Enzymes are used as biocatalysts in chemical, pharmaceutical, and food industries ^{[1, 2]}. Because the recovery and the reusability of free enzyme reactions are limited ^[3], attention has been paid to enzyme immobilization, which offers advantages over free enzymes, such as the possibility of continuous process, rapid termination of reactions, controlled product formation, ease of enzyme removal from the reaction mixture, and adaptability to various engineering designs ^[4].

The immobilization of biomolecules onto insoluble supports is an important tool to fabricate a diverse range of functional materials or devices ^[5]. It provides many distinct advantages including enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth ^[6]. Using magnetic supports for immobilization is promising. Magnetic carrier particles fulfill two functions of a magnetic material that allows ensembles to form with the species to be separated and surface properties that enable a selective separation.

The immobilization of enzymes onto nanomaterials such as nanopolymers ^[7], nanofiber ^[8], and nanoparticles ^{[9, 10]} is of high interest because the reduction in the size of the enzyme carrier materials improves the efficiency of immobilized enzymes. Moreover, for surface attachment, smaller particles can provide a larger surface area for the attachment of enzymes, leading to higher enzyme loading per unit mass of particles ^[9]. However, for industrial biotechnology applications, immobilized enzymes on nanoparticles are limited by problems in recovery, e.g., via centrifugation or filtration.

Magnetic nanoparticles (MNPs) are potentially useful supports for bioactive materials such as peptides, enzymes, antibodies, and nucleic acids, and are easily recovered for reuse ^[11-15]. They have low toxicity, and their distinct advantage is their separation from reaction mixtures using magnets. Improved enzyme activity, loading, and stability using enzyme immobilized on MNPs have been shown by Dyal et al. ^[16].

Many chemical procedures used to synthesize magnetic nanoparticles and microparticles are applicable for bioapplications, such as classical co-precipitation, reactions in constrained environments (e.g., microemulsions), sol-gel syntheses, sonochemical and microwave reactions, hydrothermal reactions, hydrolysis and thermolysis of precursors, flow injection syntheses, electrospray syntheses, and mechanochemical processes ^[17-19].

Cellulase (1, 4-(1, 3;1, 4)-D-glucan 4-glucanohydrolase, EC 3.2.1.4) can be used as a biocatalyst for cellulose hydrolysis. The cost of cellulase technology can be reduced by increasing the enzyme reusability and its stability. These may be accomplished by enzyme immobilization on suitable carriers. The immobilization of cellulase enzyme complex on magnetic supports has also reported ^[20]. The purpose of this study was to characterize the cellulase complex after direct binding to magnetic nanoparticles via glutaraldehyde cross linking and determine optimum operating conditions. Size and structure of the resultant nanoparticles were characterized by scanning electron microscopy (SEM). Binding of magnetite nanoparticles to enzyme was confirmed using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD). Operating parameters for immobilized cellulase were evaluated using varying pH and thermal conditions, in addition to the binding efficiency of enzyme to the support, to determine the conditions for optimum hydrolysis reactions.

Cellulase (from Acinetobacter sp. TSK-MASC)^[21], Glutaraldehyde (25 wt% solution in water), FeCl₃·6H₂O, FeCl₂·4H₂O carboxymethyl cellulose (CMC), 3, 5-dinitrosalicylic acid, Folin's reagent (2N), and sodium phosphate were purchased from Hi-media, Mumbai, India. All other chemicals were obtained from local suppliers and were analytical grade or better.

Magnetic nanoparticles (Fe₃O₄) were prepared by co-precipitating Fe²⁺ and Fe³⁺ ions with ammonia solution and treating under hydrothermal conditions as previously reported, but with minor modifications ^{[13, 22]}. A 2:1 molar ratio of ferric and ferrous chlorides was dissolved in water under inert conditions. Chemical precipitation was achieved at 25 ℃ under vigorous stirring by adding 28% NH₄OH solution. The precipitates were heated to 80 ℃ for 30 min, and washed three times with water and once with anhydrous ethanol. The particles were dried for 24 h.

The immobilization of cellulase on magnetic nanoparticles followed the procedure described by Verma et al. ^[23] with minor modifications. The magnetic nanoparticles were suspended in deionized water at a concentration of 5 mg/mL. This suspension was sonicated for 1 h, and suspended in 1 mol/L glutaraldehyde solution in deionized water ^[23]. The support was activated by incubating the magnetic nanoparticles for 1 h at 25 ℃ in a shaker at 250 r/min. The reaction mixture was stored at 4 ℃ and sonicated at 1-h intervals to ensure uniform dispersion. After 2 h, the mixture was sonicated a final time and heated to 25 ℃. The cellulase-bound nanoparticles were recovered by placing the container on a strong permanent magnet. They were washed twice in water and the resultant supernatants were used for protein analysis. The remaining precipitates were analyzed for enzymatic activity and stability.

