Cholesterol was purchased from Sigma Aldrich Pvt. Ltd. Chitosan (deacetylation degrees higher than 85%), Horseradish Peroxidase and single layer Graphene oxide nanopowder were purchased from the Sisco Research Laboratories, Mumbai, India. All other chemicals used were of analytical grade and used without purification.

Magnetic iron oxide nanoparticle was prepared by the thermal co-precipitation method described by Moghaddam and Aliahmad with slight modifications [18]. The synthesis of maghemite NPs was carried out using a mixture of aqueous solutions of Fe₂Cl₃ (100 mL, 1 M) and FeSO₄ (100 mL, 0.5 M). The above mixture was heated up to 90°C along with the addition of NH₄OH (25% v/v of ammonia) dropwise, with vigorous stirring till it attains the alkaline condition (pH 10.0). The color of the mixture slowly turned to black while reaching the alkaline pH. The mixture was continuously stirred for at least 30 min and centrifuged at 5,000 rpm for 5 min. The black precipitate obtained was washed with deionised water till neutral pH of the supernatant was obtained, dried in a hot air oven at 60°C and crushed using a pestle and mortar. The black powder so obtained was Fe₃O₄ NPs (magnetite). For the synthesis of Fe₂O₃ (maghemite) NPs, the black powder was kept at 200°C in a hot air oven for 14-16 h till it turned to a reddish-brown color. This powder was cooled to room temperature and crushed again by a pestle and mortar.

Chitosan was dissolved in acetic acid (25 mL, 2% v/v) with the help of a magnetic stirrer overnight, to assure no lump formation, until a clear transparent solution was obtained. The trapped air bubbles were removed by sonicating the chitosan solution for 20 min. Fe₂O₃ NPs prepared in section 2.2 were treated with ethanol in order to remove any moisture in it and air dried. Fe₂O₃ (2 g) was added slowly to the chitosan solution and vigorously stirred for 1 h till the NPs were homogeneously dispersed in the chitosan solution to form a blend. The chitosan-Fe₂O₃ blend was used for preparing chitosan beads having magnetic properties with the help of a 5 mL syringe, in NaOH (100 mL, 1 M) under continuous stirring. The beads were left in the same NaOH solution for 4 h for hardening and then washed with deionised water until neutral pH of the washings was obtained.

After extensive washing with deionised water, the Magnetic Chitosan (MC) beads (100 mg/mL) were transferred to an exfoliated and homogeneous solution of GO (stock solution 0.4 mg/mL) and was stirred overnight. The MC beads were separated from the GO solution using an external magnet. The GO solution can be reused for the second cycle of GO coating on MC beads. The MC beads were extensively washed for the removal of excess GO from beads surface. The GO coated MC beads (GOMC beads) were sonicated for 1 h in a bath sonicator, washed with deionised water and lyophilized for 6-8 h.

The activation and functionalization of GOMC beads were carried out by treatment with GA (2.5% v/v) at 25°C for 8 h. The aldehyde functionalized GOMC beads were separated using an external magnet and washed at least five times to remove any unreacted GA residues. The obtained GA activated GOMC beads were stored in potassium phosphate buffer (50mM, pH 7.0).

HRP was covalently immobilized on the surface of GOMC beads by the GA activation method [19]. The GA-activated GOMC beads were immersed in a cocktail solution of HRP (10mg/100 beads) and phosphate buffer (50mM, pH 7.0) followed by overnight incubation at 30°C, 200 rpm in an orbital shaker (Orbitek, Scigenics Biotech Pvt. Ltd., Chennai, India) for immobilization. After completing the immobilization procedure, the peroxidase immobilized Graphene Oxide coated Magnetic Chitosan beads (PGOMC) were separated using an external magnet. The PGOMC beads were washed four to five times with phosphate buffer (50mM, pH 7.0) to remove the unbound peroxidase till no protein was detected in the washings. The PGOMC beads were preserved in phosphate buffer (50mM, pH 7.0) in a refrigerator at 4°C for further studies and lyophilized (6-8 h) before structural characterization.

