J. Phys. Chem. C 2007, 111, 14091-14097
14091
Catalase Immobilization on Electrospun Nanofibers: Effects of Porphyrin Pendants and Carbon Nanotubes Ling-Shu Wan,† Bei-Bei Ke,† Jian Wu,*,‡ and Zhi-Kang Xu*,† Institute of Polymer Science, Key Laboratory of Macromolecular Synthesis and Functionalization (Ministry of Education), and State Key Laboratory of Chemical Engineering, Zhejiang UniVersity, and the Department of Chemistry, Zhejiang UniVersity, Hangzhou 310027, China ReceiVed: February 5, 2007; In Final Form: July 18, 2007
Carbon nanotube (CNT) has been generally recognized as an electron acceptor, whereas porphyrin can act as an electron donor. In this work, acrylonitrile-based copolymers bearing porphyrin pendants were therefore blended with or without CNTs and then electrospun into nanofibers on which a redox enzyme, catalase, was covalently immobilized. The uniform nanofibers diameters are around 180 nm. Field emission scanning electron microscopy and transmission electron microscopy confirmed the morphologies of the nanofibers and the distribution of CNTs. Rougher surfaces as well as obvious protruded parts induced by the blending of CNTs were observed from the studied nanofibers. Catalase was immobilized onto the nanofiber surface through the activation of carboxyl groups by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and Nhydroxysuccinimide (EDC/NHS). Results indicate that both the introduction of porphyrin pendants and CNTs obviously improve the activity and stabilities of the immobilized catalases. The facilitation of electron transfer between the immobilized catalases and the ability of both porphyrin pendants and CNTs to retain the active conformation of the catalases might be responsible for the improvements of the activity and stabilities.
Introduction Electrospinning is a simple, convenient, and versatile technique for generating fibers with diameters ranging from several micrometers to tens of nanometers.1-4 The collected nonwoven mat shows many exciting characteristics such as large surface area to volume ratio, good mechanical strength, excellent flexibility, and high porosity. Furthermore, materials in nanoscale may approach the length scale at which some specific physical or chemical interactions with their environments can occur, and then the properties differ substantially from those of the bulk materials of the same composition.5 Therefore, the study and utilization of electrospun nanofibers are of both technological and fundamental importance. In the most recent years, lots of outstanding improvements have been achieved. It is well-known that porphyrins play important roles in biological processes, for example, catalase contains ferriprotoporphyrin.6 Porphyrin polymers have also found increasing applications in many areas such as molecular recognition or molecular imprinting,7 sensors,8 light-emitting and energy/ electron-transfer materials,9 interactions with biological systems,10 and enzyme mimics for catalysis.11 Thus, combining the merits of electrospinning with the bioinspired applications of porphyrin polymers may generate functionalized nanofibers for multiple purposes. However, electrospinning of nanofibers from porphyrin-containing polymers has not received much attention yet. Most recently, acrylonitrile-based homopolymer and copolymers have been widely used for electrospinning due to their superior fiber-forming property.12-29 In our previous com* Corresponding authors. E-mail:
[email protected] (Z.-K.X.); jianwu@ zju.edu.cn (J.W.). Fax: + 86 571 8795 1773. † Institute of Polymer Science. ‡ Department of Chemistry.
