Preparation of Amidoxime Polyacrylonitrile Nanofibrous Membranes and Their Applications in Enzymatic Membrane Reactor

Transcription

1 Preparation of Amidoxime Polyacrylonitrile Nanofibrous Membranes and Their Applications in Enzymatic Membrane Reactor Quan Feng, PhD 1, Qufu Wei, PhD 2, Dayin Hou 1, Songmei Bi 1, Anfang Wei, PhD 1, Xiuzhen Xu 1 1 Anhui Polytechnic University, Wuhu, Anhui CHINA 2 Jiangnan University, Key Laboratory of Eco-Textiles, Ministry of Education CHINA Correspondence to: Quan Feng fengquan@ahpu.edu.cn ABSTRACT Ployacrylonitrile (PAN) nanofibers were formed by electrospinning. Amidoxime polyacryonitrile (AOPAN) nanofibrous membranes were formed by reaction between PAN nanofibrous membranes and hydroxylamine hydrochloride, and Fe(III)-AOPAN metal chelated nanofibrous membranes were prepared for immobilizing catalases. The enzymatic membrane reactor (EMR) with immobilized catalases was assembled, and the fluxes of membrane and conversion ratio of EMR were studied. The immobilized catalases showed better resistance to ph and temperature change than that of the free form, and the storage stabilities of immobilized catalases were higher than that of the free catalases. The flux of the membrane and the conversion ratio of EMR were calculated, and the reusability of the EMR was studied. The results of experiments show that the immobilized catalases had a high affinity with the nanofibers, and the EMR has excellent performance. INTRODUCTION Enzymatic membrane reactors will potentially be used in the textile industry, chemical industry, and food industry because they can integrate the reaction and separation process in one unit [1,2]. The use of catalases is very effective in the aspects of lower resources and energy consumption [3]. Catalases are enzymes that decompose hydrogen peroxide (H 2 O 2 ) into water and oxygen and are commonly used in various fields, including agriculture, food, textiles, and detergents. However, the applications of enzymes are suffering from various problems, such as instability, non-reusability, and high-cost [4]. Enzyme immobilization has become an effective way to overcome these limitations to some extent. Insoluble supports can be recycled much more easily than soluble enzymes [5]. Most of the transition metal ions can form stable complexes with electron-rich compounds and coordinate molecules by coordinate covalent bonds. Enzyme immobilization on the metal chelated matrix is based on multipoint interactions between chelated metal ions on the support and active sites such as the indole group of tryptophan, imidazole group of histidine, and thiol group of cysteine [6]. Electrospinning is an effective technique for preparing nanofibrous membranes. Nanofibers could provide convenient diffusion channels and more reaction sites to immobilized enzymes because of their properties, such as high surface area per unit mass and high porosity [7,8]. Polyacrylonitrile (PAN) polymers have attracted a great deal of attention due to a variety of their excellent characteristics, which include high strength, abrasion resistance, thermal stability, and resistance to bacteria, and photo irradiation. However, the relative poor hydrophilicity and biocompatibility prevent the PAN from some potential applications [9,10]. Amidoxime polyacrylonitrile (AOPAN) nanofibrous membranes could be prepared by treating PAN nanofibrous membrane in hydroxylamine hydrochloride aqueous solution, the cyano group on the surface of PAN nanofibrous membranes reacted with hydroxylamine hydrochloride and led to the formation of amidoxime groups [11]. The Journal of Engineered Fibers and Fabrics 146

