Available online at www.sciencedirect.com

Enzyme and Microbial Technology 42 (2008) 332–339

Electrospun nanofibrous membranes filled with carbon nanotubes for redox enzyme immobilization Ling-Shu Wan, Bei-Bei Ke, 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, Hangzhou 310027, China Received 15 August 2007; received in revised form 7 October 2007; accepted 24 October 2007

Abstract Catalases decompose hydrogen peroxide (H2 O2 ) into water and oxygen in an effective and environmentally friend way. The decomposition of H2 O2 by nanofibrous membrane-supported catalases is reported in this work. Poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) (PANCNVP) and polyacrylonitrile (PAN) blending with or without multi-walled carbon nanotubes (MWCNTs) were electrospun into nanofibrous membranes as enzyme immobilization matrixes and were carefully characterized. To immobilize catalase covalently, the nanofibrous membranes were treated with alkali solution to generate carboxyl groups followed by activation procedure with EDC/NHS. Results indicate that the nanofibrous membranes can bind large amount of catalases due to their superior surface area to volume ratio. The decomposition behavior of H2 O2 with these immobilized catalases was explored. The increment of activity retention induced by the NVP moieties and/or MWCNTs is remarkable. Stabilities of the immobilized catalases including the effects of substrate pH and reaction temperature, thermal stability, operational stability and storage stability were also carefully studied. Results elucidate the improved stabilities of the nanofibrous membrane-supported catalases. In conclusion, the nanofibrous membranes electrospun from this biocompatible polymer, especially those filled with MWCNTs, are suitable matrices for catalase immobilization. © 2007 Elsevier Inc. All rights reserved. Keywords: Nanofibrous membrane; Enzyme immobilization; Catalase; Multi-walled carbon nanotube (MWCNT); Hydrogen peroxide decomposition

1. Introduction Catalases are abundant enzymes in nature that decompose hydrogen peroxide (H2 O2 ) into water and oxygen [1–5]. This kind of enzyme has received considerable interest because the elimination of H2 O2 is of extreme importance in both organisms and industry. For example, H2 O2 is used in chemical bleaching in cotton textile process, in large-scale municipal water treatments and in sterilizing milk. The residual H2 O2 in all these cases should be degraded to prevent problems in subsequent treatments or applications. It is well known that enzyme is a kind of biocatalyst that exhibits a high level of catalytic efficiency and high degree of selectivity [6]. Furthermore, as pointed out by Eberhardt et al. the use of catalase for the elimination of H2 O2 is very charming in the aspects of lower energy and resource consumption as well as slight environmental impact [7].

Corresponding author. Tel.: +86 571 8795 2605; fax: +86 571 8795 1773. E-mail address: [email protected] (Z.-K. Xu).

0141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2007.10.014

In general, immobilized enzymes can offer many advantages over their free forms, which include easy recovery and possible reuse, improved stability and avoiding protein contamination of the final product. In fact, for most industrial applications, immobilized enzymes are adopted. Since in most cases the immobilization process for enzymes will inevitably result in more or less loss of their activity, improving their activity retention is a key point. The catalysis behavior of the immobilized enzymes is influenced by several factors. Conformation of the immobilized enzymes and property of the matrix are the main two; the former fundamentally determines the efficiency of the enzyme reaction centers while the latter affects the accessibility to/from the enzyme reaction centers for the substrate and product. Therefore, both the property and morphology of the matrix should be tailored for the immobilization of a certain enzyme. Firstly, it is necessary to keep the active conformation of the enzyme. It is generally recognized that biocompatible surface is able to retain the conformation of enzymes. Our previous researches on lipase immobilization confirmed that biocompatible moieties such as phospholipid benefit the enzyme activity [8]. Poly(N-vinyl-2-pyrrolidone) (PVP) is

L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339

of excellent biocompatibility and known as an additive for hemodialysis membrane preparation [9–14]. The pyrrolidone ring is amphiphilic due to the coexistence of hydrophilic carbonyl group and hydrophobic –CH2 CH2 CH2 – chain. This unique structure endows PVP with good balance between hydrophilic and hydrophobic interactions when contacting with enzymes [15,16]. Therefore, poly[acrylonitrile-co-(N-vinyl-2pyrrolidone)] (PANCNVP) was used in this work. Secondly, highly porous supports are of preference. Electrospinning is a facile technique for generating fibers with diameter in the range of several micrometers to tens of nanometers [17–20]. Such electrospun nanofibrous membranes hold much more superiority over other kinds of carrier materials due to the large surface area to volume ratio and high porosity. These two characteristics make this kind of membrane suitable for enzyme immobilization, because large surface area to volume ratio can provide large amount of bound enzyme while high porosity can enhance the enzyme activity through decreasing the diffusion resistance of substrate [21–31]. Furthermore, nanofibrous membranes can be facilely prepared in situ by means of electrospinning; the enzyme-immobilized samples therefore show great potential in development of biosensor for environmental detection. Our previous work has confirmed the enhancement of activity retention and stabilities of the immobilized catalases by multiwalled carbon nanotubes (MWCNTs) [32,33]. In this work, nanofibrous membranes filled with MWCNTs were electrospun from PANCNVP which have superior fiber-forming property and good biocompatibility. Polyacrylonitrile (PAN) nanofibrous membranes were also prepared for comparison. Subsequently, catalases were covalently immobilized onto the nanofibrous membranes and the decomposition of H2 O2 was carefully studied by exploring the activity and stability of the immobilized catalases. 2. Experimental 2.1. Materials


