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Functionalized electrospun nanofibers from poly (AN-co-MMA) for enzyme immobilization
Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 140–148
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Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Functionalized electrospun nanofibers from poly (AN-co-MMA) for enzyme immobilization M.R. El-Aassar ∗ Polymer Materials Research Department, Institute of Advanced Technology and New Material, City of Scientific Research and Technology Applications, New Borg El-Arab City 21934, Alexandria, Egypt
a r t i c l e
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Article history: Received 30 April 2012 Received in revised form 20 August 2012 Accepted 11 September 2012 Available online 18 September 2012 Keywords: Functionalization Surface modification Nanofiber Enzyme immobilization
a b s t r a c t -Galactosidase from Aspergillus oryzae was immobilized on amino functionalized poly (Acrylonitrileco-Methyl methacrylate) poly (AN-co-MMA) nanofibers using glutaraldehyde. Among the four different factor used for activation, the activation of poly (AN-co-MMA) nanofibers by glutaraldehyde followed by covalent enzyme on activated support could stabilize the enzyme -galactosidase and was found to be effective. Different factors affecting the activation process were investigated and their impact on the activity and the retention of immobilized enzyme’s activity was monitored. Concentration of glutaraldehyde and activation temperature, time, and activation pH were found of a determined effect. The optimum concentration, reaction time, reaction temperatures, and activation pH value of glutaraldehyde are 5.0, 180 min, 65 ◦ C and 11.0, respectively. The scanning electron micrographs showed the change on the poly (AN-co-MMA) nanofibers surface revealing the successful immobilization of -galactosidase. Thermal and pH stabilities were found to be increased upon immobilization. The immobilized -galactosidase had better resistance to temperature and pH inactivation than did the free form. Finally, the immobilized -galactosidase retained 35% of its initial activity when stored at 4 ◦ C for 70 days and retained 64% of its initial activity after ten consecutive reactor batch cycles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Electrospinning is a convenient method for synthesizing continuous polymeric fibers with diameters ranging from a few nanometers to several hundred nanometers [1,2]. Electrospinning is a very versatile and cost effective process for producing multifunctional nanofibers from various polymers, polymer blends and composites, etc. [3,4]. Nanofibers/nanowebs produced by electrospinning technique have several remarkable characteristics such as a very big ratio of surface area to volume, pore size within nano range, unique physical characteristics and flexibility for chemical/physical modification and functionalization. It has been shown that the unique properties and multi-functionality of the nanowebs make them very interesting for applications in various areas including biotechnology, textiles and membranes/filters, etc. [3–10]. Many types of carriers and techniques have been used for the immobilization of -galactosidase. The most important requirement for a support material is that it should be insoluble in water and have a high capacity to bind with enzyme [11–13]. Due to often conflicting requirements of a good support, various inorganic/organic materials have been used as support
∗ Corresponding author. Tel.: +20 1150526049; fax: +20 34593414. E-mail address: Mohamed [email protected] 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.09.002
for immobilization of enzyme. However, problems are associated with all of these supports whether organic or inorganic, such as low stability toward microbial attacks, involvement of various chemicals, cost, and reusability, leading to the progressive replacement of these supports [11–15]. The very large surface area-to-volume ratio resulting from such small diameters compared with other known forms is desirable for immobilizing enzymes onto the surface. In addition, the small diameter of the fibers, resulting in a small diffusion resistance of the substrate and products due to the short diffusion path, is desirable for immobilizing enzymes onto the fibers. Another desirable feature is that nonwoven fabrics composed of enzymeimmobilizing electrospun fibers can be easily applied for continuous operations in bioreactors as membrane-shaped reaction components. -Galactosidase (EC 3.2.1.23) from different sources is currently used in the production of lactose free milk products. Hydrolysis of lactose improves product sweetness, makes milk consumption by people who suffer from lactose intolerance possible, and increases product quality and process efficiency in the dairy industry. This hydrolysis reaction could also be applied to the upgrading of cheese whey, a product of cheese processing, disposal of which constitutes a problem [16,17]. Different strategies have been proposed to immobilize enzymes: adsorption, covalent binding, entrapment in polymers and cross-linking. Covalent immobilization has the advantage of
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forming strong and stable linkages between the enzyme and the carrier that result in robust biocatalyst and it also eliminates the loss of activity by enzyme leakage from the support [18,19]. Glutaraldehyde activation of the carriers is one of the widely used techniques to immobilize enzymes. The methodology is quite simple and efficient, and in some instances, it even improves enzyme stability by multipoint or multisubunit immobilization. Moreover, glutaraldehyde has also been widely used to introduce intermolecular crosslinking in proteins or to modify adsorbed proteins on aminated supports [20]. In order to immobilize an enzyme to support covalently an alternative is to use glutaraldehyde chemistry. This can be made by two ways: immobilization of enzymes on glutaraldehyde activated support or adsorption of proteins on support and then cross-linking with glutaraldehyde [21]. Furthermore, the treatment with glutaraldehyde of previously immobilized enzymes has been reported to be a useful method to get an intense multipoint crosslink between the enzyme and the support, promoting an increase in the enzyme stability [21,22]. In the present study, -galactosidase was immobilized using poly (AN-co-MMA) copolymer nanofibers. And considering the previously mentioned ideas, we will study a possible explanation of the important role that some variables play in the activity/stability achieved when immobilizing enzymes on glutaraldehyde activated supports. Thermal and pH stability of both free and immobilized enzymes were determined. Also storage stability of the immobilized system was compared with free enzyme and reusable stability of immobilized enzyme was assayed. 2. Experimental 2.1. Chemicals and reagents Methyl methacrylate (MMA) was purchased from ACROS (USA), acrylonitrile (AN) and glutaraldehyde (GA) were obtained from BDH Chemical Ltd. (England), -galactosidase (E.C.3.2.1.23), Potassium per sulfate (K2 S2 O8 ), Polyethylenimine (PEI) [low mol wt, 50% soln. in water, Mn 1.800 (GPC); Typical Mw 2.000] were supplied from the Sigma Chemical Co. (St. Louis, MO, USA), Tetrahydrofuran (THF) were obtained from Fluka AG (Switzerland), Lactose monohydrate GRG was obtained from WINLAB Chemical Co. (England), All other chemicals were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Preparation and electrospinning of poly (AN-co-MMA) copolymer Poly (AN-co-MMA) copolymer in the particle form was prepared by solution polymerization [23]. Briefly, the copolymer solutions were electrospun using needleless laboratory machine NS LAB 500S based on Nanospider TM technology developed by Elmarco s.r.o., Czech Republic. The NSLAB 500S consists of spinning head tub where rotating spinning electrode is wetted in solution under high voltage. Nanofibers are coating exchangeable substrate belt which is moving along the static collecting electrode. Internal control parameters of the process are electrode distance, high voltage, electrode speed and substrate speed. External parameters used for control of electrospinning throughput and nanofiber quality are solution characteristics (viscosity, conductivity) and air properties (temperature, relative humidity). Nanospider setup was used to spin the poly (AN-co-MMA) copolymer. The procedure was typically as follows: poly (AN-co-MMA) copolymer was dissolved in aqueous Tetrahydrofuran (THF) in concentrations 10% (w/v). Poly (AN-co-MMA) copolymer aqueous Tetrahydrofuran (THF) solutions were fed into drum of Nanospider. A drum was used to feed polymer solution into the collector. Electrospinning Parameter for
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electrospinning of poly (AN-co-MMA) copolymer nanofibers were as follows: Speed of drum = 5.3 rpm, Voltage = 60.1 kV, Electrode distance = 19 mm, Humidity = 31%, and Temperature = 20 ◦ C), a positively charged jet of the poly (AN-co-MMA) copolymer aqueous Tetrahydrofuran (THF) solution formed from the drum and sprayed to a grounded drum. Tetrahydrofuran (THF) electrospun nanofibers were deposited and collected on the grounded drum.