2 hydrophilic amidoxime groups could improve the chemical and biological properties of the PAN nanofibrous membranes. Surface modified PAN nanofibrous membranes have been widely used in biological and environmental applications such as metal ion adsorption, cell adhesion, and enzyme immobilization [12-15]. To our knowledge, the metal chelated amidoxime polyacrylonitrile nanofibrous membranes applied in EMR has not been reported. In this study, AOPAN nanofibrous membranes were obtained by treatment of electrospun PAN nanofibrous membranes in hydroxylamine hydrochloride aqueous solution. Then, Fe(III) ions were incorporated into the amidoxime groups of the AOPAN nanofibrous membranes via metal chelation. Subsequently, catalases were immobilized onto the metal chelated nanofibrous membranes, and the activity, stability of the immobilized catalases was studied. On the base of Fe(III)-AOPAN metal chelated nanofibrous membrane, the EMR was assembled. The flux and conversion ratio of EMR were calculated, and the reusability of the EMR was studied. EXPERIMENTAL Materials PAN(Mw=79100 g/mol) was obtained from Aldrich and used without further purification. N, N-dimethylformamide (DMF), Hydroxylamine hydrochloride, sodium carbonate, Coomassie brilliant blue (G250), ferric chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine liver catalases (hydrogen peroxide oxidoreduction; EC ) were purchase from Sigma. Hydrogen peroxide (30%) and the ingredients of phosphate buffer solution (PBS) such as NaCl, KCl, KH 2 PO 4, K 2 HPO 4 were of analytical grade and used as received. Water used in all experiments was de-ionized. Preparation of Amidoxime PAN Nanofibrous Membranes PAN was dissolved in DMF at room temperature with stirring. PAN solutions were placed in syringes (content of 20 ml) with a blunt needle (the nozzle diameter was about 0.7 mm), and the solution flow rate was controlled by a micro-infusion pump (JZB-1800D, Changsha, China). The high-voltage supplier (DW-P503-4AC, Tianjin, China) was used to connect the grounded collector and metal needles for forming electrostatic fields. The grounded collection roller covered with the aluminum foil was used to collect the electrospun nanofibers. The parameters of electrospinning were set as follows: voltage of 18 kv, solution flow rate of 1.2 ml/h, and collecting distance between the syringe needle tip and the grounded collector of 18 cm. Nanofibers were collected by the roller for 36 h. PAN nanofibrous membranes samples (0.12 g, 3 cm 3 cm) were added to 100 ml 0.5 mol/l hydroxylamine hydrochloride aqueous solution. The ph of the reaction solution was adjusted to 6 by adding sodium carbonate solution. The reaction was carried out at 70 o C for 2 h. After the completion of reaction, the modified PAN nanofibrous membranes taken from reaction medium were washed with distilled water and dried at 40 o C in a vacuum oven. The schematic diagram of the reaction is illustration in Figure 1. FIGURE 1. Schematic diagram of the reaction. Chelation of Fe 3+ ions on AOPAN Nanofibrous Membranes Samples of AOPAN nanofibrous membrane were put in flasks, each of them containing 30 ml ferric chloride solution. The flasks were stirred at 20 o C for 24 h. The fibers were rinsed for five minutes with de-ionized water to remove adsorbed metal ions. The metal ions concentrations were determined by atomic absorption spectrophotometer (AAS). The amounts of adsorbed metal ions were calculated by using the concentrations of the metal ions in the initial solution and in the resulting solution. The metal chelated AOPAN nanofibrous membranes were dried at 40 o C in a vacuum oven. Immobilization of Catalases on Metal Chelated AOPAN Nanofibrous Membranes Metal ion chelated AOPAN nanofibrous membranes were immersed in 30 ml of catalases solution (0.3 mg/ml) in 50 mm PBS ph 7.0 for 4 h at 20 o C in shakers (80 rpm). Then, the nanofibrous membranes were removed from solution and rinsed with the same PBS until no soluble protein was detectable. Enzyme concentration was determined by the method of Bradford [16]. The amount of bound enzymes was calculated by: Journal of Engineered Fibers and Fabrics 147

