The assisting-approach of horseradish peroxidase immobilized onto amine-functionalized superparamagnetic iron oxide nanoparticles as a biocatalyst

To enhance the removal of dye, horseradish peroxidase (HRP) was immobilized onto amine-functionalized superparamagnetic iron oxide and used as a biocatalyst for the oxidative degradation of Acid black-HC dye. The anchored enzyme was characterized by sets of techniques such as vibrating sample magnetometer, Fourier transform infrared, X-ray diffraction, thermogravimetric, scanning electron microscopy, BET and BJH methods, nitrogen adsorption-desorption measurements , Zeta potential, energy dispersive X-ray (EDX), and transmission electron microscopy. The Michaelis constant (K m ) values of free peroxidase and immobilized horseradish peroxidase were determined that equal 4.5 and 5 mM for hydrogen peroxide; 12.5 and 10 mM for guaiacol, respectively. Besides, the free peroxidase is thermally stable at 40°C however, the immobilized enzyme was up to 60°C. In the catalytic experiment, the immobilized HRP showed superior catalytic activity compared with free HRP for the oxidative decolorization and removal of Acid black-HC dye. The impacts of experimental parameters for instance catalyst dosage, pH, H 2 O 2 concentration, and temperature, were investigated. The reaction followed second-order kinetics and the thermodynamic activation parameters were determined.


Introduction
Enzymes are biocatalysts with excellent properties including high activity, selectivity, and speci city, which facilitate complex chemical reactions under the maximum benign experimental and environmental conditions (Ding et al. 2015;Min and Yoo 2014). The peroxidase enzymes are isolated from many plant sources such as horseradish and still cheapest than microbial production sources. Besides, they have a wide range of applications including Elisa kits, catalysis, and wastewater treatment. Also, they play a critical role in several industrial applications in analytical, agriculture, environmental, medical, etc.
Peroxidases also have several uses in the treatment of pollutants such as phenols. Besides, organic syntheses as well as polymer synthesis and biosensor technology and alignment of paper and pulp, development of diagnostic kits and immunoassays. They are also used for adequate methods to estimate hydrogen peroxide In industrial and biological tests (Pandey et al. 2017;Rudra et al. 2008).
Freeform of enzymes is normally unstable, di cult to reuse, and are di cultly isolated from the substrate and the product. The immobilization of enzymes is a powerful tool for circumventing these disadvantages and providing better properties for practical applications. Most current procedures for purifying HRP are expensive and time-consuming conventional protein puri cation procedures with consequently low speci c activities, yields, and folding puri cation processes (Bilal et al. 2017b). With the increased use of HRP in clinical and industrial applications, a simple, quick, and cost-effective puri cation procedure can easily be improved. Peroxidase has catalyzed pollutant transformation into other less dangerous compounds in laboratory-scale reactors in various e uents (Naghdi et al. 2018). Due to its high catalytic activity, commercial availability, broad substrates spectrum, and low costs for other enzymes, HRP is suited for dye degradation and decolorization (Yang 2017). The use of soluble and unbound enzymes is limited because they are cost-effective, reusable, balanced, sensitive to various denaturants, and are applied in non-stop reactors. To overcome these obstacles, the immobilization of enzymes has been approved in recent years as a way to promote the use of enzymes that are industrial, biological, and environmental (Sheldon and van Pelt 2013). Increased stability, activity, speci city, selectivity, decreased inhibition are not best achieved by immobilization but also enhance the enzyme's thermal stability, broaden the pH of the enzyme, and recovery of the product with a higher purity (Hoffmann et al. 2018;Min and Yoo 2014).
Different immobilization types of biocatalysts have been reported including; physical adsorption (Ali et al. 2018;Vineh et al. 2018a), covalent bonding (Vineh et al. 2018b), entrapment, cross-linking, and encapsulation (Bilal et al. 2017a;Bilal et al. 2017b). The immobilization of enzymes on the nanoparticles could increase their activities due to many reasons such as enzyme con guration, orientation, and density on the surface (Guzik et al. 2014;Zhang et al. 2011). Moreover, the morphology of nanoparticles could play a crucial role in impacting enzymatic improvement as well. Kumar et al. study demonstrated that epoxy Functional Polypropylene Films covalently immobilize HRP (Kumar et al. 2016). The lms were practically applied in the catalytic degradation of basic red dye with about 90% removal. Bilal et al. were covalently immobilized HRP on calcium alginate via glutaraldehyde as a cross-linker agent with 85% immobilization e ciency (Bilal et al. 2016). Moreover, the immobilized HRP exhibited 99% of residual activity after 10 min and was used as a biocatalyst for the degradation of dyes. Nanayakkara et al.
immobilized HRP on silica nanorods and unitized the resulting solid surface as an oxidative catalyst for the polymerization of substituted phenol (Nanayakkara et al. 2014). The supported enzyme exhibited substantial catalytic activity compared with the free enzyme. Moreover, three different shaped mesoporous silica nanoparticles were prepared and used to immobilize HRP and their catalytic activity was considered (Yang et al. 2018).
Recently, the immobilization of HRP onto magnetic supports was considered as an innovative strategy for the development of biocatalysts for wastewaters treatment (Donadelli et al. 2018). Magnetite nanoparticles' surface silanizing is the widely used approach for introducing surface functional groups (such as NH2) because of its properties, such as less-cytotoxicity, good stability, and ease of performance in organic and aqueous solvents (Hosseinifar et al. 2017;Ramalingam et al. 2018).
This work aims at developing a method that is consistent with the future application of HRP immobilization. To this effect, 3-aminopropyltrimethoxysilans were immobilized by HRP (APTMS). The biochemical characterization of the immobilized HRP was assessed and compared to those of the free HRP. Fe 3 O 4 @NH 2 was fabricated according to earlier published methods (Liu et al. 2013;Pasternack et al. 2008). Brie y, 2.5 g from the dried powder of Fe 3 O 4 NPs, which was previously prepared in (Gemeay et al.

