Immobilization of horseradish peroxidase on electrospun microfibrous membranes for biodegradation and adsorption of bisphenol A

Bioresource Technology 149 (2013) 111–116

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Immobilization of horseradish peroxidase on electrospun microfibrous membranes for biodegradation and adsorption of bisphenol A Ran Xu ⇑, Chenglong Chi, Fengting Li, Bingru Zhang State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Novel poly(methyl methacrylate-

co-ethyl acrylate) fibrous membranes were prepared.  Horseradish peroxidase immobilized on fibrous membranes retained high activity.  The stabilities and reuse capabilities of HRP greatly improved after immobilization.  Bisphenol A can be biodegraded by immobilized HRP and adsorbed by membranes.

a r t i c l e

i n f o

Article history: Received 19 July 2013 Received in revised form 3 September 2013 Accepted 6 September 2013 Available online 17 September 2013 Keywords: Fibrous membrane Enzyme immobilization Horseradish peroxidase Bisphenol A Degradation

a b s t r a c t Horseradish peroxidase (HRP) from roots of horseradish (Amoracia rusticana) was successfully immobilized on novel enzyme carriers, poly(methyl methacrylate-co-ethyl acrylate) (PMMA CEA) microfibrous membranes, and used for removal of bisphenol A from water. PMMA CEA fibrous membranes (PFM) with fiber diameters of 300–500 nm, were fabricated by electrospinning. HRP was covalently immobilized on the surface of microfibers previously activated by polyethylenimine and glutaraldehyde. HRP loading reached 285 mg/g, and enzyme activity was 70% of free HRP after immobilization. Both stabilities and reusability of HRP were greatly improved after immobilization. After six repeated runs, immobilized HRP retained about 50% of its initial activity. Immobilized HRP exhibited significantly higher removal efficiency for bisphenol A (BPA) in 3 h (93%) compared with free HRP (61%) and PFM alone (42%). The high BPA removal can be resulted by improvement of catalytic activity of immobilized HPR with adsorption on modified PMMA CEA support. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Enzymes are highly efficient biocatalysts for a series of biotransformation reactions of xenobiotics (agrochemicals, pharmaceuticals, chemicals used in household, etc.) as well as biogenic compounds (industrial spin-off materials, pollutants, toxins, etc.) used in various industries and everyday practices (Koeller and Wong, 2001). The use of free enzymes if limited due to their instability, nonreusability and high cost as well as potential hazards arising from their catalytic nature and possible allergenicity (Chaplin and Bucke, ⇑ Corresponding author. Tel.: +86 21 65980567; fax: +86 21 65986313. E-mail address: [email protected] (R. Xu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.09.030

1990). In comparison with their free-form counterparts, immobilized enzymes have the benefits of recyclability and reusability and might be applied under different reaction environments and harsh conditions significantly reducing the cost of their use (Mateo et al., 2007; Noureddini et al., 2005). Enzymes have been immobilized on various supports such as inorganic carriers, natural macromolecules (Alsarra et al., 2002), and synthetic polymers by adsorption, entrapment (Reetz et al., 1995), or covalent binding (Dyal et al., 2003). Compared with traditional enzyme supports, electrospun fibrous membranes have the following advantages: intrinsic high specific surface area and inter-fiber porosity; easy handling; a low hindrance for mass transfer; and good mechanical strength. Electrospinning is the only