The protein concentration was determined spectrophotometrically according to the method described by Bradford [20]. Bovine serum albumin was used as a standard protein for the construction of a calibration curve. The amount of immobilized protein was evaluated by calculating the difference between the initial protein concentrations of the prepared enzyme solution subjected for immobilization and the residual protein left in the same enzyme solution after immobilization.

(1)

Surface morphology of the synthesized beads was analysed with a Zeiss EVO 18 scanning electron microscope (Zeiss, Germany) using 20 kV accelerating voltage. X-Ray Diffraction (XRD) spectrum of the synthesized material was obtained for the crystallization phase analysis (Rigaku diffractometer). The measurement was carried out in the Bragg’s angle (2θ) ranging from 10°-80°, at a scanning rate of 5° min⁻¹, Cu Kα radiation (λ = 0.15418 nm) using Rigaku Miniflex 600 D Tex ultra. Magnetization curve of the synthesized magnetic nanoparticle was obtained using a Vibration Sample Magnetometer (VSM, Riken Denshi Co. Ltd., Japan) at room temperature. The analysis of the functional groups attached to the carrier support and the enzyme was performed using Fourier Transform Infrared Spectroscopy (FTIR). The samples were scanned in the range of 4000-400 cm^-1 using FTIR spectrophotometer (Shimadzu, Japan).

The estimation of peroxidase activity was done by coupling H₂O₂ with 4-aminoantipyrine and phenol in the presence of peroxidase to form quinoneimine; a chromogen having an intense pink color with the absorption maxima at 510 nm. The amount of quinoneimine produced was directly proportional to the amount of peroxidase present [21]. 1 mL of the reaction mixture consisted of 0.05 M potassium phosphate buffer at pH 7.0, 0.0025 M 4-aminoantipyrine with 0.17 M phenol and soluble or immobilized HRP, pre-incubated for 3 min at 30°C. The reaction was initiated by adding 0.0017 M H₂O₂ to the pre-incubated reaction mixture and the incubation continued for 10 min at 30°C. The reaction was terminated by heating the samples in a boiling water bath for 2 min followed by subsequently transferring the samples into an ice-bath for color development. The PGOMC beads were magnetically separated before the termination of the reaction. Blank was prepared by taking the reaction mixture containing all the components excluding HRP (free or immobilized). Absorbance was recorded at 510 nm by the discontinuous spectrophotometric method (UV 1800 Spectrophotometer, Shimadzu, Japan). One unit of activity was defined as the amount of HRP required to hydrolyze one micromole of H₂O₂ per minute at 30°C and pH 7.0.

(2)

The prepared PGOMC beads were examined for their efficacy to estimate ChoX in the reaction mixture containing cholesterol as a substrate by the modified method of Allain et al. [22]. The reaction conditions for the ChoX assay were optimized in our previous study [23]. Cholesterol (50 µL; 6 g/L) dissolved in Triton X-100 5% (v/v) and dimethylformamide was added to 1 mL of the reaction mixture containing 1mM 4-aminoantipyrine, 5 mM phenol, and sodium phosphate buffer (20 mM, pH 7.0). The prepared PGOMC beads (mentioned in section 2.5) loaded with 10 U HRP (4 beads) were added to the above reaction mixture and pre-incubated for 5 min at 30°C. 100 µL of crude ChoX extracted from the supernatant of the fermentation broth of Streptomyces olivaceus MTCC 6820 was added to the pre-incubated reaction mixture to start the reaction and the incubation continued for 10 min at 30°C. The PGOMC beads were separated by an external magnet before the termination of the reaction. The reaction was terminated by placing the samples in a boiling water bath for 2 min and then immediately placed in an ice bath for 2 min for color development. Absorbance was recorded at 500 nm (UV 1800 Spectrophotometer, Shimadzu, Japan). Blank was prepared by adding an inactivated enzyme to the reaction mixture. One unit of ChoX activity was defined as the amount of enzyme that converts 1µmol of cholesterol into H₂O₂ per minute at 30°C.