munication,28 preliminary results of the synthesis of copolymers with porphyrin pendants and the preparation of porphyrinated nanofibers have been reported. It was demonstrated that the copolymer of acrylonitrile with vinyl porphyrin monomer could be facilely synthesized by a solution polymerization process and then electrospun into nanofibers. Furthermore, in our previous work, novel nanofibers possessing reactive carboxyl groups were also fabricated from poly[acrylonitrile-co-(maleic acid)] and poly[acrylonitrile-co-(acrylic acid)] by electrospinning process.24,26 Lipase and catalase were covalently immobilized onto the nanofibers, respectively. We found the amount of immobilized enzyme was remarkably large; besides, the activity also increased compared with those immobilized on traditional matrixes such as flat membranes and hollow fiber membranes.30,31 Electrospun nanofibers for enzyme immobilization/ loading have also been sparsely reported by other groups.32-38 In view of the unique characteristics of porphyrins, the porphyrinated nanofibers may be a latent support for catalase immobilization. Especially, the possible existence of a synergistic effect by the porphyrin pendants (electron donors) and the filled carbon nanotubes (CNTs) (electron acceptors) may improve the activity of the immobilized catalase. In this work, porphyrinated nanofibers were therefore prepared by electrospinning process for the immobilization of catalase (Scheme 1). Experimental Section Materials. Terpolymer composed of acrylonitrile (AN), acrylic acid (AA), and vinyl porphyrin was synthesized in our lab according to the method reported in previous work.28 The terpolymer was employed to immobilize catalase covalently through the reactive carboxyl group, and the molar content of carboxyl group in the terpolymer was about 10% as determined by 1H NMR spectra. Multiwalled carbon nanotubes (MWCNTs)
10.1021/jp070983n CCC: $37.00 © 2007 American Chemical Society Published on Web 08/31/2007
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SCHEME 1: Schematic Representation for the Synthesis of Porphyrin-Containing Terpolymers and Enzyme Binding onto the Nanofiber Surfaces
prepared by a chemical vapor deposition process were purchased from Shenzhen Nanotech Port Co. Ltd (China). Catalase (hydrogen peroxide oxidoreductase, EC1.11.1.6, from bovine liver) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, analytical grade) were obtained form Sigma. Coumassie brilliant blue (G250) for the Bradford protein assay was purchased from Urchem. Bovine serum albumin (BSA, BP0081) was purchased from Sino-American Biotechnology. N-Hydroxysuccinimide (NHS) was biological grade, and hydrogen peroxide (30%) was analytical grade. Solvents and other chemicals were of analytical grade and used as received without further purification. Water used in all experiments was deionized and ultrafiltrated to 18.2 MΩ. Electrospinning. To purify and uniformly disperse MWCNTs in the polymer matrix, MWCNTs were treated with a mixture of concentrated sulfuric and nitric acids (3:1, v/v) at 40 °C. Carboxyl (-COOH) and hydroxyl (-OH) groups were introduced onto the oxidized MWCNTs. After chemical etching, the surface-oxidized MWCNTs were well-dispersed in N,N′dimethylformamide (DMF) after sonication for 10 h. The electrospinning procedure reported previously was used to prepare nanofibers.29 A sample of polymer was dissolved in DMF or in the suspension of MWCNTs at 60 °C. The concentration of MWCNT was 20% of the mass of the dissolved polymer. Electrospinning was performed after air bubbles were removed completely by standing for 6 h. The electrospinning apparatus consists of a plastic syringe, a blunt-end stainless steel needle (the i.d. is 1.2 mm), a ground electrode (aluminum sheet on a flat glass), and a high-voltage power supply (GDW-a, Tianjin Dongwen High-voltage Power Supply Plant, China) with a low current output (about 0.02 A). A positive voltage (10 kV) was applied to the polymer solution with a distance between the syringe tip and the collector surface being ca. 15 cm. The flow rate of the polymer solution was kept at 0.6 mL/h by a microinfusion pump (WZ-50C2, Zhejiang University Medical Instrument Co. LTD, China). The resultant nanofibers were dried to constant weight in a vacuum oven at 60 °C to remove residual solvent. The average diameter of fibers was determined from field emission scanning electron microscopy (FESEM) micrographs. Treatment of Polyacrylonitrile with NaOH and Determination of Carboxyl Contents. To generate carboxyl groups on surfaces of polyacrylonitrile (PAN) nanofibers for catalase