112

R. Xu et al. / Bioresource Technology 149 (2013) 111–116

method for fabricating fibrous carriers with fiber diameters ranging from several micrometers to several tens of nanometers. Electrospun fibers with poly(D,L-lactide) (Dai et al., 2010), poly (vinyl alcohol) (Ren et al., 2006), or poly(acrylic acids) (Chen and Hsieh, 2005) as base materials could be used as enzyme carriers. Immobilized enzymes on nanofibrous materials have been used to remove organic pollutants, such as polycyclic aromatic hydrocarbons, phenols, and azo dyes, from water (Arica et al., 2009). Poly(methyl methacrylate-co-ethyl acrylate) (PMMA CEA) has rich ester groups, which can serve as reactive sites for combining with enzyme amino groups. PMMA CEA PMMA CEA can immobilize enzymes efficiently because of its hydrophilic groups; moreover, due to its biocompatibility the use of enzymes bonded to this type of fibrous membranes might have advantages in various human practices. However, the fabrication of PMMA CEA microfibers and its application in enzyme immobilization have not been reported. Horseradish peroxidase (HRP), in which the heme iron acts as the active center, was used in the transformation of toxic compounds of industrial origin, such as phenol (Cheng and Harper, 2012) and substituted phenols, to preserve water quality. Bisphenol A (BPA) is an important chemical used to manufacture polycarbonate plastic, epoxy resin, flame retardants, and other specialty products. BPA, which shows biological toxicity and estrogenic activity in specific responses, is also released into the environment (Suzuki and Hattori, 2003; Nakagawa and Tayama, 2000). Current BPA removal methods include microbial degradation, photocatalytic degradation (Fukahori et al., 2003), sonochemical degradation (Inoue et al., 2008), and adsorption. However, these high-cost methods can generate products that are even more toxic than the original materials. Therefore, the development of a simple, safe, and economical wastewater treatment for BPA removal is needed. In this study, PMMA CEA nanofibrous membranes were prepared for the first time and used to immobilize HRP. The immobilized HRP was subsequently used to remove BPA from water. The stability of immobilized HRP and the mechanism of action of BPA removal were investigated. 2. Methods 2.1. Materials PMMCEA, tetrahydrofuran (THF), polyethylenimine (PEI), Coomassie Brilliant Blue (G250), N,N-dimethylformamide, glutaraldehyde (GA), BPA, and HRP were purchased from Sigma (Shanghai, China). Hydrogen peroxide (30%) and the chemicals for phosphate buffer solution (PBS) were of analytical grade, and were used as received. Deionized water was used in all experiments. 2.2. Preparation of PMMCEA nanofibrous membrane by electrospinning The electrospinning apparatus used in this study includes a high-voltage power supply, a plastic syringe with stainless steel needle (inner diameter, 1.2 mm), a syringe pump, and an aluminum foil collector. PMMA CEA was dissolved in 10 wt% aqueous THF at 25 °C with gentle stirring to form a homogeneous solution. The solution was placed in the syringe (15 mL), and its flow rate was controlled at 1.5 mL/h. Electrospinning was performed at 18 kV voltage with a 15 cm distance between the needle tip and the collector. Fibrous membranes were collected for 12 h and dried under vacuum for 10 h at 50 °C to obtain a non-woven mat. The surface morphology of the electrospun membranes was examined using a field emission XL-30 scanning electron microscope at 30 kV.

2.3. Immobilization of HRP For amination through PEI, 10 mg of electrospun PMMA CEA fibrous membranes were immersed in 15 mL 4 wt% PEI solution at 70 °C for 2 h. The aminated PFM (APFM) were washed three times with distilled water to remove excess PEI, and subsequently dried in an oven at 50 °C for 12 h. APFM were placed in 10 mL 2 wt% GA solution and shaken at 30 °C for 2 h. The GA-treated membranes were washed three times with distilled water to remove excess GA. After the activation process, the activated membranes were immersed in 20 mL of HRP solution (0.5 mg/mL) at 30 °C, with gentle shaking (100 rpm). The effects of pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0) and time (0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h) on HRP immobilization were analyzed. Subsequently, the PMMA CEA fibrous membranes with bonded HRP (HRP-PFM) were rinsed with PBS (pH 5.0) until no soluble protein was detectable in the washings. 2.4. Determination of immobilization efficiency Protein content in the solution was determined by Bradford’s method with a UV-1700 spectrophotometer (SHIMADZU, China). The HRP immobilization capacity was defined as the amount of protein per gram of PMMA CEA membrane. The amount of bound protein was calculated by Eq. (1):