Materials And
2019) was poured into 500 ml mixed solvents from methanol /toluene with equal value. Subsequently, the solution mixture was dispersed for 30 minutes under sonication, followed by a gentle 10 ml APTES addition. The reaction was kept under a mechanical overhead stirring at 70 o C for 6 hours. The particles obtained were frequently washed by water, the latter washed by ethanol to produce a ne powder. The powder was then dried in an oven for 12 hours at 80 o C.

Immobilization of HRP onto Fe3O4@NH2
Firstly, the Fe 3 O 4 @NH 2 NPs were washed repeatedly by doubled distilled water. The immobilization of HRP over the surface of Fe3O4@NH2. 4500 units of enzyme dissolved in 50 ml acetate buffer were contained in 5 mg enzyme, were gently mixed with the surface at room temperature (RT) overnight during an overnight. The supernatant was developed and the product (Fe3O4@NH2-HRP) dried for one night at RT (Mohamed et al. 2008).

Characterization methods
The functionalization and immobilization of HRP onto the magnetite surface were con rmed by using JASCO FT-IR-4100 (Japan) in the ύ range of 4000-200 cm -1 and Shimadzu TG-50-TA (Japan) in the temperature range from (25 -800 °C) with ow rate 5 o C/min under N 2 air. The crystalline structure of prepared samples was studied by using X-ray diffractometer GNR, APD 2000 PRO diffractometer (Italy).
When the X-ray is operated with a current of beams of 40 kV and 30 mA at 2 θ = 2 -80° at 0.05° step speeds. The JCPDS database was also used to analyze the diffraction peaks achieved. The surface measurements of catalysts were recorded on Brunauer-Emmett-Teller (BET) NONA touch 4LX (USA), Barrett-Joyner-Halenda, and (BJH) NONA touch 4LX (USA). Zeta potential Inc. Santa Barbara, Calif (USA) con rmed the dispersion of catalyst in a solution. The magnetic properties of as-prepared samples were measured using handmade VSM (Tanta, Egypt) (El-Alaily et al. 2015). The UV-VIS T80+ double-beam has been used to monitor the AB dye concentration in the solution. The morphology of catalyst surface, particle size, and elemental analysis were studied using TEM JEM-2100F (USA) and SEM FEI Quanta environmental integrated with octane silicon drift detector (SDD) delivers high-quality EDX data (EDAX, AMETEK).