3 Q e ( C0 CV ) c = (1) M d Here, Q e is the amount of catalases bound onto unit mass of nanofibrous membranes (mg/g), V c is the volume of the catalases solution, C 0 and C are the initial and equilibrium enzymes concentrations in the solution (mg/ml), and M d is the mass (dry weight) of the AOPAN nanofibrous membranes. Fe(III) can chelate with amidoxime groups of the AOPAN nanofibrous membranes and the imidazole groups, amino groups on catalases by coordination bond. Activity Assays of Free and Immobilized Catalases Catalases immobilized chelated nanofibrous membranes were mixed with 100 ml of hydrogen peroxide solution (50 mmol/l) in 50 mm PBS (ph 7.0). The reaction was kept at 35 o C for 3 min. The activity of the free and immobilized catalases was determined spectrophotometrically by measurement of decrease in the absorbance of hydrogen peroxide at 240 nm. The specific activity of enzyme was calculated by the following formula: Storage Stability of Immobilized Catalases The stability of free and immobilized enzyme at storage were measured by calculating their activity retention during 24 days at 4 o C in 50 mm PBS (ph 7.0), using 3 days intervals, then a sample was removed and determined for enzyme activity. EMR Equipment Schematic representation of the EMR is depicted in Figure 2. In this reactor system, Fe(III)-AOPAN metal chelated nanofibrous membrane contained the immobilized catalases. The configuration may contribute to increasing transport of the substrate and improvement of the reactor efficiency. ( A0 A) V v = T K Ew (2) Where A 0 and A are the initial and terminal absorbance of the solution at 240 nm, V is the volume of the hydrogen peroxide solution (ml), T is the time of reaction (min), K is the molar extinction coefficient of hydrogen peroxide at 240 nm (K= ), and E w is the amount of enzyme (mg). Activity of the immobilized and free enzymes was investigated at 35 o C and the concentrations of hydrogen peroxide ranged from 30 to 240 mmol/l. Dependence of Temperature and ph on the Activity of Immobilized and Free Catalases The immobilized and free enzymes were mixed with 20 ml of hydrogen peroxide solution (100 mmol/l) in PBS (ph 7.0) respectively, and the temperatures ranging from 15 o C to 65 o C. PBS (ph ) were used for ph dependence study. The optimum ph values of free and immobilized enzymes were investigated at 35 o C for 3 min. FIGURE 2. Schematic diagram of enzymatic membrane reactor (1. N 2 ; 2,6. H 2 O 2 solution; 3,7. magnetic stirrer; 4. immobilized catalases; 5. composite membrane; 8. filtrate) Performance of the EMR The flux of the membrane at various operation pressures was calculated by the following formula: V J = S t (3) Where J is the flux of membrane (L/m 2 min), V is the volume of the filtrate (L), S is the surface area of the membrane (m 2 ), t is the time of operation(min). The conversion ratio of hydrogen peroxide of EMR at various pressures was calculated by the following formula: C0 C p K = 100% (4) C 0 Journal of Engineered Fibers and Fabrics 148

4 K is the conversion ratio of hydrogen peroxide; C 0 and C p are concentrations of substrate in the initial solution and filtrate respectively (mg/ml). Moreover, reusability of the EMR was checked. The production capacity of the EMR with immobilization was calculated by the following formula: V (C0 C p ) P = (5) t Where P is production capacity of the EMR (mmol/min), V is the volume of the filtrate (ml), C 0 and C p are concentrations of substrate in the initial solution and filtrate respectively (mmol/ml), t is time of reaction (min). The EMR with immobilized catalases was used repeated five times within one day; the reusability of the EMR was analyzed according to production capacity. RESULTS AND DISCUSSION The Surface Morphologies of Nanofibers The SEM images of the original PAN nanofibers and AOPAN nanofibers are displayed in Figure 3. The PAN nanofibers looked very even and the average diameter of the electrospun nanofibrous membranes ranged between 220 and 320 nm, as presented in Figure 3a. The diameter of AOPAN nanofibers did not change obviously, as revealed in Figure 3b. Chelation of Fe(III) ions on AOPAN nanofibrous membrane The adsorption isotherm of AOPAN nanofibrous membranes is presented in Figure 4. It indicated that the initial metal ions adsorption increased very fast and then gradually leveled off. The initial increase in Fe(III) adsorption might be attributed to many available amidoxime groups in AOPAN nanofibrous membranes. As more sites in the amidoxime groups were filled, it became difficult for more Fe(III) to find vacant sites, and the adsorption amount of metal ions reached saturation gradually with the increase in concentration of Fe(III) solution. FIGURE 4. Adsorption isotherm of Fe(III) on the AOPAN nanofibrous membrane. Bars represent standard deviations (n=3). Effect of Temperature and ph on Enzyme Activity The effect of temperature on the activity of free and immobilized is shown in Figure 5. It can be obviously observed that the optimum temperatures for the immobilized and free catalases were at approximately 40 o C and 35 o C respectively. The immobilized catalases exhibited higher stability than the free one, indicating the improved thermal stability of the immobilized enzymes. The multipoint interactions between enzyme and support might reduce the degree of conformation change of enzyme at higher temperature, which to some extent protected it from deactivation at the high temperature. FIGURE 3. SEM images of a PAN nanofibers ( 10000), b AOPAN nanofibers ( 10000). Journal of Engineered Fibers and Fabrics 149