Ae ð%Þ ¼ ððC 0  CÞ  V  C w  V w Þ=W  100

ð1Þ

where C0 and C are the initial and final concentrations of the protein (mg/mL), and V and Vw are the volumes for the solution and washings (mL), respectively. Cw is the concentration of protein in the washings, and W is the mass of nanofibrous membrane (g). 2.5. Activity assay of free and HRP-PFM The activities of free and HRP-PFM were determined by monitoring the oxidation of ABTS in a reaction mixture under standard conditions. The mixture contained 1.6 mM ABTS, 0.8 mM H2O2 in 0.1 M PBS (pH 5.0), and a suitable amount of free or HRP-PFM. After 5 min of reaction, the absorbance changes in the solution were measured at 405 nm using a UV-1700 spectrophotometer. The kinetic parameters Vmax and Km of the free and HRP-PFM for ABTS oxidation were calculated based on Lineweaver–Burk plots. The concentrations of ABTS were between 30 and 240 mM. 2.6. Stabilities of the free and HRP-PFM The effects of temperature and pH on the activity of free and HRP-PFM were determined. A certain amount of free and immobilized enzymes were mixed with 15 mL of 1.6 mM ABTS and 0.8 mM H2O2 in 0.1 M PBS (pH 5.0) at a temperature range of 20–60 °C, and the residual activity was measured. Reactions in PBS with pH ranging from 3.0 to 6.0 were investigated to study the pH dependence of the free and HRP-PFM. The reusability of the HRP-PFM was studied determining the enzyme activity subsequently six times within 1 day under optimum conditions. After each test these membranes were washed up with PBS (pH 5.0) to eliminate any residual substrate. The storage stability of free HRP and HRP-PFM was followed for 18 days measuring the enzyme activity in 3 day intervals. HRP activity was measured as described in Section 2.5. 2.7. Removal of bisphenol A (BPA) The effects of different pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0), temperature (20, 30, 40, 50, and 60 °C), and BPA concentration (10, 20, 30, 40, 50, and 60 mg/L) on BPA removal were analyzed.

R. Xu et al. / Bioresource Technology 149 (2013) 111–116

The BPA solution was treated by PMMA CEA membrane, free HRP, and HRP immobilized on PMMA CEA fibrous membranes (HRPPFM), respectively. The reactions were terminated by adding 20 lL sodium azide (0.1 M) prior to sampling. The test materials (PMMA CEA membrane, free HRP, or HRP-PFM) were added into 15 mL solutions with 8.7 mg/L BPA and 0.019 mM H2O2. All reaction bottles were tightly closed with screw caps and placed on a horizontal shaker at 100 rpm. The concentrations of BPA in the reaction mixtures were analyzed by HPLC as described in subSection 2.8. The amount of BPA degraded by the HRP-PFM was calculated by Eq. (2):

qD ¼ q0  qs  qA

ð2Þ

where qD is the amount of BPA degraded by HRP-PFM; q0 is the initial amount of BPA in solution; qs is the amount of BPA retained in solution; and qA is the amount of BPA absorbed by HRP-PFM. All treatments were performed in triplicate. 2.8. BPA and its degradation analysis with HPLC HPLC (Autosampler Surveyor, Thermo Fisher, USA) analyses were carried out with an instrument that has two pumps, an autosampler, and a photodiode array detector. BPA and its oxidation products were extracted from the bottle with syringe and the filtered solution was analyzed by HPLC. Standard solutions of BPA were used to prepare a standard curve for the analysis of BPA and its oxidation products. The elution was conducted by pumping water and acetonitrile (55:44) isocratically at a flow rate of 1.0 mL/min. UV light absorption was measured at 224 nm. 2.9. Data analysis One-way ANOVA was used to determine statistical significance. Differences among multiple groups of data were determined by multiple comparisons using Tukey’s procedure at a family error rate of 5%. Two values were considered significantly different if p < 0.05. All statistical analyses were performed using Design Expert_Version 7.0.0. Nonlinear regression analysis using the first-order model, Eqs. (3)–(5), was used to estimate the first-order rates (k), the time required to obtain 50% of substrate degradation/adsorption (t1/2), and the BPA removal efficiency after t (REt).

