl cellulose (CMC). The high baseline activity of the native enzyme establishes a difficult benchmark for its immobilized counterparts, making it challenging for bioconjugated enzymes to maintain or match the same level of enzymatic performance.

The absence of significant differences in binding efficiency and specific activity between the two methods emphasizes the versatility of both APTES and CS in providing functional groups necessary for effective immobilization. However, the variation in binding efficiency among the different enzymes—particularly the lower binding efficiency observed for β-glucosidase—raises important questions about the nature of enzyme-surface interactions. Factors such as enzyme size, structure, and the spatial arrangement of active sites likely play a role in determining how effectively an enzyme binds to a support surface. For β-glucosidase, its relatively lower binding efficiency could be a consequence of structural or steric hindrances that limit its attachment to the nanoparticle surface, compared to exo- and endo-cellulase.

The study investigates the optimization of glucose production by combining three different levels of bioconjugates—exo-cellulase, endo-cellulase, and β-glucosidase (βG)—using two substrates: carboxymethyl cellulose (CMC) and cellulose powder. The goal was to determine the ideal ratio of these enzymes for maximizing glucose output. According to the findings, a higher concentration of endo-cellulase in the bioconjugate mix consistently resulted in better glucose production for both substrates. The bioconjugate mixture identified as “131,” which incorporated both APTES-MNPs (and CS-MNPs, showed the most promising results, producing the highest glucose levels over a 24-h period (Figure 6). Specifically, with the CMC substrate, the CS-MNPs-based bioconjugate 131 mixture generated 0.367 mg/mL of glucose. In contrast, the APTES-MNPs-based bioconjugate 131 mixture produced significantly less glucose—just 33% of the amount produced by the CS-MNPs variant, yielding 0.121 mg/mL. The results reveal important insights into the catalytic efficacy of bioconjugates and the role of surface modifications in enzyme performance. The marked difference between the glucose production of CS-MNPs and APTES-MNPs highlights the critical influence that surface chemistry exerts on colloidal dispersion stability which we discussed from Figure 4. This suggests that surface modifications on magnetic nanoparticles can either amplify or limit the enzymatic efficiency, a key consideration in industrial-scale applications where optimizing bioconversion rates is essential. The results also emphasize the importance of endo-cellulase in breaking down the internal bonds of cellulose, enhancing overall glucose yield. Most of previous studies used a cellulase mixture for immobilization on solid support and that leaves the enzyme ration unknown (Garcia et al., 1989; Tan et al., 2018; Sillu and Agnihotri, 2020; Paz-Cedeno et al., 2021; Zanuso et al., 2022; Kaur et al., 2023) In practical terms, the findings could be foundational in developing more efficient biocatalysts for biofuel production or other biotechnological processes that rely on cellulose degradation. By fine-tuning the enzyme ratios and nanoparticle modifications, industries could potentially achieve higher yields of glucose from lignocellulosic biomass, which is critical for sustainable energy production.

The glucose production of both the native enzyme mixture and the bioconjugate mixture was analyzed at 45°C, 65°C, and 85°C to assess the thermal stability of immobilized enzymes. The underlying hypothesis is that the three-dimensional structures of immobilized enzymes are stabilized at elevated temperatures due to covalent multipoint attachment to solid supports. This structural reinforcement may provide greater resistance to thermal denaturation compared to their free counterparts. Previous research has demonstrated that native exo-, endo-cellulase, and β-glucosidase (βG) are highly sensitive to heat, particularly at temperatures exceeding 50°C (Andreaus et al., 1999). As depicted in Figure 7, the native enzyme mixture exhibits a dramatic decline in activity, losing more than 95% of its functionality within 2 hours at temperatures above 65°C. This rapid loss can be attributed to the unfolding of the enzyme’s tertiary structure, a process accelerated by thermal stress. In contrast, the bioconjugates formed with chitosan-modified magnetic nanoparticles (CS-MNPs) showed a remarkable improvement in thermal stability. At 65°C, the bioconjugates retained 39.4% of their glucose production capacity, and even at the extreme temperature of 85°C, 22.2% of their initial activity was sustained after 5 hours.

This difference highlights the significant impact of immobilization on enzyme performance. The covalent attachment to the support not only anchors the enzymes but also prevents the excessive molecular flexibility that typically leads to denaturation under thermal stress. This stabilization offers substantial benefits in industrial applications, where processes often occur at elevated temperatures. Immobilized enzymes, such as the CS-MNP bioconjugates, demonstrate a more resilient structure, making them ideal for long-term use in harsh conditions, leading to both economic and practical advantages in enzymatic bioconversion processes.

The glucose production resulting from the hydrolysis of carboxymethyl cellulose (CMC) by the CS-MNPs bioconjugate was assessed in each cycle, as shown in Figure 8A. Initially, the bioconjugate generated a glucose concentration of 0.039 mg/mL, which represents roughly 10% of the yield produced by the native enzyme. Remarkably, over 80% of this initial glucose output was maintained through the 10th cycle. However, after the 10th cycle, a noticeable decline in glucose concentration was observed, dropping to 33% (0.013 mg/mL) of the original value by the 20th cycle.

Figure 8B provides a comparative analysis of glucose productivity between native enzymes and bioconjugates when acting on CMC. While the native enzymes exhibited a glucose yield approximately ten times higher than that of the bioconjugates in the initial phase, the cumulative glucose production from the bioconjugates reached parity with the native enzyme by the 10th cycle. This data highlights a significant trade-off between initial efficiency and long-term sustainability in enzyme performance. The native enzyme’s superior glucose production in the early stages emphasizes its high catalytic efficiency, a feature critical for processes that require rapid glucose output. In contrast, the bioconjugate, though initially lagging, demonstrates impressive stability over repeated cycles. The retention of 80% glucose production through 10 cycles suggests that the bioconjugate may be better suited for prolonged, repetitive use without substantial loss of activity. The decline in glucose production after the 10th cycle is a critical point for further discussion. It indicates that while bioconjugates offer extended utility, they are not immune to gradual deactivation or inhibition, likely due to enzyme denaturation, substrate fouling, or the depletion of active binding sites on the CS-MNPs surface. By the 20th cycle, the bioconjugate retains only 33% of its original glucose yield, reflecting a clear limitation for applications requiring sustained, high-level output over many cycles. Considering the fact that bioconjugates achieve cumulative productivity equivalent to native enzymes by the 10th cycle, it may offer a more sustainable and economically viable solution in processes where long-term performance and enzyme recyclability are essential.