Article pubs.acs.org/est
Immobilization of Catalase on Electrospun PVA/PA6−Cu(II) Nanofibrous Membrane for the Development of Efficient and Reusable Enzyme Membrane Reactor Quan Feng,†,‡,§ Yong Zhao,‡ Anfang Wei,† Changlong Li,† Qufu Wei,*,§ and Hao Fong*,‡ †
Key Laboratory of Textile Fabric, College of Textiles and Clothing, Anhui Polytechnic University, Wuhu, Anhui 241000, China Department of Chemistry and Applied Biological Sciences, South Dakota School of Mines and Technology, Rapid City, South Dakota 57701, United States § Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China ‡
ABSTRACT: In this study, a mat/membrane consisting of overlaid PVA/PA6−Cu(II) composite nanofibers was prepared via the electrospinning technique followed by coordination/chelation with Cu(II) ions; an enzyme of catalase (CAT) was then immobilized onto the PVA/PA6−Cu(II) nanofibrous membrane. The amount of immobilized catalase reached a high value of 64 ± 4.6 mg/g, while the kinetic parameters (Vmax and Km) of enzyme were 3774 μmol/mg·min and 41.13 mM, respectively. Furthermore, the thermal stability and storage stability of immobilized catalase were improved significantly. Thereafter, a plugflow type of immobilized enzyme membrane reactor (IEMR) was assembled from the PVA/PA6−Cu(II)−CAT membrane. With the increase of operational pressure from 0.02 to 0.2 MPa, the flux value of IEMR increased from 0.20 ± 0.02 to 0.76 ± 0.04 L/m2·min, whereas the conversion ratio of H2O2 decreased slightly from 92 ± 2.5% to 87 ± 2.1%. After 5 repeating cycles, the production capacity of IEMR was merely decreased from 0.144 ± 0.006 to 0.102 ± 0.004 mol/m2·min. These results indicated that the assembled IEMR possessed high productivity and excellent reusability, suggesting that the IEMR based on electrospun PVA/PA6−Cu(II) nanofibrous membrane might have great potential for various applications, particularly those related to environmental protection. physical−chemical interactions.6,7 Recently, some transition metal ions (e.g., Cu2+, Ni2+, Zn2+, and Fe3+) have been investigated for catalase immobilization, because the coordination bonds (formed between these ions and catalase molecules) can provide the adequate binding force for immobilization and effectively reduce the inactivation/denaturation of catalase.8−10 To date, many supports (including the beads of chitosan and its composite with poly(vinyl alcohol) (PVA),11,12 conventional fibers of Zr(IV)-modified collagen,13 glass beads with high porosity,14 and submicron-sized spheres of titania15) have been studied for catalase immobilization. However, these supports have different limitations. For example, the enzyme-loading amounts of conventional fibers are low due to small surface-to-
1. INTRODUCTION Enzymes are protein-based biocatalysts with high efficiency and superior selectivity, and the conditions for enzyme-catalyzed reactions are generally mild with low energy consumption. The catalase of H2O2 oxidoreductase, a heme-containing metalloenzyme, is a common enzyme for decomposition of H2O2 into oxygen and water,1,2 and the catalytic effect of catalase is much higher than that of light, organics, and metal ions.3,4 Although the catalase has been widely used in many fields such as food, textile, and detergent, the applications encounter several difficulties/challenges. For example, the recovery of catalase from reaction systems is usually inconvenient because the enzyme is highly water-soluble, and the catalase has to be utilized within specific a temperature range and chemical environment for achieving optimal catalysis performance.5 The immobilization of enzyme is an important/attractive approach to overcome/mitigate those difficulties/challenges, in which the catalase is adsorbed/bonded onto water-insoluble supports via © 2014 American Chemical Society
Received: Revised: Accepted: Published: 10390
April 15, 2014 July 13, 2014 August 5, 2014 August 5, 2014 dx.doi.org/10.1021/es501845u | Environ. Sci. Technol. 2014, 48, 10390−10397
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∼100 000 g/mol) ultrafiltration membranes were supplied by the Musu Membrane Technology (Shanghai, China). Bovine liver catalase (enzyme commission 1.11.1.6) was purchased from the Sigma-Aldrich Corp. (Shanghai, China). Formic acid (HCOOH, < 2.5% water as stabilizer), CuCl2, H2O2 (30 wt % in H2O), NaCl, KCl, KH2PO4, K2HPO4, and Coomassie Brilliant Blue (G250) were purchased from the Sinopharm Chemical Reagent Co. (Shanghai, China). The materials and chemicals were used without further purification. 2.2. Preparation of Electrospun Nanofibrous Membranes. Prior to electrospinning, PVA (4 g) and PA6 (12 g) were dissolved in formic acid (100 mL) at room temperature. The resulting spin dope was then loaded into a 30-mL BD Luer-Lok tip plastic syringe having an 18 gauge 90° blunt-end stainless steel needle. The electrospinning was carried out at 14 kV through a DW-P503-4AC high-voltage supplier, and the feed rate was set at 0.3 mL/h by using a WZ-50C2 microinfusion pump. Electrospun PVA/PA6 nanofibers were collected on electrically grounded aluminum foil that covered a laboratory-produced roller, and the distance between aluminum foil and the tip of needle was 15 cm. Subsequently, the aselectrospun PVA/PA6 nanofibrous membrane was coordinated/chelated with Cu(II) ions by immersion of 0.2 g of membrane into 100 mL of 0.1 M CuCl2 solution for 24 h at 20 °C under shaking condition. The resulting PVA/PA6−Cu(II) nanofibrous membrane was then rinsed with deionized water and dried at 35 °C in a vacuum oven (∼ 27 mmHg). For comparison, electrospun neat PVA nanofibrous membrane was also prepared from the spin dope containing 8 wt % PVA alone. The PVA nanofibrous membrane coordinated/chelated with Cu(II) ions (i.e., PVA−Cu(II) nanofibrous membrane) was then prepared with the same procedure. The prepared PVA/PA6−Cu(II) and PVA−Cu(II) nanofibrous membranes had similar thickness of ∼70 μm and mass per unit area of ∼20 g/m2. Atomic absorption spectroscopy (AAS) was employed to determine the adsorption amounts of Cu(II) ions in the nanofibrous membranes. A Hitachi S-4800 field-emission scanning electron microscope (SEM) was used to examine the morphological structures of PVA/PA6, PVA/ PA6−Cu(II), PVA, and PVA−Cu(II) nanofibrous membranes. 2.3. Immobilization of Catalase on PVA/PA6−Cu(II) and PVA−Cu(II) Nanofibrous Membranes. The bovine liver catalase (0.3 g) was first dissolved in 1.0 L of 50 mM phosphate buffer solution (PBS) with pH value of 7.0. Thereafter, 0.2 g of PVA/PA6−Cu(II) nanofibrous membrane (3.03 mmol/g of coordinated Cu2+ ions, determined by AAS) was placed in 200 mL of catalase solution (0.3 mg/mL) for 4 h at 20 °C under shaking condition. The PVA/PA6−Cu(II) nanofibrous membrane was then rinsed with the same PBS until no enzyme could be detected in the rinsing PBS. For comparison, PVA−Cu(II) nanofibrous membrane (2.15 mmol/ g of coordinated Cu2+ ions, determined by AAS) was also treated by the same procedure for catalase immobilization. To examine the distributions of immobilized catalase on the surfaces of nanofibrous membranes, fluorescein isothiocyanate (FITC) was used to label the catalase.32 Specifically, 0.1 mL of FITC (2 mM) was added into 20 mL of catalase solution (0.3 mg/mL) under magnetic stirring condition for 4 h in a brown vial, and the mixture was then placed at 4 °C for 24 h. This was followed by centrifugation at 3000 rpm for 30 min to remove any precipitates. Subsequently, the resulting catalase (labeled with FITC) was immobilized on the surface of PVA/PA6− Cu(II) or PVA−Cu(II) nanofibrous membranes with the same
mass ratios. Another example, although the enzyme-loading amounts of microporous beads/spheres are quite high, the spatial/steric hindrances are usually serious. Electrospun nanofibrous membranes have recently been investigated for filtration, drug delivery, tissue engineering, dental composite, and enzyme immobilization.16−22 Note that these membranes possess a large number of interaction sites for enzyme immobilization due to high surface-to-mass ratios. Meanwhile, the membranes are highly porous and mechanically flexible, providing more diffusion channels and thus allowing for faster flux rates than conventional membranes.23,24 PVA is a nontoxic, hydrophilic, and biocompatible polymer with each repeating unit having a hydroxyl group, and it has been widely utilized for cell and enzyme immobilization.25 However, electrospun PVA nanofibers in the membranes swell substantially during the coordination/chelation with transition metal ions in an aqueous environment, resulting in partial or complete loss of the morphological structure of the nanofibrous membranes. Recently, the method of chemical cross-linking with glutaraldehyde has been studied to improve the stability of electrospun PVA nanofibrous membranes.26 On the other hand, electrospun composite nanofibrous membranes of PVA and polyamide 6 (PA6) also exhibit excellent structural stability in aqueous solutions;27,28 and the following is the explanation: If a spin dope contains two polymers (e.g., PVA and PA6, both having high concentrations), microphase separations will occur in the spin dope; after the phase-separated domains are stretched during bending instability,29 followed by rapid solidification of the jet/filament, the composite nanofiber with both polymer components being semigyroid or cocontinuous can be acquired.30 This would further result in the macromolecular interactions between PVA and PA6 being strong; consequently, the PVA component in the composite nanofibers would be unlikely to leach away when immersed in an aqueous environment. The goal of this study is to develop a plug-flow type of immobilized enzyme membrane reactor (IEMR) with elevated productivity from an electrospun nanofibrous membrane with high loading amount of catalase. Note that IEMRs have been broadly adopted for various applications, particularly those related to environmental protection. Practical applications of IEMRs are determined upon several criteria such as large amount of immobilized enzyme, high productivity, and excellent reusability.31 During this study, an electrospun PVA/PA6 nanofibrous membrane coordinated/chelated with Cu(II) ions was first prepared and then used for the immobilization of catalase (CAT). Experimental results indicated that the PVA/PA6−Cu(II)−CAT nanofibrous membrane possessed superior morphological stability, while the immobilized CAT exhibited high thermal stability and excellent storage stability. Thereafter, a plug-flow type of IEMR was assembled from the prepared nanofibrous membrane. The flux value and H2O2 conversion ratio of this IEMR were calculated, and the IEMR’s operational stability and reusability were evaluated. This study demonstrated that the assembled IEMR would be promising for various applications related to environmental protection (such as wastewater treatment in making foods, textiles, and detergents).