C t ¼ C 0 expðktÞ

ð3Þ

C0 and Ct are the substrate concentrations at the beginning of the run at the time (t), and k is the first-order reaction constant. From the k values, two efficiency factors can be calculated: t1/2 and REt.

t 1=2 ¼ ln 2=k

ð4Þ

REt ¼ ðC 0  C t Þ=C 0  100

ð5Þ

lower glass transition temperature was obtained during the first heating cycle compared with PMMA powder based on differential scanning calorimetry results. Therefore, the expansion of PMMA CEA fibers may be due to swelling in water during enzyme immobilization. SEM images also show the attachment of enzyme molecules on the surface of PMMA CEA fibers after enzyme immobilization, which demonstrates that the HRP molecules were covalently bound to the surface of PMMA CEA fibers and were not rinsed off by the PBS. From the FTIR spectra in the range of 500–4000 cm1 for the PMMA CEA nanofiber, APFM, and activated PMMA CEA nanofiber with GA (Supporting information Fig. S2), the characteristic bands at 1737 cm1 corresponded to the C@O vibrational peaks of the PMMA CEA material. The adsorption bands at 3344 and 1576 cm1 were due to –NH2 stretching vibration and N–H deformation, respectively. This result indicates that the amine groups were introduced into the PMMA CEA membranes after PEI amination. The characteristic band at 1637 cm1 for –CHO proves that the PMMA CEA membranes were successfully modified with GA. 3.2. Effect of reaction time and pH on HRP activity and its loading on the PFM HRP immobilization was significantly affected by pH and time (p < 0.05). Fig. 1 shows that the optimum pH for HRP immobilization was between 4.0 and 5.5, whereas pH values lower than 4 or higher than 5.5 caused a significant decrease in HRP loading (p < 0.05). Extreme pH conditions normally result in the loss of binding sites between enzyme and carrier, resulting in the reduction in enzyme loading on the surface of the carrier. HRP loading reached 285 mg/g membranes after 3 h of immobilization at pH 5.0 and 30 °C, and this value is higher than that for previously reported carriers (Takahashi et al., 2001; Lai and Lin, 2005). High HRP loading may be due to the high specific surface area of the PMMA CEA fibrous membranes and the effectiveness of the immobilization method. The retention of HRP-PFM activity was approximately 70% of the free HRP. The loss of HRP catalytic activity in the immobilized system may be due to changes in the enzyme diffusion rate and variations in the microenvironment (Wang et al., 2009). The Km and Vmax values were 0.34 mmol/L and 133.1 lmol/mg/min for free HRP, and 0.62 mmol/L and 99.8 lmol/mg/min for HRP-PFM, respectively. Compared with free HRP, the changes in Km and Vmax values after enzyme immobilization could be due to the decreased access of substrate molecules to the active points of the immobilized enzyme (Bromberg et al., 2008). The Vmax values of HRP-PFM

C0 and Ct are the BPA concentrations at the onset and time (t) of reaction. 3. Results and discussion 3.1. Characterization of electrospun PMMA CEA fibrous membranes before and after HRP immobilization SEM images of PMMA CEA fibrous membranes before and after HRP immobilization (Supporting information Fig. S1) show almost homogenous nanofibers with diameters ranging from 300 to 500 nm. The fibers expanded from nanometer to micrometer level after modification with functional groups. Carrizales et al. (2008) found that PMMA electrospun nanofibers could absorb water. A

113

Fig. 1. Effect of time and pH on HRP immobilization efficiency.

114

R. Xu et al. / Bioresource Technology 149 (2013) 111–116

were 70% of free HRP, indicating that the PMMA CEA fibrous support and immobilization method used in this study efficiently retained the catalytic ability of HRP.