The cellulase activity was measured using CMC, and the amount of released glucose equivalent during the hydrolysis of CMC solution ^[24]. A 1% (w/v) CMC solution in acetate buffer pH 5 was used as the substrate. First, 1 mL of substrate solution and 1 mL of the immobilized enzyme were incubated at 37 ℃ for 1 h. The reaction was stopped by putting the enzyme reaction tubes in a boiling water bath for 15 min. The amount of reducing sugar was measured using the 3, 5-dinitrosalicylic acid method ^[25]. One unit of enzyme activity is defined as the amount of enzyme producing 1 mol/L of glucose equivalent per min at 37 ℃ and pH 5. All experiments were conducted in triplicate, and reported as mean ± standard deviation.

Response surface methodology combined with Box-Behnken design (BBD) was established using Design Expert software (9.0.0.7 trial version). Four factors, enzyme concentration, magnetic nanoparticle concentration, cross linking (glutaraldehyde %), and cross linking time were optimized for immobilization. Using BBD, the factors were analyzed at two levels: -1 for the low level and +1 for the high level. A total of 29 runs were performed to optimize the process parameters, and experiments were performed in accordance with the experimental design matrix. The results were assessed by applying the coefficient of determination (R²), analysis of variance, and response plots. Response surface methodology was employed with the most widely used second-order polynomial equation developed to fit the experimental results and identify the relevant model terms

$Y~=~{{\beta }_{0}}~\Sigma ~{{\beta }_{i}}{{X}_{i}}+\text{ }\Sigma ~{{\beta }_{i}}{{X}_{i}}{{\beta }_{ij}}~+\text{ }\Sigma \text{ }{{X}_{i}}{{X}_{j}}~$

(1)

where Y is the predicted response, β₀, β_i, and β_ij are constant regression coefficients of the model, and X_i and X_j represent independent variables.

SEM (Jeol JSM 6390 model) was performed to study the surface morphology of magnetic nanoparticles before and after cross linking of cellulase. Samples were dehydrated and mounted on stubs and sputter coated with gold for 300 s in a high vacuum and analyzed at a voltage acceleration of 10 kV.

The binding of cellulase onto MNPs was analyzed by FTIR spectrometer (Shimadzu). The samples were obtained by direct transmittance using the KBr pellet technique. The spectra of 400-4000 cm^-1 were analyzed at a spectra resolution of 1 cm^-1.

The crystalline structure of the MNPs was characterized using X-ray diffractometer (XRD6000, Shimadzu) measurements performed at 40 kV and 30 mA. The θ-2θ method was applied to collect the diffraction spectra.

The effect of pH on free and immobilized cellulase activity was analyzed by measuring the reaction at different pH levels (5-11). The effects of incubation temperature on free and immobilized cellulase activities were determined by performing the reaction at 40-80 ℃. The stabilities of free and immobilized cellulase was determined after storage in phosphate buffer (0.02 mol/L, pH 7.0) at 4 ℃ for 100 d. Retained activities were measured as described above, and the activity of each preparation was expressed as a percentage of its retained activity compared with its initial activity. To evaluate the reusability of the immobilized cellulase, the matrices were washed with water and buffer after use and suspended again in a fresh reaction mixture to measure the enzymatic activity.

Magnetic nanoparticles were prepared by the co- precipitation method. The cellulase was immobilized onto Fe₃O₄ magnetic nanoparticles using glutaraldehyde as the cross linking agent. BBD was implemented to identify the optimal conditions for cellulase immobilization. The experimental design is shown in Table 1. Analysis of variance of the quadratic regression model (Table 2) exhibits that it was a highly significant model as was evident from the Fisher's F-test with a very low probability value (F-value = 6.33). Values of ‘Probability > F' (0.0007) indicate that the term of the model was significant. The Model F-value of 6.33 implies that the model was significant. There was only a 0.01% chance that a model F-value could occur because of noise. The predicted R² (0.2219) and adjusted R² (0.7271) values for optimal conditions were in reasonable agreement with the value of R² (0.8636), which is closer to 1.0, indicating the better fit of the model in the experimental data. The model for optimal conditions for cellulase immobilization, three different tests, sequential model sum of squares, lack of fit tests, and model summary statistics were performed.

Contour plots graphical representations were generated (Fig. 1). The results demonstrate that there was a significant relationship among enzyme concentration, magnetic nanoparticle concentration, cross linking (glutaraldehyde %), and crosslinking time. The optimum levels of the variables were obtained using BBD. The model predicted a maximum cellulase activity recovery of 92% for an enzyme concentration of 0.0625 mg/mL, magnetic nanoparticle concentration of 300 mg/mL, cross linking with glutaraldehyde at 0.3%, and a cross linking time of 3 h. The predicted model was validated and experiments were conducted using these optimal conditions. The predicted model values were in good agreement with the values measured in these experiments, mitigating the validity of the response model and the necessity for optimal conditions. Graphs highlighted the parts played by the variables for the production of cellulase. The coefficients of the regression equation were calculated, and the following regression equation was achieved:

$\begin{array}{*{35}{l}} Y~=+90.80-2.08A+0.67B+4.00C+1.25D+3.75AB+ \\ 1.00AC+0.50AD+1.25BC-1.00BD+3.25CD- \\ 9.57{{A}^{2}}-7.44{{B}^{2}}-4.19{{C}^{2}}-6.82{{D}^{2}} \\ \end{array}$

(2)

where Y is cellulase activity, A is enzyme concentration, B is magnetic nanoparticle concentration, C is cross linking (glutaraldehyde %), and D is cross linking time. A high degree of similarity of experimental values were observed, reflecting the precision and applicability of response surface methodology to optimize the enzyme immobilization.