Kinetics measurements
The addition of either H 2 O 2 or the Fe 3 O 4 @NH 2 -HRP separately to the AB dye solution does not affect the decolorization of the AB dye. In contrast, if the H 2 O 2 solution and the Fe 3 O 4 @NH 2 -HRP were added at the same time to the AB solution the decolorization process becomes remarkable. The decreasing of AB concentration was followed using UV-Vis spectrophotometer at λmax = 575 nm. Moreover, the absorption time-output data were also tted to the kinetic plot of the second order, Equation (1). Where the oxidation of AB was executed in 250 mL Erlenmeyer ask present in shaker thermostat with speed 150 rpm. In detail, 300 mg of Fe 3 O 4 @NH 2 -HRP, 2.4x10 -4 M of AB, and 0.16 M of H 2 O 2 were added with shaking at the desired temperature. The supernatant was separated using a magnet at time intervals to follow the dye consumption calculated from Eq. (2).
Where Aο is the dye absorbance at time 0 and A t is the dye absorbance at any time t (min).

Partial puri cation of Horseradish Peroxidase
We partially puri ed Egyptian HRP (Mohamed et al. 2008). HRP enzyme was extracted from horseradish roots about (1 kg) was cut into a cube and juiced using a blender for 10 min. The extract was ltered with Whomman ltering paper and a centrifuged lter was made at 8000 rpm, 25ºC for 30 minutes. After centrifugation, the supernatant was identi ed as the crude extract, that fallen by ammonium sulfate (75 percent saturation). Centrifugation at 12000 Xg for 20 minutes collected the precipitate, dissolved in 50 ml of distilled water, and dialyzed against successive distilled water change. The dialysate was centrifuged by a non-bound protein lter (porous size 0.45 μM, Millipore) at 15,000 Xg for 20 min to remove cell debris. The ltrate was lyophilized using Christ Alpha 1-5 lyophilizer overnight to obtain a powder form of horseradish peroxidase (Mohamed et al. 2008).

Determination of peroxidase activity
Peroxidase activity was examined spectrophotometrically by oxidation of guaiacol (2-methoxyphenyl) to tetraguaiacol at 470 nm. (Tonami et al. 2004). The reaction mixture contained 1 mL 50 mM acetate buffer (pH 5.5), and 40 mm guaiacol, 8 mM H 2 O 2 , and enough enzyme. The absorption was considerably changed at 470 nm at RT because of the guaiacol oxidation. The reaction mixture was used as a control without the enzyme. Enzyme activity is indicated as a unit; the amount of the enzyme that can catalyze the oxidation of 1 μmol of guaiacol per minute at 30 o C is de ned as 1 unit of peroxidase activity.

Determination of protein
The protein concentration in puri ed HRP was assessed using bovine serum albumin that was used as a standard by using the Bradford method (He 2011) Knowing that the protein concentration in the immobilized HRP was estimated by eliminating from immobilization the concentration of protein calculated in the supernatant before immobilization.

The Immobilization of HRP onto amino-functionalized magnetite nanoparticles
The enzyme was immobilized by carefully mixing the 3-aminopropyl-trimethoxysilane (APTMS) end-overend at RT overnight and then by re-washing the enzyme (5 mg contained 4,500 unit) with distilled water dissolved in 50 ml of 0.05 M sodium acetate buffer pH 5.6 overnight. the supernatant aliquots were pulled and dried for further analysis at RT. (Mohamed et al. 2008).