2. EXPERIMENTAL SECTION 2.1. Materials. PVA (Mw ∼86 000 g/mol) and PA6 (Mw ∼18 000 g/mol) were obtained from the Shanghai Kanghu Chemical Co. in China. Polyoxyphenylene sulfone (PS, Mw 10391
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K 1 1 1 = m × + Vi Vmax [S] Vmax
procedure. Finally, the distribution of the FITC-labeled immobilized catalase was examined by an inverted fluorescence microscope (IFM, Nikon Eclipse Ti). During the immobilization of catalase as schematically shown in Figure 1, Cu(II) ions were first coordinated/chelated with
where Vi and Vmax are the initial and maximal rates of the enzyme-catalyzed reaction, Km is the Michaelis constant, and [S] is the concentration of H2O2. Note that kinetic parameters were determined at 35 °C, and the concentration of H2O2 was 30−200 mM. 2.5. Analysis of Thermal Stability and Storage Stability of the Immobilized Catalase. Thermal stability was investigated by incubating the free and immobilized catalase at different temperatures (i.e., 30, 35, 40, 45, 50, 55, and 60 °C) in PBS (50 mM, pH 7.0) for 5 h; thereafter, the free and immobilized catalase was studied for enzymatic decomposition of H2O2 in the same conditions as described in Section 2.4. Storage stability of the free and immobilized catalase was evaluated upon calculating the residual activity of PVA/PA6− Cu(II)−CAT, PVA−Cu(II)−CAT, and free catalase after being stored at 4 °C in PBS for 20 days, and the highest value of each set was assigned as 100%. 2.6. Equipment Design of IEMR. An IEMR was assembled from the PVA/PA6−Cu(II)−CAT nanofibrous membrane. As shown in Figure 2, the reactor with capacity
Figure 1. Schematic showing the immobilization of a catalase molecule on the PVA/PA6−Cu(II) nanofibrous membrane.
hydroxyl and/or carbonyl groups on the PVA/PA6 nanofibers; thereafter, these Cu(II) ions would further form coordination bonds with imidazole and/or amino groups on catalase molecules, leading to the catalase immobilization on the PVA/PA6−Cu(II) nanofibrous membrane.8 The Bradford assay was adopted to determine the immobilization amount upon the following equation:33 Qe =
(C0 − Ce)V0 − CrVr Md
(1)
where Qe is the amount of catalase immobilized on unit mass of membrane (mg/g), C0 and Ce are initial and equilibrium enzyme concentrations in the solution (mg/mL), Cr is the enzyme concentration in the rinsing PBS (mg/mL), Vo is the volume (mL) of catalase solution, Vr is the volume (mL) of rinsing PBS, and Md is the mass (g) of membrane. 2.4. Activity Assay of Catalase under the Free and Immobilized Conditions. To test the respective activities of catalase under the free and immobilized conditions, 0.3 mL of catalase solution (0.3 mg/mL) or 0.01 g of PVA/PA6−Cu(II)− CAT nanofibrous membrane was mixed with 50 mL of 100 mM H2O2 solution. The system was then kept at 35 °C for 3 min, and 3 replicates were tested for each sample. The activities of catalase under the free or immobilized conditions were determined spectrophotometrically by measuring the decrease of absorbance at 240 nm, as a consequence of H 2 O 2 consumption. The specific activity of enzyme was then calculated by using the following equation:34 v=
(A 0 − A ) × V T × K × Ew
(3)
Figure 2. Diagram showing the assembled IEMR.
of 500 mL was placed on a hot plate, while a PVA/PA6− Cu(II)−CAT nanofibrous membrane with diameter of 8.0 cm was overlaid on a PS ultrafiltration membrane with the same size to form the reaction layer. The H2O2 solution was supplied via a peristaltic pump, and the operational pressure was adjusted with a N2 regulator to control the flow rate. The filtrate was then collected for subsequent analyses. The O2 gas generated during the reaction was released from an overpressure valve. For comparison, another reactor was also assembled from the PVA/PA6−Cu(II) nanofibrous membrane without immobilization of catalase. Note that in this reactor the catalase (with the concentration of 0.05 mg/mL) was suspended in H2O2 solution. 2.7. Determination of Flux Values of the Assembled IEMRs. To determine the flux value of an IEMR, 300 mL of 0.2 M H2O2 aqueous solution was first added into the IEMR, and the additional H2O2 solution was then supplied intermittently by the peristaltic pump. The following conditions were adopted during the test: the operational pressure was 0.2 MPa, the stirring speed was 100 rad/min, the temperature was 35 °C, and the collection time of filtrate was 4 h. For comparison, the flux value of the reactor with free enzyme was also determined under the same conditions. The flux values of IEMRs were calculated by using the following equation:
(2)
where v is the specific activity of free or immobilized catalase (μmol/mg·min), A0 and A are the initial and final absorbances of the solution at 240 nm, V is the volume of H2O2 solution (mL), T is the reaction time (min), K is the molar extinction coefficient of H2O2 at 240 nm (K = 0.033 L/mmol·cm, and the optical path-length is 1 cm),35 and Ew is the enzyme amount (mg). The kinetic parameters (i.e., Vmax and Km) of enzyme under the free and immobilized conditions were calculated from Lineweaver−Burk plot of the Michaelis−Menten equation.36 10392
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Figure 3. SEM images of PVA/PA6 nanofibrous membrane (A), PVA/PA6−Cu(II) nanofibrous membrane (B), PVA nanofibrous membrane (C), and PVA−Cu(II) nanofibrous membrane (D). IFM images of FITC-labeled immobilized catalase on the PVA/PA6−Cu(II) nanofibrous membrane (E) and the PVA−Cu(II) nanofibrous membrane (F).
J=
V S×t
operational pressure was 0.2 MPa, and the reaction temperature was 35 °C. Subsequently, the nanofibrous membrane and PS ultrafiltration membrane were preserved in PBS (50 mM, pH = 7) and 0.5% formaldehyde solution at 4 °C, respectively. The productivity of IEMR was calculated by using the following equation:
(4)
where J is the flux of membrane (L/m2·min), V is the volume of filtrate (L), S is the size of membrane (m2), and t is the time of operation (min). 2.8. Analysis of the Conversion Ratios of H2O2. Conversion ratios of H2O2 under varied operational pressures with different flux values of IEMR were determined. The operational pressure was from 0.02 to 0.2 MPa (note that the pressure was adjusted once every 10 min, and the increment was 0.02 MPa), the concentration of H2O2 aqueous solution was set at 0.2 M, and the temperature was maintained at 35 °C. The H2O2 conversion ratios were calculated by using the following equation: K=
C0 − Cp C0
× 100%
P=
V × (C0 − Cp) t×S
(6)
where P is the productivity of IEMR (mol/m2·min), V is the volume of filtrate (L), C0 and Cp are concentrations of H2O2 in the initial solution and in the filtrate (M), respectively, t is the reaction time (min), and S is the size of membrane (m2). For examining the reusability of an IEMR, the productivity of this IEMR was tested once every day for 5 days, and the reaction time was set at 4 h. After examination of reusability, the PVA/PA6−Cu(II)−CAT nanofibrous membrane (before test and after the tests for 5 times) was collected. Thereafter, the Brunauer−Emmett−Teller (BET) specific surface area, pore volume, and average pore size of the collected membrane were determined by N2 adsorption at −196 °C.