2.3. Poly (AN-co-MMA) electrospun nanofibers surface modification Poly (AN-co-MMA) copolymer nanofibers were aminated by treating it with excess of an aqueous solution of Polyethylenimine (PEI). The nanofibers were submerged in PEI solution at 70 ◦ C for 1 h with gentle shaking. The nanofibers were collected and washed successively with hot distilled water to remove the excess PEI. The aminated nanofibers were dried in an oven at 55 ◦ C for 24 h. Poly (AN-co-MMA) modified nanofibers was activated using 20 ml of glutaraldehyde. After completion of the activation process, the poly (AN-co-MMA) activated nanofibers were taken out and rinsed with distilled water to remove unreached glutaraldehyde. The activated Poly (AN-co-MMA) nanofibers were then transferred to enzyme phosphate–citrate buffer solution (Fig. 1). -Galactosidase solution (5.0 mg/20 ml) was prepared by adding appropriate amount of -galactosidase powder to phosphate–citrate buffer (pH 4.4). A designed weight of nanofibers were submerged in 20 ml of galactosidase solution in a vertical orientation and shaken gently in a water bath at 40 ◦ C for 1 h. Then, for 16 h at 4 ◦ C to complete the immobilization process, the nanofibers were taken out and rinsed with buffer until no soluble protein was detectable in washings.
2.4. Activity assays of free and immobilized ˇ-galactosidase The catalytic activity of the free enzyme activity was measured by using -galactosidase solution (5.0 mg/20 ml) was prepared by adding 5.0 mg of -galactosidase powder to 20 ml phosphate–citrate buffer (pH 4.4), and using 50 ml of 100 mM lactose as the substrate in 50 mM phosphate buffer (pH 4.4). And the activity of the immobilized -galactosidase on the nanofibers in aqueous medium was determined according to the method reported previously [24]. The catalytic activity of the immobilized enzyme was determined by mixing 13.7 cm2 of catalytic poly (AN-co-MMA) nanofibers with 50 ml of 100 mM lactose in phosphate–citrate buffer solution (pH 4.4 at 40 ◦ C) in a vertical orientation and shaken gently in a water bath, for 30 min. Samples (0.1 ml each) were withdrawn every 5 min to assess the produced glucose using glucose Kit. The enzymatic activity was determined by the angular coefficient of the liner plot of the glucose production as a function of time.
2.5. Retention of activity, R.A (%) The success of any immobilization process is governed basically by keeping almost, if not all, of the enzyme activity after completion of the immobilization process, this factor known as retention of activity % Eq. (1): Retention of Activity (%) =
Measured Activity of Immobilized Enzyme (A) × 100 Measured Activity of Free Enzyme (B)
(1)
where (B) is the measure activity of free enzyme for the same amount of immobilized one.
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Fig. 1. The scheme of strategy for surface activation with glutaraldehyde on amino-groups for -galactosidase immobilization.
2.6. Thermal and pH stability measurements Free and immobilized -galactosidase preparations were stored in phosphate–citrate buffer solutions (20 mM, pH 4.4) at 30 ◦ C, 50 ◦ C and 70 ◦ C for 300 min, respectively. 0.005 g free -galactosidase solutions (0.005/20 mg/ml) or a certain amount of immobilized nanofibers were periodically withdrawn for activity assay. The residual activity was determined as above. The pH stabilities of the free and immobilized -galactosidase were assayed by immersing them in phosphate–citrate buffer solutions (20 mM) in the pH 3.0, 4.4 and 8.0 for 300 min at 40 ◦ C and then determining their activities.
2.7. Storage stability and reusability assay Activities of the free and immobilized -galactosidase were determined after storage in 50 mM phosphate–citrate buffer solution (pH 4.4) at 4 and 28 ◦ C. The measurements were performed at intervals of 1 week and 2 week within a period of 70 days. The reusability of bound -galactosidase was examined by conducting the activity measurement of bound -galactosidase at time intervals of 15 min. After each activity measurement, the bound -galactosidase was washed three times with 20 mM phosphate–citrate buffer, and the recycled supports subjected to the activity assay for the second cycle and so on.
2.8. Characterization of poly (AN-co-MMA) nanofiber 2.8.1. Morphological characterization (SEM) The surface morphology of poly (AN-co-MMA) nanofiber, and glutaraldehyde-activated nanofiber observed with the help of a scanning electron microscopy (Joel JSM-6380 LA, Japan) at an accelerated voltage of 20 kV. The surfaces were vacuum coated with gold for SEM. The diameters of the electrospun poly (AN-coMMA) nanofibers were measured directly from the printed SEM micrographs of fibers. The average diameters of the electrospun nanofibers was determined by measuring and averaging the diameter of approximately 50 random nanofibers in each sample using scanning electron microscopy software (SMILE VIEW SOFTWARE developed by JOEL on SEM- Model JEOL JSM-6380 LA) after sputtering by gold.