Enzyme Characterization
Enzyme characterization was performed on the free and immobilized enzyme, like Lineweaver-Burk reciprocal tracks for the Km peroxidase activity (Lineweaver and Burk 1934). The values from the slopes and intercepts on the curves have been calculated with Different H2O2 and guaiacol substrates, ranging between 1.5 and 8 mM and between 8 and 40 mM respectively in acetate buffers (pH 5.6). To add to this, the pH pro le was analyzed in various buffers types, including 50mM sodium citrate buffer (pH 3.0-4.0); sodium acetate buffer (pH 5.0-5.6); sodium phosphate buffer (pH6.0-8.0), as well as Tris-HCl buffer (pH 9) at 40 °C. Also, the temperature effect of the activity was analyzed from 10-70 °C and the optimal temperature for soluble and immobilized HRP was also measured. The residual activities of the enzyme were also measured at different temperatures between 30 and 80 °C, with thermal stability, after 30 minutes of incubation. Time intervals for the free and immobilized enzyme were also evaluated at the optimum temperature at 30 °C and the immobilized peroxidase enzyme was evaluated for reusability.  (111), (2 2 0), (3 1 1), (4 0 0), (511) and (4 4 0) planes, respectively. These results agree with the magnetite database (JCPDS-19-0629) and con rm as-prepared magnetite is spinal cubic. The highly crystalline magnetite was kept stable after functionalization by APTES and HRP immobilization Because both materials have an amorphous structure (Phan and Jones 2006). Figure 3 shows the magnetization performance of Fe 3 O 4 , Fe 3 O 4 @NH 2, and Fe 3 O 4 @NH 2 -HRP with zero remanences and coercively con rmed the prepared samples poses superparamagnetic behavior (Mutneja et al. 2016). Also, the magnetization saturation (Ms) values of Fe 3 O 4 , Fe 3 O 4 @NH 2, and Fe 3 O 4 @NH 2 -HRP are 60.7 emu/g, 59.1 emu/g, and 63.6 emu/g respectively. The coating of pristine magnetite by a non-magnetic substance like APTES leads to a decrease Ms value and in contrast after immobilization HRP that contains Fe ion in its chemical structure leads to an increased Ms value than pristine (Petcharoen and Sirivat 2012). These phenomena con rm successful functionalization and immobilization steps. In any case, this does not lose the samples their advantage in separation under the in uence of magnets.

TGA analysis
Functionalization of APTES and immobilization of HRP over Fe 3 O 4 surface were con rmed by TGA curves illustrated in Figure 4. For all three samples, general behavior appeared above 570 o C corresponds to phase transition from magnetite (Fe 3 O 4 ) to hematite (FeO) (Mahdavi et al. 2013 Figures 5a, 5b, 5c, respectively. The images revealed a quasi-spherical structure with particle sizes below 70 nm. The

The Michaelis constant (Km) value of HRP activity
The process of immobilization affects the distribution of the substrate to the active site of the enzyme (Mielgo et al. 2003). In this study, the km free and immobilized HRP values were determined to be 4.5 and 5 mM for H 2 O 2 , and 12.5, and 10 mM respectively for guaiacol (Figure 6 a,b) this indicated a greater substrate a nity than free enzymes to immobilized HRP.

Effect of Temperature and pH on free and immobilized HRP activities
The optima temperature on the HRP activity for the free and immobilized status of about 40°C was shown in (Figure 7a). This showed that the immobilized HRP was subject to temperature increase denaturation resistance. The immobilized enzyme maintained greater fractions of catalytic activities at higher temperatures compared to the free enzyme (Kulshrestha and Husain 2006). Figure (7b) shows that the pH shows the essential ionization status of the signi cant proton groups that donate or accept in the catalyst site of the enzyme. The free and immobilized HRP pH activity pro le with the same pH optimal as the acetate buffer is displayed at pH 5.6. But the immobilized enzyme maintained a relative HRP activity less than soluble at lower and high pH levels, but pH changes were susceptible with the immobile peroxidase compared to soluble peroxidase. This data agrees with (Diao et al. 2014) that most plant peroxidase has reported studies that have shown optimum activity between 4.5 and 6.0 in acidic areas.

Impact of Temperature on HRP stability
To assess the thermal stability of the free and immobilized HRP, both enzymes were exposed to different temperatures (30-80 °C) were incubated for 30 min (Figure 8). The data demonstrated that HRPs are soluble and immobilized, respectively, at 40 and 50°C. Whereas the soluble form showed a full loss for 30 minutes at 70°C after incubation, 25% of the HRP's activity remained immobilized. The immobilization thus resulted in signi cant thermostability increases. Furthermore, immobilized HRP on APTMS maintained full activity at 60°C for at least 2 hours, while free HRP activity decreased to nearly zero at the same temperature (Tupper et al. 2010). Likewise, immobilized turnip peroxidase which was incubated at 60°C for 2 h retained 54% of the initial enzyme activity, while in similar incubation conditions the soluble enzyme lost almost 83 percent of the original activity. (Xu and Wang 2010). The relative activity (%) was calculated from the ratio of its activity after incubation to the activity under the optimum temperature of incubation.
3.2.5. Time intervals in optimum temp at 30 °C for free and immobilized HRP (Figure 9).