(5)
where K is the conversion ratio of H2O2 (%), and C0 and Cp are the concentrations of H2O2 in the initial solution and in the filtrate, respectively (mol/L). 2.9. Evaluation of the Productivity and Reusability of IEMR. The productivity of an IEMR under varied operational pressures was evaluated under the following conditions: the 10393
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3. RESULTS AND DISCUSSION 3.1. Morphologies of Nanofibrous Membranes and Distributions of Immobilized Catalase. Figure 3A, B, C, and D are SEM images showing the morphologies of PVA/ PA6, PVA/PA6−Cu(II), PVA, and PVA−Cu(II) nanofibrous membranes, respectively. As shown in Figure 3A, electrospun PVA/PA6 composite nanofibers were overlaid in the membrane with diameters from 100 to 500 nm. The nanofibrous structure of PVA/PA6 membrane was wellretained after immersion in aqueous solution containing Cu(II) ions for 24 h (Figure 3B). In contrast, the morphological structure of neat PVA nanofibrous membrane showed substantial variation/discrepancy after immersion in the solution for 24 h (Figure 3C and D). Unlike the sample of PVA/PA6−Cu(II) which was a typical nanofibrous membrane, the sample of PVA−Cu(II) partially lost the morphological structure of nanofibrous membrane; i.e., the nanofibers in the membrane appeared to fuse/conglutinate together into a meshlike structure. Such results indicated that the morphological/ structural stability of PVA/PA6 nanofibrous membranes was significantly improved upon the incorporation of PA6 into the nanofibers. The IFM images of the immobilized catalase (labeled with FITC) on the surface of PVA/PA6−Cu(II) and PVA−Cu(II) nanofibrous membranes are depicted in Figure 3E and F, respectively. The immobilized catalase molecules were attached on the surface of PVA/PA6−Cu(II) nanofibers uniformly (Figure 3E), and the density/amount of immobilized catalase on PVA/PA6−Cu(II) nanofibers was distinguishably higher than that on PVA−Cu(II) nanofibers (Figure 3F). Such IFM results were consistent with the results acquired from SEM, further confirming that the PVA/PA6−Cu(II) nanofibrous membrane could well-retain the nanofibrous morphological structure after the coordination/chelation of Cu(II) ions in aqueous environment, and the resulting PVA/PA6−Cu(II) membrane would have more interaction sites for immobilization of catalase molecules than the PVA−Cu(II) membrane. 3.2. Kinetic Parameters of Immobilized and Free Catalase. As shown in Table 1, the amount of immobilized
contacts between membrane and active sites of catalase. Nevertheless, the specific activities of catalase immobilized on PVA/PA6−Cu(II) and PVA−Cu(II) membranes were lower than that of free catalase. This could be easily understood, since the spatial/steric barriers would arise upon the immobilization of catalase molecules on the PVA/PA6−Cu(II) and PVA− Cu(II) nanofibrous membranes. Such a condition could hinder the diffusion (i.e., decrease the accessibility) of H2O2 molecules to the active sites of immobilized enzyme. Similar observations have also been reported by other researchers.39,40 For the immobilized catalase Vmax was smaller, while the Km value was higher than those of free catalase. Note that the Vmax is defined as the highest possible rate when an enzyme is saturated with substrate, which reflects the intrinsic characteristics of immobilized enzyme. On the other hand, the Km is defined as the H2O2 concentration at the reaction rate of 1/2 V max , which represents the affinity of an enzyme to substrate.41−43 According to the kinetic parameters of Vmax and Km, the PVA/PA6−Cu(II) membrane exhibited desired biological compatibility to immobilized catalase.44,45 3.3. Thermal Stability and Storage Stability of Immobilized Catalase. Thermal stability is an important parameter in consideration of a practical application. Figure 4
Figure 4. Effect of temperature on catalytic activity of free and immobilized catalase (the lines showing nonlinear regression curve fittings). Error bars show one standard deviation (n = 3).
Table 1. Amounts of Immobilized Catalase on Different Nanofibrous Membranes and the Kinetic Parameters of Catalase under the Immobilized and Free Conditions
free CAT PVA/ PA6− Cu(II)− CAT PVA− Cu(II)− CAT
immobilized amount of catalase (mg/g)
specific activity (unit/mg)
64 ± 4.6
3400 ± 116 2150 ± 85
4878 3774
26.8 41.1
35 ± 3.1
1720 ± 98
3106
43.5
shows the relative activities of immobilized and free catalases after incubation at various temperatures (30−60 °C) in PBS (50 mM, pH = 7.0) for 5 h, and the results can reflect the thermal stability of free and immobilized catalase. Note that the capacity of free or immobilized catalase after incubation at 30 °C in the same PBS for 5 h was set as 100%. The activity of free catalase decreased significantly with the increase of incubation temperature, whereas the activity decrease of immobilized catalase was considerably lower. The samples of PVA/PA6− Cu(II)−CAT and PVA−Cu(II)−CAT retained approximately half of the activity after the incubation at 60°C for 5 h, while free catalase lost ∼90% of the activity under the same conditions. The residual activities of the PVA/PA6−Cu(II)− CAT and PVA−Cu(II)−CAT samples were higher than those of free catalase, showing their higher thermal stability. For the free catalase, the increase of temperature might vary the tertiary structure, resulting in the reduction of activity. However, the immobilization of enzyme could strengthen the interactions between catalase molecules and supports (i.e., PVA/PA6− Cu(II) and PVA−Cu(II) nanofibrous membranes), and thus limit the freedom of conformational changes for catalase.
Km Vmax (μmol/mg·min) (mM)
catalase for the sample of PVA/PA6−Cu(II)−CAT reached a high value of 64 ± 4.6 mg/g, which was almost twice as much as that of the PVA−Cu(II)−CAT sample; such a value was also higher than the reported values in the literature.8,11,13,14,37,38 This was because the PVA/PA6−Cu(II) sample retained the morphological structure of nanofibrous membrane (Figure 3B), which would provide large surface area for effective catalase immobilization, result in a uniform dispersion of catalase molecules on the membrane, and allow/ensure optimal 10394
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Figure 5), indicating the excellent stability and permeability of this IEMR. 3.5. IEMR’s Conversion Ratios of H2O2. As the operational pressure increased from 0.02 to 0.2 MPa, the flux values of IEMR increased from 0.20 ± 0.02 to 0.76 ± 0.04 L/ m2·min (black curve in Figure 6), and the conversion ratios of
Hence, the thermal stability of catalase would be effectively increased.46,47 The relative activities of PVA/PA6−Cu(II)−CAT, PVA− Cu(II)−CAT, and free catalase after being stored at 4 °C in PBS for 20 days were 54%, 56%, and 19%, respectively (note that initial activities of the free and immobilized catalases were set as 100%). These results indicated that the storage stability of immobilized catalase was distinguishably higher than that of free catalase; and this was because the immobilization of enzyme could preserve its tertiary structure, and thus could prevent/mitigate the inactivation/denaturation upon long-term storage.48 3.4. Flux Values of the Membrane Reactors. Flux values of the IEMR based on electrospun PVA/PA6−Cu(II) nanofibrous membrane and the other reactor with free enzyme (under the operational pressure of 0.2 MPa) are shown in Figure 5. The following modified Darcy’s Law was adopted to
Figure 6. IEMR’s conversion ratios of H2O2 under varied operational pressures. Error bars show one standard deviation (n = 3).
H2O2 slightly decreased from 92 ± 2.5% to 87 ± 2.1% (red curve in Figure 6). Hence, the IEMR maintained its high conversion ratio of H2O2 when the operational pressure and the flux value of IEMR were increased by several times; this clearly demonstrated the superior stability and efficiency of this IEMR. Note that during practical applications, the high productivity with stable conversion ratio of H2O2 could be achieved via increase of operational pressure. 3.6. Reusability of the IEMR. Reusability is another important parameter of an IEMR, and it can be expressed by production capacity of IEMR. The production capacity of the assembled IEMR was tested once every day for 5 days (with the reaction time of 4 h) to examine the reusability. The production capacity of IEMR was decreased from the initial 0.144 ± 0.006 to 0.102 ± 0.004 mol/m2·min after 5 repeating cycles. For ease of presentation, the initial production capacity was set as 100%, and the relative production capacities of each repeating cycle are shown in Figure 7. The IEMR still maintained ∼70% production capacity in the fifth repeating cycle, indicating high stability of immobilized catalase and excellent reusability of IEMR.