immobilization, the PAN nanofibers were hydrolyzed in 1.0 M NaOH at 40 °C for 10 min to convert the nitrile groups (-Ct N) to carboxylic groups (-COOH).39 Afterward, the nanofibers were rinsed with ultrapure water to remove unreacted NaOH and then with 2 M HCl to obtain -COOH from -COONa. The treated nanofibers were dried to constant weight in a vacuum oven at 60 °C for further uses. The content of carboxyl groups was determined according to the method reported by Gupta et al.40 A sample of nanofibers was placed in 0.50 M KCl solution for 18 h at ambient temperature. The solution was then titrated against 0.01 M NaOH solution using phenolphthalein as indicator. The carboxyl content was represented as the mmol/g of the dry nanofibers. Characterization. Absorption spectra measurement was carried out on a UV-vis spectrophotometer (756PC, Shanghai Spectrum Instruments, Co., Ltd., China) using matched quartz cells of 1 cm path length. A field emission scanning electron microscope (FESEM, Sirion-100, FEI, U.S.A.) was applied to observe morphologies of the nanofibrous membranes. A transmission electron microscope (TEM, JEM-1200EX, Japan) was utilized to study morphologies of the nanofibers and the distribution of MWCNTs on 200-mesh Cu grids with an accelerating voltage of 120 kV. Immobilization of Catalase. Catalase was covalently immobilized onto the nanofibers with the EDC/NHS activation procedure, as described previously.26 A weighed amount (proximately 2.00 mg) of nanofibers (in the form of nonwoven meshes) was thoroughly washed with deionized water and then rinsed with phosphate buffer solution (PBS, 50 mM, pH 7.0). After this, the pretreated nanofibers were submerged into an EDC/NHS solution (10 g/L in PBS buffer, 50 mM, pH 7.0, the molar ratio of EDC to NHS ) 1:1) and shaken gently for 2 h at 25 °C. The activated nanofibers were then taken out, washed several times with PBS (pH 7.0), and mixed with a catalase solution (0.10 mg/mL in PBS, pH 7.0). Enzyme immobilization was conducted at 25 °C for a required time. The resultant catalase-immobilized nanofibers were washed with PBS (50 mM, pH 7.0) until no protein was detected in the washings. Immobilization Efficiency and Activity of the Catalases. To determinate the immobilization efficiency, protein contents in the solution were determined by the method of Bradford41 using BSA as the protein standard on a UV spectrophotometer. The amount of immobilized enzyme protein was estimated by
Catalase Immobilization on Electrospun Nanofibers
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Figure 1. FESEM images of the electrospun nanofibers: (A) PAN (154 ( 30 nm); (B) PANAACoPP (180 ( 30 nm); (C) PAN/CNT (180 ( 34 nm); (D) PANAACoPP/CNT (165 ( 37 nm) (×50 000).
subtracting the amount of protein determined in the residual solutions and washings from the total amount of protein used in the immobilization procedure. The activity of free and immobilized catalases was determined spectrophotometrically, by the direct measurement of the decrease in the absorbance of hydrogen peroxide at 240 nm with a specific absorption coefficient of 39.21 M-1 cm-1. A sample of 3 mL of reaction mixture containing 9.7 mM substrate in 50 mM PBS at pH 7.0 was preincubated at 25 °C for 10 min, and the reaction was started by adding 0.025 mL of catalase solution (0.10 mg enzyme/mL). The decrease in absorbance at 240 nm was recorded for 3 min. The rate of change in absorbance was calculated from the initial 2 min portion with the help of the absorbance versus time curve. A sample of about 2.00 mg of enzyme-immobilized nanofibers was introduced into the assay mixture to initiate the reaction as above. After 2 min, the reaction was terminated by removing the nanofibers from the reaction mixture. The absorbance of the reaction mixture was recorded, and the immobilized catalase activity was calculated. One unit of activity was defined as the decomposition of 1 µmol of hydrogen peroxide per min at 25 °C and pH 7.0. The activity of free catalase was given as µmol H2O2/(mg enzyme)‚min and immobilized catalase’s activity as µmol H2O2/ (mg immobilized enzyme)‚min at 25 °C and pH 7.0. Stability of the Catalases. The thermal stabilities of the free and immobilized catalases were determined according to the following procedure. Free and immobilized preparations were stored in PBS (50 mM, pH 7.0) at 50 °C, respectively. Free catalase solution (0.025 mL, 0.1 mg/mL) or a certain amount of immobilized enzyme was withdrawn at same-timed intervals (20 min) during incubation, and the residual activity was measured. The effects of reaction temperature and substrate pH were investigated by measuring the activity of free and immobilized catalases at different temperatures and pHs of H2O2 solution, respectively. The operational stabilities of the free and immobilized catalases were determined according to the following procedure. The activity of immobilized enzyme was measured as described
in the activity assays of catalase. After each reaction run, the immobilized preparation was taken out and washed with PBS (50 mM, pH 7.0) to remove any residual substrate on the nanofiber mesh. It was then reintroduced into a fresh reaction medium, and the enzyme activity was detected at optimum conditions. The storage stabilities of the free and immobilized catalases were determined according to the following procedure. Free catalase was stored as a solution of 0.1 mg/mL in PBS (50 mM, pH 7.0), and the immobilized catalases were stored as the wet form at 4 °C. Results and Discussion Preparation of Porphyrinated Nanofibers via Electrospinning. In order to immobilize a redox enzyme, terpolymer PANAACoPP from acrylonitrile, acrylic acid, and metalloporphyrin with Co2+ was synthesized in this work. Nanofibers can be conveniently fabricated from