3.3. Stabilities of free and HRP-PFM The stability of an immobilized enzyme is important for various biotechnological applications. Fig. 2(a) shows that both free and HRP-PFM retained above 90% of initial activity at temperatures between 30 and 40 °C. The reduction in the activity of free HRP was more significant than that of the HRP-PFM (p < 0.05) at temperatures above or below this range. For example, the relative activity of free HRP at 60 °C was about 62%, whereas that of HRPPFM was about 75%. Fig. 2(b) shows that the optimum working pH of both free and HRP-PFM was between 4.0 and 5.0, in which the enzyme activity retained was above 85% of the initial values. When pH level was lower than 4.0 or higher than 5.0, HRP-PFM retained significantly higher activity than free HRP (p < 0.05). At pH 3.0, the relative activity of free HRP was about 48%, whereas that of HRP-PFM was about 73%. HRP-PFM showed higher resistance to the changes in temperature and pH compared with free HRP. Fig. 3 shows the storage stability of free and HRP-PFM. The activity of free HRP dropped significantly faster than HRP-PFM under the same storage conditions (p < 0.05). After 18 d, HRPPFM retained approximately 95% of the initial enzyme activity in the buffer solution, whereas free HRP retained only 25%. Therefore, the storage stability of HRP-PFM was significantly higher than free HRP (p < 0.05), which may be attributed to the limited conformational changes of enzyme molecules in the matrix of PMMA CEA fibrous membranes (Wan et al., 2008). The retained enzyme activity of HRP-PFM after incubation for 18 d was much higher than

Fig. 2. Effect of temperature (a) and pH (b) on HRP activity.

that on other supports, e.g., 60% on silica sol–gel (Li et al., 1996), 70% on poly (ethylene terephthalate) grafted acrylamide fiber (Temoçin and Yigitoglu, 2009), and 80% on aluminum-pillared interlayered clay (Yu et al., 2006). Unlike free enzyme, immobilized enzyme is easily separated from the reaction solution and reused, which greatly reduces the cost of operations in practical applications. Fig. 4 shows the operational stabilities of HRP-PFM. After six repeated runs, HRP-PFM retained about 50% of its initial activity. This result is better than those of HRP immobilized on other materials, such as macroporous glycidyl methacrylate-based copolymers (45% activity retained after 4 cycles) (Olivera et al., 2012) and P(DEA-co-AA) microgels (50% activity retained after 5 cycles) (Zhang et al., 2012). The decrease in activity of HRP-PFM after repeated use may be due to enzyme protein denaturation and membrane damage (Huang et al., 2008).

3.4. Removal of BPA from water by HRP Fig. 5 shows the effect of pH and temperature on the removal of BPA by PMMCEA fibrous membranes (PFM) and HRP-PFM, as well as the biodegradation of BPA by free and HRP-PFM. The removal rates of BPA by PFM adsorption was between 30% and 36% under the conditions of 30 °C and pH 3.0–6.0, and between 36% and 45% at 20–60 °C and pH 5.0. Statistical analysis shows that pH did not significantly affect the adsorption of BPA by PFM in this experiment (p > 0.05). The PFM have no functional groups that may be affected by pH in the experimental range. The adsorption efficiency of BPA by PFM decreased significantly at 20 °C compared with 40 °C (p > 0.05), suggesting that the adsorption process was an endothermic reaction. The adsorption of BPA onto PFM may be due to the nano-sized electrospun fibers and the porous structure of the membranes, indicating the involvement of physical adsorption. Fig. 5a shows a significant variation in BPA degradation by free HRP at different pH (p < 0.05). This result may be attributed to the generation of H2O2 (Cho et al., 2010; Khan and Nicell, 2007). By contrast, the immobilization of HRP on the PMMA CEA fibrous membranes significantly enhanced pH stability (p < 0.05) and BPA degradation (p < 0.05). Fig. 5b shows that the removal rates of BPA at 40 °C for HRP-PFM and free HRP were 52% and 58%, respectively. The removal efficiencies of both free HRP and HRPPFM decreased significantly at 20 °C than that at 40 °C (p < 0.05), suggesting that low temperature inhibited the effect on the degradation of BPA by HRP. An increase in temperature lowered the concentration of dissolved oxygen and the self-decomposition of H2O2 (Wang et al., 2008). Therefore, the optimal temperature for the catalytic removal of BPA was 40 °C.