were well-dispersed in N,N -dimethyl-formamide (DMF) after sonication for 10 h. An electrospinning procedure reported previously was used to prepare the nanofibrous membranes [19]. A sample of polymer was dissolved in DMF or the suspension of MWCNTs at 60 ◦ C. The mass ratio of polymer to MWCNT was 5:1. 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 inner diameter is 1.2 mm), a ground electrode (aluminium 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 (17 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 1.0 mL/h by a microinfusion pump (WZ-50C2, Zhejiang University Medical Instrument Co., Ltd., China). The resultant nanofibrous membranes were dried to constant weight in vacuum oven at 60 ◦ C to remove residual solvent. The average diameter of fibers was determined from field emission scanning electron microscope (FESEM) micrographs.

2.3. Treatment of the membranes with NaOH and determination of carboxyl contents To generate carboxyl groups on the surfaces of nanofibers, the membranes were hydrolyzed in 1.0 M NaOH at 40 ◦ C for 10 min to convert part of the nitrile groups (–C N) to carboxylic groups (–COOH) [35]. Afterwards, the membranes were rinsed with de-ionized water to remove unreacted NaOH and then with 2.0 M HCl solution to obtain –COOH from –COONa. The treated membranes were dried to constant weight in vacuum oven at 60 ◦ C for further use. The content of carboxyl groups on the fiber surfaces was determined according to the method reported by Gupta et al. [36]. A sample of membrane was placed in 0.5 M KCl solution for 18 h at ambient temperature. The solution was then titrated against 0.01 M NaOH solution by using phenolphthalein as indicator. The carboxyl content was represented as mmol/g of the dry membrane.

2.4. Characterization Absorption spectra measurement was carried out on a UV–vis spectrophotometer (756PC, Shanghai Spectrum Instruments, Co. Ltd., China) matched quartz cells of 1 cm path length. FESEM (Sirion-100, FEI, USA) was applied to observe the morphologies of nanofibrous membranes. Transmission electron microscope (TEM, JEM-1200EX, Japan) was utilized to study the morphology of nanofibers and the distribution of MWCNTs on 200-mesh Cu grids with an accelerating voltage of 120 kV. For TEM observation, nanofibers were directly electrospun on Cu grids.

PAN and PANCNVP were synthesized in our lab according to the method reported in previous work [34]. The content of NVP in the PANCNVP is about 22 wt.%. These polymers were electrospun into nanofibrous membranes and then employed to immobilize catalase covalently. MWCNTs 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 purchase from Sigma. Coumassie brilliant blue (G250) was purchased from Urchem and filtered before use. Bovine serum albumin (BSA, BP0081) was purchased from Sino-American Biotechnology (China). N-Hydroxysuccinimide (NHS) was of biological grade and hydrogen peroxide (30%) was of analytical grade. Solvents and other chemicals were of analytical grade and used as received. Water used in all experiments was de-ionized and ultrafiltrated to 18.2 M.

Catalase was covalently immobilized onto the nanofibrous membranes with the EDC/NHS activation procedure, as described previously [33]. A weighted amount (proximately 2 mg) of nanofibrous membrane was thoroughly washed with de-ionized water and phosphate buffer solution (PBS, 50 mM, pH 7.0). Afterwards, the pre-treated membrane was 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 at 25 ◦ C for 2 h. The activated membrane was then taken out and washed several times with PBS (pH 7.0); and submerged into a catalase solution (0.10 mg/mL in PBS, pH 7.0). Enzyme immobilization was conducted at 25 ◦ C for a required time (3 h). The resultant catalase-immobilized membrane was washed with PBS (50 mM, pH 7.0) until no protein was detected in the washings.