the terpolymer via the electrospinning technique. It is well-known that many factors influence the diameters and morphology of the electrospun nanofibers, which include solution concentration, applied voltage, solution velocity, tip-to-collector distance, and solution properties (polarity, surface tension, and electric conductivity, etc.). Among them, solution concentration is one of the most important factors for a certain polymer solution. In our cases, solution with 5 wt % polymer in DMF results in microspheres with diameters between 0.5 and 2 µm.28 This type of morphology has been reported by some groups, and it was mainly attributed to the low solution concentration.42-44 By increasing the concentration of solution from 5 to 12 wt %, uniform nanofibers with diameter around 180 nm were fabricated from PANAACoPP solution as indicated in Figure 1B. By changing the parameters such as the solution concentration and the molecular weight of polymer, fibers with different diameters and morphology can be prepared to meet the requirements for various purposes. Nanofibers filled with CNTs were also prepared. Typical FESEM images for the obtained nanofibers are shown in
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Figure 2. TEM images of (A) PAN/CNT and (B) PANAACoPP/CNT electrospun nanofibers.
TABLE 1: Carboxyl Content of the Nanofibers and Activity and Kinetic Parameters for the Free and Immobilized Catalases sample
carboxyl content (mmol/g)
bound enzyme (mg/g fibers)
specific activity (Units)
activity retention (%)
Km (mM)
Vmax (Units)
free catalase PAN PAN/CNT PANAACoPP PANAACoPP/CNT
0.18 ( 0.03 0.25 ( 0.04 0.14 ( 0.04 0.20 ( 0.06
24.45 ( 2.33 29.81 ( 3.76 18.93 ( 4.03 22.81 ( 4.22
2491.03 ( 50.21 807.63 ( 107.82 1127.91 ( 103.17 979.46 ( 120.24 1207.39 ( 134.66
32.4 45.3 39.3 48.5
34.1 82.3 65.1 73.9 64.8
12124.7 9077.4 10063.9 9641.0 11097.4
Figure 1, parts C and D. In comparison with the corresponding nanofibers without CNTs, both PAN/CNT and PANAACoPP/ CNT nanofibers exhibit rougher surfaces, which indicates that there are some CNTs not completely embedded into the nanofibers matrix. CNTs will mostly be covered with a thin polymer film and will not be exposed onto the air because the polymer solution is of lower surface tension than the nanotubes and will thus cover the surface. Similarly, TEM images presented in Figure 2 reveal some obvious protruded parts. These results are consistent with those reported by Hou et al.23 although the TEM images are obscure to some extent because of the small difference between CNTs and noncarbonized PAN-based polymer. The terpolymer PANAACoPP was selected in this work to prepare composite nanofibers with CNTs since (1) this terpolymer can be dissolved in DMF, which has been proved to be a good solvent for suspending CNTs that were surface-modified with carboxylic groups, as pointed out by Hou et al.;23 (2) charge-transfer complexes between the negatively charged nitrile groups and CNTs can be formed, which leads to composite nanofibers with enhanced electrical conductivity and interfacial interaction;15 (3) the introduced acrylic acid can offer reactive group for covalent immobilization of enzyme; (4) above all, it is speculated that synergistic effect might be induced by the porphyrin pendants (that can act as an electron donor) at the nanofiber surface cooperating with the embedded CNTs (electron acceptor). Therefore, catalase, a kind of redox enzyme, was chemically immobilized on the porphyrinated nanofibers with and without blending of CNTs. PAN nanofibers entrusted with carboxyl groups were also explored for comparison. Activity and Stability of the Catalase Immobilized on Porphyrinated Nanofibers. To covalently immobilize catalase onto these nanofibers, a two-step process was employed. First, the carboxyl groups on the nanofiber surfaces were activated with EDC/NHS. Second, condensation reaction between the amino groups of catalase with the activated carboxyl groups was carried out. The contents of carboxyl groups on the nanofiber surfaces are listed in Table 1 for all studied samples. It can be seen that the nanofibers filled with CNTs possess more carboxyl groups than those without CNTs. It is speculated that
the protruded CNTs (see the SEM and TEM images in Figures 1 and 2) treated with acids also offer some carboxyl groups. However, the increase of carboxyl groups may be mainly induced by the rougher nanofiber surface, which indicates larger surface area and thus more surface carboxyl groups. Therefore, the increase of content of carboxyl group is reasonable. Accordingly, as shown in Table 1, the amounts of catalase immobilized on the CNTs-filled nanofibers are larger than those on the nanofibers without CNTs. That means part of the catalases may be directly immobilized on the protruded CNTs. In fact, it has been reported that proteins including enzyme can be chemically immobilized onto the CNT surface by a similar process.45 The activity of the immobilized catalases as well as the kinetic parameters were measured for comparison with free catalase. It can be seen from Table 1 that the blending of CNTs indeed increases the activity of the immobilized catalase, as demonstrated in our