2.8.2. FT-IR spectroscopy FT-IR spectra were recorded using FT-IR spectrometer, Bruker, TENSOR Series FT-IR Spectrometer, Germany, connected to a PC, and analysis the data by IR Solution software, analytical methods are standard in OPUSTM software. 2.8.3. Thermal characterization (TGA) The thermal degradation behaviors of the poly (AN-co-MMA) fiber, aminated poly (AN-co-MMA) nanofiber, and activated poly (AN-co-MMA) nanofiber were studied using Thermo Gravimetric Analyzer (TA Instruments, Q500 TGA, United States); instrument in the temperature range from 20 ◦ C to 800 ◦ C under nitrogen at a flow rate of 40 ml/min and at a heating rate of 10 ◦ C/min. 3. Results and discussion The aminated surface of the poly (AN-co-MMA) nanofibers was specifically designed to be used with functional crosslinkers to covalently couple to the functional groups of biomolecules. In this study, poly (AN-co-MMA) nanofibers were synthesized and used as carriers for -galactosidase immobilization. These carriers have ester groups that are suitable for modification. Hence, these carriers were modified with diamines in order to introduce amino groups to their structure. The presence of amino groups is needed for glutaraldehyde modification. Glutaraldehyde can readily react with amino groups; therefore the aldehyde group content should be close to the amino group content on the poly (ANco-MMA) nanofibers. Factors affecting the activation step namely; glutaraldehyde concentration, reaction time, reaction temperature and reaction pH have been investigated and the obtained results are mentioned in the following. 3.1. Glutaraldehyde concentration Glutaraldehyde activation of aminated supports is one of the most popular techniques for immobilizing enzymes [22]. The precise control of the conditions during support activation with glutaraldehyde has enabled the modification of the amino groups of the matrix with one or two glutaraldehyde molecules [20]. Fig. 2 shows the effect of various concentrations of a glutaraldehyde solution on the activity and retention activity of immobilized galactosidase. The glutaraldehyde concentration varied from 0.5%
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Fig. 2. Effect of glutaraldehyde concentration on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
to 7.5%. As can be seen from Fig. 2, the amount of -galactosidase bound to the surface in the final stage of the procedure increased with increasing glutaraldehyde concentrations up to 2.5%. At concentrations over 2.5%, however, minimal increases in amount of bound -galactosidase were observed. Thus, in consideration of increases the number of available end aldehyde groups of glutaraldehyde to bind with a higher number of covalent bond with enzyme molecules. Fixation of enzyme there dimensional structure may leads to reduction of its active center availability for substrates. On the other hand, cause like “protein–protein” interaction could happened which consequently leads to reduce the activity of immobilization enzyme [25]. Higher mobility of immobilized enzyme with high concentration of glutaraldehyde could be easier due to removing from surface where easy to react with the substrate, resulting in the increase of the retention activity. The leveling off all activity and retention of activity at glutaraldehyde concentration above 7.5% is expected since all the enzyme molecules kept almost of its catalytic activity; 76% retention of activity. 3.2. Effect of activation temperature with glutaraldehyde Effect of variation activation temperature with glutaraldehyde on the catalytic activity, and retention of activity of immobilized galactosidase is shown in Fig. 3. From illustrated data it is clear that the activity linearly increase with temperature to reach its maximum value at 50 ◦ C. Further increase of temperature beyond 50 ◦ C, the activity linearly decreased to reach the lowest value at 80 ◦ C. According to the published results by Roberto Fernˇıandez-Lafuente et al. [20], proceeding of glutaraldehyde reaction at higher temperature may promoted the polymerization and yielded dimeric and cyclic forms which are less reactive toward amine groups. This is may explain the linear decrease of activity beyond 50 ◦ C. 3.3. Effect of activation time with glutaraldehyde Fig. 4 shows the influence of coupling time on the amount of immobilized -galactosidase, and retention of activity of immobilized enzyme. The catalytic activity of immobilized -galactosidase increased progressively with increasing concentration of glutaraldehyde, as can be observed in Fig. 4. Liner increment of activity has been observed with increasing reaction time up to 60 min after which, exponential increment obtained with reaction time increase up to 120 min which the maximum activity was obtained. It can be ascribed to the reason that at higher reaction time with glutaraldehyde, higher amount of -galactosidase was introduced
onto the surface of the poly (AN-co-MMA) nanofibers, also. The effect of reaction time with glutaraldehyde on the retention activity of immobilized -galactosidase illustrated in Fig. 4. By increasing the reaction time with glutaraldehyde, the amount of immobilized enzyme increased onto the surface of the poly (AN-co-MMA) nanofibers, and the retention activity increasing. The behavior of the catalytic activity and retention activity very similar, they showed approximately the same behavior when the reaction time increase. 3.4. Effect of activation pH with glutaraldehyde The effect of variation pH value of glutaraldehyde activating solution on the catalytic activity, and retention of activity of immobilized -galactosidase is presented in Fig. 5, linearly increase with pH up to its highest value at 10.0. This behavior may be referred to the deprotonation effect of alkaline medium on the amine groups which increase its reaction rate with end aldehyde groups of glutaraldehyde. This consequently leads to increase the amount of immobilized enzyme and hence the catalytic activity [21]. Further linear increase of the retention activity of immobilized -galactosidase has been observed with glutaraldehyde pH increase up to pH 10.0. This behavior may be explained according to assume that with low density of aldehyde groups, the enzyme molecules were linked not in “Optimum” structure. 3.5. Morphology of activated electrospun fibers The performance and morphology of the electrospun fiber were affected by the electrospinning process parameters. In the present study, the changes of chemical structure of nanofibers always reflect on its morphological characters. The scanning electron microscope examination of poly (AN-co-MMA) nanofiber, and glutaraldehyde-activated nanofiber was shown in Fig. 6. It is clear that the morphological structure of poly (AN-co-MMA) nanofiber differ from glutaraldehyde-activated poly (AN-co-MMA) nanofiber. The surface morphology of poly (AN-co-MMA) nanofiber before modification with glutaraldehyde (Fig. 6A) shows relatively smooth, defect-free fibers. Also the fibers obtained had cylindrical morphology and no fiber bundles, and the average fiber diameter of the poly (AN-co-MMA) nanofiber were in the range of 320 ± 100 nm, which have been changed to porous form after modification (Fig. 6B). The porous degree increases with increase of glutaraldehyde used in the modification step, but the total nanofiber structure were maintained.
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Fig. 3. Effect of activation temperature with glutaraldehyde on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
Fig. 4. Effect of activation time with glutaraldehyde on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
Fig. 5. Effect of Activation pH with glutaraldehyde on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
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Fig. 6. SEM photographs of poly (AN-co-MMA) nanofibers electrospun from THF solutions: (A) poly (AN-co-MMA) nanofiber, and (B) activated poly (AN-co-MMA) with GA.