Figure 5. Flux values of the IEMR based on electrospun PVA/PA6− Cu(II) nanofibrous membrane and the other reactor with free enzyme (operational pressure: 0.2 MPa). Error bars show one standard deviation (n = 3).
understand the relationship between resistance and flux of the membranes:49−51 R m + Rc =
Δp Jμ
(7)
where Rm is the resistance of membrane, Rc is the resistance of sediments on the membrane, J is the flux of membrane, and μ is the solution viscosity. As shown in eq 7, the Rm + Rc and J are in the inverse relationship. For the free enzyme membrane reactor, the PVA/ PA6−Cu(II) nanofibrous membrane with diameter of 8.0 cm was overlaid on PS ultrafiltration membrane to form the reaction layer, and the flux values were reduced from 0.84 ± 0.04 to 0.44 ± 0.02 L/m2·min as the operational time was prolonged to 240 min (black curve in Figure 5). Because the catalases were dispersed into H2O2 aqueous solution in the free enzyme membrane reactor, the catalase molecules would gradually penetrate the PVA/PA6−Cu(II) nanofibrous membrane, adhere to the PS ultrafiltration membrane, and lead to the fouling of membrane. For the IEMR, since the catalase molecules were immobilized on the PVA/PA6−Cu(II) membrane, thus the movement of catalase molecules were restricted, and the flux values of IEMR were merely reduced from 0.76 ± 0.02 to 0.72 ± 0.02 L/m2·min (i.e., 94.7% capacity of initial flux was maintained, as shown by the red curve in
Figure 7. Reusability of the IEMR (upon being tested once every day for 5 days). Error bars show one standard deviation (n = 3). 10395
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(4) Yuan, A.; Wang, J.; Chen, T. Room-temperature bleaching of cotton fabrics with chitosan with chitosan-metal complexes/H2O2. J. Text. Res. 2010, 31, 66−69. (5) Hong, J.; Xu, D.; Gong, P.; Yu, J.; Ma, H.; Yao, S. Covalentbonded immobilization of enzyme on hydrophilic polymer covering magnetic nanogels. Microporous Mesoporous Mater. 2008, 109, 470− 477. (6) Ricca, E.; Calabro, V.; Curcio, S.; Basso, A.; Gardossi, L.; Iorio, G. Fructose production by inulinase covalently immobilized on sepabeads in batch and fluidized bed bioreactor. Int. J. Mol. Sci. 2010, 11, 1180− 1189. (7) Mazzei, R.; Drioli, E.; Giorno, L. Enzyme membrane reactor with heterogenized-glucosidase to obtain phytotherapic compound: Optimization study. J. Membr. Sci. 2012, 391, 121−129. (8) Bayramoglu, G.; Arica, M. Y. Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane. J. Mol. Catal. B: Enzym. 2010, 62, 297−304. (9) Akgol, S.; Denizli, A. Novel metal-chelate affinity sorbents for reversible use in catalase adsorption. J. Mol. Catal. B: Enzym. 2004, 28, 7−14. (10) Chen, C. I.; Ko, Y. M.; Liu, Y. C. Direct penicillin G acylase immobilization by using the self-prepared immobilized metal affinity membrane. J. Membr. Sci. 2011, 380, 34−40. (11) Ran, J.; Jia, S.; Liu, Y.; Zhang, W.; Wu, S.; Pan, X. A facile method for improving the covalent crosslinking adsorption process of catalase immobilization. Bioresour. Technol. 2010, 101, 6285−6290. (12) Zang, J.; Jia, S.; Liu, Y.; Wu, X.; Zhang, Y. A facile method to prepare chemically crosslinked and efficient polyvinyl alcohol/chitosan beads for catalase immobilization. Catal. Commun. 2012, 27, 73−77. (13) Song, N.; Chen, S.; Huang, X.; Liao, X.; Shi, B. Immobilization of catalase by using Zr(IV)-modified collagen fiber as the supporting matrix. Process Biochem. 2011, 46, 2187−2193. (14) Alptekin, O.; Tukel, S. S.; Yıldırım, D.; Alagoz, D. Characterization and properties of catalase immobilized onto controlled pore glass and its application in batch and plug-flow type reactors. J. Mol. Catal. B: Enzym. 2009, 58, 124−131. (15) Wu, H.; Liang, Y.; Shi, J.; Wang, X.; Yang, D.; Jiang, Z. Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres. Mater. Sci. Eng., C 2013, 33, 1438−1445. (16) Bui, N. N.; McCutcheon, J. R. Hydrophilic nanofibers as new supports for thin film composite membranes for engineered osmosis. Environ. Sci. Technol. 2013, 47, 1761−1769. (17) Ku, S. H.; Lee, S. H.; Park, C. B. Synergic effects of nanofiber alignment and electroactivity on myoblast differentiation. Biomaterials 2012, 33, 6098−6104. (18) Wang, M.; Meng, G.; Huang, Q.; Qian, Y. Electrospun 1,4DHAQ-doped cellulose nanofiber films for reusable fluorescence detection of trace Cu2+ and further for Cr3+. Environ. Sci. Technol. 2012, 46, 367−373. (19) Kim, J. H.; Choi, D. C.; Yeon, K. M.; Kim, S. R.; Lee, C. H. Enzyme-immobilized nanofiltration membrane to mitigate biofouling based on quorum quenching. Environ. Sci. Technol. 2011, 45, 1601− 1607. (20) Fong, H.; Dickens, S. H.; Flaim, G. M. Evaluation of dental restorative composites containing polyhedral oligomeric silsesquioxane methacrylate. Dent. Mater. 2005, 21, 520−529. (21) Ding, B.; Wang, M.; Wang, X.; Yu, J.; Sun, G. Electrospun nanomaterials for ultrasensitive sensors. Mater. Today 2010, 13, 16− 27. (22) Feng, Q.; Wang, Q.; Tang, B.; Wei, A.; Wang, X.; Wei, Q.; Huang, F.; Cai, Y.; Hou, D.; Bi, M. Immobilization of catalases on the amidoxime polyacrylonitrile nanofibrous membranes. Polym. Int. 2013, 62, 251−256. (23) Wang, X.; Ding, B.; Sun, G.; Wang, M.; Yu, J. Electro-spinning/ netting: A strategy for the fabrication of three-dimensional polymer nano-fiber/nets. Prog. Mater. Sci. 2013, 58, 1173−1243. (24) Wang, R.; Liu, Y.; Lia, B.; Hsiao, B. S.; Chu, B. Electrospun nanofibrous membranes for high flux microfiltration. J. Membr. Sci. 2012, 392−393, 167−174.
Additionally, to examine the structural stability of PVA/ PA6−Cu(II) nanofibrous membrane in IEMR, the specific surface area, pore volume, and average pore size of the membrane were characterized. As shown in Table 2, all of the parameters were only decreased slightly after the reuse of IEMR for 5 times, indicating that the PVA/PA6−Cu(II) nanofibrous membrane in IEMR was structurally stable. Table 2. BET Specific Surface Area, Pore Volume, and Average Pore Size of the PVA/PA6−Cu(II)−CAT Nanofibrous Membrane in IEMR before Test and after Tests for Five Times sample before test after tests for 5 times
BET specific surface area (m2/g)
pore volume (mL/g)
average pore size (nm)
14.15 ± 2.01 12.11 ± 1.86
0.032 ± 0.004 0.031 ± 0.004
9.7 ± 2.2 8.8 ± 1.8
In summary, the thermal stability and storage stability of immobilized catalase on PVA/PA6−Cu(II)−CAT nanofibrous membrane were significantly higher than those of free catalase, and the specific activity and kinetic parameters (Vmax and Km) of immobilized catalase also showed the desired affinity between catalase molecules and PVA/PA6−Cu(II) membrane. More importantly, the IEMR based on PVA/PA6−Cu(II)− CAT nanofibrous membrane exhibited high productivity and excellent reusability, indicating that the prepared PVA/PA6− Cu(II) nanofibrous membrane and the developed IEMR would be promising for various applications, particularly those related to environmental protection (such as wastewater treatment in making foods, textiles, and detergents).
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +86-510-8591-2007; fax: +86-510-8591-2002; e-mail: [email protected]. *Phone: +1-605-394-1229; fax: +1-605-394-1232; e-mail: Hao. [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (Grant 21377004), the Natural Science Foundation of Anhui Province (Grant 1408085ME87), and the Research Foundation of Key Laboratory for Eco-Textiles of Ministry of Education (Grant KLET1204). We also acknowledge the Biomedical Engineering Program at the South Dakota School of Mines and Technology.
