Degradation of Bisphenol a Using Horseradish Peroxidase Immobilized on Fe-based MOFs

In this study, we synthesized a water-stable Fe-based metal-organic framework, MIL-88B (Fe) by a solvothermal method, and for the rst time, MIL-88B (Fe)/HRP composite was prepared for the degradation of bisphenol A (BPA) by immobilizing horseradish peroxidase on MIL-88B (Fe) using covalent xation method. The material was characterized via XRD, FTIR, TG, and SEM methods. The results showed that the composite could remove bisphenol A quickly and eciently by adding the hydrophilic agent polyethylene glycol, with the removal rate of BPA up to 99.2% within 1 hour. In addition, the initial concentration of bisphenol A, the dosage of the immobilized enzyme and the amount of H 2 O 2 added had a great inuence on the degradation eciency. It was found that immobilized HRP could be reused, and its storage stability and thermal stability were better than free HRP. These show that immobilized enzymes have a broad application prospect in waste-water treatment.


Introduction
Bisphenol A (BPA) is a raw material for synthetic plastics, which can be made into a variety of common plastic products, such as water bottles, sports equipment, medical devices and so on. For the unreasonable use, it can be extensively found in industrial waste-water, surface water, groundwater and even drinking water ). BPA has neurotoxicity, immunotoxicity, carcinogenicity and teratogenicity, which can affect the endocrine system of humans and animals, leading to the feminization of male animals and the reduction of reproductive ability (Zhang et al. 2020b). Therefore, the degradation of BPA has a great signi cance to the ecological environment and human health. Among them, enzymatic treatment has been widely used due to the high degree of speed and high selectivity (Besharati Vineh et al. 2018). Horseradish peroxidase (HRP) is a heme protein with an iron porphyrin active center, which can oxidize and degrade bisphenol A in the presence of H 2 O 2 . After oxidized to free radicals, the substrate molecules can be rearranged and coupled to form corresponding polymers and other compounds, which are non-toxic and can be easily removed from water (Pylypchuk et al. 2020). Nevertheless, the free HRP has many drawbacks including poor thermal and storage stability, high cost for no reusability and recyclability. Thus, researchers have been looking for materials with superior properties to overcome the shortcomings in enzyme applications.
Metal organic frameworks (MOFs) is a crystalline porous material with periodic network structure formed by self-assembly of metal ions and organic ligands through complexation. It has been proved that MOFs can be used for enzyme covalent immobilization. Shih

Characterization
X-ray diffraction (XRD) patterns of MIL-88B(Fe) powder and MIL-88B(Fe)/HRP composite were obtained via using a Bruker D8 Advance X-ray diffractometer. Fourier transform infrared (FTIR) spectra of the products were carried on a Thermo-Nicolet spectrometer (CCR-1; USA). And then, scanning electron microscope (SEM, Carl Zeiss Microscopy, Germany) was employed to observe the morphologies of samples. The thermal stability was measured on a TG-DTG 6300 thermogravimetric analyzer (EXSTAR, Japan) by heating samples to 600℃ at a rate of 5 ℃/min in 60 mL/min air ow. 60mL DMF were mixed and stirred for 30 min to form a homogenous solution. The mixture was transferred into the Te on autoclave and placed in the oven at 150 ℃ for 2 hours. When the reaction nished, the product was gathered by centrifugation and washed several times with ethanol and dried under vacuum at 60 ℃ overnight.

Enzyme immobilization
MIL-88B(Fe) (10 mg), EDC (40mg), NHS (20mg) were dispersed in enzyme solution (0.04mg/ml, 10ml) and stirred with a magnetic stirrer for 8 hours at 30℃. The mixture was gathered by centrifugal collection and rinsed with deionized water several times to remove non-speci cally unbound enzyme. The supernatant with residual enzyme was collected to measure the quantity of protein loading with UV/Visible spectroscopy (λ=595nm) using the Bradford method (Wang et al. 2015). The MIL-88B(Fe)/HRP was dispersed in deionized water and stored in refrigerator at 4 °C.