2.2. Preparation of the nanofibrous membranes

2.6. Immobilization efficiency and decomposition of H2 O2

To purify and uniformly disperse MWCNTs in the nanofibrous membranes, 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 MWCNTs

To determine the immobilization efficiency, the amount of catalase in the solution was assayed by the method of Bradford [37] using BSA as protein standard on UV spectrophotometer. The amount of immobilized catalase was then calculated by subtracting the amount of catalase determined in the residual solu-

2.5. Immobilization of catalase


L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339

tions and washings from the total amount of catalase used in the immobilization procedure. The activity of free or immobilized catalases for H2 O2 decomposition was spectrophotometrically determined by measuring the decrease in the absorbance of H2 O2 at 240 nm with a specific absorption coefficient of 0.03921 cm2 (␮mol H2 O2 )−1 . A sample of 3.0 mL reaction mixture in 50 mM PBS (pH 7.0) containing 9.7 mM of substrate was pre-incubated at 25 ◦ C for 10 min and the reaction was started by adding 0.025 mL of catalase solution (0.1 mg enzyme/mL). The decrease in the 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 enzyme-immobilized membrane was introduced into the assay mixture to initiate the reaction. After 2 min, the reaction was terminated by removing the membrane 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 ␮mol hydrogen peroxide per min at 25 ◦ C and pH 7.0. The activity of free catalase was given as ␮mol H2 O2 /(mg enzyme) min and immobilized catalase’s activity as ␮mol H2 O2 /(mg immobilized enzyme) min at 25 ◦ C and pH 7.0. Activity retention was defined as the ratio of the activity of the amount of the enzyme coupled on the supports to the activity of the same amount of free enzyme.

2.7. Stability of the catalases The thermal stabilities of the free and immobilized catalases were determined according to the following procedure. Free and immobilized enzymes were stored in PBS (50 mM, pH 7.0) at 50 ◦ C. Free catalase solution (0.025 mL, 0.1 mg/mL) or a certain amount of immobilized catalases was withdrawn at a certain 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 temperature and pH of H2 O2 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 catalase was taken out and washed with PBS (50 mM, pH

7.0) to remove any residual substrate on the nanofibrous membrane. 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 wet form at 4 ◦ C.

3. Results and discussion 3.1. Preparation of nanofibrous membranes as matrixes for catalase immobilization Nanofibrous membranes were prepared by electrospinning technique according to the procedure reported in our previous paper [19]. 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, electric conductivity, etc.). Among them, solution concentration is one of the most important factors for a certain polymer solution. According to the optimized conditions reported previously [19], PAN and PANCNVP nanofibrous membranes consisting of uniform nanofibers were prepared in this work. Nanofibrous membranes filled with MWCNTs were also prepared by electrospinning from polymer/MWCNTs mixing solutions. Typical FESEM images for the obtained membranes are shown in Fig. 1. Compared with those without MWCNTs, both PAN/MWCNTs and PANCNVP/MWCNTs nanofibers exhibit rough surfaces, which indicates that not all MWCNTs are completely embedded into the polymer nanofibers. Sim-

Fig. 1. FESEM images of nanofibrous membranes electrospun from (A) PAN; (B) PANCNVP; (C) PAN/MWCNTs and (D) PANCNVP/MWCNTs. Flow rate for electrospinning is 1.0 mL/h (50,000×).

L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339


Fig. 2. TEM images of (A) PAN/MWCNTs and (B) PANCNVP/MWCNTs composite electrospun nanofibers.

ilarly, TEM images presented in Fig. 2 reveal some obvious protruded parts. These results are consistent with those reported by Hou et al. [38] although the TEM images are obscure to some extent because of the slight difference between MWCNTs and non-carbonized PAN-based polymer. The copolymer PANCNVP was selected in this work to prepare composite nanofibrous membranes with MWCNTs since: (1) our previous work and other researches have confirmed that PANCNVP is a biocompatible polymer [39–42], which may keep the active conformation of the immobilized enzyme and then promote its activity retention and stability; (2) poly(Nvinyl-2-pyrrolidone) (PVP) has good compatibility with carbon nanotube (CNT) and can well disperse CNT [43], besides, PVP has been widely used as a protecting polymer for nanoparticles [44]; (3) this copolymer 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 proposed by Hou et al. [38]; (4) charge-transfer complexes between the negatively charged nitrile groups and CNTs can be formed, which leads to composite nanofibrous membrane with enhanced electrical conductivity and interfacial interaction [45]. Therefore, PANCNVP

is a suitable candidate for preparing MWCNTs-filled nanofibrous membrane as catalase immobilization matrix. 3.2. Activity of the immobilized catalases for H2 O2 decomposition To covalently immobilize catalase, the as-spun nanofibrous membranes were treated with alkali according to the reported procedure followed by a two-step immobilization process [35], as illustrated in Scheme 1. First, the introduced carboxyl groups on the surfaces of nanofibers 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 are listed in Table 1 for all studied membranes. It can be seen that the nanofibrous membranes filled with MWCNTs possess more carboxyl groups than those without MWCNTs. The increase of carboxyl groups is mainly caused by the MWCNTs-induced rougher nanofiber surface as confirmed by the FESEM and TEM micrographs, which indicates larger surface area and thus more surface carboxyl groups. Accordingly, as shown in Table 1, the amounts of catalase