SEM was used to study the surface morphology of MNPs with and without bonded cellulase. The surface morphology of MNPs was changed after cellulase immobilization. These changes can easily be distinguished in the SEM (Fig. 2). The micrographs showed the surface of MNPs before immobilization of enzyme and after immobilization. There was a slight aggregation of enzyme particles present on the MNP surface.

The crystal structures of the composite magnetic nanoparticles were characterized by XRD and are shown in Fig. 3. For the Fe₃O₄ MNPs before immobilization, six characteristic peaks (2θ = 30.08°, 35.42°, 43.08°, 53.56°, 56.98°, and 62.62°) marked by their indices ((220), (311), (400), (422), (511), and (440), respectively) were observed ^[26].

The binding of cellulase onto activated nanoparticles was confirmed by FTIR spectroscopy. Fig. 4 represents the FTIR spectra of activated nanoparticles, cellulase-bound nanoparticles, and cellulase. FTIR bands at 500-700 cm^-1 were the vibrations of Fe-O bonds of iron oxide (Moon et al., 2000). The characteristic absorption peak of Fe₃O₄ is at 563.75 cm^{-1 [27]}. After immobilization, new absorption bands at 1512.56 and 3180.92 cm^-1 appeared, which are the NH₄⁺ bending vibration and N-H bond, respectively, in the enzyme structure and confirm enzyme immobilization.

The effect of pH on the activity of free and immobilized cellulase was examined (Fig. 5(a)). The immobilized enzyme showed increasing activity at higher pH values compared with that of the free enzyme. The binding between cellulase and the amino bonds on nanoparticles improves resistance to a medium with high alkalinity. The alkaline medium affected the free enzyme by placing the enzyme in an electrostatic state that reduced activity. The binding onto MNPs allows the enzyme to retain the conformation and structural arrangement for activity and stability.

The effect of temperature on the activity of free and immobilized cellulase was examined (Fig. 5(b)). The optimum temperature for the free cellulase was observed at 50 ℃, whereas the maximum activity of both immobilized cellulase preparations showed an optimum temperature at 55 ℃. However, both immobilized cellulase preparations showed higher activities over a wider range of temperatures than those of the free enzyme. The increase in optimum temperature for the immobilized cellulase preparation may be caused by the changing physical and chemical properties of the enzyme. An earlier study conducted on immobilization using cellulase binding to magnetic nanoparticle supports showed an optimum activity at 50 ℃ ^[28] and most of the immobilized cellulase exhibited higher optimum temperature values than their free counterpart ^{[29, 30]}.

The storage stabilities at 4 ℃ in the dry state were investigated by measuring the enzyme activities at certain time intervals (0-100 d), and the results are given in Fig. 5(c). The enzyme activity was determined at 37 ℃ in phosphate buffer pH 7. The free cellulase maintained 15% from its original activity, and the immobilized cellulase on MNPs maintained 70% from its initial activity after 100 d. High storage stabilities have been reported for immobilized enzymes with a magnetic support ^{[31, 32]}.

Reuse of enzymes in industrial processes is advantageous from an economic point of view. The reuse of cellulase-immobilized MNPs was investigated because the magnetic properties and enhanced enzyme stability and reusability were intended to facilitate multiple cycles. Fig. 5(d) shows the activity of cellulase-immobilized MNPs after multiple cycles of magnetic separation and reuse. Although the activity of the immobilized cellulase began to decrease after three cycles, more than 70% of its initial activity was retained after 10 cycles. Earlier studies of cellulase immobilization on polyamidoamine-grafted silica reported that 75% activity was retained after three cycles. In another study, 41% activity was retained after six cycles when the enzyme was attached using adsorption, and 67% when the enzyme was covalently crosslinked ^[33]. Some reports have indicated that the gradual loss of enzyme activity after only a few cycles occurs because of factors such as product inhibition, structural modification of the enzyme, protein denaturation, and inactivation of the enzyme ^[28]. Because the nanoparticle was magnetic in nature, it facilitated easy separation and recovery of the immobilized enzyme from the reaction mixture, supporting its reusability.

The immobilization of cellulase onto a functionalized nanoparticle was achieved, and the optimal immobilization conditions using response surface methodology were evaluated. The binding efficiency onto MNPs was about 92%. The binding of cellulase onto MNPs was supported by FTIR spectroscopy. The characterization of nanoparticles was determined by XRD and SEM methods. The activity of the enzyme was further confirmed by the CMC activity method. This study showed that the immobilization of cellulase enzyme via glutaraldehyde activation onto magnetic nanoparticles improved stability over free enzyme, particularly at higher pH values.