Assays of enzymatic activity
A colorimetric method was used to determine the activity of the enzyme using phenol, 4-AAP, and H 2 O 2 as substrates (Chang &Tang 2014). The catalytic reaction was monitored by recording the absorbance of the red product at 510 nm. One unit of activity was de ned as the amount of HRP required to hydrolyze 1 × 10 −6 mol of H 2 O 2 per minute under the conditions stated above.

Degradation experiments
Degradation experiments were conducted in conical asks (150 mL) and vigorously stirred with magnetic stirrers. Reagents were added in the following order: bisphenol A (10 -100 mg/L) solution, 2. What's more, the decline in peak intensity indicated that the enzyme was successfully loaded.
In order to further ascertain the molecular structure and functional groups of MIL-88B(Fe), FTIR spectrum was performed. As displayed in Fig. 2 The TGA curves showed that MIL-88B (Fe) had good thermal stability. When the temperature was above 350℃, the structure collapsed (rapid weight loss). The TGA curve of MIL-88(Fe)/HRP showed two distinct weightlessness steps (Fig. 2c). The rst weight loss was due to evaporation of adsorptive and bound water and enzyme decomposition (Zhang et al. 2020c). The second weight loss was the collapse of the MOF skeleton, which was consistent with the trend of MIL-88B (Fe). When the temperature reached 450℃, the immobilized enzyme retained 29.9% of its initial weight, which was slightly higher than 25.3% of MIL-88B(Fe). This was due to some organic residues left in the pyrolysis process of immobilized HRP The degradation experiments for BPA (20 mg/L) at pH 7.0 were performed to evaluate the catalytic nature of as-synthesized materials. As shown in Fig. 3a, MIL-88(Fe) exhibited poor adsorption performance, only 22.6% of bisphenol A removal within 3 h. Under the same conditions, almost 50.5% of BPA was removed when MIL-88(Fe)/HRP served as the catalyst in an hour, but then the reaction was terminated because the enzyme was inactivated. It has been reported that the inactivation of horseradish peroxidase is due to the attachment of free radicals and polymeric products to the enzyme in the catalytic cycle (Dalal &Gupta 2007). In order to prevent this, some researchers have suggested that highly hydrophilic additives such as PEG can be added to the reaction system. PEG can form a protective layer in the vicinity of the HRP active center, limiting the free phenoxy radicals formed during the reaction to inactivate enzyme. Also, PEG has a stronger a nity with degradation products than enzyme, which prevents HRP from being absorbed (Cheng et al. 2006). With the aid of PEG, MIL-88(Fe)/HRP materials successfully achieved signi cant degradation of BPA and showed excellent catalytic properties compared with previous reports (Table 1). The effect of reaction time: When the catalyst dosage was 0.06g/L and the initial concentration of BPA was 20mg/L, it could be seen that the degradation rate occurred speedily in the initial 60 min, then slowed down with the increase of time and the degradation equilibrium was basically reached at 3 h with a removal rate of 98.4%. Therefore, the optimal reaction time for the degradation experiments was determined to be 3 h.
The effect of PEG dosage: When the mass ratio of PEG/BPA was 0.1, the degradation rate of BPA was 72.7% (Fig. 3b). With the increased amount of PEG, the removal rate of BPA was signi cantly increased. This was due to a decrease in the amount of enzymatic inactivation and the degradation reaction was allowed to continue. When the value was 0.4, the degradation rate peaked at 98.4% and remained unchanged for the further addition of PEG. Therefore, subsequent experiments were all carried out in the presence of PEG and the mass ratio of PEG/BPA was equal to 0.4.