Reusability
To assess the reusability of the immobilized HRP ( Figure 10). The reaction mixture of the immobilized HRP after a single reaction cycle contained 1.0 ml of 8 mM H 2 O 2 , 40 mM guaiacol, 50 mM sodium acetate buffer pH 5.6, and 0.1 unit of the enzyme was collected and washes with a sodium acetate buffer pH 5.6 for removal of any residual substrate and added to the following reaction cycle. In each succeeding reaction cycle, the percentage of relative activity was calculated using the ratio of the enzyme activity to the rst reaction cycle. The important advantages of the use of an immobilized biocatalyst are increased stability and easy separation of the bound peroxidase during washes (Mohamed et al. 2014 .6) and (Glycine -HCl buffer, pH=2.6). As the oxidation rate of AB dye is decreased with increasing pH as revealed in Figure   11. This result will return to the Fenton reaction mechanism (Pereira et al. 2012).

Effect of [AB]
The dependency of the reaction rate on the [AB] o was calculated by changing its concentration between 1.6×10 −4 mol L -1 and 4×10 -4 mol L -1 , whereas concentrations of other reaction constituents were kept constant. The dye consumption in the presence of Fe 3 O 4 @NH2-HRP is shown as a function of time and a second-order graph for the reaction of oxidative degradation of AB to 37° C is shown in Figure 12. Where values of the rate constants that are derived from the second-order plot were noted to decrease with increasing the concentration of AB from 1.6×10 −4 mol L -1 to 4×10 -4 mol L -1 . This performance can be attributed to the restricted number of radicals concerned in the oxidation process (Dulman et al. 2012).

Effect of [H 2 O 2 ]
AB dye's oxidation rate depends on the rst H 2 O 2 concentration so; it was calculated by changing H 2 O 2 concentration between 0.08 and 0.32 mol L −1 while keeping the concentration of other reactants constant. The second-order plots for the oxidative degradation reaction of AB are shown in Figure 13.
Here in the oxidation rate slightly decrease and increased again with increased [H 2 O 2 ] o . The tolerable explanation for this phenomena is the immobilized HRP consume a higher concentration from H 2 O 2 to reach its maximum activity for ( ) production consumed in AB dye degradation (Zhu et al. 2011).

Effect of temperature
The effect of temperature on AB dye decolorization was investigated over a temperature range of 20-37°C . The concentrations of AB, H 2 O 2 , and Fe 3 O 4 @NH 2 -HRP were set up constant at 0.16 mol L -1 , 2.4 x10 -4 mol L -1 , and 300 mg L -1 , respectively. Figure (14) displays increasing consumption of AB as a function of time with the increasing temperature that con rms the oxidation process of AB using Fe 3 O 4 @NH 2 -HRP catalyst is endothermic.
On other hand, the thermodynamic activation parameters, ∆S # , and ∆H # are computed from the intercept and the slope respectively based on Eq.
(3) and Eyring plot, Figure (15). Besides, the activation energy (E a ) and free energy (∆G # ) were estimated from Eqs. (4-5) and recorded in Table 3. Wherever the negative sign of entropy value speci ed the reaction from endothermic type.
As h, k B and R are constants of Planck, Boltzmann, and ideal gas respectively. Anywhere T exp is the average experimental temperature.        Graphical representation of the thermal stability of soluble and immobilized HRP by the temperature effect. In the 1.0 ml, a 50 mM sodium acetate buffer pH of 5.6, 40 mM guaiacol, 8 mM H2O2, and an enzyme unit of 0.1 included a reaction mixture. At different temperatures between 10 and 80°C before the addition of the substrate for 30 min, the reaction mix was pre-incubated then cooled down in an ice bath.
100 percent activity was taken as a zero-time activity for soluble and immobilized HRP.
Page 21/24 Figure 9 Graphical representation of the time intervals in optimum temp at 30 °C for immobilized and free enzyme Figure 10 Graphic representation of immobilized peroxidase enzyme reusability on APTMS by being washed repeatedly following each test. A 1.0 ml of 8 mM H2O2, 40 mM guaiacol, and 50 mM sodium acetate buffer pH 5.6 were included in the reaction mix.   The consumption of AB as a function of time at different temperatures.