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REFERENCES
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Immobilization of Catalase on Electrospun PVA/PA6−Cu(II) Nanofibrous Membrane for the Development of Efficient and Reusable Enzyme Membrane Reactor Quan Feng,†,‡,§ Yong Zhao,‡ Anfang Wei,† Changlong Li,† Qufu Wei,*,§ and Hao Fong*,‡ †
Key Laboratory of Textile Fabric, College of Textiles and Clothing, Anhui Polytechnic University, Wuhu, Anhui 241000, China Department of Chemistry and Applied Biological Sciences, South Dakota School of Mines and Technology, Rapid City, South Dakota 57701, United States § Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China ‡
ABSTRACT: In this study, a mat/membrane consisting of overlaid PVA/PA6−Cu(II) composite nanofibers was prepared via the electrospinning technique followed by coordination/chelation with Cu(II) ions; an enzyme of catalase (CAT) was then immobilized onto the PVA/PA6−Cu(II) nanofibrous membrane. The amount of immobilized catalase reached a high value of 64 ± 4.6 mg/g, while the kinetic parameters (Vmax and Km) of enzyme were 3774 μmol/mg·min and 41.13 mM, respectively. Furthermore, the thermal stability and storage stability of immobilized catalase were improved significantly. Thereafter, a plugflow type of immobilized enzyme membrane reactor (IEMR) was assembled from the PVA/PA6−Cu(II)−CAT membrane. With the increase of operational pressure from 0.02 to 0.2 MPa, the flux value of IEMR increased from 0.20 ± 0.02 to 0.76 ± 0.04 L/m2·min, whereas the conversion ratio of H2O2 decreased slightly from 92 ± 2.5% to 87 ± 2.1%. After 5 repeating cycles, the production capacity of IEMR was merely decreased from 0.144 ± 0.006 to 0.102 ± 0.004 mol/m2·min. These results indicated that the assembled IEMR possessed high productivity and excellent reusability, suggesting that the IEMR based on electrospun PVA/PA6−Cu(II) nanofibrous membrane might have great potential for various applications, particularly those related to environmental protection. physical−chemical interactions.6,7 Recently, some transition metal ions (e.g., Cu2+, Ni2+, Zn2+, and Fe3+) have been investigated for catalase immobilization, because the coordination bonds (formed between these ions and catalase molecules) can provide the adequate binding force for immobilization and effectively reduce the inactivation/denaturation of catalase.8−10 To date, many supports (including the beads of chitosan and its composite with poly(vinyl alcohol) (PVA),11,12 conventional fibers of Zr(IV)-modified collagen,13 glass beads with high porosity,14 and submicron-sized spheres of titania15) have been studied for catalase immobilization. However, these supports have different limitations. For example, the enzyme-loading amounts of conventional fibers are low due to small surface-to-
1. INTRODUCTION Enzymes are protein-based biocatalysts with high efficiency and superior selectivity, and the conditions for enzyme-catalyzed reactions are generally mild with low energy consumption. The catalase of H2O2 oxidoreductase, a heme-containing metalloenzyme, is a common enzyme for decomposition of H2O2 into oxygen and water,1,2 and the catalytic effect of catalase is much higher than that of light, organics, and metal ions.3,4 Although the catalase has been widely used in many fields such as food, textile, and detergent, the applications encounter several difficulties/challenges. For example, the recovery of catalase from reaction systems is usually inconvenient because the enzyme is highly water-soluble, and the catalase has to be utilized within specific a temperature range and chemical environment for achieving optimal catalysis performance.5 The immobilization of enzyme is an important/attractive approach to overcome/mitigate those difficulties/challenges, in which the catalase is adsorbed/bonded onto water-insoluble supports via © 2014 American Chemical Society
Received: Revised: Accepted: Published: 10390
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∼100 000 g/mol) ultrafiltration membranes were supplied by the Musu Membrane Technology (Shanghai, China). Bovine liver catalase (enzyme commission 1.11.1.6) was purchased from the Sigma-Aldrich Corp. (Shanghai, China). Formic acid (HCOOH, < 2.5% water as stabilizer), CuCl2, H2O2 (30 wt % in H2O), NaCl, KCl, KH2PO4, K2HPO4, and Coomassie Brilliant Blue (G250) were purchased from the Sinopharm Chemical Reagent Co. (Shanghai, China). The materials and chemicals were used without further purification. 2.2. Preparation of Electrospun Nanofibrous Membranes. Prior to electrospinning, PVA (4 g) and PA6 (12 g) were dissolved in formic acid (100 mL) at room temperature. The resulting spin dope was then loaded into a 30-mL BD Luer-Lok tip plastic syringe having an 18 gauge 90° blunt-end stainless steel needle. The electrospinning was carried out at 14 kV through a DW-P503-4AC high-voltage supplier, and the feed rate was set at 0.3 mL/h by using a WZ-50C2 microinfusion pump. Electrospun PVA/PA6 nanofibers were collected on electrically grounded aluminum foil that covered a laboratory-produced roller, and the distance between aluminum foil and the tip of needle was 15 cm. Subsequently, the aselectrospun PVA/PA6 nanofibrous membrane was coordinated/chelated with Cu(II) ions by immersion of 0.2 g of membrane into 100 mL of 0.1 M CuCl2 solution for 24 h at 20 °C under shaking condition. The resulting PVA/PA6−Cu(II) nanofibrous membrane was then rinsed with deionized water and dried at 35 °C in a vacuum oven (∼ 27 mmHg). For comparison, electrospun neat PVA nanofibrous membrane was also prepared from the spin dope containing 8 wt % PVA alone. The PVA nanofibrous membrane coordinated/chelated with Cu(II) ions (i.e., PVA−Cu(II) nanofibrous membrane) was then prepared with the same procedure. The prepared PVA/PA6−Cu(II) and PVA−Cu(II) nanofibrous membranes had similar thickness of ∼70 μm and mass per unit area of ∼20 g/m2. Atomic absorption spectroscopy (AAS) was employed to determine the adsorption amounts of Cu(II) ions in the nanofibrous membranes. A Hitachi S-4800 field-emission scanning electron microscope (SEM) was used to examine the morphological structures of PVA/PA6, PVA/ PA6−Cu(II), PVA, and PVA−Cu(II) nanofibrous membranes. 2.3. Immobilization of Catalase on PVA/PA6−Cu(II) and PVA−Cu(II) Nanofibrous Membranes. The bovine liver catalase (0.3 g) was first dissolved in 1.0 L of 50 mM phosphate buffer solution (PBS) with pH value of 7.0. Thereafter, 0.2 g of PVA/PA6−Cu(II) nanofibrous membrane (3.03 mmol/g of coordinated Cu2+ ions, determined by AAS) was placed in 200 mL of catalase solution (0.3 mg/mL) for 4 h at 20 °C under shaking condition. The PVA/PA6−Cu(II) nanofibrous membrane was then rinsed with the same PBS until no enzyme could be detected in the rinsing PBS. For comparison, PVA−Cu(II) nanofibrous membrane (2.15 mmol/ g of coordinated Cu2+ ions, determined by AAS) was also treated by the same procedure for catalase immobilization. To examine the distributions of immobilized catalase on the surfaces of nanofibrous membranes, fluorescein isothiocyanate (FITC) was used to label the catalase.32 Specifically, 0.1 mL of FITC (2 mM) was added into 20 mL of catalase solution (0.3 mg/mL) under magnetic stirring condition for 4 h in a brown vial, and the mixture was then placed at 4 °C for 24 h. This was followed by centrifugation at 3000 rpm for 30 min to remove any precipitates. Subsequently, the resulting catalase (labeled with FITC) was immobilized on the surface of PVA/PA6− Cu(II) or PVA−Cu(II) nanofibrous membranes with the same
mass ratios. Another example, although the enzyme-loading amounts of microporous beads/spheres are quite high, the spatial/steric hindrances are usually serious. Electrospun nanofibrous membranes have recently been investigated for filtration, drug delivery, tissue engineering, dental composite, and enzyme immobilization.16−22 Note that these membranes possess a large number of interaction sites for enzyme immobilization due to high surface-to-mass ratios. Meanwhile, the membranes are highly porous and mechanically flexible, providing more diffusion channels and thus allowing for faster flux rates than conventional membranes.23,24 PVA is a nontoxic, hydrophilic, and biocompatible polymer with each repeating unit having a hydroxyl group, and it has been widely utilized for cell and enzyme immobilization.25 However, electrospun PVA nanofibers in the membranes swell substantially during the coordination/chelation with transition metal ions in an aqueous environment, resulting in partial or complete loss of the morphological structure of the nanofibrous membranes. Recently, the method of chemical cross-linking with glutaraldehyde has been studied to improve the stability of electrospun PVA nanofibrous membranes.26 On the other hand, electrospun composite nanofibrous membranes of PVA and polyamide 6 (PA6) also exhibit excellent structural stability in aqueous solutions;27,28 and the following is the explanation: If a spin dope contains two polymers (e.g., PVA and PA6, both having high concentrations), microphase separations will occur in the spin dope; after the phase-separated domains are stretched during bending instability,29 followed by rapid solidification of the jet/filament, the composite nanofiber with both polymer components being semigyroid or cocontinuous can be acquired.30 This would further result in the macromolecular interactions between PVA and PA6 being strong; consequently, the PVA component in the composite nanofibers would be unlikely to leach away when immersed in an aqueous environment. The goal of this study is to develop a plug-flow type of immobilized enzyme membrane reactor (IEMR) with elevated productivity from an electrospun nanofibrous membrane with high loading amount of catalase. Note that IEMRs have been broadly adopted for various applications, particularly those related to environmental protection. Practical applications of IEMRs are determined upon several criteria such as large amount of immobilized enzyme, high productivity, and excellent reusability.31 During this study, an electrospun PVA/PA6 nanofibrous membrane coordinated/chelated with Cu(II) ions was first prepared and then used for the immobilization of catalase (CAT). Experimental results indicated that the PVA/PA6−Cu(II)−CAT nanofibrous membrane possessed superior morphological stability, while the immobilized CAT exhibited high thermal stability and excellent storage stability. Thereafter, a plug-flow type of IEMR was assembled from the prepared nanofibrous membrane. The flux value and H2O2 conversion ratio of this IEMR were calculated, and the IEMR’s operational stability and reusability were evaluated. This study demonstrated that the assembled IEMR would be promising for various applications related to environmental protection (such as wastewater treatment in making foods, textiles, and detergents).