Scheme 1. Schematic representation for the reactions of membrane activation and enzyme immobilization. Table 1 Carboxyl contents of the support samples and the activities and kinetic parameters for the free and immobilized catalases Samples

Carboxyl content (mmol/g)

Bound enzyme (mg/g)

Specific activity (units)


– 0.18 ± 0.25 ± 0.13 ± 0.21 ±

– 24.45 ± 29.81 ± 18.39 ± 25.77 ±

2491.03 807.63 1127.91 933.46 1212.91

0.03 0.04 0.02 0.05

2.33 3.76 3.29 4.10

± ± ± ± ±

50.21 107.82 103.17 94.64 112.79

Activity retention (%)

Km (mM)

– 32.4 ± 45.3 ± 37.5 ± 48.7 ±

34.1 82.3 65.1 80.4 66.8

3.2 3.5 2.7 3.6

± ± ± ± ±

1.3 3.5 3.0 3.4 2.5

Vmax (Units) 12124.7 9077.4 10063.9 9550.7 11044.8

± ± ± ± ±

101.3 90.1 107.2 88.4 98.3


L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339

immobilized on the MWCNTs-filled nanofibrous membranes are larger than those without MWCNTs. The decomposition of H2 O2 solution by the immobilized catalases was then carefully examined through studying the activity of the enzymes and the kinetic parameters. It can be seen from Table 1 that filling with MWCNTs indeed increases the activity retention of the immobilized catalases. Catalysis process of redox enzymes (catalase in this work) often involves electron transfer between the supports and the enzymes. It is well known that CNT possesses superb electrical conductivity. Therefore, the increase of activity retention might be attributed to the enhanced electrical conductivity of the nanofibers. Also, the incorporation of biocompatible NVP moieties benefits the activity retention. For example, the activity retention of catalases immobilized on PANCNVP membrane increases from 32.4% to 37.5% when compared with that on the PAN membrane. At the same time, the activity retention of catalases on PANCNVP/MWCNTs membrane (48.7%) is larger than that on PAN/MWCNTs (45.3%). Although the activity increment induced by the NVP moieties is not very large, this result is still encouraging because the improvement confirmed our principal idea, i.e. the biocompatible 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 double reciprocal plot. When compared with free catalases, the immobilized catalases 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, while Km reflects the effective characteristics of the enzyme and depends upon both partition and diffusion effects. The higher Km value of the immobilized catalase is due to either the conformational changes of the enzyme, which results in a lower possibility of forming a substrate–enzyme complex, or a less accessibility of the substrate to the active sites of the immobilized enzyme. 3.3. Stability of the immobilized catalases To practically decompose H2 O2 using the immobilized catalases, their stability is very important. Therefore, measurements including the effect of substrate pH and reaction temperature, thermal stability, operational stability and storage stability were carried out. Fig. 3 shows the effects of pH while no maximum pH shift is observed. It is reasonable because the charge density on the nanofibrous membrane surfaces is not large enough to change the microenvironment for catalase catalysis as other polycation such as chitosan does [46,47]. It is also found that the immobilized catalases show less sensitivity to pH than the free form, although there is little difference between different immobilized enzymes in the sensitivity extent. The effects of temperature on the activity of free and immobilized catalases are shown in Fig. 4. It was found the optimum temperature for free catalase is about 25 ◦ C, while it shifts to nearly 35 ◦ C for the two immobilized catalases. In addition, at higher temperature range, the immobilized enzymes reveal

Fig. 3. Effect of pH on the activity of free and immobilized catalases.

Fig. 4. Effect of temperature on the activity of free and immobilized catalases.

obviously higher stability to temperature than the free one. It reflects the increased thermal property of the immobilized enzymes, which is attributed to the conformational limitation on the enzyme as a result of covalent bond formation between

Fig. 5. Thermal stability of free catalases and immobilized catalases after preincubation at 50 ◦ C for different times.