3.6. FT-IR spectroscopic analysis Infrared spectroscopy has been frequently used to investigate the conformational changes of proteins such as poly (AN-coMMA) nanofiber and glutaraldehyde-activated nanofiber. Fig. 7 shows typical FTIR spectra in the range 600–4000 cm−1 for electrospun poly (AN-co-MMA) nanofiber and glutaraldehyde-activated nanofiber. As shown in Fig. 7(A), the structure of the poly (ANco-MMA) is characterized by typical absorption bands at around 1730 cm−1 for C O and 2241 cm−1 for C N. The secondary structure of the glutaraldehyde-activated nanofiber; from the chart it is clear the appearance of characteristic band of CHO groups at 1637 cm−1 . Fig. 7(B), the presence of such band proved the fictionalization of the surface with aldhyde groups as a result of modification with glutaraldehyde. 3.7. Thermal analysis Fig. 8 shows the thermogram of electrospun poly (AN-co-MMA) nanofiber, PEI aminated nanofiber, and glutaraldehyde-activated nanofiber. It is clear that attaching of glutaraldehyde molecules onto the surface of the nanofibers backbone enhances the thermal stability of the poly (AN-co-MMA) nanofiber. This was reflected on the shift of half weight loss temperature (T50 ) of poly
(AN-co-MMA) nanofiber from 360 ◦ C to 379 ◦ C of glutaraldehydeactivated nanofibers. Also in the weight loss, percent at temperature ranged from 100 ◦ C to 400 ◦ C. Such behavior confirms the formation of new chemical structure different from the native poly (AN-co-MMA) nanofiber structure. Possible cross-linking is also expected. 3.8. Effect of ˇ-galactosidase concentration on immobilization efficiency Immobilization of proteins on glutaraldehyde activated supports is a very versatile technique. However, several critical variables must be considered when designing either the support or the immobilization conditions. The immobilization capacity of galactosidase for glutaraldehyde groups functionalized nanofibers were determined by changing the initial concentration of galactosidase between 0.001 g and 0.015 g (Fig. 9). An increase the -galactosidase concentration in immobilization medium led to a linear increase in the amount of immobilized -galactosidase onto poly (AN-co-MMA) nanofibers up to 0.015 g -galactosidase in the immobilization medium. This increase in the observed activity must be associated with an increase in enzyme molecules available to bind with fixed aldehyde groups on the surface of nanoparticles. This leads consequently to reduce the formation of multi
Fig. 7. FTIR Spectra for: (A) poly (AN-co-MMA) nanofiber and (B) activated poly (AN-co-MMA) nanofiber in the range of 500–4000 cm−1 .
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Fig. 8. TGA Analysis: for (A) poly (AN-co-MMA) nanofiber, (B) poly (AN-co-MMA) nanofiber +PEI, and (C) poly (AN-co-MMA) nanofiber +PEI + GA.
attachment points and increase the number of immobilized enzyme molecules which in turn leading to rise of “protein–protein” interaction. This explanation is confirmed by the obtained behavior of enzyme retained activity [24].
immobilized enzyme being protected from conformational changes causing effect of the environment. Similar results have been previously reported for various covalently immobilized enzymes [25–28].
3.9. Effect of temperature on ˇ-galactosidase stability
3.10. Effect of pH on ˇ-galactosidase stability
The effect of immobilization on stability at several temperatures was evaluated in experiments in which -galactosidase was incubated for 5 h at temperatures 30 ◦ C, 50 ◦ C, and 70 ◦ C. The results are presented in Fig. 10. It can be seen that the free galactosidase lost about 100% of its initial activity at 70 ◦ C after a 300 min incubation period, after a 300 min heat treatment at 80 ◦ C; immobilized -galactosidase lost about 80% of its initial activity at the same temperature. The immobilized form onto 30◦ C and 50 ◦ C shows higher stability which lost only 16% and 36%, respectively, of its activity after 300 min. These results suggest that the immobilization led to a considerable increase of thermostability for -galactosidase and the thermal stability of immobilized -galactosidase becomes significantly higher than that of the free enzyme at high temperature. This is to the covalently
The pH stability of immobilized -galactosidase on poly (AN-co-MMA) nanofibers has also increased compared to free galactosidase. Fig. 11 shows the pH stability of free and immobilized enzyme in pH 3, pH 4.4, and pH 8. Inspecting the figure shows that the stability of immobilized form is higher in general. Almost linear decrement of the immobilized enzyme relative activity has been noticed at pH 4.4 where the immobilized form kept around 56% of its activity after 5 h incubation compared to about 48.5% of free form. Real advantage of immobilization is clear at pH 8.0 where the free from keeps around 20% of its original activity while the immobilized form keeps 40%. The presence of covalent bonds between the enzyme molecules and the nanofibers surface contributes in inducing structural stability which restricts the conformational changes leads to inactivation effect [29].
Fig. 9. Effect of -galactosidase concentration on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
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Fig. 10. Temperature stability of free -galactosidase and immobilized -galactosidase on the catalytic activity.
Fig. 11. pH stability of free -galactosidase and immobilized -galactosidase on the catalytic activity.
Fig. 12. Storage stability of the immobilized -galactosidase on the poly (AN-co-MMA) nanofibers.
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Fig. 13. Effect of repeated use on residual activity of immobilized -galactosidase on the poly (AN-co-MMA) nanofibers.
3.11. Storage stability of the immobilized ˇ-galactosidase An important advantage of immobilized enzymes over free enzymes is their improved stability for repeated use and storage. To investigate the stability of immobilized -galactosidase on the poly (AN-co-MMA) nanofibers, the ability of the immobilized enzyme to remain active was tested by monitoring the changes in activity after a predetermined length of time. Fig. 12 shows that the immobilized -galactosidase was stored in phosphate–citrate buffer solution at 4 ◦ C and 28 ◦ C separately and activities were measured periodically over duration of 70 days. Upon 70 days of storage, the catalytic activity of immobilized enzyme was retained 65% at 4 ◦ C and 23% at 28 ◦ C. This indicates that the immobilized enzymes effectively decrease denaturation, as well as obviously increase the endurance and stability of immobilized -galactosidase. 3.12. Operational stability of the immobilized ˇ-galactosidase In addition, reusability of immobilized enzymes that was important for their practical application was carried out by measuring the activity of the immobilized enzyme successive times. As shown in Fig. 13, shows the effect of repeated use on activity of the immobilized -galactosidase. After 10 repeated uses, the immobilized -galactosidase maintained more than 60% of its original activity after ten reuses. The loss of catalytic activity may be explained by the following two reasons. First, there may be some -galactosidase molecules, which were not chemically bonded on poly (AN-coMMA) nanofibers but only physically embedded, were lost during the process of measurement. Second, the diameter of poly (ANco-MMA) nanofibers may become larger and the surface area got smaller after several arrays due to the hydrophilicity of poly (ANco-MMA) nanofibers [30].