Fig. 3. Storage stability of free HRP and HRP-PFM at 4 °C.

115

R. Xu et al. / Bioresource Technology 149 (2013) 111–116

Fig. 4. Reuse capacity of HRP-PFM. Fig. 6. Degradation kinetics of BPA by free and HRP-PFM (at 30 °C and pH 5.0 with 0.038 mmol/L BPA and 0.019 mmol/L H2O2 initially).

Table 1 k, t1/2, RE6 of free HRP, HRP-PFM, and PFM toward BPA. Sample

K (h)

t1/2 (h)

RE6 (%)

Free HRP HRP-PFM PFM Biodegradation by HRP-PFM

0.29 0.93 0.16 0.24

2.39 0.74 4.33 2.88

61 93 42 58

HRP-PFM: PMMA CEA fibrous membranes with bonded HRP, PFM: PMMA CEA fibrous membranes, Biodegradation by HRP-PFM: biodegradation by bonded HRP.

Fig. 5. (a) Effect of pH on the removal efficiency of BPA by free and HRP-PFM (3 h at 30 °C with 0.038 mmol/L BPA and 0.019 mmol/L H2O2 initially). (b) Effect of temperature on the removal efficiency of BPA by free and HRP-PFM (3 h at pH 5.0 with 0.038 mmol/L BPA and 0.019 mmol/L H2O2 initially). (j HRP-PFM; d PFM; N Free HRP; . Biodegradation by HRP-PFM).

removal efficiencies of BPA at initial concentrations of 10 and 60 mg/L by HRP-PFM were 57% and 95%, respectively. The results were consistent with those reported by Nicell and Wright (1997), in which the degradation of phenolic compounds by HRP had a close relationship with the initial concentrations of the phenolic compounds. Fig. 6 shows the rapid degradation of BPA by free HRP. The degradation percentage reached approximately 38% in the first 30 min and 61% after 3 h. The difference between the removal efficiencies of HRP-PFM and PFM may be due to the catalysis of HRP. The kinetic curves of BPA removal followed a first-order reaction. Table 1 shows that the BPA degradation rate of HRP immobilized on PFM was slower than that of free HRP. This result may be due to the spatial limitations for substrate diffusion and protein flexibility after enzyme immobilization on the carrier, suggesting that HRP-PFM was partly inactivated during the immobilization process. Obviously, the total removal percentage (93%) of BPA by HRP-PFM after 3 h was much higher than in the other two treatments. The reason may be explained by PFM adsorption and enzyme catalysis (Cheung et al., 2007). A similar report by Liu et al. (2012) claimed that nearly 20% of the removal of phenolic compounds is due to the adsorption by the mesoporous support.

4. Conclusion In Fig. 5a and b, the removal rates of BPA by HRP-PFM were significantly higher than free HRP (p < 0.05) and PFM (p < 0.05) because of the combined advantages of the adsorption function of microfibrous structures of PFM and the biocatalytic property of HRP in HRP-PFM. The maximal removal percentages of free HRP and HRP-PFM were 60% and 93%, respectively. Therefore, the HRP-PFM was more suitable for BPA removal in practical industrial effluents with low pH compared with free HRP. BPA removal catalyzed by free HRP and HRP-PFM increased with increasing initial BPA concentration from 10 to 60 mg/L. The

PMMA CEA microfibrous membranes were successfully fabricated by electrospinning and employed as matrices for the immobilization of HRP. The PFM and immobilization methods efficiently retained the catalytic activity of HRP. The stabilities and reuse capabilities of the HRP-PFM were significantly higher compared with free HRP. The HRP-PFM conducted catalytic and adsorption functions, thereby exhibiting a significantly higher BPA removal efficiency than free HRP and PFM. Therefore, immobilized enzymes on PFM may have a great application potential in the field of wastewater treatment.