previous communication.26 Also, the introduction of porphyrin pendants benefits the activity of the immobilized catalase. For example, the activity retention of catalases on PANAACoPP nanofibers increases to 39.3% from 32.4%, compared with that on the PAN nanofibers. At the same time, the activity retention of catalase on PANAACoPP/CNT nanofibers, 48.5%, is larger than that on PAN/CNT, 45.3%. Although the activity increment induced by the porphyrin pendants is not very large, the result is still encouraging because the improvement confirmed our principle idea, i.e., porphyrin moieties have positive effects on the immobilized enzyme. The kinetics parameters, maximum reaction rates (Vmax) and Michaelis-Menten constants (Km) for the free and immobilized catalases, were calculated from a double-reciprocal plot (Lineweaver-Burk method). In comparison with free catalase, the immobilized preparations reveal lower Vmax and higher Km values. It is well-known that Vmax reflects the intrinsic characteristics of the immobilized enzyme and can be affected by diffusion constrains, whereas Km reflects the effective characteristics of the enzyme and depends upon both partition and diffusion effects. In comparison with the free enzyme, the higher Km value of immobilized one is either due to the conformational changes of the enzyme resulting in a lower possibility of forming
Catalase Immobilization on Electrospun Nanofibers a substrate-enzyme complex or less accessibility of the substrate to the active sites of the immobilized enzymes caused by the increased diffusion limitation. It has been demonstrated in our previous work24 that, as a matrix for enzyme immobilization, electrospun nanofibers can not only offer high enzyme loading due to the large surface area to volume ratio but also enhance the enzyme activity because the high porosity, which can decrease the diffusion limitation during the catalytic reaction. However, in this work, extra improvement of the activity of immobilized catalase is expected and has been actually achieved. This improvement should be first attributed to the facilitation of electron transfer between the matrix and immobilized catalases. It has been proposed that the improved electronic conductivity induced by the unique electronic properties of CNTs is in favor of the immobilized glucose oxidase.46 Zhao et al. prepared multibilayer film from CNT and heme protein by layer-by-layer assembly and found effective electron transfer of the proteins was greatly facilitated in the microenvironment of multibilayer films.47 Also, porphyrin has been widely applied in light-emitting or energytransfer materials, and the possibility of electron/energy transfer of porphyrin has been proved.9,48 Especially, CNT is generally recognized as an electron acceptor, whereas porphyrin is an electron donor;49 thus, the composites of CNTs with porphyrin have been studied by some researchers, and the unique interactions have been confirmed.50,51 Since catalase is a kind of redox enzyme, the facilitation of electron transfer will unambiguously increase the enzyme activity. The second factor for the increase of catalase activity might be the biocompatibility of the porphyrinated nanofibers filled with or without CNTs, namely, the ability to maintain the active conformation of the catalases. It was found by Karajanagi et al. that soybean peroxidase, a kind of heme protein, retained its native shape and a large fraction of its native secondary structure on CNTs and high activity could be obtained.52 Gan et al. studied the interactions between CNTs and lactate dehydrogenase, and they concluded that CNTs could increase the enzyme activity of lactate dehydrogenase.53 Most recently, results of Zhao et al. also confirmed the near-active conformation of the heme protein retained in the microenvironment of CNTs.47 On the other hand, the steric and electronic properties, which can be greatly influenced in our case by the porphyrin pendants on the nanofiber surface, of the support might be very important for supported catalysis.11,54 Zhou et al. suggested that certain tetraphenylphorphyrins could bind to complementary protein surfaces with considerable selectivity in which the hydrophobic core of the porphyrin primarily contributed to the binding affinity.10 The porphyrin pendants also show remarkable steric effects. In fact, the improvement of enzyme retention activity can be attributed to several causes. For example, the CNTs-induced morphological features that enhance the access to/from the enzyme reaction centers may be partly responsible for the enhanced enzyme activity retention. Facilitation of electrontransfer process might also be one of such, but this is not certain because the process is too difficult to monitor or observe directly unless in the case of a biosensor. Up to now, experimental evidence for electron transfer was mostly collected from biosensor researches. Therefore, the above-mentioned experimental results can only preliminarily support the facts that the introduction of both CNTs and porphyrin pendants are in favor of the immobilized catalases. Enzyme immobilization on nanofibers in the form of a biosensor should be carried out to find more evidence.