immobilized -galactosidase had better resistance to temperature and pH inactivation than did the free form. A high storage stability obtained with the immobilized -galactosidase indicates that the stability of -galactosidase increased upon immobilization on the poly (AN-co-MMA) nanofibers, the immobilized -galactosidase retained 35% of its initial activity when stored at 4 ◦ C for 70 days and retained 64% of its initial activity after ten consecutive reactor batch cycles. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
4. Conclusion One of the most important aims of enzyme technology is to enhance the conformational stability of the enzyme. The extent of stabilization depends on the enzyme structure, the immobilization methods, and type of support. In the present study, poly (AN-coMMA) nanofibers could be fabricated by electrospinning and the enzyme molecules could be covalently bound to the nanofiber with glutaraldehyde. The aminated nanofibers were used for immobilization of -galactosidase via glutaraldehyde coupling. The
[24] [25] [26] [27] [28] [29] [30]
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Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Functionalized electrospun nanofibers from poly (AN-co-MMA) for enzyme immobilization M.R. El-Aassar ∗ Polymer Materials Research Department, Institute of Advanced Technology and New Material, City of Scientific Research and Technology Applications, New Borg El-Arab City 21934, Alexandria, Egypt
a r t i c l e
i n f o
Article history: Received 30 April 2012 Received in revised form 20 August 2012 Accepted 11 September 2012 Available online 18 September 2012 Keywords: Functionalization Surface modification Nanofiber Enzyme immobilization
a b s t r a c t -Galactosidase from Aspergillus oryzae was immobilized on amino functionalized poly (Acrylonitrileco-Methyl methacrylate) poly (AN-co-MMA) nanofibers using glutaraldehyde. Among the four different factor used for activation, the activation of poly (AN-co-MMA) nanofibers by glutaraldehyde followed by covalent enzyme on activated support could stabilize the enzyme -galactosidase and was found to be effective. Different factors affecting the activation process were investigated and their impact on the activity and the retention of immobilized enzyme’s activity was monitored. Concentration of glutaraldehyde and activation temperature, time, and activation pH were found of a determined effect. The optimum concentration, reaction time, reaction temperatures, and activation pH value of glutaraldehyde are 5.0, 180 min, 65 ◦ C and 11.0, respectively. The scanning electron micrographs showed the change on the poly (AN-co-MMA) nanofibers surface revealing the successful immobilization of -galactosidase. Thermal and pH stabilities were found to be increased upon immobilization. The immobilized -galactosidase had better resistance to temperature and pH inactivation than did the free form. Finally, the immobilized -galactosidase retained 35% of its initial activity when stored at 4 ◦ C for 70 days and retained 64% of its initial activity after ten consecutive reactor batch cycles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Electrospinning is a convenient method for synthesizing continuous polymeric fibers with diameters ranging from a few nanometers to several hundred nanometers [1,2]. Electrospinning is a very versatile and cost effective process for producing multifunctional nanofibers from various polymers, polymer blends and composites, etc. [3,4]. Nanofibers/nanowebs produced by electrospinning technique have several remarkable characteristics such as a very big ratio of surface area to volume, pore size within nano range, unique physical characteristics and flexibility for chemical/physical modification and functionalization. It has been shown that the unique properties and multi-functionality of the nanowebs make them very interesting for applications in various areas including biotechnology, textiles and membranes/filters, etc. [3–10]. Many types of carriers and techniques have been used for the immobilization of -galactosidase. The most important requirement for a support material is that it should be insoluble in water and have a high capacity to bind with enzyme [11–13]. Due to often conflicting requirements of a good support, various inorganic/organic materials have been used as support
∗ Corresponding author. Tel.: +20 1150526049; fax: +20 34593414. E-mail address: Mohamed [email protected] 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.09.002
for immobilization of enzyme. However, problems are associated with all of these supports whether organic or inorganic, such as low stability toward microbial attacks, involvement of various chemicals, cost, and reusability, leading to the progressive replacement of these supports [11–15]. The very large surface area-to-volume ratio resulting from such small diameters compared with other known forms is desirable for immobilizing enzymes onto the surface. In addition, the small diameter of the fibers, resulting in a small diffusion resistance of the substrate and products due to the short diffusion path, is desirable for immobilizing enzymes onto the fibers. Another desirable feature is that nonwoven fabrics composed of enzymeimmobilizing electrospun fibers can be easily applied for continuous operations in bioreactors as membrane-shaped reaction components. -Galactosidase (EC 3.2.1.23) from different sources is currently used in the production of lactose free milk products. Hydrolysis of lactose improves product sweetness, makes milk consumption by people who suffer from lactose intolerance possible, and increases product quality and process efficiency in the dairy industry. This hydrolysis reaction could also be applied to the upgrading of cheese whey, a product of cheese processing, disposal of which constitutes a problem [16,17]. Different strategies have been proposed to immobilize enzymes: adsorption, covalent binding, entrapment in polymers and cross-linking. Covalent immobilization has the advantage of
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forming strong and stable linkages between the enzyme and the carrier that result in robust biocatalyst and it also eliminates the loss of activity by enzyme leakage from the support [18,19]. Glutaraldehyde activation of the carriers is one of the widely used techniques to immobilize enzymes. The methodology is quite simple and efficient, and in some instances, it even improves enzyme stability by multipoint or multisubunit immobilization. Moreover, glutaraldehyde has also been widely used to introduce intermolecular crosslinking in proteins or to modify adsorbed proteins on aminated supports [20]. In order to immobilize an enzyme to support covalently an alternative is to use glutaraldehyde chemistry. This can be made by two ways: immobilization of enzymes on glutaraldehyde activated support or adsorption of proteins on support and then cross-linking with glutaraldehyde [21]. Furthermore, the treatment with glutaraldehyde of previously immobilized enzymes has been reported to be a useful method to get an intense multipoint crosslink between the enzyme and the support, promoting an increase in the enzyme stability [21,22]. In the present study, -galactosidase was immobilized using poly (AN-co-MMA) copolymer nanofibers. And considering the previously mentioned ideas, we will study a possible explanation of the important role that some variables play in the activity/stability achieved when immobilizing enzymes on glutaraldehyde activated supports. Thermal and pH stability of both free and immobilized enzymes were determined. Also storage stability of the immobilized system was compared with free enzyme and reusable stability of immobilized enzyme was assayed. 2. Experimental 2.1. Chemicals and reagents Methyl methacrylate (MMA) was purchased from ACROS (USA), acrylonitrile (AN) and glutaraldehyde (GA) were obtained from BDH Chemical Ltd. (England), -galactosidase (E.C.3.2.1.23), Potassium per sulfate (K2 S2 O8 ), Polyethylenimine (PEI) [low mol wt, 50% soln. in water, Mn 1.800 (GPC); Typical Mw 2.000] were supplied from the Sigma Chemical Co. (St. Louis, MO, USA), Tetrahydrofuran (THF) were obtained from Fluka AG (Switzerland), Lactose monohydrate GRG was obtained from WINLAB Chemical Co. (England), All other chemicals were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Preparation and electrospinning of poly (AN-co-MMA) copolymer Poly (AN-co-MMA) copolymer in the particle form was prepared by solution polymerization [23]. Briefly, the copolymer solutions were electrospun using needleless laboratory machine NS LAB 500S based on Nanospider TM technology developed by Elmarco s.r.o., Czech Republic. The NSLAB 500S consists of spinning head tub where rotating spinning electrode is wetted in solution under high voltage. Nanofibers are coating exchangeable substrate belt which is moving along the static collecting electrode. Internal control parameters of the process are electrode distance, high voltage, electrode speed and substrate speed. External parameters used for control of electrospinning throughput and nanofiber quality are solution characteristics (viscosity, conductivity) and air properties (temperature, relative humidity). Nanospider setup was used to spin the poly (AN-co-MMA) copolymer. The procedure was typically as follows: poly (AN-co-MMA) copolymer was dissolved in aqueous Tetrahydrofuran (THF) in concentrations 10% (w/v). Poly (AN-co-MMA) copolymer aqueous Tetrahydrofuran (THF) solutions were fed into drum of Nanospider. A drum was used to feed polymer solution into the collector. Electrospinning Parameter for
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electrospinning of poly (AN-co-MMA) copolymer nanofibers were as follows: Speed of drum = 5.3 rpm, Voltage = 60.1 kV, Electrode distance = 19 mm, Humidity = 31%, and Temperature = 20 ◦ C), a positively charged jet of the poly (AN-co-MMA) copolymer aqueous Tetrahydrofuran (THF) solution formed from the drum and sprayed to a grounded drum. Tetrahydrofuran (THF) electrospun nanofibers were deposited and collected on the grounded drum.