5 Storage Stability The storage stability of immobilized and free enzymes is presented in Figure 7. Immobilized catalases lost 48% of its activity within 24 days, and the free catalase lost 80% of its activity during the same period. The results indicated that the storage stability of immobilized catalases was better than that of free catalases, which might be attributed to the immobilization of enzyme to a matrix limitation of their freedom to undergo conformational changes, resulting in increased stability towards denaturation [19-21]. FIGURE 5. Effect of temperature on activity: ( ) immobilized catalases; ( ) free catalases. Bars represent standard deviations (n=3). Changes in ph values could affect the enzyme conformation, and thus affect the binding and catalysis between the enzyme molecules and substrate [17]. The effect of ph on the activity of free and immobilized catalases is shown in Figure 6. FIGURE 7. Storage stability of catalases: ( ) immobilized catalases, ( ) free catalases. FIGURE 6. Effect of ph on activity: ( ) immobilized catalases: ( ) free catalases. Bars represent standard deviations (n=3). Performance of the EMR The flux of membrane and the conversion ratio of hydrogen peroxide of EMR at various pressures were presented in Figure 8. They indicated that the flux of the membrane increased very fast with the rise of the operation pressure, while the conversion ratio of hydrogen peroxide decreased mildly at the same condition. The optimal ph value was found at about 7.0 for free and immobilized enzyme, but the residual relative activity of the immobilized catalases was higher than that of the free catalases in the ph range between 5 and 8.5. Such results may be attributed to diffusion limitations of nanofibrous membranes and to the multipoint stabilization of catalases on the surface of the nanofibrous membranes that restrict the corresponding change of immobilized catalases conformation against ph inactivation [18]. FIGURE 8. Flux of membrane and conversion ratio of H 2 O 2 at various pressures. Journal of Engineered Fibers and Fabrics 150

6 Reusability of the EMR was estimated according to the production capacity of the EMR, it is shown in Figure 9. FIGURE 9. Reusability of the EMR. After 5 repeated uses, the production capacity of the EMR is about 68% of its initial value, which shows its potential value in actual application. CONCLUSION Catalases were successfully immobilized on the Fe(III) chelated AOPAN nanofibrous membranes, and the effects of ph and temperature on the catalases activity were studied. The residual relative activity of the immobilized and free catalase were 52% and 20% respectively, after storage for 24 days. The EMR was assembled on the base of Fe(III)-AOPAN nanofibrous membranes. Moreover, the flux and conversion ratio of EMR were calculated. As for reusability of EMR, the production capacity retained 68% after 5 repeated uses. These results showed that the immobilized catalases had a high affinity with the support, and the EMR has excellent performance. ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (Grant No.: ), the Natural Science Foundation of Anhui Province (Grant No.: ME87), and the Natural Science Foundation of Anhui Provincial Education Department (Grant No.: KJ2012A039). REFERNCES [1] Wang Y.J.; Hu Y.; Xu J.; et al; Immobilization of lipase with a special microstructure in composite hydrophilic CA/hydrophobic PTFE membrane for the chial separation of racemic ibuprofen; J. Membrane. Sci. 2007, 293, [2] Rios G.M.; Belleville M.P.; Paolucci D.; Sanchez J.; Progress in enzymatic membrane reactors- a review; J. Membrane. Sci. 2004, 242, [3] Akgol S.; Denizli A.; Novel metal-chelated affinity sorbents for reversible use in catalase adsorption; J. Mol. Catal. B-Enzym. 2004, 28, [4] Hong J.; Xu D.M.; Gong P.J.; Yu J.H.; Ma H.J.; Yao S.D.; Covalent-bonded immobilization of enzyme on hydrophilic polymercovering magnetic cnanogels; Micropor. Mesopor. Mat. 2008, 109, [5] Feng Q.; Xia X.; Wei A.F.; Wang X.Q.; Wei Q.F.; Preparation of Cu(II)-chelated poly(vinyl alcohol) nanofibrous membranes for catalase immobilization; J. Appl, Polym, Sci. 2011, 120, [6] 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, [7] Fong H.; Electrospun nylon6 nanofiber reinforced BIS-GMA/TEGDMA dental restorative composite resins; Polym. 2004, 45, [8] Sakai S.; Antoku K.; Yamaguchi T.; Development of electrospun poly(vinyl alcohol) fibers immobilizing lipase highly activated by alkyl-silicate for flow-through reactors; J. Membrane. Sci. 2008, 325, [9] Wang Z.G.; Wan L.S.; Xu Z.K.; Surface engineering of polyacrylonitrile-based asymmetric membranes towards biomedical applications: an overview; J. Membrane. Sci. 2007, 304, [10] Liu X.; Chen H.; Wang C.C.; Qu R.J.; Ji C.N.; Sun C.M.; Zhang Y.; Synthesis of porous acrylonitrile/methyl acrylate copolymer beads by suspended emulsion polymerization and their adsorption properties after amidoximation; J. Hazard. Mater. 2010, 175, Journal of Engineered Fibers and Fabrics 151

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