The use of immobilized HRP in the enzymatic assay method of ChoX can be explained by the following biochemical reaction. The oxidation of cholesterol is catalyzed by the ChoX enzyme to produce H₂O₂; the degradation of H₂O₂ is further catalyzed by HRP to produce quinoneimine dye having absorption maxima at 510 nm.

(3)

In the present study, we prepared GO coated magnetic chitosan beads for HRP immobilization and characterized the immobilized enzyme. The magnetic chitosan beads were prepared, coated with GO and activated with GA. The immobilization of HRP was successful with nearly 80 ± 1.35% immobilization efficiency. The maximum amount of bound protein was 0.08 mg/bead. The immobilized enzyme

Fig. (1). SEM image of (a) HRP immobilized GO coated magnetic chitosan bead (b) Surface morphology of the GOMC bead (20.00 KX magnification) (c) Surface morphology of the GOMC bead (500 X magnification).

Fig. (2). FT-IR Spectra of (a) Fe2O3 (b) Chitosan (c) Fe2O3-Chitosan (d) Fe2O3-Chitosan-Graphene Oxide and (e) Fe2O3-Chitosan-Graphene Oxide-HRP.

was GA for optimum functional range and stability. GA acted as a cross-linking agent and the activation of chitosan beads by GA generated multipoint attachment sites for the enzyme. Literature suggests that the conjugate of Fe₂O₃-GO mimics peroxidase-like activity, which may be the reason to enhance the HRP activity [24]. Due to the mimicking of Fe₂O₃-GO support matrix as peroxidase-like activity, it showed a positive synergistic effect over HRP immobilization than any other support matrix. Moreover, coating GO on the magnetic chitosan beads with sonication allowed the small-sized GO to well-disperse onto the chitosan beads surface, providing uniformity in active sites throughout the beads. With the high immobilization efficiency of ~80%, the GO coated magnetic chitosan beads proved to be an excellent carrier support for HRP immobilization.

Enzymes often exhibit altered kinetic behavior after immobilization which may be attributed to the changes in the microenvironment of the immobilized enzyme provided by the support matrix.

3.3.1. Effect of pH and Temperature on Free and Immobilized HRP

The effect of pH on the activity of free and immobilized HRP was studied by incubating with different buffers in the pH range 4 - 9 at 30°C and the results are presented in Fig. (5a). The pH profile of the immobilized HRP showed that it was mostly active between pH 6-8. However, the optimum pH values for the activity of free and immobilized HRP were 6.5 and 7.0, respectively. Similar results were found, where the pH 6.5 for free HRP shifted to 7.0 for immobilized HRP [33]. A possible reason for this pH shift may be attributed to the conformational changes in the three-dimensional structure of the enzyme protein due to the surface properties of the carrier support after immobilization. The outer surface of the PGOMC bead is rich in free amino groups due to the presence of chitosan, which absorbs H⁺ ions from the reaction mixture resulting in lower pH value in the vicinity of the immobilized enzyme. In order to account for the conformational change brought about by the immobilization, the pH is raised to a certain degree, facilitating the enzyme to function properly [30]. This shift in the pH optima of the immobilized HRP shows its adaptability towards the environmental alkalinity, which is favorable for its application in the ChoX assay [23].

Fig. (5). (a) Effect of pH on activity of free and Immobilized HRP. (b) Effect of temperature on activity of free and Immobilized HRP.

The optimum temperature of the free and immobilized HRP was determined by measuring the enzyme activity at a variable temperature ranging from 25°C to 85°C at pH 7.0 (Fig. 5b). The immobilized enzyme was active over a wide range of temperature viz. 35°C - 65°C. An elevation in the optimum reaction temperature of the free HRP (50°C), as compared to the immobilized HRP (55°C), was observed which was in accordance with the reported literature [34]. This increased heat resistance was possibly because of the support matrix providing a higher degree of cross-linking sites for the attachment of enzyme protein in their active orientation, which shields the HRP from inappropriate structural changes at high temperature. It is well established that if subjected to thermal exposure, enzymes in the soluble form are more prone to the unfolding of protein native three-dimensional conformation thus affecting the protein structure-function relationship which is minimized in case of immobilized enzymes [35].