116

R. Xu et al. / Bioresource Technology 149 (2013) 111–116

Acknowledgements This work was funded by the National Natural Science Foundation of China (51208368). The authors are also grateful for the financial support from the Ministry of Science and Technology, China (Nos. 2010DFA92820 and 2010DFA92800). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 09.030. References Alsarra, I.A., Betigeri, S.S., Zhang, H., Evans, B.A., Neau, S.H., 2002. Molecular weight and degree of deacetylation effects on lipase-loaded chitosan bead characteristics. Biomaterials 23, 3637–3644. Arica, M.Y., Altintas, B., Bayramog˘lu, G., 2009. Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation. Bioresour. Technol. 100, 665–669. Bromberg, A., Marx, S., Frishman, G., 2008. Kinetic study of the thermal inactivation of cholinesterase enzymes immobilized in solid matrices. BBA-Proteins Proteom. 1784, 961–966. Carrizales, C., Pelfrey, S., Rincon, R., Eubanks, T.M., Kuang, A., McClure, M.J., Bowlin, G.L., Macossay, J., 2008. Thermal and mechanical properties of electrospun PMMA, PVC, Nylon 6, and Nylon 6,6. Polym. Adv. Technol. 19, 124–130. Chaplin, M.F., Bucke, C., 1990. Enzyme Technology. Cambridge University Press. Chen, H., Hsieh, Y., 2005. Enzyme immobilization on ultrafine cellulose fibers via poly(acrylic acid) electrolyte grafts. Biotechnol. Bioeng. 90, 405–413. Cheng, W., Harper, W.F., 2012. Chemical kinetics and interactions involved in horseradish peroxidase-mediated oxidative polymerization of phenolic compounds. Enzyme Microb. Technol. 50, 204–208. Cheung, W.H., Szeto, Y.S., McKay, G., 2007. Intraparticle diffusion processes during acid dye adsorption onto chitosan. Bioresour. Technol. 98, 2897–2904. Cho, S.H., Jang, A., Bishop, P.L., Moon, S.H., 2010. Kinetics determination of electrogenerated hydrogen peroxide (H2O2) using carbon fiber microelectrode in electroenzymatic degradation of phenolic compounds. J. Hazard. Mater. 175, 253–257. Dai, Y., Niu, J., Liu, J., Yin, L., Xu, J., 2010. In situ encapsulation of laccase in microfibers by emulsion electrospinning: preparation, characterization, and application. Bioresour. Technol. 101, 8942–8947. Dyal, A., Loos, K., Noto, M., Chang, S.W., Spagnoli, C., Shafi, K.V.P.M., Ulman, A., Cowman, M., Gross, R.A., 2003. Activity of Candida rugosa lipase immobilized on g-Fe2O3 magnetic nanoparticles. J. Am. Chem. Soc. 125, 1684–1685. Fukahori, S., Fukahori, H., Kitaoka, T., Tanaka, H., 2003. Capturing of bisphenol-A photodecomposition intermediates by composite TiO2-zeolite sheets. Appl. Catal. B 46, 453–462. Huang, X.J., Yu, A.G., Xu, Z.K., 2008. Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application. Bioresour. Technol. 99, 5459–5465. Inoue, M., Masuda, Y., Okada, F., Sakurai, A., Takahashi, I., Sakakibara, M., 2008. Degradation of BPA using sonochemical reactions. Water Res. 42, 1379–1386.