L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339


Fig. 6. FESEM images of (a) PAN and (b) PANCNVP nanofibrous membranes after immersed in 50 ◦ C water bath for 1 h (50,000×).

the enzyme and the matrix or lower restriction to the diffusion of the substrate at higher temperature or both. However, as shown in Fig. 5, differing from the effects of temperature, the thermal stability of the catalases immobilized on membranes containing NVP moieties is poor. On the contrary, the thermal stability of immobilized catalases without hydrophilic NVP moieties shows a more or less increase. Therefore, it is speculated that the decrease is caused by the hydrophilic NVP moieties. Our previous results have confirmed the significant overshoot behavior of PANCNVP nanofibrous membranes for water adsorption [19]. Fig. 6 shows the FESEM images of PAN and PANCNVP nanofibrous membranes treated in 50 ◦ C water bath for 1 h. It is clear that the PAN membrane almost keeps its porous structure well after the treatment. Nevertheless, for the PANCNVP membrane the nanofibers entangles each other. This entanglement will increase the diffusion resistance and even embed the immobilized catalases. But this decrease is not fatal because most of the applications for immobilized catalases are at room temperature at which the morphology of nanofibrous membranes can be well kept. The difference between the effects of temperature (Fig. 4) and the thermal stability (Fig. 5) may be induced by the immersion time in hot water; in the former case, the catalase-immobilized nanofibrous membranes only underwent hot water for several minutes. Fig. 7 shows the operational stability of the immobilized catalases. The

Fig. 8. Storage stability of free catalases and immobilized catalases.

behaviors of the four kinds of catalase-immobilized nanofibrous membranes are similar with each other and receive about 50% of residual activity. Storage stability is one of the significant indexes to evaluate the properties of enzyme [48,49]. Fig. 8 shows the residual activity of the studied catalases after different periods of storage. It is obvious that there is a remarkable difference in the activity retentions with storage time. In other words, catalases immobilized on the PANCNVP membranes, especially on that filled with MWCNTs, show high stability while free catalase loses most of its initial activity very soon. So far, the mechanisms underlying the storage stability of immobilized enzyme are complex and still unclear. The ability of the nanofibrous membranes to keep the active conformation of enzyme may be a factor. 4. Conclusion

Fig. 7. Operational stability of the immobilized catalases.

Nanofibrous membranes filled with MWCNTs were electrospun from PAN and biocompatible PANCNVP. The flow rate of electrospinning only has a slight effect on the fiber diameter and morphologies while the incorporation of MWCNT induces rougher fiber surface and then larger surface area to volume ratio. Catalase was successfully immobilized onto the alkalitreated membranes through an EDC/NHS activation process.


L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339

Results indicate that the catalases immobilized on the PANCNVP membranes, especially on those filled with MWCNTs, show improved activity retention compared with those on PAN membranes. Furthermore, the nanofibrous membrane-supported catalases exhibit significantly improved storage stability. Therefore, this kind of nanofibrous membrane-supported catalases may be used to decompose H2 O2 effectively in various fields including environmental monitor and industrial wastewater treatments. Acknowledgements 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 SciTech University), Ministry of Education (Grant No. 2007005) are gratefully acknowledged. References [1] Betancor L, Hidalgo A, Fernandez-Lorente G, Mateo C, FernandezLafuente R, Guisan JM. Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques. Biotechnol Prog 2003;19(3):763–7. [2] Yoshimoto M, Sakamoto H, Yoshimoto N, Kuboi R, Nakao K. Stabilization of quaternary structure and activity of bovine liver catalase through encapsulation in liposomes. Enzyme Microb Technol 2007;41(6–7):849–58. [3] Cetinus SA, Oztop HN, Saraydin D. Immobilization of catalase onto chitosan and cibacron blue F3GA attached chitosan beads. Enzyme Microb Technol 2007;41(4):447–54. [4] Akgol S, Denizli A. Novel metal-chelate affinity sorbents for reversible use in catalase adsorption. J Mol Catal B: Enzym 2004;28(1):7–14. [5] Jurgen-Lohmann DL, Legge RL. Immobilization of bovine catalase in sol–gels. Enzyme Microb Technol 2006;39(4):626–33. [6] Kayali HA, Tarhan L. The relationship between the levels of total sialic acid, lipid peroxidation and superoxide dismutase, catalase, glutathione peroxidase, ascorbate antioxidant in urea supplemented medium by Fusarium species. Enzyme Microb Technol 2006;39(4):697–702. [7] Eberhardt AM, Pedroni V, Volpe M, Ferreira ML. Immobilization of catalase from Aspergillus niger on inorganic and biopolymeric supports for H2 O2 decomposition. Appl Catal B: Environ 2004;47(3):153–63. [8] Deng HT, Xu ZK, Huang XJ, Wu J, Seta P. Adsorption and activity of Candida rugosa lipase on polypropylene hollow fiber membrane modified with phospholipid analogous polymers. Langmuir 2004;20(23):10168–73. [9] Hayama M, Yamamoto K, Kohori F, Uesaka T, Ueno Y, Sugaya H, et al. Nanoscopic behavior of polyvinylpyrrolidone particles on polysulfone/polyvinylpyrrolidone film. Biomaterials 2004;25(6):1019–28. [10] Hayama M, Yamamoto K, Kohori F, Sakai K. How polysulfone dialysis membranes containing polyvinylpyrrolidone achieve excellent biocompatibility? J Membr Sci 2004;234(1–2):41–9. [11] Wan LS, Xu ZK, Wang ZG. Leaching of PVP from polyacrylonitrile/PVP blending membranes: a comparative study of asymmetric and dense membranes. J Polym Sci Part B: Polym Phys 2006;44(10):1490–8. [12] Higuchi A, Shirano K, Harashima M, Yoon BO, Hara M, Hattori M, et al. Chemically modified polysulfone hollow fibers with vinylpyrrolidone having improved blood compatibility. Biomaterials 2002;23(13):2659–66. [13] Wetzels GMR, Koole LH. Photoimmobilization of poly(Nvinylpyrrolidinone) as a means to improve haemocompatibility of polyurethane biomaterials. Biomaterials 1999;20(20):1879–87. [14] Wan LS, Xu ZK, Huang XJ, Che AF, Wang ZG. A novel process for the post-treatment of polyacrylonitrile-based membranes: performance improvement and possible mechanism. J Membr Sci 2006;277(1–2):157–64.