2.3. Poly (AN-co-MMA) electrospun nanofibers surface modification Poly (AN-co-MMA) copolymer nanofibers were aminated by treating it with excess of an aqueous solution of Polyethylenimine (PEI). The nanofibers were submerged in PEI solution at 70 ◦ C for 1 h with gentle shaking. The nanofibers were collected and washed successively with hot distilled water to remove the excess PEI. The aminated nanofibers were dried in an oven at 55 ◦ C for 24 h. Poly (AN-co-MMA) modified nanofibers was activated using 20 ml of glutaraldehyde. After completion of the activation process, the poly (AN-co-MMA) activated nanofibers were taken out and rinsed with distilled water to remove unreached glutaraldehyde. The activated Poly (AN-co-MMA) nanofibers were then transferred to enzyme phosphate–citrate buffer solution (Fig. 1). -Galactosidase solution (5.0 mg/20 ml) was prepared by adding appropriate amount of -galactosidase powder to phosphate–citrate buffer (pH 4.4). A designed weight of nanofibers were submerged in 20 ml of galactosidase solution in a vertical orientation and shaken gently in a water bath at 40 ◦ C for 1 h. Then, for 16 h at 4 ◦ C to complete the immobilization process, the nanofibers were taken out and rinsed with buffer until no soluble protein was detectable in washings.
2.4. Activity assays of free and immobilized ˇ-galactosidase The catalytic activity of the free enzyme activity was measured by using -galactosidase solution (5.0 mg/20 ml) was prepared by adding 5.0 mg of -galactosidase powder to 20 ml phosphate–citrate buffer (pH 4.4), and using 50 ml of 100 mM lactose as the substrate in 50 mM phosphate buffer (pH 4.4). And the activity of the immobilized -galactosidase on the nanofibers in aqueous medium was determined according to the method reported previously [24]. The catalytic activity of the immobilized enzyme was determined by mixing 13.7 cm2 of catalytic poly (AN-co-MMA) nanofibers with 50 ml of 100 mM lactose in phosphate–citrate buffer solution (pH 4.4 at 40 ◦ C) in a vertical orientation and shaken gently in a water bath, for 30 min. Samples (0.1 ml each) were withdrawn every 5 min to assess the produced glucose using glucose Kit. The enzymatic activity was determined by the angular coefficient of the liner plot of the glucose production as a function of time.
2.5. Retention of activity, R.A (%) The success of any immobilization process is governed basically by keeping almost, if not all, of the enzyme activity after completion of the immobilization process, this factor known as retention of activity % Eq. (1): Retention of Activity (%) =
Measured Activity of Immobilized Enzyme (A) × 100 Measured Activity of Free Enzyme (B)
(1)
where (B) is the measure activity of free enzyme for the same amount of immobilized one.
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Fig. 1. The scheme of strategy for surface activation with glutaraldehyde on amino-groups for -galactosidase immobilization.
2.6. Thermal and pH stability measurements Free and immobilized -galactosidase preparations were stored in phosphate–citrate buffer solutions (20 mM, pH 4.4) at 30 ◦ C, 50 ◦ C and 70 ◦ C for 300 min, respectively. 0.005 g free -galactosidase solutions (0.005/20 mg/ml) or a certain amount of immobilized nanofibers were periodically withdrawn for activity assay. The residual activity was determined as above. The pH stabilities of the free and immobilized -galactosidase were assayed by immersing them in phosphate–citrate buffer solutions (20 mM) in the pH 3.0, 4.4 and 8.0 for 300 min at 40 ◦ C and then determining their activities.
2.7. Storage stability and reusability assay Activities of the free and immobilized -galactosidase were determined after storage in 50 mM phosphate–citrate buffer solution (pH 4.4) at 4 and 28 ◦ C. The measurements were performed at intervals of 1 week and 2 week within a period of 70 days. The reusability of bound -galactosidase was examined by conducting the activity measurement of bound -galactosidase at time intervals of 15 min. After each activity measurement, the bound -galactosidase was washed three times with 20 mM phosphate–citrate buffer, and the recycled supports subjected to the activity assay for the second cycle and so on.
2.8. Characterization of poly (AN-co-MMA) nanofiber 2.8.1. Morphological characterization (SEM) The surface morphology of poly (AN-co-MMA) nanofiber, and glutaraldehyde-activated nanofiber observed with the help of a scanning electron microscopy (Joel JSM-6380 LA, Japan) at an accelerated voltage of 20 kV. The surfaces were vacuum coated with gold for SEM. The diameters of the electrospun poly (AN-coMMA) nanofibers were measured directly from the printed SEM micrographs of fibers. The average diameters of the electrospun nanofibers was determined by measuring and averaging the diameter of approximately 50 random nanofibers in each sample using scanning electron microscopy software (SMILE VIEW SOFTWARE developed by JOEL on SEM- Model JEOL JSM-6380 LA) after sputtering by gold.