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Figure 3. Effect of pH on the activity of free and immobilized catalases.
Figure 4. Thermal stability of free catalases and immobilized catalases after preincubation at 50 °C for different time.
Measurements including the effect of substrate pH and reaction temperature, thermal stability, operational stability, and storage stability were also performed to further study the immobilization of catalase. Figure 3 shows the effects of pH while no maximum pH shift was observed. It is reasonable that the charge density on the nanofiber surfaces is not so high as other polycations such as chitosan to change the microenvironment for catalase catalysis.55,56 It was also found that the immobilized catalases show less sensitivity to pH than the free one, although there is little difference between the immobilization preparations in the sensitivity extent. Figures 4 and 5 present the thermal stability and operational stability of the catalases, respectively. It can be seen that the stability of immobilized catalases on the nanofibers is improved to some extent. The effects of temperature on the activity of free and immobilized catalases are shown in Figure 6. It was found that the optimum temperature for free catalase is about 25 °C, whereas it shifts to nearly 35 °C for the two immobilized enzymes. In addition, at higher temperature range, the immobilized enzymes reveal obviously higher stability to temperature than the free one. This reflects the antithermal property of the immobilized preparations, which might be attributed to the conformational limitation on the enzyme movement as a result of covalent bond formation between the enzyme and the
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Wan et al. whereas free catalase losses most of its initial activity very soon. So far, the mechanisms interpreting the storage stability of immobilized enzyme are complex and still unclear. However, from Figure 5, it appeared that the covalent bonding improved the enzyme’s stability against structural denaturation. Conclusion
Figure 5. Operational stability of the immobilized catalases.
Acrylonitrile-based terpolymers bearing porphyrin pendants were synthesized according to our previously reported process. Porphyrinated nanofibers with diameters around 180 nm could be conveniently fabricated from these terpolymers with an electrospinning technique. The blending of CNTs induces rougher nanofiber surfaces with protruded parts as confirmed by TEM observation. Nonwoven mats formed by the nanofibers were found to be proper supports for redox enzyme such as catalase immobilization. Catalase can be immobilized onto the nanofiber surface through the activation of carboxyl groups by EDC/NHS. Results indicate that both the introduction of porphyrin pendants and CNTs obviously improve the activity and stabilities of the immobilized catalases. The facilitation of electron transfer between the immobilized catalases and the ability of both porphyrin pendants and CNTs to maintain the active conformation of the catalases might be responsible for the improvements of the activity and stabilities. Acknowledgment. Financial support from the National Natural Science Foundation of China for Distinguished Young Scholars (Grant No. 50625309) and the Foundation of Key Laboratory of Advanced Textile Materials and Manufacturing Technology (Zhejiang Sci-Tech University), Ministry of Education (Grant No. 2007005) is gratefully acknowledged. References and Notes
Figure 6. Effect of temperature on the activity of free and immobilized catalases.
Figure 7. Storage stability at 4 °C.
matrix, or lower restriction to the diffusion of the substrate at higher temperature, or both. Storage stability is one of the significant indexes to evaluate the properties of an enzyme.57,58 Figure 7 shows the residual activity of the catalases. It is obvious that there is a remarkable difference in the activity retentions with storage time. In other words, catalases immobilized on the porphyrinated nanofibers, especially on those filled with CNTs, show high stability,
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