[15] Kitano H, Tada S, Mori T, Takaha K, Gemmei-Ide M, Tanaka M, et al. Correlation between the structure of water in the vicinity of carboxybetaine polymers and their blood-compatibility. Langmuir 2005;21(25):11932– 40. [16] Wan LS, Huang XJ, Xu ZK. Diffusion and structure of water in polymers containing N-vinyl-2-pyrrolidone. J Phys Chem B 2007;111(5):922–8. [17] Li D, Xia YN. Electrospinning of nanofibers: reinventing the wheel? Adv Mater 2004;16(14):1151–70. [18] Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol 2003;63(15):2223–53. [19] Wan LS, Xu ZK, Jiang HL. Fibrous membranes electrospinning from acrylonitrile-based polymers: specific absorption behaviors and states of water. Macromol Biosci 2006;6(5):364–72. [20] Wan LS, Wu J, Xu ZK. Porphyrinated nanofibers via copolymerization and electrospinning. Macromol Rapid Commun 2006;27(18):1533–8. [21] Patel AC, Li SX, Yuan JM, Wei Y. In situ encapsulation of horseradish peroxidase in electrospun porous silica fibers for potential biosensor applications. Nano Lett 2006;6(5):1042–6. [22] Lee KH, Ki CS, Baek DH, Kang GD, Ihm DW, Park YH. Application of electrospun silk fibroin nanofibers as an immobilization support of enzyme. Fiber Polym 2005;6(3):181–5. [23] Huang XJ, Xu ZK, Wan LS, Innocent C, Seta P. Electrospun nanofibers modified with phospholipid moieties for enzyme immobilization. Macromol Rapid Commun 2006;27(16):1341–5. [24] Wu LL, Yuan XY, Sheng J. Immobilization of cellulase in nanofibrous PVA membranes by electrospinning. J Membr Sci 2005;250(1–2):167–73. [25] Xie JB, Hsieh YL. Ultra-high surface fibrous membranes from electrospinning of natural proteins: casein and lipase enzyme. J Mater Sci 2003;38(10):2125–33. [26] Jia HF, Zhu GY, Vugrinovich B, Kataphinan W, Reneker DH, Wang P. Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts. Biotechnol Prog 2002;18(5):1027–32. [27] Kim BC, Nair S, Kim J, Kwak JH, Grate JW, Kim SH, et al. Preparation of biocatalytic nanofibres with high activity and stability via enzyme aggregate coating on polymer nanofibres. Nanotechnology 2005;16(7):S382–8. [28] Wang ZG, Wang JQ, Xu ZK. Immobilization of lipase from Candida rugosa on electrospun polysulfone nanofibrous membranes by adsorption. J Mol Catal B: Enzym 2006;42(1–2):45–51. [29] Ye P, Xu ZK, Wu J, Innocent C, Seta P. Nanofibrous membranes containing reactive groups: electrospinning from poly(acrylonitrile-co-maleic acid) for lipase immobilization. Macromolecules 2006;39(3):1041–5. [30] Ye P, Xu ZK, Wu J, Innocent C, Seta P. Nanofibrous poly(acrylonitrile-comaleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization. Biomaterials 2006;27(22):4169–76. [31] Wan LS, Ke BB, Wu J, Xu ZK. Catalase immobilization on electrospun nanofibers: effects of porphyrin pendants and carbon nanotubes. J Phys Chem C 2007;111(38):14091–7. [32] Wang ZG, Ke BB, Xu ZK. Covalent immobilization of redox enzyme on electrospun nonwoven poly(acrylonitrile-co-acrylic acid) nanofiber mesh filled with carbon nanotubes: a comprehensive study. Biotechnol Bioeng 2007;97(4):708–20. [33] Wang ZG, Xu ZK, Wan LS, Wu J, Innocent C, Seta P. Nanofibrous membranes containing carbon nanotubes: electrospun for redox enzyme immobilization. Macromol Rapid Commun 2006;27(7):516–21. [34] Wan LS, Xu ZK, Huang XJ, Wang ZG, Wang HL. Copolymerization of acrylonitrile with N-vinyl-2-pyrrolidone to improve the hemocompatibility of polyacrylonitrile. Polymer 2005;46(18):7715–23. [35] Lin WC, Liu TY, Yang MC. Hemocompatibility of polyacrylonitrile dialysis membrane immobilized with chitosan and heparin conjugate. Biomaterials 2004;25(10):1947–57. [36] Gupta B, Jain R, Anjum N, Singh H. Preparation of antimicrobial sutures by preirradiation grafting of acrylonitrile onto polypropylene monofilament part III. Hydrolysis of the grafted suture. J Appl Polym Sci 2004;94(6):2509–16. [37] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54.