2. EXPERIMENTAL SECTION 2.1. Materials. PVA (Mw ∼86 000 g/mol) and PA6 (Mw ∼18 000 g/mol) were obtained from the Shanghai Kanghu Chemical Co. in China. Polyoxyphenylene sulfone (PS, Mw 10391
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K 1 1 1 = m × + Vi Vmax [S] Vmax
procedure. Finally, the distribution of the FITC-labeled immobilized catalase was examined by an inverted fluorescence microscope (IFM, Nikon Eclipse Ti). During the immobilization of catalase as schematically shown in Figure 1, Cu(II) ions were first coordinated/chelated with
where Vi and Vmax are the initial and maximal rates of the enzyme-catalyzed reaction, Km is the Michaelis constant, and [S] is the concentration of H2O2. Note that kinetic parameters were determined at 35 °C, and the concentration of H2O2 was 30−200 mM. 2.5. Analysis of Thermal Stability and Storage Stability of the Immobilized Catalase. Thermal stability was investigated by incubating the free and immobilized catalase at different temperatures (i.e., 30, 35, 40, 45, 50, 55, and 60 °C) in PBS (50 mM, pH 7.0) for 5 h; thereafter, the free and immobilized catalase was studied for enzymatic decomposition of H2O2 in the same conditions as described in Section 2.4. Storage stability of the free and immobilized catalase was evaluated upon calculating the residual activity of PVA/PA6− Cu(II)−CAT, PVA−Cu(II)−CAT, and free catalase after being stored at 4 °C in PBS for 20 days, and the highest value of each set was assigned as 100%. 2.6. Equipment Design of IEMR. An IEMR was assembled from the PVA/PA6−Cu(II)−CAT nanofibrous membrane. As shown in Figure 2, the reactor with capacity
Figure 1. Schematic showing the immobilization of a catalase molecule on the PVA/PA6−Cu(II) nanofibrous membrane.
hydroxyl and/or carbonyl groups on the PVA/PA6 nanofibers; thereafter, these Cu(II) ions would further form coordination bonds with imidazole and/or amino groups on catalase molecules, leading to the catalase immobilization on the PVA/PA6−Cu(II) nanofibrous membrane.8 The Bradford assay was adopted to determine the immobilization amount upon the following equation:33 Qe =
(C0 − Ce)V0 − CrVr Md
(1)
where Qe is the amount of catalase immobilized on unit mass of membrane (mg/g), C0 and Ce are initial and equilibrium enzyme concentrations in the solution (mg/mL), Cr is the enzyme concentration in the rinsing PBS (mg/mL), Vo is the volume (mL) of catalase solution, Vr is the volume (mL) of rinsing PBS, and Md is the mass (g) of membrane. 2.4. Activity Assay of Catalase under the Free and Immobilized Conditions. To test the respective activities of catalase under the free and immobilized conditions, 0.3 mL of catalase solution (0.3 mg/mL) or 0.01 g of PVA/PA6−Cu(II)− CAT nanofibrous membrane was mixed with 50 mL of 100 mM H2O2 solution. The system was then kept at 35 °C for 3 min, and 3 replicates were tested for each sample. The activities of catalase under the free or immobilized conditions were determined spectrophotometrically by measuring the decrease of absorbance at 240 nm, as a consequence of H 2 O 2 consumption. The specific activity of enzyme was then calculated by using the following equation:34 v=
(A 0 − A ) × V T × K × Ew
(3)
Figure 2. Diagram showing the assembled IEMR.
of 500 mL was placed on a hot plate, while a PVA/PA6− Cu(II)−CAT nanofibrous membrane with diameter of 8.0 cm was overlaid on a PS ultrafiltration membrane with the same size to form the reaction layer. The H2O2 solution was supplied via a peristaltic pump, and the operational pressure was adjusted with a N2 regulator to control the flow rate. The filtrate was then collected for subsequent analyses. The O2 gas generated during the reaction was released from an overpressure valve. For comparison, another reactor was also assembled from the PVA/PA6−Cu(II) nanofibrous membrane without immobilization of catalase. Note that in this reactor the catalase (with the concentration of 0.05 mg/mL) was suspended in H2O2 solution. 2.7. Determination of Flux Values of the Assembled IEMRs. To determine the flux value of an IEMR, 300 mL of 0.2 M H2O2 aqueous solution was first added into the IEMR, and the additional H2O2 solution was then supplied intermittently by the peristaltic pump. The following conditions were adopted during the test: the operational pressure was 0.2 MPa, the stirring speed was 100 rad/min, the temperature was 35 °C, and the collection time of filtrate was 4 h. For comparison, the flux value of the reactor with free enzyme was also determined under the same conditions. The flux values of IEMRs were calculated by using the following equation:
(2)
where v is the specific activity of free or immobilized catalase (μmol/mg·min), A0 and A are the initial and final absorbances of the solution at 240 nm, V is the volume of H2O2 solution (mL), T is the reaction time (min), K is the molar extinction coefficient of H2O2 at 240 nm (K = 0.033 L/mmol·cm, and the optical path-length is 1 cm),35 and Ew is the enzyme amount (mg). The kinetic parameters (i.e., Vmax and Km) of enzyme under the free and immobilized conditions were calculated from Lineweaver−Burk plot of the Michaelis−Menten equation.36 10392
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Figure 3. SEM images of PVA/PA6 nanofibrous membrane (A), PVA/PA6−Cu(II) nanofibrous membrane (B), PVA nanofibrous membrane (C), and PVA−Cu(II) nanofibrous membrane (D). IFM images of FITC-labeled immobilized catalase on the PVA/PA6−Cu(II) nanofibrous membrane (E) and the PVA−Cu(II) nanofibrous membrane (F).
J=
V S×t
operational pressure was 0.2 MPa, and the reaction temperature was 35 °C. Subsequently, the nanofibrous membrane and PS ultrafiltration membrane were preserved in PBS (50 mM, pH = 7) and 0.5% formaldehyde solution at 4 °C, respectively. The productivity of IEMR was calculated by using the following equation:
(4)
where J is the flux of membrane (L/m2·min), V is the volume of filtrate (L), S is the size of membrane (m2), and t is the time of operation (min). 2.8. Analysis of the Conversion Ratios of H2O2. Conversion ratios of H2O2 under varied operational pressures with different flux values of IEMR were determined. The operational pressure was from 0.02 to 0.2 MPa (note that the pressure was adjusted once every 10 min, and the increment was 0.02 MPa), the concentration of H2O2 aqueous solution was set at 0.2 M, and the temperature was maintained at 35 °C. The H2O2 conversion ratios were calculated by using the following equation: K=
C0 − Cp C0
× 100%
P=
V × (C0 − Cp) t×S
(6)
where P is the productivity of IEMR (mol/m2·min), V is the volume of filtrate (L), C0 and Cp are concentrations of H2O2 in the initial solution and in the filtrate (M), respectively, t is the reaction time (min), and S is the size of membrane (m2). For examining the reusability of an IEMR, the productivity of this IEMR was tested once every day for 5 days, and the reaction time was set at 4 h. After examination of reusability, the PVA/PA6−Cu(II)−CAT nanofibrous membrane (before test and after the tests for 5 times) was collected. Thereafter, the Brunauer−Emmett−Teller (BET) specific surface area, pore volume, and average pore size of the collected membrane were determined by N2 adsorption at −196 °C.