2.8.2. FT-IR spectroscopy FT-IR spectra were recorded using FT-IR spectrometer, Bruker, TENSOR Series FT-IR Spectrometer, Germany, connected to a PC, and analysis the data by IR Solution software, analytical methods are standard in OPUSTM software. 2.8.3. Thermal characterization (TGA) The thermal degradation behaviors of the poly (AN-co-MMA) fiber, aminated poly (AN-co-MMA) nanofiber, and activated poly (AN-co-MMA) nanofiber were studied using Thermo Gravimetric Analyzer (TA Instruments, Q500 TGA, United States); instrument in the temperature range from 20 ◦ C to 800 ◦ C under nitrogen at a flow rate of 40 ml/min and at a heating rate of 10 ◦ C/min. 3. Results and discussion The aminated surface of the poly (AN-co-MMA) nanofibers was specifically designed to be used with functional crosslinkers to covalently couple to the functional groups of biomolecules. In this study, poly (AN-co-MMA) nanofibers were synthesized and used as carriers for -galactosidase immobilization. These carriers have ester groups that are suitable for modification. Hence, these carriers were modified with diamines in order to introduce amino groups to their structure. The presence of amino groups is needed for glutaraldehyde modification. Glutaraldehyde can readily react with amino groups; therefore the aldehyde group content should be close to the amino group content on the poly (ANco-MMA) nanofibers. Factors affecting the activation step namely; glutaraldehyde concentration, reaction time, reaction temperature and reaction pH have been investigated and the obtained results are mentioned in the following. 3.1. Glutaraldehyde concentration Glutaraldehyde activation of aminated supports is one of the most popular techniques for immobilizing enzymes [22]. The precise control of the conditions during support activation with glutaraldehyde has enabled the modification of the amino groups of the matrix with one or two glutaraldehyde molecules [20]. Fig. 2 shows the effect of various concentrations of a glutaraldehyde solution on the activity and retention activity of immobilized galactosidase. The glutaraldehyde concentration varied from 0.5%
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Fig. 2. Effect of glutaraldehyde concentration on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
to 7.5%. As can be seen from Fig. 2, the amount of -galactosidase bound to the surface in the final stage of the procedure increased with increasing glutaraldehyde concentrations up to 2.5%. At concentrations over 2.5%, however, minimal increases in amount of bound -galactosidase were observed. Thus, in consideration of increases the number of available end aldehyde groups of glutaraldehyde to bind with a higher number of covalent bond with enzyme molecules. Fixation of enzyme there dimensional structure may leads to reduction of its active center availability for substrates. On the other hand, cause like “protein–protein” interaction could happened which consequently leads to reduce the activity of immobilization enzyme [25]. Higher mobility of immobilized enzyme with high concentration of glutaraldehyde could be easier due to removing from surface where easy to react with the substrate, resulting in the increase of the retention activity. The leveling off all activity and retention of activity at glutaraldehyde concentration above 7.5% is expected since all the enzyme molecules kept almost of its catalytic activity; 76% retention of activity. 3.2. Effect of activation temperature with glutaraldehyde Effect of variation activation temperature with glutaraldehyde on the catalytic activity, and retention of activity of immobilized galactosidase is shown in Fig. 3. From illustrated data it is clear that the activity linearly increase with temperature to reach its maximum value at 50 ◦ C. Further increase of temperature beyond 50 ◦ C, the activity linearly decreased to reach the lowest value at 80 ◦ C. According to the published results by Roberto Fernˇıandez-Lafuente et al. [20], proceeding of glutaraldehyde reaction at higher temperature may promoted the polymerization and yielded dimeric and cyclic forms which are less reactive toward amine groups. This is may explain the linear decrease of activity beyond 50 ◦ C. 3.3. Effect of activation time with glutaraldehyde Fig. 4 shows the influence of coupling time on the amount of immobilized -galactosidase, and retention of activity of immobilized enzyme. The catalytic activity of immobilized -galactosidase increased progressively with increasing concentration of glutaraldehyde, as can be observed in Fig. 4. Liner increment of activity has been observed with increasing reaction time up to 60 min after which, exponential increment obtained with reaction time increase up to 120 min which the maximum activity was obtained. It can be ascribed to the reason that at higher reaction time with glutaraldehyde, higher amount of -galactosidase was introduced
onto the surface of the poly (AN-co-MMA) nanofibers, also. The effect of reaction time with glutaraldehyde on the retention activity of immobilized -galactosidase illustrated in Fig. 4. By increasing the reaction time with glutaraldehyde, the amount of immobilized enzyme increased onto the surface of the poly (AN-co-MMA) nanofibers, and the retention activity increasing. The behavior of the catalytic activity and retention activity very similar, they showed approximately the same behavior when the reaction time increase. 3.4. Effect of activation pH with glutaraldehyde The effect of variation pH value of glutaraldehyde activating solution on the catalytic activity, and retention of activity of immobilized -galactosidase is presented in Fig. 5, linearly increase with pH up to its highest value at 10.0. This behavior may be referred to the deprotonation effect of alkaline medium on the amine groups which increase its reaction rate with end aldehyde groups of glutaraldehyde. This consequently leads to increase the amount of immobilized enzyme and hence the catalytic activity [21]. Further linear increase of the retention activity of immobilized -galactosidase has been observed with glutaraldehyde pH increase up to pH 10.0. This behavior may be explained according to assume that with low density of aldehyde groups, the enzyme molecules were linked not in “Optimum” structure. 3.5. Morphology of activated electrospun fibers The performance and morphology of the electrospun fiber were affected by the electrospinning process parameters. In the present study, the changes of chemical structure of nanofibers always reflect on its morphological characters. The scanning electron microscope examination of poly (AN-co-MMA) nanofiber, and glutaraldehyde-activated nanofiber was shown in Fig. 6. It is clear that the morphological structure of poly (AN-co-MMA) nanofiber differ from glutaraldehyde-activated poly (AN-co-MMA) nanofiber. The surface morphology of poly (AN-co-MMA) nanofiber before modification with glutaraldehyde (Fig. 6A) shows relatively smooth, defect-free fibers. Also the fibers obtained had cylindrical morphology and no fiber bundles, and the average fiber diameter of the poly (AN-co-MMA) nanofiber were in the range of 320 ± 100 nm, which have been changed to porous form after modification (Fig. 6B). The porous degree increases with increase of glutaraldehyde used in the modification step, but the total nanofiber structure were maintained.
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Fig. 3. Effect of activation temperature with glutaraldehyde on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
Fig. 4. Effect of activation time with glutaraldehyde on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
Fig. 5. Effect of Activation pH with glutaraldehyde on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
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Fig. 6. SEM photographs of poly (AN-co-MMA) nanofibers electrospun from THF solutions: (A) poly (AN-co-MMA) nanofiber, and (B) activated poly (AN-co-MMA) with GA.