L.-S. Wan et al. / Enzyme and Microbial Technology 42 (2008) 332–339 [38] Hou HQ, Ge JJ, Zeng J, Li Q, Reneker DH, Greiner A, et al. Electrospun polyacrylonitrile nanofibers containing a high concentration of well-aligned multi-wall carbon nanotubes. Chem Mater 2005;17(5):967– 73. [39] Wan LS, Xu ZK, Huang XJ, Wang ZG, Ye P. Hemocompatibility of poly(acrylonitrile-co-N-vinyl-2-pyrrolidone)s: swelling behavior and water states. Macromol Biosci 2005;5(3):229–36. [40] Krasteva N, Harms U, Albrecht W, Seifert B, Hopp M, Altankov G, et al. Membranes for biohybrid liver support systems—investigations on hepatocyte attachment, morphology and growth. Biomaterials 2002;23(12):2467–78. [41] Groth T, Seifert B, Malsch G, Albrecht W, Paul D, Kostadinova A, et al. Interaction of human skin fibroblasts with moderate wettable polyacrylonitrile-copolymer membranes. J Biomed Mater Res 2002;61(2):290–300. [42] Wan LS, Xu ZK, Huang XJ, Huang XD, Yao K. Cytocompatibility of poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) membranes with human endothelial cells and macrophages. Acta Biomater 2007;3(2):183– 90. [43] Gao JB, Yu AP, Itkis ME, Bekyarova E, Zhao B, Niyogi S, et al. Largescale fabrication of aligned single-walled carbon nanotube array and




[47] [48]



hierarchical single-walled carbon nanotube assembly. J Am Chem Soc 2004;126(51):16698–9. Gao Y, Jiang P, Liu DF, Yuan HJ, Yan XQ, Zhou ZP, et al. Evidence for the monolayer assembly of poly(vinylpyrrolidone) on the surfaces of silver nanowires. J Phys Chem B 2004;108(34):12877–81. Ge JJ, Hou HQ, Li Q, Graham MJ, Greiner A, Reneker DH, et al. Assembly of well-aligned multi-walled carbon nanotubes in confined polyacrylonitrile environments: electrospun composite nanofiber sheets. J Am Chem Soc 2004;126(48):15754–61. Godjevargova T, Dayal R, Marinov I. Simultaneous covalent immobilization of glucose oxidase and catalase onto chemically modified acrylonitrile copolymer membranes. J Appl Polym Sci 2004;91(6):4057–63. Cetinus SA, Oztop HN. Immobilization of catalase into chemically crosslinked chitosan beads. Enzyme Microb Technol 2003;32(7):889–94. Godjevargova T, Dayal R, Turmanova S. Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane. Macromol Biosci 2004;4(10):950–6. Ozyilmaz G, Tukel SS, Alptekin O. Activity and storage stability of immobilized glucose oxidase onto magnesium silicate. J Mol Catal B: Enzym 2005;35(4–6):154–60.

Electrospun nanofibrous membranes filled with carbon ...

over their free forms, which include easy recovery and possible reuse, improved stability and ..... both partition and diffusion effects. The higher Km value of.

1MB Sizes 0 Downloads 278 Views

Recommend Documents

Nanofibrous Membranes Containing Carbon Nanotubes
co-acrylic acid) (PANCAA) solutions containing MWCNTs. These nanofibrous ..... Chen, Y. G. Zhang, D. W. Wang, H. J. Dai, J. Am. Chem. Soc. 2001, 123, 3838.

An electrospun triphasic nanofibrous scaffold for bone ...
May 8, 2007 - 80 nm particle size) into the solution mixture for co- electrospinning [26]. ... synthesized by chemical precipitation at room temperature using Ca(OH)2 and ... were fully enclosed within a clear Lexan box in order to minimize ...