pH values
The effect of pH values on BPA degradation with MIL-88(Fe)/HRP was investigated. The consequences are shown in Fig. 4a. When pH ranged from 5 to 7, the removal rate was all at a high level. The optimum pH was observed at pH = 6, with maximum catalytic effect of 98.5% in 3 h. The increased pH had an adverse effect on materials, for the degradation rate slightly decreased when pH was 9. This was caused by the denaturation of enzymes under alkaline conditions. However, there was still above 90% of BPA removed, which demonstrated that the immobilized enzyme could better withstand pH changes. This might be owing to the multi-point covalent bonding on the MIL-88B(Fe) carrier to stabilize the enzyme molecules (Wang et al. 2014).
It is worth noting that at pH =5, the degradation rate of BPA was slightly lower at the beginning, but increased after 40 minutes. This may be due to the leaching of iron ions, resulting in a Fenton reaction in the presence of H 2 O 2 . It has been reported that at pH =5, the iron leaching rate of MIL-88B(Fe) is 0.5 mg/L at 30 min and the concentration increases with reaction time (Gao et al. 2017 (Lu et al. 2017). Fig. 4b shows that the highest catalytic e ciency appears at n (H 2 O 2 ): n (BPA) = 1.5:1. When the molar ratio was lower than 1.5, the reaction was ine cient and the amount of BPA removed raised signi cantly with an increase in hydrogen peroxide concentration, which showed that H 2 O 2 was the limiting factor in this range. It could also be seen that the molar ratio of H 2 O 2 and BPA ranged from 1.5:1 to 2:1, the removal rate was basically the same, both at the highest value. This is consistent with the results reported in previous literature that the optimal ratio on the treatment of BPA by horseradish peroxidase is 1.5 or greater than 1.5 (Hong-Mei &Nicell 2008, Xiao et al. 2020).

Reaction temperature
The optimum temperature of BPA degradation using synthesized materials was investigated. Fig. 4c showed that the maximum degradation rate for BPA was reached at 25°C. This was due to the high catalytic activity of HRP under this condition. At the temperature of 55°C, it could be seen that the catalyst e ciency of BPA by the immobilized HRP slowed down and the removal rate was at a low level. This was because enzymes were inactivated by prolonged exposure to high temperatures. Therefore, the optimal temperature for the reaction is 25°C.

Bisphenol A concentration
The bisphenol A concentration in the aqueous phase can affect HRP mediated reactions and the results are shown in Fig. 4d. Apparently, MIL-88B(Fe)/HRP exhibited the highest degradation e ciency when the concentration of BPA was 10 mg/L, and the removal e ciency decreased with increasing BPA concentration from 10 to 100 mg/L. The reason might be that the BPA molecules adsorbed on the surface of the material blocked the active site on the HRP, resulting in the reduction of the formation of active substances (Cao et al. 2020). In addition, with the increase of BPA concentration, the enzymecatalyzed reaction rate tended to be balanced, thus the removal rate of BPA began to slow down. It is noticed that even at a concentration of 100 mg/L, 93.1% of BPA was removed within 4 hours. This shows the excellent degradation performance of MIL-88B(Fe)/HRP and its good prospects in wastewater treatment applications.

Optimum catalyst dose
The effect of catalyst dosages on BPA removal was also studied. As illustrated in Fig. 4e, the degradation e ciency of BPA improved rapidly when the materiel dose increased from 0.02 to 0.10 g/L, which was because of the increase of catalytic activity sites for BPA degradation. At the catalyst dosage of 0.10 g/L, 99.2% of BPA was removed within one hour. The most expensive part of enzymatic treatment of wastewater depends on the enzyme, so the balance between the catalytic e ciency and the cost should be considered. In addition, in order to make the reaction not to proceed too fast and easy to measure, 0.06 g/L is chosen as the optimum catalyst dose for BPA degradation in the experiments.

Michaelis-Menten kinetics
The kinetic parameters (K m and V max ) of the free and immobilized HRP were calculated by Michaelis-Menten and Lineweaver-Burk plots and the results were shown in Fig. 5. With the increase of substrate concentration, the enzymatic reaction rate rst increased and then tended to be stable (Fig. 5a).
Compared with free enzyme, immobilized HRP had a lower reaction rate due to the limited mass transfer (Bilal et al. 2020). K m refers to the a nity between the enzyme and the substrate, and the smaller the K m value is, the higher the a nity is. The K m of free HRP was 0.34 mM, while immobilized enzyme increased to 0.47 mM, and the V max decreased from 19.23 mM ·min -1 to 14.28 mM ·min -1 (Fig. 5b). This was because the covalent xation changed the conformation of enzyme, hindered the contact between the substrate and HRP active site, reduced the a nity and the maximum reaction rate.