(5)
where K is the conversion ratio of H2O2 (%), and C0 and Cp are the concentrations of H2O2 in the initial solution and in the filtrate, respectively (mol/L). 2.9. Evaluation of the Productivity and Reusability of IEMR. The productivity of an IEMR under varied operational pressures was evaluated under the following conditions: the 10393
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3. RESULTS AND DISCUSSION 3.1. Morphologies of Nanofibrous Membranes and Distributions of Immobilized Catalase. Figure 3A, B, C, and D are SEM images showing the morphologies of PVA/ PA6, PVA/PA6−Cu(II), PVA, and PVA−Cu(II) nanofibrous membranes, respectively. As shown in Figure 3A, electrospun PVA/PA6 composite nanofibers were overlaid in the membrane with diameters from 100 to 500 nm. The nanofibrous structure of PVA/PA6 membrane was wellretained after immersion in aqueous solution containing Cu(II) ions for 24 h (Figure 3B). In contrast, the morphological structure of neat PVA nanofibrous membrane showed substantial variation/discrepancy after immersion in the solution for 24 h (Figure 3C and D). Unlike the sample of PVA/PA6−Cu(II) which was a typical nanofibrous membrane, the sample of PVA−Cu(II) partially lost the morphological structure of nanofibrous membrane; i.e., the nanofibers in the membrane appeared to fuse/conglutinate together into a meshlike structure. Such results indicated that the morphological/ structural stability of PVA/PA6 nanofibrous membranes was significantly improved upon the incorporation of PA6 into the nanofibers. The IFM images of the immobilized catalase (labeled with FITC) on the surface of PVA/PA6−Cu(II) and PVA−Cu(II) nanofibrous membranes are depicted in Figure 3E and F, respectively. The immobilized catalase molecules were attached on the surface of PVA/PA6−Cu(II) nanofibers uniformly (Figure 3E), and the density/amount of immobilized catalase on PVA/PA6−Cu(II) nanofibers was distinguishably higher than that on PVA−Cu(II) nanofibers (Figure 3F). Such IFM results were consistent with the results acquired from SEM, further confirming that the PVA/PA6−Cu(II) nanofibrous membrane could well-retain the nanofibrous morphological structure after the coordination/chelation of Cu(II) ions in aqueous environment, and the resulting PVA/PA6−Cu(II) membrane would have more interaction sites for immobilization of catalase molecules than the PVA−Cu(II) membrane. 3.2. Kinetic Parameters of Immobilized and Free Catalase. As shown in Table 1, the amount of immobilized
contacts between membrane and active sites of catalase. Nevertheless, the specific activities of catalase immobilized on PVA/PA6−Cu(II) and PVA−Cu(II) membranes were lower than that of free catalase. This could be easily understood, since the spatial/steric barriers would arise upon the immobilization of catalase molecules on the PVA/PA6−Cu(II) and PVA− Cu(II) nanofibrous membranes. Such a condition could hinder the diffusion (i.e., decrease the accessibility) of H2O2 molecules to the active sites of immobilized enzyme. Similar observations have also been reported by other researchers.39,40 For the immobilized catalase Vmax was smaller, while the Km value was higher than those of free catalase. Note that the Vmax is defined as the highest possible rate when an enzyme is saturated with substrate, which reflects the intrinsic characteristics of immobilized enzyme. On the other hand, the Km is defined as the H2O2 concentration at the reaction rate of 1/2 V max , which represents the affinity of an enzyme to substrate.41−43 According to the kinetic parameters of Vmax and Km, the PVA/PA6−Cu(II) membrane exhibited desired biological compatibility to immobilized catalase.44,45 3.3. Thermal Stability and Storage Stability of Immobilized Catalase. Thermal stability is an important parameter in consideration of a practical application. Figure 4
Figure 4. Effect of temperature on catalytic activity of free and immobilized catalase (the lines showing nonlinear regression curve fittings). Error bars show one standard deviation (n = 3).
Table 1. Amounts of Immobilized Catalase on Different Nanofibrous Membranes and the Kinetic Parameters of Catalase under the Immobilized and Free Conditions
free CAT PVA/ PA6− Cu(II)− CAT PVA− Cu(II)− CAT
immobilized amount of catalase (mg/g)
specific activity (unit/mg)
64 ± 4.6
3400 ± 116 2150 ± 85
4878 3774
26.8 41.1
35 ± 3.1
1720 ± 98
3106
43.5
shows the relative activities of immobilized and free catalases after incubation at various temperatures (30−60 °C) in PBS (50 mM, pH = 7.0) for 5 h, and the results can reflect the thermal stability of free and immobilized catalase. Note that the capacity of free or immobilized catalase after incubation at 30 °C in the same PBS for 5 h was set as 100%. The activity of free catalase decreased significantly with the increase of incubation temperature, whereas the activity decrease of immobilized catalase was considerably lower. The samples of PVA/PA6− Cu(II)−CAT and PVA−Cu(II)−CAT retained approximately half of the activity after the incubation at 60°C for 5 h, while free catalase lost ∼90% of the activity under the same conditions. The residual activities of the PVA/PA6−Cu(II)− CAT and PVA−Cu(II)−CAT samples were higher than those of free catalase, showing their higher thermal stability. For the free catalase, the increase of temperature might vary the tertiary structure, resulting in the reduction of activity. However, the immobilization of enzyme could strengthen the interactions between catalase molecules and supports (i.e., PVA/PA6− Cu(II) and PVA−Cu(II) nanofibrous membranes), and thus limit the freedom of conformational changes for catalase.
Km Vmax (μmol/mg·min) (mM)
catalase for the sample of PVA/PA6−Cu(II)−CAT reached a high value of 64 ± 4.6 mg/g, which was almost twice as much as that of the PVA−Cu(II)−CAT sample; such a value was also higher than the reported values in the literature.8,11,13,14,37,38 This was because the PVA/PA6−Cu(II) sample retained the morphological structure of nanofibrous membrane (Figure 3B), which would provide large surface area for effective catalase immobilization, result in a uniform dispersion of catalase molecules on the membrane, and allow/ensure optimal 10394
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Figure 5), indicating the excellent stability and permeability of this IEMR. 3.5. IEMR’s Conversion Ratios of H2O2. As the operational pressure increased from 0.02 to 0.2 MPa, the flux values of IEMR increased from 0.20 ± 0.02 to 0.76 ± 0.04 L/ m2·min (black curve in Figure 6), and the conversion ratios of
Hence, the thermal stability of catalase would be effectively increased.46,47 The relative activities of PVA/PA6−Cu(II)−CAT, PVA− Cu(II)−CAT, and free catalase after being stored at 4 °C in PBS for 20 days were 54%, 56%, and 19%, respectively (note that initial activities of the free and immobilized catalases were set as 100%). These results indicated that the storage stability of immobilized catalase was distinguishably higher than that of free catalase; and this was because the immobilization of enzyme could preserve its tertiary structure, and thus could prevent/mitigate the inactivation/denaturation upon long-term storage.48 3.4. Flux Values of the Membrane Reactors. Flux values of the IEMR based on electrospun PVA/PA6−Cu(II) nanofibrous membrane and the other reactor with free enzyme (under the operational pressure of 0.2 MPa) are shown in Figure 5. The following modified Darcy’s Law was adopted to
Figure 6. IEMR’s conversion ratios of H2O2 under varied operational pressures. Error bars show one standard deviation (n = 3).
H2O2 slightly decreased from 92 ± 2.5% to 87 ± 2.1% (red curve in Figure 6). Hence, the IEMR maintained its high conversion ratio of H2O2 when the operational pressure and the flux value of IEMR were increased by several times; this clearly demonstrated the superior stability and efficiency of this IEMR. Note that during practical applications, the high productivity with stable conversion ratio of H2O2 could be achieved via increase of operational pressure. 3.6. Reusability of the IEMR. Reusability is another important parameter of an IEMR, and it can be expressed by production capacity of IEMR. The production capacity of the assembled IEMR was tested once every day for 5 days (with the reaction time of 4 h) to examine the reusability. The production capacity of IEMR was decreased from the initial 0.144 ± 0.006 to 0.102 ± 0.004 mol/m2·min after 5 repeating cycles. For ease of presentation, the initial production capacity was set as 100%, and the relative production capacities of each repeating cycle are shown in Figure 7. The IEMR still maintained ∼70% production capacity in the fifth repeating cycle, indicating high stability of immobilized catalase and excellent reusability of IEMR.