Electrospun Nanofibers Modified with Phospholipid ...
purified by vacuum distillation before use. Lipase (from. Candida rugosa, protein concentration was 6.8 wt.-%), Brad- ford reagent, bovine serum albumin (BSA, ...

Electrospun for Redox Enzyme Immobilization
ever, for redox enzymes such as catalase, a direct electron- transfer path should be .... protoporphyrin ring and a central Fe atom, i.e., ferriproto- porphyrin, where ...

membranes with human endothelial cells and ...
All data presented are simple mean values from the results of four parallel ... idone-iodine- poly(epsilon-caprolactone) blends to adherence of. Escherichia coli.

Sc(OTf)3 catalyzed carbon-carbon and carbon-heteroatom ... - Arkivoc
Dec 4, 2016 - Himachal Pradesh-174301, India ..... The author is thankful to the Indus International University, Una, Himachal Pradesh, India for support and ...

Resonant Oscillators with Carbon-Nanotube ... - Semantic Scholar
Sep 27, 2004 - The bold type indicates consistency with the expected shear modulus of nanotubes. .... ment may be found in the online article's HTML refer-.

polydiphenylamine composite electrospun membrane ...
a Department of Chemistry Graduate School, Kyungpook National University, Daegu 702-701, South Korea b Nano ... Electrospinning technology has recently been extended ..... cells under open-circuit conditions provide useful information on.

Filled Tutorial
... WhereIt Hurts RemodelAnd Fraud - PaintingNiche.. Enhancing Painting FauxChunk Video And. Image Filled TutorialStep-By-Step FauxBrick Techniques Are ...

Waves in a cylindrical borehole filled with micropolar ...
Received 27 December 2007; accepted 17 April 2008; published online 23 June 2008. Dispersion ..... dial displacement, microrotation, radial force stress, shear.

Proton-conducting membranes from phosphotungstic ...
Mar 19, 2009 - eight scans of 64 K data points were acquired with a spectral width of ... system; conductivity lower than 6 9 10-8 X-1 cm-1), with appropriate ...

Proton-conducting membranes from phosphotungstic ...
Mar 19, 2009 - computer-connected Sartorius analytical balance with a sensitivity of 10-5 g ..... chlorogenic and in cider apple juices by 1H-RMN spectrometry.

Fibrous Membranes Electrospinning from Acrylonitrile ...
Fax: юю86 571 8795 1773; E-mail: [email protected]. Received: January ... non-freezable bound water and free water can be distinguished in the fibrous ...

Asymmetric membranes fabricated from poly ...
DOI 10.1002/app.25045 ..... Kang, J. S.; Lee, Y. M. J Appl Polym Sci 2002, 85, 57. 5. Kurkuri ... Kang, J. S.; Kim, K. Y.; Lee, Y. M. J Membr Sci 2003, 214, 311. 23.

Cheap 2001).pdf3000.5mm With 100% Real Carbon Fiber plate ...
Cheap 2001).pdf3000.5mm With 100% Real Carbon Fibe ... Plain Weave 3K and Gloss Surface of Both Sides.pdf. Cheap 2001).pdf3000.5mm With 100% Real ...

Resonant Oscillators with Carbon-Nanotube ... - APS Link Manager
Sep 27, 2004 - Page 1 ... difficult lithographically to create uniform small high- aspect-ratio suspended beams like the MWNT in Fig. 1. Furthermore, the current ...

Fabrication of Functional Nanofibrous Ammonia Sensor
of NH gas at room temperature. Inset: Plot of concentration of NH gas Vs resistance of FNFM. due to the thermal degradation of head-to-head linkages and un-.

Screen Printed Carbon Electrode Modified with Poly(L ...
electron microscopy and transmission electron microscopy revealed the size and distribution of gold nanoparticles. The PLA – AuNP/SPE was applied effectively ...

Tunable Water Channels with Carbon Nanoscrolls
hydrogen-bonding network of water. For case ... rates are greatly enhanced in comparison with the .... [4] G. Hummer, J. C. Rasaiah, J. P. Noworyta, Nature. 2001 ...

pdf-147\fluorine-carbon-and-fluoride-carbon-materials-chemistry ...
... of the apps below to open or edit this item. pdf-147\fluorine-carbon-and-fluoride-carbon-materials-chemistry-physics-and-applications-from-crc-press.pdf.

Comparison of tribological properties of carbon and carbon nitride ...
Laboratory of Tribology, School of Mechanical Engineering, Tohoku University, Sendai 980-77, Japan. (Received 14 December 1995; accepted 7 June 1996). According to theoretical predictions made in 1989–1990, carbon nitride can be even harder than di