3.6. FT-IR spectroscopic analysis Infrared spectroscopy has been frequently used to investigate the conformational changes of proteins such as poly (AN-coMMA) nanofiber and glutaraldehyde-activated nanofiber. Fig. 7 shows typical FTIR spectra in the range 600–4000 cm−1 for electrospun poly (AN-co-MMA) nanofiber and glutaraldehyde-activated nanofiber. As shown in Fig. 7(A), the structure of the poly (ANco-MMA) is characterized by typical absorption bands at around 1730 cm−1 for C O and 2241 cm−1 for C N. The secondary structure of the glutaraldehyde-activated nanofiber; from the chart it is clear the appearance of characteristic band of CHO groups at 1637 cm−1 . Fig. 7(B), the presence of such band proved the fictionalization of the surface with aldhyde groups as a result of modification with glutaraldehyde. 3.7. Thermal analysis Fig. 8 shows the thermogram of electrospun poly (AN-co-MMA) nanofiber, PEI aminated nanofiber, and glutaraldehyde-activated nanofiber. It is clear that attaching of glutaraldehyde molecules onto the surface of the nanofibers backbone enhances the thermal stability of the poly (AN-co-MMA) nanofiber. This was reflected on the shift of half weight loss temperature (T50 ) of poly
(AN-co-MMA) nanofiber from 360 ◦ C to 379 ◦ C of glutaraldehydeactivated nanofibers. Also in the weight loss, percent at temperature ranged from 100 ◦ C to 400 ◦ C. Such behavior confirms the formation of new chemical structure different from the native poly (AN-co-MMA) nanofiber structure. Possible cross-linking is also expected. 3.8. Effect of ˇ-galactosidase concentration on immobilization efficiency Immobilization of proteins on glutaraldehyde activated supports is a very versatile technique. However, several critical variables must be considered when designing either the support or the immobilization conditions. The immobilization capacity of galactosidase for glutaraldehyde groups functionalized nanofibers were determined by changing the initial concentration of galactosidase between 0.001 g and 0.015 g (Fig. 9). An increase the -galactosidase concentration in immobilization medium led to a linear increase in the amount of immobilized -galactosidase onto poly (AN-co-MMA) nanofibers up to 0.015 g -galactosidase in the immobilization medium. This increase in the observed activity must be associated with an increase in enzyme molecules available to bind with fixed aldehyde groups on the surface of nanoparticles. This leads consequently to reduce the formation of multi
Fig. 7. FTIR Spectra for: (A) poly (AN-co-MMA) nanofiber and (B) activated poly (AN-co-MMA) nanofiber in the range of 500–4000 cm−1 .
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Fig. 8. TGA Analysis: for (A) poly (AN-co-MMA) nanofiber, (B) poly (AN-co-MMA) nanofiber +PEI, and (C) poly (AN-co-MMA) nanofiber +PEI + GA.
attachment points and increase the number of immobilized enzyme molecules which in turn leading to rise of “protein–protein” interaction. This explanation is confirmed by the obtained behavior of enzyme retained activity [24].
immobilized enzyme being protected from conformational changes causing effect of the environment. Similar results have been previously reported for various covalently immobilized enzymes [25–28].
3.9. Effect of temperature on ˇ-galactosidase stability
3.10. Effect of pH on ˇ-galactosidase stability
The effect of immobilization on stability at several temperatures was evaluated in experiments in which -galactosidase was incubated for 5 h at temperatures 30 ◦ C, 50 ◦ C, and 70 ◦ C. The results are presented in Fig. 10. It can be seen that the free galactosidase lost about 100% of its initial activity at 70 ◦ C after a 300 min incubation period, after a 300 min heat treatment at 80 ◦ C; immobilized -galactosidase lost about 80% of its initial activity at the same temperature. The immobilized form onto 30◦ C and 50 ◦ C shows higher stability which lost only 16% and 36%, respectively, of its activity after 300 min. These results suggest that the immobilization led to a considerable increase of thermostability for -galactosidase and the thermal stability of immobilized -galactosidase becomes significantly higher than that of the free enzyme at high temperature. This is to the covalently
The pH stability of immobilized -galactosidase on poly (AN-co-MMA) nanofibers has also increased compared to free galactosidase. Fig. 11 shows the pH stability of free and immobilized enzyme in pH 3, pH 4.4, and pH 8. Inspecting the figure shows that the stability of immobilized form is higher in general. Almost linear decrement of the immobilized enzyme relative activity has been noticed at pH 4.4 where the immobilized form kept around 56% of its activity after 5 h incubation compared to about 48.5% of free form. Real advantage of immobilization is clear at pH 8.0 where the free from keeps around 20% of its original activity while the immobilized form keeps 40%. The presence of covalent bonds between the enzyme molecules and the nanofibers surface contributes in inducing structural stability which restricts the conformational changes leads to inactivation effect [29].
Fig. 9. Effect of -galactosidase concentration on the catalytic activity and retention of activity of immobilized enzyme onto amino-nanofibers surfaces.
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Fig. 10. Temperature stability of free -galactosidase and immobilized -galactosidase on the catalytic activity.
Fig. 11. pH stability of free -galactosidase and immobilized -galactosidase on the catalytic activity.
Fig. 12. Storage stability of the immobilized -galactosidase on the poly (AN-co-MMA) nanofibers.
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Fig. 13. Effect of repeated use on residual activity of immobilized -galactosidase on the poly (AN-co-MMA) nanofibers.
3.11. Storage stability of the immobilized ˇ-galactosidase An important advantage of immobilized enzymes over free enzymes is their improved stability for repeated use and storage. To investigate the stability of immobilized -galactosidase on the poly (AN-co-MMA) nanofibers, the ability of the immobilized enzyme to remain active was tested by monitoring the changes in activity after a predetermined length of time. Fig. 12 shows that the immobilized -galactosidase was stored in phosphate–citrate buffer solution at 4 ◦ C and 28 ◦ C separately and activities were measured periodically over duration of 70 days. Upon 70 days of storage, the catalytic activity of immobilized enzyme was retained 65% at 4 ◦ C and 23% at 28 ◦ C. This indicates that the immobilized enzymes effectively decrease denaturation, as well as obviously increase the endurance and stability of immobilized -galactosidase. 3.12. Operational stability of the immobilized ˇ-galactosidase In addition, reusability of immobilized enzymes that was important for their practical application was carried out by measuring the activity of the immobilized enzyme successive times. As shown in Fig. 13, shows the effect of repeated use on activity of the immobilized -galactosidase. After 10 repeated uses, the immobilized -galactosidase maintained more than 60% of its original activity after ten reuses. The loss of catalytic activity may be explained by the following two reasons. First, there may be some -galactosidase molecules, which were not chemically bonded on poly (AN-coMMA) nanofibers but only physically embedded, were lost during the process of measurement. Second, the diameter of poly (ANco-MMA) nanofibers may become larger and the surface area got smaller after several arrays due to the hydrophilicity of poly (ANco-MMA) nanofibers [30].
immobilized -galactosidase had better resistance to temperature and pH inactivation than did the free form. A high storage stability obtained with the immobilized -galactosidase indicates that the stability of -galactosidase increased upon immobilization on the poly (AN-co-MMA) nanofibers, the immobilized -galactosidase retained 35% of its initial activity when stored at 4 ◦ C for 70 days and retained 64% of its initial activity after ten consecutive reactor batch cycles. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]
4. Conclusion One of the most important aims of enzyme technology is to enhance the conformational stability of the enzyme. The extent of stabilization depends on the enzyme structure, the immobilization methods, and type of support. In the present study, poly (AN-coMMA) nanofibers could be fabricated by electrospinning and the enzyme molecules could be covalently bound to the nanofiber with glutaraldehyde. The aminated nanofibers were used for immobilization of -galactosidase via glutaraldehyde coupling. The
[24] [25] [26] [27] [28] [29] [30]
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