Khan, U., Nicell, J.A., 2007. Horseradish peroxidase-catalyzed oxidation of aqueous natural and synthetic estrogens. J. Chem. Technol. Biotechnol. 82, 818–830. Koeller, K.M., Wong, C.H., 2001. Enzymes for chemical synthesis. Nature 409, 232– 240. Lai, Y.C., Lin, S.C., 2005. Application of immobilized horseradish peroxidase for the removal of p-chlorophenol from aqueous solution. Process Biochem. 40, 1167– 1174. Li, J., Tan, S.N., Ge, H., 1996. Silica sol–gel immobilized amperometric biosensor for hydrogen peroxide. Anal. Chem. Acta 335, 137–145. Liu, Y., Zeng, Z., Zeng, G., Tang, L., Pang, Y., Li, Z., Liu, C., Lei, X., Wu, M., Ren, P., Liu, Z., Chen, M., Xie, G., 2012. Immobilization of laccase on magnetic bimodal mesoporous carbon and the application in the removal of phenolic compounds. Bioresour. Technol. 115, 21–26. Mateo, C., Palomo, J.M., Gloria, F.M., Guisan, J.M., Roberto, F.L., 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451–1463. Nakagawa, Y., Tayama, S., 2000. Metabolism and cytotoxicity of BPA and other bisphenols in isolated rat hepatocytes. Arch. Toxicol. 74, 99–105. Nicell, J.A., Wright, H., 1997. A model of peroxidase activity with inhibition by hydrogen peroxide. Enzyme Microb. Technol. 21, 302–310. Noureddini, H., Gao, X., Philkana, R.S., 2005. Immobilized Pseudomonas cepacia lipase for bio-diesel fuel production from soybean oil. Bioresour. Technol. 96, 769–777. Olivera, P., Milos, P., Dragica, S., Zeljko, S., Ksenija, R., Zorica, K.J., Radivoje, P., 2012. Improved covalent immobilization of horseradish peroxidase on macroporous glycidyl methacrylate-based copolymers. Appl. Biochem. Biotechnol. 168, 1288–1301. Reetz, M.T., Zonta, A., Simpelkamp, J., 1995. Efficient heterogeneous biocatalysts by entrapment of lipases in hydrophobic sol–gel materials. Angew. Chem. Int. Ed. Engl. 34, 301–303. Ren, G., Xu, X., Liu, Q., Cheng, J., Yuan, X., Wu, L., Wan, Y., 2006. Electrospun poly(vinyl alcohol)/glucose oxidase biocomposite membranes for biosensor applications. React. Funct. Polym. 66, 1559–1564. Suzuki, N., Hattori, A., 2003. BPA suppresses osteoclastic and osteoblastic activities in the cultured scales of goldfish. Life Sci. 73, 2237–2247. Takahashi, H., Li, B., Sasaki, T., Miyazaki, C., Kajino, T., Inagaki, S., 2001. Immobilized enzymes in ordered mesoporous silica materials and improvement of their stability and catalytic activity in an organic solvent. Microporous Mesoporous Mater. 44, 755–762. Temoçin, Z., Yig˘itog˘lu, M., 2009. Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber. Bioprocess Biosyst. Eng. 32, 467–474. Wan, L.S., Ke, B.B., Xu, Z.K., 2008. Electrospun nanofibrous membranes filled with carbon nanotubes for redox enzyme immobilization. Enzyme Microb. Technol. 42, 332. Wang, C.T., Hua, J.L., Chou, W.L., Kuo, Y.M., 2008. Removal of color from real dyeing wastewater by Electro-Fenton technology using a three-dimensional graphite cathode. J. Hazard. Mater. 152, 601–606. Wang, Z.G., Wan, L.S., Liu, Z.M., Huang, X.J., Xu, Z.K., 2009. Enzyme immobilization on electrospun polymer nanofibers: an overview. J. Mol. Catal. B Enzym. 56, 189–195. Yu, S.M., Cheng, J., Zuo, P., 2006. Horseradish peroxidase immobilized on aluminumpillared interlayered clay for the catalytic oxidation of phenolic wastewater. Water Res. 40, 283–290. Zhang, Y.P., Liu, T.H., Wang, Q., Zhao, J.H., Fang, J., Shen, W.G., 2012. Synthesis of novel poly(N,N-diethylacrylamide-co-acrylic acid) (P(DEA-co-AA)) microgels as carrier of horseradish peroxidase immobilization for pollution treatment. Macromol. Res. 5, 484–489.