Figure 5. Flux values of the IEMR based on electrospun PVA/PA6− Cu(II) nanofibrous membrane and the other reactor with free enzyme (operational pressure: 0.2 MPa). Error bars show one standard deviation (n = 3).
understand the relationship between resistance and flux of the membranes:49−51 R m + Rc =
Δp Jμ
(7)
where Rm is the resistance of membrane, Rc is the resistance of sediments on the membrane, J is the flux of membrane, and μ is the solution viscosity. As shown in eq 7, the Rm + Rc and J are in the inverse relationship. For the free enzyme membrane reactor, the PVA/ PA6−Cu(II) nanofibrous membrane with diameter of 8.0 cm was overlaid on PS ultrafiltration membrane to form the reaction layer, and the flux values were reduced from 0.84 ± 0.04 to 0.44 ± 0.02 L/m2·min as the operational time was prolonged to 240 min (black curve in Figure 5). Because the catalases were dispersed into H2O2 aqueous solution in the free enzyme membrane reactor, the catalase molecules would gradually penetrate the PVA/PA6−Cu(II) nanofibrous membrane, adhere to the PS ultrafiltration membrane, and lead to the fouling of membrane. For the IEMR, since the catalase molecules were immobilized on the PVA/PA6−Cu(II) membrane, thus the movement of catalase molecules were restricted, and the flux values of IEMR were merely reduced from 0.76 ± 0.02 to 0.72 ± 0.02 L/m2·min (i.e., 94.7% capacity of initial flux was maintained, as shown by the red curve in
Figure 7. Reusability of the IEMR (upon being tested once every day for 5 days). Error bars show one standard deviation (n = 3). 10395
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(4) Yuan, A.; Wang, J.; Chen, T. Room-temperature bleaching of cotton fabrics with chitosan with chitosan-metal complexes/H2O2. J. Text. Res. 2010, 31, 66−69. (5) Hong, J.; Xu, D.; Gong, P.; Yu, J.; Ma, H.; Yao, S. Covalentbonded immobilization of enzyme on hydrophilic polymer covering magnetic nanogels. Microporous Mesoporous Mater. 2008, 109, 470− 477. (6) Ricca, E.; Calabro, V.; Curcio, S.; Basso, A.; Gardossi, L.; Iorio, G. Fructose production by inulinase covalently immobilized on sepabeads in batch and fluidized bed bioreactor. Int. J. Mol. Sci. 2010, 11, 1180− 1189. (7) Mazzei, R.; Drioli, E.; Giorno, L. Enzyme membrane reactor with heterogenized-glucosidase to obtain phytotherapic compound: Optimization study. J. Membr. Sci. 2012, 391, 121−129. (8) Bayramoglu, G.; Arica, M. Y. Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane. J. Mol. Catal. B: Enzym. 2010, 62, 297−304. (9) Akgol, S.; Denizli, A. Novel metal-chelate affinity sorbents for reversible use in catalase adsorption. J. Mol. Catal. B: Enzym. 2004, 28, 7−14. (10) Chen, C. I.; Ko, Y. M.; Liu, Y. C. Direct penicillin G acylase immobilization by using the self-prepared immobilized metal affinity membrane. J. Membr. Sci. 2011, 380, 34−40. (11) Ran, J.; Jia, S.; Liu, Y.; Zhang, W.; Wu, S.; Pan, X. A facile method for improving the covalent crosslinking adsorption process of catalase immobilization. Bioresour. Technol. 2010, 101, 6285−6290. (12) Zang, J.; Jia, S.; Liu, Y.; Wu, X.; Zhang, Y. A facile method to prepare chemically crosslinked and efficient polyvinyl alcohol/chitosan beads for catalase immobilization. Catal. Commun. 2012, 27, 73−77. (13) Song, N.; Chen, S.; Huang, X.; Liao, X.; Shi, B. Immobilization of catalase by using Zr(IV)-modified collagen fiber as the supporting matrix. Process Biochem. 2011, 46, 2187−2193. (14) Alptekin, O.; Tukel, S. S.; Yıldırım, D.; Alagoz, D. Characterization and properties of catalase immobilized onto controlled pore glass and its application in batch and plug-flow type reactors. J. Mol. Catal. B: Enzym. 2009, 58, 124−131. (15) Wu, H.; Liang, Y.; Shi, J.; Wang, X.; Yang, D.; Jiang, Z. Enhanced stability of catalase covalently immobilized on functionalized titania submicrospheres. Mater. Sci. Eng., C 2013, 33, 1438−1445. (16) Bui, N. N.; McCutcheon, J. R. Hydrophilic nanofibers as new supports for thin film composite membranes for engineered osmosis. Environ. Sci. Technol. 2013, 47, 1761−1769. (17) Ku, S. H.; Lee, S. H.; Park, C. B. Synergic effects of nanofiber alignment and electroactivity on myoblast differentiation. Biomaterials 2012, 33, 6098−6104. (18) Wang, M.; Meng, G.; Huang, Q.; Qian, Y. Electrospun 1,4DHAQ-doped cellulose nanofiber films for reusable fluorescence detection of trace Cu2+ and further for Cr3+. Environ. Sci. Technol. 2012, 46, 367−373. (19) Kim, J. H.; Choi, D. C.; Yeon, K. M.; Kim, S. R.; Lee, C. H. Enzyme-immobilized nanofiltration membrane to mitigate biofouling based on quorum quenching. Environ. Sci. Technol. 2011, 45, 1601− 1607. (20) Fong, H.; Dickens, S. H.; Flaim, G. M. Evaluation of dental restorative composites containing polyhedral oligomeric silsesquioxane methacrylate. Dent. Mater. 2005, 21, 520−529. (21) Ding, B.; Wang, M.; Wang, X.; Yu, J.; Sun, G. Electrospun nanomaterials for ultrasensitive sensors. Mater. Today 2010, 13, 16− 27. (22) Feng, Q.; Wang, Q.; Tang, B.; Wei, A.; Wang, X.; Wei, Q.; Huang, F.; Cai, Y.; Hou, D.; Bi, M. Immobilization of catalases on the amidoxime polyacrylonitrile nanofibrous membranes. Polym. Int. 2013, 62, 251−256. (23) Wang, X.; Ding, B.; Sun, G.; Wang, M.; Yu, J. Electro-spinning/ netting: A strategy for the fabrication of three-dimensional polymer nano-fiber/nets. Prog. Mater. Sci. 2013, 58, 1173−1243. (24) Wang, R.; Liu, Y.; Lia, B.; Hsiao, B. S.; Chu, B. Electrospun nanofibrous membranes for high flux microfiltration. J. Membr. Sci. 2012, 392−393, 167−174.
Additionally, to examine the structural stability of PVA/ PA6−Cu(II) nanofibrous membrane in IEMR, the specific surface area, pore volume, and average pore size of the membrane were characterized. As shown in Table 2, all of the parameters were only decreased slightly after the reuse of IEMR for 5 times, indicating that the PVA/PA6−Cu(II) nanofibrous membrane in IEMR was structurally stable. Table 2. BET Specific Surface Area, Pore Volume, and Average Pore Size of the PVA/PA6−Cu(II)−CAT Nanofibrous Membrane in IEMR before Test and after Tests for Five Times sample before test after tests for 5 times
BET specific surface area (m2/g)
pore volume (mL/g)
average pore size (nm)
14.15 ± 2.01 12.11 ± 1.86
0.032 ± 0.004 0.031 ± 0.004
9.7 ± 2.2 8.8 ± 1.8
In summary, the thermal stability and storage stability of immobilized catalase on PVA/PA6−Cu(II)−CAT nanofibrous membrane were significantly higher than those of free catalase, and the specific activity and kinetic parameters (Vmax and Km) of immobilized catalase also showed the desired affinity between catalase molecules and PVA/PA6−Cu(II) membrane. More importantly, the IEMR based on PVA/PA6−Cu(II)− CAT nanofibrous membrane exhibited high productivity and excellent reusability, indicating that the prepared PVA/PA6− Cu(II) nanofibrous membrane and the developed IEMR would be promising for various applications, particularly those related to environmental protection (such as wastewater treatment in making foods, textiles, and detergents).
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +86-510-8591-2007; fax: +86-510-8591-2002; e-mail: [email protected]. *Phone: +1-605-394-1229; fax: +1-605-394-1232; e-mail: Hao. [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (Grant 21377004), the Natural Science Foundation of Anhui Province (Grant 1408085ME87), and the Research Foundation of Key Laboratory for Eco-Textiles of Ministry of Education (Grant KLET1204). We also acknowledge the Biomedical Engineering Program at the South Dakota School of Mines and Technology.
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