Thermal and storage stability
In industrial applications, the denaturation of enzymes caused by high temperature is also an urgent problem to be solved. Therefore, the thermostability of the immobilized and free HRP was conducted by measuring the residual activity after incubating in the temperature range from 25°C to 60 °C for an hour. It can be seen from Figure 6a that after 60℃ heat treatment, the residual activity of free HRP decreased to 55.9%, while the immobilized HRP still maintained 70.2% of the initial value. What's more, the activity of MIL-88B(Fe)/HRP was higher than that of free enzyme at different temperatures. These results indicate that immobilized enzyme is more heat resistant than free enzyme, which may be due to limited conformational changes of enzyme. The protective effect of MIL-88B(Fe) can constrain the unfolding and nonspeci c aggregation of the enzyme molecules and thus prevent thermal denaturation of HRP (Vineh et al. 2020).
The storage stability of synthesized samples is an important index to assess the immobilized e ciency. Both MIL-88B(Fe)/HRP and free HRP were evaluated by measuring the residual activity after 30 days of storage in deionized water at 4°C (Fig. 6b). As expected, the activity of MIL-88B(Fe)/HRP declined much slower than that of free enzyme. The immobilized HRP retained as high as 90% of its initial viability during storage for 15 d, whereas the free HRP decreased to 64% of enzyme activity. After 30 days, only 26.2% initial activity could remain for free HRP, while for the synthesized materials, the activity reached over 70%. The loss of activity of free HRP might be caused by conformational changes during storage, and the active sites could not react with the substrates (Chang et al. 2016). On the contrary, MIL-88B(Fe)/HRP showed great resistance to conformational changes due to the formation of strong covalent bonds with enzyme. This could be further interpreted as the protective effect of samples on the potential adverse warping effect of aqueous solution on HRP active sites (Zhai et al. 2013). Therefore, immobilized enzyme is more stable and has a longer shelf life than free HRP. The result shows that MIL-88B(Fe)/HRP has great durability and shows extraordinary potential in industrial application.

Reusability of immobilized HRP
One of the main purposes of enzyme immobilization is the performance of its reuse. This is because enzymes are mostly expensive and unstable compared to conventional chemical catalysts. Enzyme immobilization can restore the catalytic samples easily and stabilize the active conformation of the enzyme. The reusability of MIL-88B(Fe)/HRP was studied by assessing the degradation rate of BPA after successive service times. As shown in Fig. 6c, the immobilized HRP can be reused for up to two cycles with little change in its retained activity, which may be connected with the strong covalent bond formed by HRP and MIL-88B(Fe) through EDC/NHS. This may inhibit the shedding of enzyme resulting from repeated washing, thus effectively preventing reduction of activity (Hu et al. 2018). After 5 cycles, the residual activity still retained 55.2%, indicating that the catalytic samples are stable and can be recycled. What's more, the relative activity of synthesized materials is reduced with the increase of reuse times.
There are several possible reasons for this phenomenon. (1) Due to the trace amounts of samples, part of the MIL-88B(Fe)/HRP are lost in the recycling process. (2) The accumulation of the products on the materials during the reaction may hinder the active sites of HRP and have an adverse effect on the next reaction cycle (Chang et al. 2015). (3) Part of HRP is folded or denatured during the reaction with the substrate, resulting in enzyme inactivation (Shakerian et al. 2020). In conclusion, the immobilized enzyme has the advantages of simple separation and good stability, which can greatly reduce the actual application cost of the enzyme system and can be successfully used in continuous preparation.

Conclusion
In summary, we immobilized HRP on MIL-88B(Fe) through EDC/NHS covalent cross-linking system to obtain MIL-88B(Fe)/HRP composite and used it for the degradation of BPA. The results showed that the hydrophilic substance PEG could greatly improve the degradation e ciency, and 99.2% BPA with an initial concentration of 20mg/L could be degraded within 1 hour with the addition of 0.1g/L immobilized enzyme. The factors in uencing the removal of BPA were also investigated. When the molar ratio of H 2 O 2 and BPA was 1.5, the maximum removal effect could be achieved within 3 hours, pH had little in uence on the removal rate of BPA, and 25℃ was the optimal temperature for the degradation reaction. The Michaelis-Menten kinetics showed that the a nity between enzyme and substrate decreased after immobilization. MIL-88B (Fe)/HRP has better thermal stability, storage stability and reusability than free HRP, which provides a possibility in industrial application.