ChoX assay essentially requires the use of various non-ionic detergents and organic solvents for the dissolution of cholesterol (water-insoluble) in the reaction mixture. Therefore, the stability studies of enzymes in the presence of organic solvents and detergents are very important. The activity of free and the immobilized HRP was determined in the presence of ethanol, methanol, isopropanol, acetonitrile, Triton X-100, Tween - 20 and Tween - 80. The results are illustrated in Tables 1 and 2. The free and the immobilized HRP showed a very similar behavioral pattern for the organic solvents and detergents, however, the immobilized HRP exhibited more stability in the presence of these chemicals. The relative activity of the immobilized HRP increased in the presence of methanol, ethanol and isopropanol, whereas it reduced in the presence of acetonitrile. Triton X-100 and Tween-20 enhanced the relative activity of both the free and immobilized HRP. The significant stability of the immobilized HRP in ethanol, Triton X-100 and Tween-20 with relative activity ≥ 150% proved that the PGOMC beads were highly suited for assaying ChoX, as they are the most commonly used solvents in the ChoX assay.

Fig. (6). Thermostability of (a) Immobilized HRP (b) Free HRP.

The effects of different metals on free HRP and HRP immobilized on GOMC beads were studied and summarized in Table 2. Mg²⁺, Ca²⁺, Zn²⁺, and Mn²⁺ showed strong activation effects on immobilized HRP. Zn²⁺ strongly inhibited the free HRP while the inhibition by Cs²⁺ was strong for both free and immobilized HRP. Pb²⁺ inhibition effect was more on free HRP while Hg²⁺ and Cu²⁺ completely inhibited both the free and immobilized HRP. The results indicated that the HRP immobilized on GOMC beads had more resistance towards metal ions than the free HRP.

One of the major advantages of the immobilized enzyme is the reusability, which can effectively reduce the cost of its applications. In this study, the operational stability of the HRP immobilized on GOMC beads was evaluated by performing repeated cycles of ChoX assay. Fig. (7) displays the reusability assay of immobilized HRP for estimation of ChoX. The relative activity of the immobilized HRP reduced with the increasing number of reuses. The immobilized HRP retained ~90% of the residual activity after 12 times repeated reuse in ChoX assay. The enzyme activity obtained in the first cycle was considered as 100% activity. The number of reuses represent a more satisfying performance when compared to recent studies (Table 3). These results may be attributed to the covalent attachment of enzyme to the support after GA activation. Also, the GO coated on the magnetic chitosan beads enhanced the mechanical integrity of the beads, which made them non-brittle and allowed easy separation from the reaction mixture [36]. GO sheet has also been reported as an excellent matrix for enzyme immobilization, which prevented the HRP from easy leach out from the surface of the GOMC beads [37]. The magnetic separation and the enhanced mechanical strength of the chitosan beads proved very practical in the reusability assay.

The study of storage stability is a very essential factor to be taken into account for reducing the cost of the immobilized enzyme by the assessment of immobilization efficiency [38]. Free and immobilized HRP were stored in a refrigerator at 4ºC in potassium phosphate buffer (pH = 7) and the enzyme activity was examined at every 10 days interval. The enzyme activity at day zero was considered as 100% and the relative activity was determined. It is evident in Fig. (8), after 30 days of storage; the relative activity of immobilized HRP was 93.72%, whereas that of the free HRP, was ~72%. After 60 days of storage, the relative activity of the immobilized HRP was 60.97%, which was quite better than the free HRP (~50%). The free HRP lost about 72% activity, whereas the immobilized HRP retained more than 70% of its relative activity and showed good stability upon storage for 90 days. Table 4 displays a comparative analysis of the storage stability of HRP immobilized on different carriers, which demonstrated excellent storage stability of HRP immobilized on GOMC beads. These results indicated that the storage stability of the HRP immobilized on GOMC beads greatly enhanced after immobilization, due to the covalent attachment of the enzyme with the carrier support.

Fig. (7). Effect of repeated use of immobilized enzyme on activity.

Fig. (8). Storage stability of free and immobilized HRP.